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Sample records for genes increases extracellular

  1. A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity of Bacillus brevis

    Kajino, Tsutomu; Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Udaka, Shigezo; Yamada, Yukio; Takahashi, Haruo

    2000-01-01

    We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system. PMID:10653729

  2. Silencing of acidic pathogenesis-related PR-1 genes increases extracellular beta-(1 -> 3)-glucanase activity at the onset of tobacco defence reactions

    Riviere, M.P.; Marais, A.; Ponchet, M.

    2008-01-01

    silenced. Plants lacking extracellular PR-1s were more susceptible than wild-type plants to the oomycete Phytophthora parasitica but displayed unaffected systemic acquired resistance and developmental resistance to this pathogen. Treatment with salicylic acid up-regulates the PR-1g gene, encoding a basic...... protein of the PR-1 family, in PR-1-deficient tobacco, indicating that PR-1 expression may repress that of PR-1g. This shows that acidic PR-1s are dispensable for expression of salicylic acid-dependent acquired resistances against P. parasitica and may reveal a functional overlap in tobacco defence......The class 1 pathogenesis-related (PR) proteins are thought to be involved in plant defence responses, but their molecular functions are unknown. The function of PR-1 was investigated in tobacco by generating stable PR-1a-silenced lines in which other acidic PR-1 genes (PR-1b and PR-1c) were...

  3. High-fat diet decreases energy expenditure and expression of genes controlling lipid metabolism, mitochondrial function and skeletal system development in the adipose tissue, along with increased expression of extracellular matrix remodelling- and inflammation-related genes.

    Choi, Myung-Sook; Kim, Young-Je; Kwon, Eun-Young; Ryoo, Jae Young; Kim, Sang Ryong; Jung, Un Ju

    2015-03-28

    The aim of the present study was to identify the genes differentially expressed in the visceral adipose tissue in a well-characterised mouse model of high-fat diet (HFD)-induced obesity. Male C57BL/6J mice (n 20) were fed either HFD (189 % of energy from fat) or low-fat diet (LFD, 42 % of energy from fat) for 16 weeks. HFD-fed mice exhibited obesity, insulin resistance, dyslipidaemia and adipose collagen accumulation, along with higher levels of plasma leptin, resistin and plasminogen activator inhibitor type 1, although there were no significant differences in plasma cytokine levels. Energy intake was similar in the two diet groups owing to lower food intake in the HFD group; however, energy expenditure was also lower in the HFD group than in the LFD group. Microarray analysis revealed that genes related to lipolysis, fatty acid metabolism, mitochondrial energy transduction, oxidation-reduction, insulin sensitivity and skeletal system development were down-regulated in HFD-fed mice, and genes associated with extracellular matrix (ECM) components, ECM remodelling and inflammation were up-regulated. The top ten up- or down-regulated genes include Acsm3, mt-Nd6, Fam13a, Cyp2e1, Rgs1 and Gpnmb, whose roles in the deterioration of obesity-associated adipose tissue are poorly understood. In conclusion, the genes identified here provide new therapeutic opportunities for prevention and treatment of diet-induced obesity.

  4. Eosinophils enhance WNT-5a and TGF-β1 genes expression in airway smooth muscle cells and promote their proliferation by increased extracellular matrix proteins production in asthma.

    Januskevicius, Andrius; Vaitkiene, Simona; Gosens, Reinoud; Janulaityte, Ieva; Hoppenot, Deimante; Sakalauskas, Raimundas; Malakauskas, Kestutis

    2016-06-13

    Recent studies have suggested that eosinophils may have a direct effect on airway smooth muscle cells (ASMC), causing their proliferation in patients with asthma, but the precise mechanism of the interaction between these cells remains unknown. We propose that changes in Wnt signaling activity and extracellular matrix (ECM) production may help explain these findings. Therefore, the aim of this study was to investigate the effect of eosinophils from asthmatic and non-asthmatic subjects on Wnt-5a, transforming growth factor β1 (TGF-β1), and ECM protein (fibronectin and collagen) gene expression and ASMC proliferation. A total of 18 subjects were involved in the study: 8 steroid-free asthma patients and 10 healthy subjects. Peripheral blood eosinophils were isolated using centrifugation and magnetic separation. An individual co-culture of eosinophils with human ASMC was prepared for each study subject. Adhesion of eosinophils to ASMC (evaluated by assaying eosinophil peroxidase activity) was determined following various incubation periods (30, 45, 60, 120, and 240 min). The expression of Wnt-5a, TGF-β1, and ECM protein genes in ASMC was measured using quantitative real-time polymerase chain reaction (PCR) after 24 h of co-culture. Proliferation of ASMC was measured using the Alamar blue method after 48 h and 72 h of co-culture with eosinophils. Eosinophils from asthmatic subjects demonstrated increased adhesion to ASMC compared with eosinophils from healthy subjects (p eosinophils from asthmatic subjects, while co-culture of ASMC with eosinophils from healthy subjects increased only TGF-β1 and fibronectin gene expression. ASMC proliferation was augmented after co-culture with eosinophils from asthma patients compared with co-culture with eosinophils from healthy subjects (p Eosinophils enhance Wnt-5a, TGF-β1, fibronectin, and collagen gene expression in ASMC and promote proliferation of these cells in asthma. ClinicalTrials.gov Identifier: NCT02648074 .

  5. Stimulation of Suicidal Erythrocyte Death by Increased Extracellular Phosphate Concentrations

    Jakob Voelkl

    2014-02-01

    Full Text Available Background/Aim: Anemia in renal insufficiency results in part from impaired erythrocyte formation due to erythropoietin and iron deficiency. Beyond that, renal insufficiency enhances eryptosis, the suicidal erythrocyte death characterized by phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be stimulated by increase of cytosolic Ca2+-activity ([Ca2+]i. Several uremic toxins have previously been shown to stimulate eryptosis. Renal insufficiency is further paralleled by increase of plasma phosphate concentration. The present study thus explored the effect of phosphate on erythrocyte death. Methods: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, and [Ca2+]i from Fluo3-fluorescence. Results: Following a 48 hours incubation, the percentage of phosphatidylserine exposing erythrocytes markedly increased as a function of extracellular phosphate concentration (from 0-5 mM. The exposure to 2 mM or 5 mM phosphate was followed by slight but significant hemolysis. [Ca2+]i did not change significantly up to 2 mM phosphate but significantly decreased at 5 mM phosphate. The effect of 2 mM phosphate on phosphatidylserine exposure was significantly augmented by increase of extracellular Ca2+ to 1.7 mM, and significantly blunted by nominal absence of extracellular Ca2+, by additional presence of pyrophosphate as well as by presence of p38 inhibitor SB203580. Conclusion: Increasing phosphate concentration stimulates erythrocyte membrane scrambling, an effect depending on extracellular but not intracellular Ca2+ concentration. It is hypothesized that suicidal erythrocyte death is triggered by complexed CaHPO4.

  6. How does the extracellular matrix direct gene expression

    Bissell, M J; Hall, H G; Parry, G

    1982-01-01

    Based on the existing literature, a model is presented that postulates a ''dynamic reciprocity'' between the extracellular matrix (ECM) on the one hand and the cytoskeleton and the nuclear matrix on the other hand. The ECM is postulated to exert physical and chemical influences on the geometry and the biochemistry of the cell via transmembrane receptors so as to alter the pattern of gene expression by changing the association of the cytoskeleton with mRNA and the interaction of the chromatin with the nuclear matrix. This, in turn, would affect the ECM, which would affect the cell.

  7. Activation of retinal glial (Müller cells by extracellular ATP induces pronounced increases in extracellular H+ flux.

    Boriana K Tchernookova

    Full Text Available Small alterations in extracellular acidity are potentially important modulators of neuronal signaling within the vertebrate retina. Here we report a novel extracellular acidification mechanism mediated by glial cells in the retina. Using self-referencing H+-selective microelectrodes to measure extracellular H+ fluxes, we show that activation of retinal Müller (glial cells of the tiger salamander by micromolar concentrations of extracellular ATP induces a pronounced extracellular H+ flux independent of bicarbonate transport. ADP, UTP and the non-hydrolyzable analog ATPγs at micromolar concentrations were also potent stimulators of extracellular H+ fluxes, but adenosine was not. The extracellular H+ fluxes induced by ATP were mimicked by the P2Y1 agonist MRS 2365 and were significantly reduced by the P2 receptor blockers suramin and PPADS, suggesting activation of P2Y receptors. Bath-applied ATP induced an intracellular rise in calcium in Müller cells; both the calcium rise and the extracellular H+ fluxes were significantly attenuated when calcium re-loading into the endoplasmic reticulum was inhibited by thapsigargin and when the PLC-IP3 signaling pathway was disrupted with 2-APB and U73122. The anion transport inhibitor DIDS also markedly reduced the ATP-induced increase in H+ flux while SITS had no effect. ATP-induced H+ fluxes were also observed from Müller cells isolated from human, rat, monkey, skate and lamprey retinae, suggesting a highly evolutionarily conserved mechanism of potential general importance. Extracellular ATP also induced significant increases in extracellular H+ flux at the level of both the outer and inner plexiform layers in retinal slices of tiger salamander which was significantly reduced by suramin and PPADS. We suggest that the novel H+ flux mediated by ATP-activation of Müller cells and of other glia as well may be a key mechanism modulating neuronal signaling in the vertebrate retina and throughout the brain.

  8. Co-transfection of decorin and interleukin-10 modulates pro-fibrotic extracellular matrix gene expression in human tenocyte culture

    Abbah, Sunny A.; Thomas, Dilip; Browne, Shane; O'Brien, Timothy; Pandit, Abhay; Zeugolis, Dimitrios I.

    2016-02-01

    Extracellular matrix synthesis and remodelling are driven by increased activity of transforming growth factor beta 1 (TGF-β1). In tendon tissue repair, increased activity of TGF-β1 leads to progressive fibrosis. Decorin (DCN) and interleukin 10 (IL-10) antagonise pathological collagen synthesis by exerting a neutralising effect via downregulation of TGF-β1. Herein, we report that the delivery of DCN and IL-10 transgenes from a collagen hydrogel system supresses the constitutive expression of TGF-β1 and a range of pro-fibrotic extracellular matrix genes.

  9. Smoking is associated with increased levels of extracellular peptidylarginine deiminase 2 (PAD2) in the lungs

    Damgaard, Dres; Friberg Bruun Nielsen, Michael; Quisgaard Gaunsbaek, Maria

    2015-01-01

    lavage (BAL) fluid from smokers, but intracellularly located PAD cannot be responsible for citrullination of extracellular self-antigens. We aimed to establish a link between smoking and extracellular PAD2 in the lungs. METHODS: BAL fluid samples were obtained from 13 smokers and 11 nonsmoking controls...... fluids from smokers as compared to non-smokers (p=0.018). The PAD2 content correlated with the overall CRP levels (p=0.009) and cell count (p=0.016). CONCLUSIONS: This first demonstration of increased levels of extracellular PAD2 in the lungs of smokers supports the hypothesis that smoking promotes...

  10. Transient extracellular application of gold nanostars increases hippocampal neuronal activity.

    Salinas, Kirstie; Kereselidze, Zurab; DeLuna, Frank; Peralta, Xomalin G; Santamaria, Fidel

    2014-08-20

    With the increased use of nanoparticles in biomedical applications there is a growing need to understand the effects that nanoparticles may have on cell function. Identifying these effects and understanding the mechanism through which nanoparticles interfere with the normal functioning of a cell is necessary for any therapeutic or diagnostic application. The aim of this study is to evaluate if gold nanoparticles can affect the normal function of neurons, namely their activity and coding properties. We synthesized star shaped gold nanoparticles of 180 nm average size. We applied the nanoparticles to acute mouse hippocampal slices while recording the action potentials from single neurons in the CA3 region. Our results show that CA3 hippocampal neurons increase their firing rate by 17% after the application of gold nanostars. The increase in excitability lasted for as much as 50 minutes after a transient 5 min application of the nanoparticles. Further analyses of the action potential shape and computational modeling suggest that nanoparticles block potassium channels responsible for the repolarization of the action potentials, thus allowing the cell to increase its firing rate. Our results show that gold nanoparticles can affect the coding properties of neurons by modifying their excitability.

  11. Transcription activator-like effector-mediated regulation of gene expression based on the inducible packaging and delivery via designed extracellular vesicles

    Lainšček, Duško; Lebar, Tina; Jerala, Roman

    2017-01-01

    Transcription activator-like effector (TALE) proteins present a powerful tool for genome editing and engineering, enabling introduction of site-specific mutations, gene knockouts or regulation of the transcription levels of selected genes. TALE nucleases or TALE-based transcription regulators are introduced into mammalian cells mainly via delivery of the coding genes. Here we report an extracellular vesicle-mediated delivery of TALE transcription regulators and their ability to upregulate the reporter gene in target cells. Designed transcriptional activator TALE-VP16 fused to the appropriate dimerization domain was enriched as a cargo protein within extracellular vesicles produced by mammalian HEK293 cells stimulated by Ca-ionophore and using blue light- or rapamycin-inducible dimerization systems. Blue light illumination or rapamycin increased the amount of the TALE-VP16 activator in extracellular vesicles and their addition to the target cells resulted in an increased expression of the reporter gene upon addition of extracellular vesicles to the target cells. This technology therefore represents an efficient delivery for the TALE-based transcriptional regulators. - Highlights: • Inducible dimerization enriched cargo proteins within extracellular vesicles (EV). • Farnesylation surpassed LAMP-1 fusion proteins for the EV packing. • Extracellular vesicles were able to deliver TALE regulators to mammalian cells. • TALE mediated transcriptional activation was achieved by designed EV.

  12. Indication of Horizontal DNA Gene Transfer by Extracellular Vesicles.

    Stefanie Fischer

    Full Text Available The biological relevance of extracellular vesicles (EV in intercellular communication has been well established. Thus far, proteins and RNA were described as main cargo. Here, we show that EV released from human bone marrow derived mesenchymal stromal cells (BM-hMSC also carry high-molecular DNA in addition. Extensive EV characterization revealed this DNA mainly associated with the outer EV membrane and to a smaller degree also inside the EV. Our EV purification protocol secured that DNA is not derived from apoptotic or necrotic cells. To analyze the relevance of EV-associated DNA we lentivirally transduced Arabidopsis thaliana-DNA (A.t.-DNA as indicator into BM-hMSC and generated EV. Using quantitative polymerase chain reaction (qPCR techniques we detected high copy numbers of A.t.-DNA in EV. In recipient hMSC incubated with tagged EV for two weeks we identified A.t.-DNA transferred to recipient cells. Investigation of recipient cell DNA using quantitative PCR and verification of PCR-products by sequencing suggested stable integration of A.t.-DNA. In conclusion, for the first time our proof-of-principle experiments point to horizontal DNA transfer into recipient cells via EV. Based on our results we assume that eukaryotic cells are able to exchange genetic information in form of DNA extending the known cargo of EV by genomic DNA. This mechanism might be of relevance in cancer but also during cell evolution and development.

  13. The effect of extracellular alkalinization on lactate metabolism of breast cancer stem cells: Overview of LDH-A, LDH-B, MCT1 and MCT4 gene expression

    Neolaka, G. M. G.; Yustisia, I.; Sadikin, M.; Wanandi, S. I.

    2017-08-01

    Changes in the metabolic status of cancer cells are presumed to be correlated with the adjustment of these cells to extracellular changes. Cell glycolysis increases the production of intracellular lactate catalyzed by the lactate dehydrogenases, both LDH-A and LDH-B. An increase in intracellular lactate can affect extracellular pH balance through monocarboxylate transporters, particularly MCT1 and MCT4. This study aimed to analyze the effects of extracellular alkalinization on the lactate metabolism of human breast cancer stem cells (BCSCs). In this study, human primary BCSCs (CD24-/CD44+ cells) were treated with 100 mM sodium bicarbonate for 0.5, 24, and 48 h in DMEM F12/HEPES. After incubation, extracellular pH was measured and cells were harvested to extract the total RNA and protein. The expression of LDH-A, LDH-B, MCT1, and MCT4 mRNA genes were analyzed using qRT-PCR method. Our study shows that administration of sodium bicarbonate in the BCSC culture medium could increase extracellular pH. To balance the increase of extracellular pH, BCSCs regulated the expression of LDH-A, LDH-B, MCT1, and MCT4 genes. As the extracellular pH increases, the expression of LDH-A that converts pyruvate to lactate increased along with the increase of MCT 4 and MCT 1 expression, which act as lactate transporters. As the incubation time increases, the pH decreases, leading to the suppression of LDH-A and increase of LDH-B expression that converts lactate into pyruvate. Therefore, we suggest that the extracellular alkalinization by sodium bicarbonate in BCSCs affected the genes that regulate lactate metabolism.

  14. Indomethacin abolishes cerebral blood flow increase in response to acetazolamide-induced extracellular acidosis

    Wang, Qian; Paulson, O B; Lassen, N A

    1993-01-01

    by acetazolamide (Az), a drug that induces brain extracellular acidosis, which triggers its effect on CBF. We compared the results to the inhibitory effect of indomethacin on the CBF increase during hypercapnia. Indomethacin but not diclofenac, another potent cyclooxygenase inhibitor, was found to block almost...... completely the CBF increase caused by Az-induced extracellular acidosis or by CO2, but it did not influence the CBF increase produced by sodium nitroprusside or papaverine. The results suggest that indomethacin exerts its action on CO2 reactivity by a nonprostaglandin-mediated mechanism that directly......Indomethacin is known to attenuate quite markedly the increase in CBF during hypercapnia. Hypercapnia is, in all likelihood, mediated by the acid shift at the level of the smooth muscle cells of the cerebral arterioles. We therefore investigated the effect of indomethacin on the CBF increase caused...

  15. Gene expression in gut symbiotic organ of stinkbug affected by extracellular bacterial symbiont.

    Futahashi, Ryo; Tanaka, Kohjiro; Tanahashi, Masahiko; Nikoh, Naruo; Kikuchi, Yoshitomo; Lee, Bok Luel; Fukatsu, Takema

    2013-01-01

    The bean bug Riptortus pedestris possesses a specialized symbiotic organ in a posterior region of the midgut, where numerous crypts harbor extracellular betaproteobacterial symbionts of the genus Burkholderia. Second instar nymphs orally acquire the symbiont from the environment, and the symbiont infection benefits the host by facilitating growth and by occasionally conferring insecticide resistance. Here we performed comparative transcriptomic analyses of insect genes expressed in symbiotic and non-symbiotic regions of the midgut dissected from Burkholderia-infected and uninfected R. pedestris. Expression sequence tag analysis of cDNA libraries and quantitative reverse transcription PCR identified a number of insect genes expressed in symbiosis- or aposymbiosis-associated patterns. For example, genes up-regulated in symbiotic relative to aposymbiotic individuals, including many cysteine-rich secreted protein genes and many cathepsin protease genes, are likely to play a role in regulating the symbiosis. Conversely, genes up-regulated in aposymbiotic relative to symbiotic individuals, including a chicken-type lysozyme gene and a defensin-like protein gene, are possibly involved in regulation of non-symbiotic bacterial infections. Our study presents the first transcriptomic data on gut symbiotic organ of a stinkbug, which provides initial clues to understanding of molecular mechanisms underlying the insect-bacterium gut symbiosis and sheds light on several intriguing commonalities between endocellular and extracellular symbiotic associations.

  16. Extracellular Histones Increase Tissue Factor Activity and Enhance Thrombin Generation by Human Blood Monocytes.

    Gould, Travis J; Lysov, Zakhar; Swystun, Laura L; Dwivedi, Dhruva J; Zarychanski, Ryan; Fox-Robichaud, Alison E; Liaw, Patricia C

    2016-12-01

    Sepsis is characterized by systemic activation of inflammatory and coagulation pathways in response to infection. Recently, it was demonstrated that histones released into the circulation by dying/activated cells may contribute to sepsis pathology. Although the ability of extracellular histones to modulate the procoagulant activities of several cell types has been investigated, the influence of histones on the hemostatic functions of circulating monocytes is unknown. To address this, we investigated the ability of histones to modulate the procoagulant potential of THP-1 cells and peripheral blood monocytes, and examined the effects of plasmas obtained from septic patients to induce a procoagulant phenotype on monocytic cells. Tissue factor (TF) activity assays were performed on histone-treated THP-1 cells and blood monocytes. Exposure of monocytic cells to histones resulted in increases in TF activity, TF antigen, and phosphatidylserine exposure. Histones modulate the procoagulant activity via engagement of Toll-like receptors 2 and 4, and this effect was abrogated with inhibitory antibodies. Increased TF activity of histone-treated cells corresponded to enhanced thrombin generation in plasma determined by calibrated automated thrombography. Finally, TF activity was increased on monocytes exposed to plasma from septic patients, an effect that was attenuated in plasma from patients receiving unfractionated heparin (UFH). Our studies suggest that increased levels of extracellular histones found in sepsis contribute to dysregulated coagulation by increasing TF activity of monocytes. These procoagulant effects can be partially ameliorated in sepsis patients receiving UFH, thereby identifying extracellular histones as a potential therapeutic target for sepsis treatment.

  17. Trichomonas vaginalis NTPDase and ecto-5'-nucleotidase hydrolyze guanine nucleotides and increase extracellular guanosine levels under serum restriction.

    Menezes, Camila Braz; Durgante, Juliano; de Oliveira, Rafael Rodrigues; Dos Santos, Victor Hugo Jacks Mendes; Rodrigues, Luiz Frederico; Garcia, Solange Cristina; Dos Santos, Odelta; Tasca, Tiana

    2016-05-01

    Trichomonas vaginalis is the aethiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease in the world. The purinergic signaling pathway is mediated by extracellular nucleotides and nucleosides that are involved in many biological effects as neurotransmission, immunomodulation and inflammation. Extracellular nucleotides can be hydrolyzed by a family of enzymes known as ectonucleotidases including the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) family which hydrolyses nucleosides triphosphate and diphosphate as preferential substrates and ecto-5'-nucleotidase which catalyzes the conversion of monophosphates into nucleosides. In T. vaginalis the E-NTPDase and ecto-5'-nucleotidase activities upon adenine nucleotides have already been characterized in intact trophozoites but little is known concerning guanine nucleotides and nucleoside. These enzymes may exert a crucial role on nucleoside generation, providing the purine sources for the synthesis de novo of these essential nutrients, sustaining parasite growth and survival. In this study, we investigated the hydrolysis profile of guanine-related nucleotides and nucleoside in intact trophozoites from long-term-grown and fresh clinical isolates of T. vaginalis. Knowing that guanine nucleotides are also substrates for T. vaginalis ectoenzymes, we evaluated the profile of nucleotides consumption and guanosine uptake in trophozoites submitted to a serum limitation condition. Results show that guanine nucleotides (GTP, GDP, GMP) were substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. Different T. vaginalis isolates (two from the ATCC and nine fresh clinical isolates) presented a heterogeneous hydrolysis profile. The serum culture condition increased E-NTPDase and ecto-5'-nucleotidase activities with high consumption of extracellular GTP generating enhanced GDP, GMP and guanosine levels as demonstrated by HPLC, with final

  18. Acute isoproterenol induces anxiety-like behavior in rats and increases plasma content of extracellular vesicles.

    Leo, Giuseppina; Guescini, Michele; Genedani, Susanna; Stocchi, Vilberto; Carone, Chiara; Filaferro, Monica; Sisti, Davide; Marcoli, Manuela; Maura, Guido; Cortelli, Pietro; Guidolin, Diego; Fuxe, Kjell; Agnati, Luigi Francesco

    2015-04-01

    Several clinical observations have demonstrated a link between heart rate and anxiety or panic disorders. In these patients, β-adrenergic receptor function was altered. This prompted us to investigate whether the β-adrenergic receptor agonist isoproterenol, at a dose that stimulates peripheral β-adrenergic system but has no effects at the central nervous system, can induce anxiety-like behavior in rats. Moreover, some possible messengers involved in the peripheral to brain communication were investigated. Our results showed that isoproterenol (5 mg kg(-1) i.p.) increased heart rate, evoked anxiety-like behavior, did not result in motor impairments and increased extracellular vesicle content in the blood. Plasma corticosterone level was unmodified as well as vesicular Hsp70 content. Vesicular miR-208 was also unmodified indicating a source of increased extracellular vesicles different from cardiomyocytes. We can hypothesize that peripheral extracellular vesicles might contribute to the β-adrenergic receptor-evoked anxiety-like behavior, acting as peripheral signals in modulating the mental state. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Extracellular Matrix-Regulated Gene Expression RequiresCooperation of SWI/SNF and Transcription Factors

    Xu, Ren; Spencer, Virginia A.; Bissell, Mina J.

    2006-05-25

    Extracellular cues play crucial roles in the transcriptional regulation of tissue-specific genes, but whether and how these signals lead to chromatin remodeling is not understood and subject to debate. Using chromatin immunoprecipitation (ChIP) assays and mammary-specific genes as models, we show here that extracellular matrix (ECM) molecules and prolactin cooperate to induce histone acetylation and binding of transcription factors and the SWI/SNF complex to the {beta}- and ?-casein promoters. Introduction of a dominant negative Brg1, an ATPase subunit of SWI/SNF complex, significantly reduced both {beta}- and ?-casein expression, suggesting that SWI/SNF-dependent chromatin remodeling is required for transcription of mammary-specific genes. ChIP analyses demonstrated that the ATPase activity of SWI/SNF is necessary for recruitment of RNA transcriptional machinery, but not for binding of transcription factors or for histone acetylation. Coimmunoprecipitation analyses showed that the SWI/SNF complex is associated with STAT5, C/EBP{beta}, and glucocorticoid receptor (GR). Thus, ECM- and prolactin-regulated transcription of the mammary-specific casein genes requires the concerted action of chromatin remodeling enzymes and transcription factors.

  20. Neuropeptide Y infusion into the shell region of the rat nucleus accumbens increases extracellular levels of dopamine

    Sørensen, Gunnar; Wegener, Gregers; Hasselstrøm, Jørgen

    2009-01-01

    Increases in extracellular dopamine in the shell region of the nucleus accumbens are centrally involved in mediating reinforcement of addictive drugs. Neuropeptide Y (NPY) and its receptors are present in the nucleus accumbens and have been implicated in addiction mechanisms. This study further...... explored the potential role of NPY in addiction mechanisms using microdialysis to measure extracellular dopamine in vivo after infusion of NPY directly into the accumbal shell region of adult rats. NPY was found to dose-dependently increase extracellular dopamine levels, indicating that NPY could play...... an important role in drug reinforcement by modulating accumbal dopamine levels...

  1. Increased Interleukin-32 Levels in Obesity Promote Adipose Tissue Inflammation and Extracellular Matrix Remodeling: Effect of Weight Loss.

    Catalán, Victoria; Gómez-Ambrosi, Javier; Rodríguez, Amaia; Ramírez, Beatriz; Valentí, Víctor; Moncada, Rafael; Landecho, Manuel F; Silva, Camilo; Salvador, Javier; Frühbeck, Gema

    2016-12-01

    Interleukin (IL)-32 is a recently described cytokine involved in the regulation of inflammation. We aimed to explore whether IL-32 could function as an inflammatory and angiogenic factor in human obesity and obesity-associated type 2 diabetes. Samples obtained from 90 subjects were used in the study. Obese patients exhibited higher expression levels of IL-32 in visceral adipose tissue (AT) as well as in subcutaneous AT and peripheral blood mononuclear cells. IL32 was mainly expressed by stromovascular fraction cells, and its expression was significantly enhanced by inflammatory stimuli and hypoxia, whereas no changes were found after the incubation with anti-inflammatory cytokines. The addition of exogenous IL-32 induced the expression of inflammation and extracellular matrix-related genes in human adipocyte cultures, and IL32-silenced adipocytes showed a downregulation of inflammatory genes. Furthermore, adipocyte-conditioned media obtained from obese patients increased IL32 gene expression in human monocyte cultures, whereas the adipocyte-conditioned media from lean volunteers had no effect on IL32 mRNA levels. These findings provide evidence, for the first time, about the inflammatory and remodeling properties of IL-32 in AT, implicating this cytokine in obesity-associated comorbidities. © 2016 by the American Diabetes Association.

  2. Novel phosphate-activated macrophages prevent ectopic calcification by increasing extracellular ATP and pyrophosphate

    Villa-Bellosta, Ricardo; Hamczyk, Magda R.; Andrés, Vicente

    2017-01-01

    Purpose Phosphorus is an essential nutrient involved in many pathobiological processes. Less than 1% of phosphorus is found in extracellular fluids as inorganic phosphate ion (Pi) in solution. High serum Pi level promotes ectopic calcification in many tissues, including blood vessels. Here, we studied the effect of elevated Pi concentration on macrophage polarization and calcification. Macrophages, present in virtually all tissues, play key roles in health and disease and display remarkable plasticity, being able to change their physiology in response to environmental cues. Methods and results High-throughput transcriptomic analysis and functional studies demonstrated that Pi induces unpolarized macrophages to adopt a phenotype closely resembling that of alternatively-activated M2 macrophages, as revealed by arginine hydrolysis and energetic and antioxidant profiles. Pi-induced macrophages showed an anti-calcifying action mediated by increased availability of extracellular ATP and pyrophosphate. Conclusion We conclude that the ability of Pi-activated macrophages to prevent calcium-phosphate deposition is a compensatory mechanism protecting tissues from hyperphosphatemia-induced pathologic calcification. PMID:28362852

  3. Inflammaging and Frailty Status Do Not Result in an Increased Extracellular Vesicle Concentration in Circulation

    Ainhoa Alberro

    2016-07-01

    Full Text Available In the last decades extracellular vesicles (EVs have emerged as key players for intercellular communication. In the case of inflammation, several studies have reported that EV levels are increased in circulation during inflammatory episodes. Based on this, we investigated whether aging results in elevated EV number, as a basal proinflammatory status termed “inflammaging” has been described in aged individuals. Moreover, we also hypothesized that frailty and dependence conditions of the elderly could affect EV concentration in plasma. Results showed that inflammaging, frailty or dependence status do not result in EV increase, at least in the total number of EVs in circulation. These results open a new perspective for investigating the role of EVs in human aging and in the inflammaging process.

  4. Inflammaging and Frailty Status Do Not Result in an Increased Extracellular Vesicle Concentration in Circulation.

    Alberro, Ainhoa; Sáenz-Cuesta, Matías; Muñoz-Culla, Maider; Mateo-Abad, Maider; Gonzalez, Esperanza; Carrasco-Garcia, Estefania; Araúzo-Bravo, Marcos J; Matheu, Ander; Vergara, Itziar; Otaegui, David

    2016-07-20

    In the last decades extracellular vesicles (EVs) have emerged as key players for intercellular communication. In the case of inflammation, several studies have reported that EV levels are increased in circulation during inflammatory episodes. Based on this, we investigated whether aging results in elevated EV number, as a basal proinflammatory status termed "inflammaging" has been described in aged individuals. Moreover, we also hypothesized that frailty and dependence conditions of the elderly could affect EV concentration in plasma. Results showed that inflammaging, frailty or dependence status do not result in EV increase, at least in the total number of EVs in circulation. These results open a new perspective for investigating the role of EVs in human aging and in the inflammaging process.

  5. Mutations in the collagen XII gene define a new form of extracellular matrix-related myopathy.

    Hicks, Debbie; Farsani, Golara Torabi; Laval, Steven; Collins, James; Sarkozy, Anna; Martoni, Elena; Shah, Ashoke; Zou, Yaqun; Koch, Manuel; Bönnemann, Carsten G; Roberts, Mark; Lochmüller, Hanns; Bushby, Kate; Straub, Volker

    2014-05-01

    Bethlem myopathy (BM) [MIM 158810] is a slowly progressive muscle disease characterized by contractures and proximal weakness, which can be caused by mutations in one of the collagen VI genes (COL6A1, COL6A2 and COL6A3). However, there may be additional causal genes to identify as in ∼50% of BM cases no mutations in the COL6 genes are identified. In a cohort of -24 patients with a BM-like phenotype, we first sequenced 12 candidate genes based on their function, including genes for known binding partners of collagen VI, and those enzymes involved in its correct post-translational modification, assembly and secretion. Proceeding to whole-exome sequencing (WES), we identified mutations in the COL12A1 gene, a member of the FACIT collagens (fibril-associated collagens with interrupted triple helices) in five individuals from two families. Both families showed dominant inheritance with a clinical phenotype resembling classical BM. Family 1 had a single-base substitution that led to the replacement of one glycine residue in the triple-helical domain, breaking the Gly-X-Y repeating pattern, and Family 2 had a missense mutation, which created a mutant protein with an unpaired cysteine residue. Abnormality at the protein level was confirmed in both families by the intracellular retention of collagen XII in patient dermal fibroblasts. The mutation in Family 2 leads to the up-regulation of genes associated with the unfolded protein response (UPR) pathway and swollen, dysmorphic rough-ER. We conclude that the spectrum of causative genes in extracellular matrix (ECM)-related myopathies be extended to include COL12A1.

  6. Altered expression of mitochondrial and extracellular matrix genes in the heart of human fetuses with chromosome 21 trisomy

    Olla Carlo

    2007-08-01

    Full Text Available Abstract Background The Down syndrome phenotype has been attributed to overexpression of chromosome 21 (Hsa21 genes. However, the expression profile of Hsa21 genes in trisomic human subjects as well as their effects on genes located on different chromosomes are largely unknown. Using oligonucleotide microarrays we compared the gene expression profiles of hearts of human fetuses with and without Hsa21 trisomy. Results Approximately half of the 15,000 genes examined (87 of the 168 genes on Hsa21 were expressed in the heart at 18–22 weeks of gestation. Hsa21 gene expression was globally upregulated 1.5 fold in trisomic samples. However, not all genes were equally dysregulated and 25 genes were not upregulated at all. Genes located on other chromosomes were also significantly dysregulated. Functional class scoring and gene set enrichment analyses of 473 genes, differentially expressed between trisomic and non-trisomic hearts, revealed downregulation of genes encoding mitochondrial enzymes and upregulation of genes encoding extracellular matrix proteins. There were no significant differences between trisomic fetuses with and without heart defects. Conclusion We conclude that dosage-dependent upregulation of Hsa21 genes causes dysregulation of the genes responsible for mitochondrial function and for the extracellular matrix organization in the fetal heart of trisomic subjects. These alterations might be harbingers of the heart defects associated with Hsa21 trisomy, which could be based on elusive mechanisms involving genetic variability, environmental factors and/or stochastic events.

  7. Identification of Plagl1/Zac1 binding sites and target genes establishes its role in the regulation of extracellular matrix genes and the imprinted gene network.

    Varrault, Annie; Dantec, Christelle; Le Digarcher, Anne; Chotard, Laëtitia; Bilanges, Benoit; Parrinello, Hugues; Dubois, Emeric; Rialle, Stéphanie; Severac, Dany; Bouschet, Tristan; Journot, Laurent

    2017-10-13

    PLAGL1/ZAC1 undergoes parental genomic imprinting, is paternally expressed, and is a member of the imprinted gene network (IGN). It encodes a zinc finger transcription factor with anti-proliferative activity and is a candidate tumor suppressor gene on 6q24 whose expression is frequently lost in various neoplasms. Conversely, gain of PLAGL1 function is responsible for transient neonatal diabetes mellitus, a rare genetic disease that results from defective pancreas development. In the present work, we showed that Plagl1 up-regulation was not associated with DNA damage-induced cell cycle arrest. It was rather associated with physiological cell cycle exit that occurred with contact inhibition, growth factor withdrawal, or cell differentiation. To gain insights into Plagl1 mechanism of action, we identified Plagl1 target genes by combining chromatin immunoprecipitation and genome-wide transcriptomics in transfected cell lines. Plagl1-elicited gene regulation correlated with multiple binding to the proximal promoter region through a GC-rich motif. Plagl1 target genes included numerous genes involved in signaling, cell adhesion, and extracellular matrix composition, including collagens. Plagl1 targets also included 22% of the 409 genes that make up the IGN. Altogether, this work identified Plagl1 as a transcription factor that coordinated the regulation of a subset of IGN genes and controlled extracellular matrix composition. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Extracellular matrix influence in Streptococcus mutans gene expression in a cariogenic biofilm.

    Florez Salamanca, E J; Klein, M I

    2018-04-01

    Caries etiology is biofilm-diet-dependent. Biofilms are highly dynamic and structured microbial communities enmeshed in a three-dimensional extracellular matrix. The study evaluated the expression dynamics of Streptococcus mutans genes associated with exopolysaccharides (EPS) (gtfBCD, gbpB, dexA), lipoteichoic acids (LTA) (dltABCD, SMU_775c) and extracellular DNA (eDNA) (lytST, lrgAB, ccpA) during matrix development within a mixed-species biofilm of S. mutans, Actinomyces naeslundii and Streptococcus gordonii. Mixed-species biofilms using S. mutans strains UA159 or ΔgtfB formed on saliva-coated hydroxyapatite discs were submitted to a nutritional challenge (providing an abundance of sucrose and starch). Biofilms were removed at eight developmental stages for gene expression analysis by quantitative polymerase chain reaction. The pH of spent culture media remained acidic throughout the experimental periods, being lower after sucrose and starch exposure. All genes were expressed at all biofilm developmental phases. EPS- and LTA-associated genes had a similar expression profile for both biofilms, presenting lower levels of expression at 67, 91 and 115 hours and a peak of expression at 55 hours, but having distinct expression magnitudes, with lower values for ΔgtfB (eg, fold-difference of ~382 for gtfC and ~16 for dltB at 43 hours). The eDNA-associated genes presented different dynamics of expression between both strains. In UA159 biofilms lrgA and lrgB genes were highly expressed at 29 hours (which were ~13 and ~5.4 times vs ΔgtfB, respectively), whereas in ΔgtfB biofilms an inverse relationship between lytS and lrgA and lrgB expression was detected. Therefore, the deletion of gtfB influences dynamics and magnitude of expression of genes associated with matrix main components. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Changes in cortical cytoskeletal and extracellular matrix gene expression in prostate cancer are related to oncogenic ERG deregulation

    Schulz, Wolfgang A; Ingenwerth, Marc; Djuidje, Carolle E; Hader, Christiane; Rahnenführer, Jörg; Engers, Rainer

    2010-01-01

    The cortical cytoskeleton network connects the actin cytoskeleton to various membrane proteins, influencing cell adhesion, polarity, migration and response to extracellular signals. Previous studies have suggested changes in the expression of specific components in prostate cancer, especially of 4.1 proteins (encoded by EPB41 genes) which form nodes in this network. Expression of EPB41L1, EPB41L2, EPB41L3 (protein: 4.1B), EPB41L4B (EHM2), EPB41L5, EPB49 (dematin), VIL2 (ezrin), and DLG1 (summarized as „cortical cytoskeleton' genes) as well as ERG was measured by quantitative RT-PCR in a well-characterized set of 45 M0 prostate adenocarcinoma and 13 benign tissues. Hypermethylation of EPB41L3 and GSTP1 was compared in 93 cancer tissues by methylation-specific PCR. Expression of 4.1B was further studied by immunohistochemistry. EPB41L1 and EPB41L3 were significantly downregulated and EPB41L4B was upregulated in cancer tissues. Low EPB41L1 or high EPB41L4B expression were associated with earlier biochemical recurrence. None of the other cortical cytoskeleton genes displayed expression changes, in particular EPB49 and VIL2, despite hints from previous studies. EPB41L3 downregulation was significantly associated with hypermethylation of its promoter and strongly correlated with GSTP1 hypermethylation. Protein 4.1B was detected most strongly in the basal cells of normal prostate epithelia. Its expression in carcinoma cells was similar to the weaker one in normal luminal cells. EPB41L3 downregulation and EPB41L4B upregulation were essentially restricted to the 22 cases with ERG overexpression. Expression changes in EPB41L3 and EPB41L4B closely paralleled those previously observed for the extracellular matrix genes FBLN1 and SPOCK1, respectively. Specific changes in the cortical cytoskeleton were observed during prostate cancer progression. They parallel changes in the expression of extracellular matrix components and all together appear to be associated with

  10. Acute extracellular fluid volume changes increase ileocolonic resistance to saline flow in anesthetized dogs

    Santiago Jr. A.T.

    1997-01-01

    Full Text Available We determined the effect of acute extracellular fluid volume changes on saline flow through 4 gut segments (ileocolonic, ileal, ileocolonic sphincter and proximal colon, perfused at constant pressure in anesthetized dogs. Two different experimental protocols were used: hypervolemia (iv saline infusion, 0.9% NaCl, 20 ml/min, volume up to 5% body weight and controlled hemorrhage (up to a 50% drop in mean arterial pressure. Mean ileocolonic flow (N = 6 was gradually and significantly decreased during the expansion (17.1%, P<0.05 and expanded (44.9%, P<0.05 periods while mean ileal flow (N = 7 was significantly decreased only during the expanded period (38%, P<0.05. Mean colonic flow (N = 7 was decreased during expansion (12%, P<0.05 but returned to control levels during the expanded period. Mean ileocolonic sphincter flow (N = 6 was not significantly modified. Mean ileocolonic flow (N = 10 was also decreased after hemorrhage (retracted period by 17% (P<0.05, but saline flow was not modified in the other separate circuits (N = 6, 5 and 4 for ileal, ileocolonic sphincter and colonic groups, respectively. The expansion effect was blocked by atropine (0.5 mg/kg, iv both on the ileocolonic (N = 6 and ileal (N = 5 circuits. Acute extracellular fluid volume retraction and expansion increased the lower gastrointestinal resistances to saline flow. These effects, which could physiologically decrease the liquid volume being supplied to the colon, are possible mechanisms activated to acutely balance liquid volume deficit and excess.

  11. Suramin Inhibits Osteoarthritic Cartilage Degradation by Increasing Extracellular Levels of Chondroprotective Tissue Inhibitor of Metalloproteinases 3.

    Chanalaris, Anastasios; Doherty, Christine; Marsden, Brian D; Bambridge, Gabriel; Wren, Stephen P; Nagase, Hideaki; Troeberg, Linda

    2017-10-01

    Osteoarthritis is a common degenerative joint disease for which no disease-modifying drugs are currently available. Attempts to treat the disease with small molecule inhibitors of the metalloproteinases that degrade the cartilage matrix have been hampered by a lack of specificity. We aimed to inhibit cartilage degradation by augmenting levels of the endogenous metalloproteinase inhibitor, tissue inhibitor of metalloproteinases (TIMP)-3, through blocking its interaction with the endocytic scavenger receptor, low-density lipoprotein receptor-related protein 1 (LRP1). We discovered that suramin (C 51 H 40 N 6 O 23 S 6 ) bound to TIMP-3 with a K D value of 1.9 ± 0.2 nM and inhibited its endocytosis via LRP1, thus increasing extracellular levels of TIMP-3 and inhibiting cartilage degradation by the TIMP-3 target enzyme, adamalysin-like metalloproteinase with thrombospondin motifs 5. NF279 (8,8'-[carbonyl bis (imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)] bis -1,3,5-naphthalenetrisulfonic acid hexasodium salt), a structural analog of suramin, has an increased affinity for TIMP-3 and increased ability to inhibit TIMP-3 endocytosis and protect cartilage. Suramin is thus a promising scaffold for the development of novel therapeutics to increase TIMP-3 levels and inhibit cartilage degradation in osteoarthritis. Copyright © 2017 by The Author(s).

  12. Increased postdialysis systolic blood pressure is associated with extracellular overhydration in hemodialysis outpatients.

    Nongnuch, Arkom; Campbell, Neil; Stern, Edward; El-Kateb, Sally; Fuentes, Laura; Davenport, Andrew

    2015-02-01

    Recently, intradialytic hypertension was reported to be associated with increased mortality for hemodialysis patients. To determine whether volume status plays a role in dialysis-associated hypertension, we prospectively audited 531 patients that had volume assessments measured by multiple-frequency bioelectrical impedance during their midweek dialysis session. Mean pre- and postdialysis weights were 73.2 vs 71.7 kg, and systolic blood pressures (SBPs) 140.5 vs. 130.3 mm Hg, respectively. Patients were divided into groups based on a fall in SBP of 20 mm Hg or more (32%), an increased SBP of 10 mm Hg or more (18%), and a stable group (50%). There were no differences in patient demographics, dialysis prescriptions, predialysis weight, total body (TBW), and extracellular (ECW) and intracellular water (ICW). However, the change in weight was significantly less in the increased blood pressure group (1.01 kg vs. stable 1.65, and 1.7 hypotensive). The ratio of ECW to TBW was significantly higher in the increased blood pressure group, particularly post dialysis (39.1 vs. stable 38.7% and fall in blood pressure group 38.7%). ECW overhydration was significantly greater in the increased blood pressure group post dialysis (0.7 (0.17 to 1.1) vs. stable 0.39 (-0.2 to 0.95) and fall in blood pressure group 0.38 (-0.19 to 0.86) liter). We found that patients who had increased blood pressure post dialysis had greater hydration status, particularly ECW. Thus, patients who increase their blood pressure post dialysis should have review of target weight, consideration of lowering the post-dialysis weight, and may benefit from increasing dialysis session time or frequency.

  13. Increased level of extracellular ATP at tumor sites: in vivo imaging with plasma membrane luciferase.

    Patrizia Pellegatti

    2008-07-01

    Full Text Available There is growing awareness that tumour cells build up a "self-advantageous" microenvironment that reduces effectiveness of anti-tumour immune response. While many different immunosuppressive mechanisms are likely to come into play, recent evidence suggests that extracellular adenosine acting at A2A receptors may have a major role in down-modulating the immune response as cancerous tissues contain elevated levels of adenosine and adenosine break-down products. While there is no doubt that all cells possess plasma membrane adenosine transporters that mediate adenosine uptake and may also allow its release, it is now clear that most of extracellularly-generated adenosine originates from the catabolism of extracellular ATP.Measurement of extracellular ATP is generally performed in cell supernatants by HPLC or soluble luciferin-luciferase assay, thus it generally turns out to be laborious and inaccurate. We have engineered a chimeric plasma membrane-targeted luciferase that allows in vivo real-time imaging of extracellular ATP. With this novel probe we have measured the ATP concentration within the tumour microenvironment of several experimentally-induced tumours.Our results show that ATP in the tumour interstitium is in the hundreds micromolar range, while it is basically undetectable in healthy tissues. Here we show that a chimeric plasma membrane-targeted luciferase allows in vivo detection of high extracellular ATP concentration at tumour sites. On the contrary, tumour-free tissues show undetectable extracellular ATP levels. Extracellular ATP may be crucial for the tumour not only as a stimulus for growth but also as a source of an immunosuppressive agent such as adenosine. Our approach offers a new tool for the investigation of the biochemical composition of tumour milieu and for development of novel therapies based on the modulation of extracellular purine-based signalling.

  14. Increased extracellular matrix metalloproteinase inducer (EMMPRIN) expression in the conjunctival epithelium exposed to antiglaucoma treatments.

    Labbé, Antoine; Gabison, Eric; Brignole-Baudouin, Françoise; Riancho, Luisa; Menashi, Suzanne; Baudouin, Christophe

    2015-01-01

    To analyze the effect of preserved antiglaucoma eye drops on the expression of extracellular matrix (ECM) metalloproteinase inducer (EMMPRIN) in conjunctival epithelial cells. A total of 18 patients treated for primary open-angle glaucoma with benzalkonium chloride (BAK) preserved eye drops and eight age-matched controls were included in this study. Glaucoma patients were divided into two groups according to their daily exposure to BAK: high-exposure (HE) group and low-exposure (LE) group. HLA-DR and EMMPRIN were quantified on conjunctival impression cytology specimens using flow cytometry. In parallel, IOBA-NHC conjunctival epithelial cells were exposed to different BAK concentrations, in the presence or absence of cyclosporine A (CsA), and their total and surface expressions of EMMPRIN were assessed by flow cytometry and results are given in relative fluorescence intensities (RFIs). Compared to the control group (1.71 ± 0.39 RFI), EMMPRIN was significantly increased in the HE (4.19 ± 1.50 RFI, p EMMPRIN (R(2) = 0.875, p EMMPRIN, which was proportional to the concentration of BAK. The surface expression of EMMPRIN was inhibited by CsA. The increased expression of EMMPRIN in patients topically treated with multiple antiglaucoma BAK-preserved eye drops suggests a matrix metalloproteinase-related modification of conjunctival ECM remodeling. In vitro results suggest that CsA has the potential to limit BAK effects on EMMPRIN.

  15. The study on mutations of the gene of extracellular domain of human thyrotropin receptor in the patients with thyroid diseases

    Zhang Zuncheng; Fang Peihua; Tan Jian; Lu Mei

    2002-01-01

    Objective: To define the sequence of the gene of extracellular domain of normal human thyrotropin receptor (hTSHR) and to investigate the mutations of the gene in the patients with thyroid diseases. Methods: Total RNAs were extracted from the thyroid tissue of four normal controls, twelve Graves' disease, four Hashimoto's thyroiditis and eleven nodular goiter patients. The extracellular domain of hTSHR genes were amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced with CEQ 2000 Genetic Analyzer. Results: The normal controls and the patients with thyroid disease had the same gene sequences of the extracellular domain of hTSHR. No mutation was found, except a silent base exchange in exon 7 (Asn187) at 661 base, in which 20 samples were 'T', 11 samples were 'C', without changes of amino acid of the TSHR. Conclusions: This study has not revealed mutations in the gene of extracellular domain of hTSHR. Other molecular pathogenetic mechanisms may be involved and more research is demanded

  16. Increasing extracellular matrix collagen level and MMP activity induces cyst development in polycystic kidney disease.

    Liu, Bin; Li, Chenghai; Liu, Zijuan; Dai, Zonghan; Tao, Yunxia

    2012-09-11

    Polycystic Kidney Disease (PKD) kidneys exhibit increased extracellular matrix (ECM) collagen expression and metalloproteinases (MMPs) activity. We investigated the role of these increases on cystic disease progression in PKD kidneys. We examined the role of type I collagen (collagen I) and membrane bound type 1 MMP (MT1-MMP) on cyst development using both in vitro 3 dimensional (3D) collagen gel culture and in vivo PCK rat model of PKD. We found that collagen concentration is critical in controlling the morphogenesis of MDCK cells cultured in 3D gels. MDCK cells did not form 3D structures at collagen I concentrations lower than 1 mg/ml but began forming tubules when the concentration reaches 1 mg/ml. Significantly, these cells began to form cyst when collagen I concentration reached to 1.2 mg/ml, and the ratios of cyst to tubule structures increased as the collagen I concentration increased. These cells exclusively formed cyst structures at a collagen I concentration of 1.8 mg/ml or higher. Overexpression of MT1-MMP in MDCK cells significantly induced cyst growth in 3D collagen gel culture. Conversely, inhibition of MMPs activity with doxycycline, a FDA approved pan-MMPs inhibitor, dramatically slowed cyst growth. More importantly, the treatment of PCK rats with doxycycline significantly decreased renal tubule cell proliferation and markedly inhibited the cystic disease progression. Our data suggest that increased collagen expression and MMP activity in PKD kidneys may induce cyst formation and expansion. Our findings also suggest that MMPs may serve as a therapeutic target for the treatment of human PKD.

  17. Increasing extracellular matrix collagen level and MMP activity induces cyst development in polycystic kidney disease

    Liu Bin

    2012-09-01

    Full Text Available Abstract Background Polycystic Kidney Disease (PKD kidneys exhibit increased extracellular matrix (ECM collagen expression and metalloproteinases (MMPs activity. We investigated the role of these increases on cystic disease progression in PKD kidneys. Methods We examined the role of type I collagen (collagen I and membrane bound type 1 MMP (MT1-MMP on cyst development using both in vitro 3 dimensional (3D collagen gel culture and in vivo PCK rat model of PKD. Results We found that collagen concentration is critical in controlling the morphogenesis of MDCK cells cultured in 3D gels. MDCK cells did not form 3D structures at collagen I concentrations lower than 1 mg/ml but began forming tubules when the concentration reaches 1 mg/ml. Significantly, these cells began to form cyst when collagen I concentration reached to 1.2 mg/ml, and the ratios of cyst to tubule structures increased as the collagen I concentration increased. These cells exclusively formed cyst structures at a collagen I concentration of 1.8 mg/ml or higher. Overexpression of MT1-MMP in MDCK cells significantly induced cyst growth in 3D collagen gel culture. Conversely, inhibition of MMPs activity with doxycycline, a FDA approved pan-MMPs inhibitor, dramatically slowed cyst growth. More importantly, the treatment of PCK rats with doxycycline significantly decreased renal tubule cell proliferation and markedly inhibited the cystic disease progression. Conclusions Our data suggest that increased collagen expression and MMP activity in PKD kidneys may induce cyst formation and expansion. Our findings also suggest that MMPs may serve as a therapeutic target for the treatment of human PKD.

  18. Increased Urinary Extracellular Vesicle Sodium Transporters in Cushing's Syndrome with Hypertension.

    Salih, Mahdi; Bovée, Dominique M; van der Lubbe, Nils; Danser, Alexander H J; Zietse, Robert; Feelders, Richard A; Hoorn, Ewout J

    2018-05-02

    Increased renal sodium reabsorption contributes to hypertension in Cushing's syndrome (CS). Renal sodium transporters can be analyzed non-invasively in urinary extracellular vesicles (uEVs). To analyze renal sodium transporters in uEVs of patients with CS and hypertension. Observational study. University hospital. uEVs were isolated by ultracentrifugation and analyzed by immunoblotting in 10 CS patients and 7 age-matched healthy subjects. In 7 CS patients uEVs were analyzed before and after treatment. uEV protein abundance. The 10 CS patients were divided in those with suppressed and non-suppressed renin-angiotensin-aldosterone system (RAAS, n = 5/group). CS patients with suppressed RAAS had similar blood pressure but significantly lower serum potassium than CS patients with non-suppressed RAAS. Compared to healthy subjects, only those with suppressed RAAS had higher phosphorylated Na+-K+-Cl- cotransporter type 2 (pNKCC2) and higher total and phosphorylated Na+-Cl- cotransporter (NCC) in uEVs. Serum potassium but not urinary free cortisol correlated with pNKCC2, pNCC, and NCC in uEVs. Treatment of CS reversed the increases in pNKCC2, NCC, and pNCC. CS increases renal sodium transporter abundance in uEVs especially in patients with hypertension and suppressed RAAS. As potassium has recently been identified as an important driver of NCC activity, low serum potassium may also contribute to increased renal sodium reabsorption and hypertension in CS. These results may also be relevant for hypertension induced by exogenous glucocorticoids.

  19. Expression of Genes Involved in Cellular Adhesion and Extracellular Matrix Remodeling Correlates with Poor Survival of Patients with Renal Cancer.

    Boguslawska, Joanna; Kedzierska, Hanna; Poplawski, Piotr; Rybicka, Beata; Tanski, Zbigniew; Piekielko-Witkowska, Agnieszka

    2016-06-01

    Renal cell carcinoma is the most common highly metastatic kidney malignancy. Adhesion has a crucial role in the metastatic process. TGF (transforming growth factor)-β1 is a pleiotropic cytokine that influences cancerous transformation. We hypothesized that 1) changes in the expression of adhesion related genes may influence survival rate of patients with renal cell carcinoma and 2) TGF-β1 may contribute to changed expression of adhesion related genes. Two-step quantitative real-time polymerase chain reaction arrays were used to analyze the expression of adhesion related genes in 77 tumors and matched pair controls. The prognostic significance of genes was evaluated in TCGA (The Cancer Genome Atlas) data on 468 patients with renal cell carcinoma. Quantitative real-time polymerase chain reaction and Western blot were applied for TGF-β1 analysis. TGF-β1 mediated regulation of gene expression was analyzed by TGF-β1 supplementation of Caki-2 cells and quantitative real-time polymerase chain reaction. The expression of 19 genes related to adhesion and extracellular matrix remodeling was statistically significantly disturbed in renal cell carcinoma compared with controls. The 10-gene expression signature (COL1A1, COL5A1, COL11A1, FN1, ICAM1, ITGAL, ITGAM, ITGB2, THBS2 and TIMP1) correlated with poor survival (HR 2.85, p = 5.7e-10). TGF-β1 expression was 22 times higher in renal cell carcinoma than in controls (p adhesion and extracellular matrix remodeling develops early during renal cell carcinoma carcinogenesis and correlates with poor survival. TGF-β1 contributes to changed expression of extracellular matrix and adhesion related genes. Bioinformatic analysis performed on a broad panel of cancers of nonkidney origin suggests that disturbed expression of genes related to extracellular matrix and adhesion may be a universal feature of cancerous progression. Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All

  20. Modulation of sibutramine-induced increases in extracellular noradrenaline concentration in rat frontal cortex and hypothalamus by α2-adrenoceptors

    Wortley, K E; Heal, D J; Stanford, S C

    1999-01-01

    The effects of sibutramine (0.25–10 mg kg−1 i.p.) on extracellular noradrenaline concentration in the frontal cortex and hypothalamus of freely-moving rats were investigated using microdialysis. The role of presynaptic α2-adrenoceptors in modulating the effects of sibutramine in these brain areas was also determined.Sibutramine induced an increase in extracellular noradrenaline concentration, the magnitude of which paralleled dose, in both brain areas. In the cortex, this increase was gradual and sustained, whereas in the hypothalamus it was more rapid and of shorter duration.In both the cortex and hypothalamus, pretreatment of rats with the α2-adrenoceptor antagonist RX821002 (3 mg kg−1 i.p.) potentiated increases in the accumulation of extracellular noradrenaline induced by sibutramine (10 mg kg−1 i.p.), by 7 and 10 fold respectively. RX821002 also reduced the latency of sibutramine to reach its maximum effect in the cortex, but not in the hypothalamus.Infusion of RX821002 (1 μM) via the probe increased the accumulation of extracellular noradrenaline induced by sibutramine (10 mg kg−1 i.p.) in both brain areas. In the hypothalamus, the effects of RX821002 on the accumulation of noradrenaline induced by sibutramine were 2 fold greater than those in the cortex.These findings support evidence that sibutramine inhibits the reuptake of noradrenaline in vivo, but that the accumulation of extracellular noradrenaline is limited by noradrenergic activation of presynaptic α2-adrenoceptors. Furthermore, the data suggest that terminal α2-adrenoceptors in the hypothalamus exert a greater inhibitory effect over the control of extracellular noradrenaline accumulation than do those in the cortex. PMID:10516646

  1. Variation in extracellular matrix genes is associated with weight regain after weight loss in a sex-specific manner

    Roumans, Nadia J T; Vink, Roel G; Gielen, Marij

    2015-01-01

    The extracellular matrix (ECM) of adipocytes is important for body weight regulation. Here, we investigated whether genetic variation in ECM-related genes is associated with weight regain among participants of the European DiOGenes study. Overweight and obese subjects (n = 469, 310 females, 159 m.......40-5.63). Concluding, variants of ECM genes are associated with weight regain after weight loss in a sex-specific manner....

  2. TOL plasmid carriage enhances biofilm formation and increases extracellular DNA content in Pseudomonas putida KT2440

    D'Alvise, Paul; Sjoholm, O.R.; Yankelevich, T.

    2010-01-01

    laser scanning microscopy. The TOL-carrying strains formed pellicles and thick biofilms, whereas the same strains without the plasmid displayed little adherent growth. Microscopy using fluorescent nucleic acid-specific stains revealed differences in the production of extracellular polymeric substances......: TOL carriage leads to more extracellular DNA (eDNA) in pellicles and biofilms. Pellicles were dissolved by DNase I treatment. Enhanced cell lysis due to plasmid carriage was ruled out as the mechanism for eDNA release. We report, for the first time, that carriage of a conjugative plasmid leads...

  3. Genes responsible for vaginal extracellular matrix metabolism are modulated by women's reproductive cycle and menopause

    Oksana Shynlova

    2013-04-01

    Full Text Available Objectives To analyze the expression of genes involved in extracellular matrix (ECM biogenesis and remodeling in vaginal tissue of women with clinically normal pelvic floor support (defined as controls according to the phase of menstrual cycle and postmenopausal women with and without pelvic organ prolapse (POP. Materials and Methods This study examined the expression of matrix metalloproteinases (MMPs, their tissue inhibitors (TIMPs, and the Lysyl oxidase (LOX family genes in the anterior vaginal wall of Caucasian women by real-time RT-PCR. Initially, mRNA expression was assessed in premenopausal controls in the secretory (group 1, n = 10 vs. proliferative (group 2, n = 8 phase of menstrual cycle. In addition, we compared premenopausal controls in the proliferative phase (group 2 vs. postmenopausal controls (group 3, n = 5. Finally, we analyzed postmenopausal controls (group 3 vs. postmenopausal women with advanced POP (group 4, n = 13. Results According to the phase of menstrual cycle, MMP1 was significantly reduced (p = 0.003, whereas the expression of TIMP1 and LOXL4 was significantly up-regulated during proliferative phase (both p < 0.01 when compared to the secretory phase in premenopausal control women. Regarding menopausal status/ageing, all MMPs were down-regulated, while TIMP3, TIMP4 and LOXL2 were significantly up-regulated in postmenopausal control women when compared to premenopausal controls (p = 0.005, p = 0.01 and p < 0.001, correspondingly. TIMP4 and LOXL2 mRNA levels were significantly decreased in postmenopausal POP patients compared to asymptomatic postmenopausal controls (p < 0.01 for both. Conclusions Our results indicate that ovarian cycle and age-related changes influence the expression of genes encoding proteins responsible for ECM metabolism in human vagina. Moreover, POP is associated with alteration in vaginal ECM components after menopause.

  4. Identification of Bicarbonate as a Trigger and Genes Involved with Extracellular DNA Export in Mycobacterial Biofilms

    Sasha J. Rose

    2016-12-01

    Full Text Available Extracellular DNA (eDNA is an integral biofilm matrix component of numerous pathogens, including nontuberculous mycobacteria (NTM. Cell lysis is the source of eDNA in certain bacteria, but the source of eDNA remains unidentified for NTM, as well as for other eDNA-containing bacterial species. In this study, conditions affecting eDNA export were examined, and genes involved with the eDNA export mechanism were identified. After a method for monitoring eDNA in real time in undisturbed biofilms was established, different conditions affecting eDNA were investigated. Bicarbonate positively influenced eDNA export in a pH-independent manner in Mycobacterium avium, M. abscessus, and M. chelonae. The surface-exposed proteome of M. avium in eDNA-containing biofilms revealed abundant carbonic anhydrases. Chemical inhibition of carbonic anhydrases with ethoxzolamide significantly reduced eDNA export. An unbiased transposon mutant library screen for eDNA export in M. avium identified many severely eDNA-attenuated mutants, including one not expressing a unique FtsK/SpoIIIE-like DNA-transporting pore, two with inactivation of carbonic anhydrases, and nine with inactivation of genes belonging to a unique genomic region, as well as numerous mutants involved in metabolism and energy production. Complementation of nine mutants that included the FtsK/SpoIIIE and carbonic anhydrase significantly restored eDNA export. Interestingly, several attenuated eDNA mutants have mutations in genes encoding proteins that were found with the surface proteomics, and many more mutations are localized in operons potentially encoding surface proteins. Collectively, our data strengthen the evidence of eDNA export being an active mechanism that is activated by the bacterium responding to bicarbonate.

  5. [Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli].

    Inoue, Masaharu; Kikuchi, Maho; Komoriya, Tomoe; Watanabe, Kunitomo; Kouno, Hideki

    2007-01-01

    Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.

  6. A High-Fat Meal, or Intraperitoneal Administration of a Fat Emulsion, Increases Extracellular Dopamine in the Nucleus Accumbens

    Bartley G. Hoebel

    2012-06-01

    Full Text Available Evidence links dopamine (DA in the nucleus accumbens (NAc shell to the ingestion of palatable diets. Less is known, however, about the specific relation of DA to dietary fat and circulating triglycerides (TG, which are stimulated by fat intake and promote overeating. The present experiments tested in Sprague-Dawley rats whether extracellular levels of NAc DA increase in response to acute access to fat-rich food or peripheral injection of a fat emulsion and, if so, whether this is related to caloric intake or elevated circulating lipids. When rats consumed more calories of a high-fat meal compared with a low-fat meal, there was a significant increase in extracellular accumbens DA (155% vs. 119%. Systemic injection of a fat emulsion, which like a high-fat diet raises circulating TG but eliminates the factor of taste and allows for the control of caloric intake, also significantly increased extracellular levels of DA (127% compared to an equicaloric glucose solution (70% and saline (85%. Together, this suggests that a rise in circulating TG may contribute to the stimulatory effect of a high-fat diet on NAc DA.

  7. INCREASE OF GLYCOSAMINOGLYCANS AND METALLOPROTEINASES 2 AND 9 IN LIVER EXTRACELLULAR MATRIX ON EARLY STAGES OF EXTRAHEPATIC CHOLESTASIS

    Pedro Luiz Rodrigues GUEDES

    2014-12-01

    Full Text Available Context Cholestasis produces hepatocellular injury, leukocyte infiltration, ductular cells proliferation and fibrosis of liver parenchyma by extracellular matrix replacement. Objective Analyze bile duct ligation effect upon glycosaminoglycans content and matrix metalloproteinase (MMPs activities. Methods Animals (6-8 weeks; n = 40 were euthanized 2, 7 or 14 days after bile duct ligation or Sham-surgery. Disease evolution was analyzed by body and liver weight, seric direct bilirubin, globulins, gamma glutamyl transpeptidase (GGT, alkaline phosphatase (Alk-P, alanine and aspartate aminotransferases (ALT and AST, tissue myeloperoxidase and MMP-9, pro MMP-2 and MMP-2 activities, histopathology and glycosaminoglycans content. Results Cholestasis caused cellular damage with elevation of globulins, GGT, Alk-P, ALT, AST. There was neutrophil infiltration observed by the increasing of myeloperoxidase activity on 7 (P = 0.0064 and 14 (P = 0.0002 groups which leads to the magnification of tissue injuries. Bile duct ligation increased pro-MMP-2 (P = 0.0667, MMP-2 (P = 0.0003 and MMP-9 (P<0.0001 activities on 14 days indicating matrix remodeling and establishment of inflammatory process. Bile duct ligation animals showed an increasing on dermatan sulfate and/or heparan sulfate content reflecting extracellular matrix production and growing mitosis due to parenchyma depletion. Conclusions Cholestasis led to many changes on rats’ liver parenchyma, as so as on its extracellular matrix, with major alterations on MMPs activities and glycosaminoglycans content.

  8. Increased extracellular dopamine and 5-hydroxytryptamine levels contribute to enhanced subthalamic nucleus neural activity during exhausting exercise

    Y Hu

    2015-09-01

    Full Text Available The purpose of the study was to explore the mechanism underlying the enhanced subthalamic nucleus (STN neural activity during exhausting exercise from the perspective of monoamine neurotransmitters and changes of their corresponding receptors. Rats were randomly divided into microdialysis and immunohistochemistry study groups. For microdialysis study, extracellular fluid of the STN was continuously collected with a microdialysis probe before, during and 90 min after one bout of exhausting exercise. Dopamine (DA and 5-hydroxytryptamine (5-HT levels were subsequently detected with high-performance liquid chromatography (HPLC. For immunohistochemistry study, the expression of DRD 2 and HT 2C receptors in the STN, before, immediately after and 90 min after exhaustion was detected through immunohistochemistry technique. Microdialysis study results showed that the extracellular DA and 5-HT neurotransmitters increased significantly throughout the procedure of exhausting exercise and the recovery period (P0.05. Our results suggest that the increased extracellular DA and 5-HT in the STN might be one important factor leading to the enhanced STN neural activity and the development of fatigue during exhausting exercise. This study may essentially offer useful evidence for better understanding of the mechanism of the central type of exercise-induced fatigue.

  9. Effect of SLC34A2 gene mutation on extracellular phosphorus transport in PAM alveolar epithelial cells.

    Ma, Tiangang; Qu, Danhua; Yan, Bingdi; Zhang, Qinghua; Ren, Jin; Hu, Yanbing

    2018-01-01

    A mutation in the IIb sodium phosphate transporter SLC34A2 gene has recently been described in pulmonary alveolar microlithiasis (PAM) patients. Experiments in this study were aimed at confirming the role of the gene product in PAM by comparing phosphorylated products in extracellular fluid of alveolar epithelial cells overexpressing the SLC34A2 gene or its mutated version. Eukaryotic expression vectors were constructed and transfected into A549 human alveolar epithelial cells. There were three groups of cells including those transfected with empty vector plasmid pcDNA3.1(+) (plasmid control group), those transfected with normal SLC34A2 gene expressed from pcDNA3.1 (normal control group), and those transfected with a version of the PAM SLC34A2 gene linked to the pcDNA3.1(+) (PAM group). Transfection efficiencies were detected by reverse transcription-polymerase chain reaction (RT-PCR). At 48 h after transfection, the concentration of inorganic phosphorus in the culture medium was detected using an automatic biochemical analyzer. Our results showed the concentration of inorganic phosphorus in the supernatant of the normal control group was significantly lower than that in the plasmid control and PAM groups (PPAM group was significantly lower than that in the plasmid control group (PPAM patients, given that the function of the phosphate transporter seems to be affected and it is conceivable that it would lead to extracellular fluid alterations in vivo .

  10. NG2 proteoglycan increases mesangial cell proliferation and extracellular matrix production

    Xiong Jing; Wang Yang; Zhu, Zhonghua; Liu Jianshe; Wang Yumei; Zhang Chun; Hammes, Hans-Peter; Lang, Florian; Feng Yuxi

    2007-01-01

    As a membrane-spanning protein, NG2 chondroitin sulfate proteoglycan interacts with molecules on both sides of plasma membrane. The present study explored the role of NG2 in the pathogenesis of diabetic nephropathy. In the normal kidneys, NG2 was observed predominantly in glomerular mesangium, Bowman's capsule and interstitial vessels. Both mRNA and protein expression in kidneys was significantly higher in strepozotocin-induced diabetic rats than that in normal rats. In the cultured rat mesangial cell line HBZY-1, overexpression of NG2 promoted mesangial cell proliferation and extracellular matrix (ECM) production, such as type VI collagen and laminin. Furthermore, target knockdown of NG2 resulted in decreased cell proliferation and ECM formation. The observations suggest that NG2 is up-regulated in diabetic nephropathy. It actively participates in the development and progression of glomerulosclerosis by stimulating proliferation of mesangial cells and deposition of ECM

  11. Increase of extracellular glutamate concentration increases its oxidation and diminishes glucose oxidation in isolated mouse hippocampus: reversible by TFB-TBOA.

    Torres, Felipe Vasconcelos; Hansen, Fernanda; Locks-Coelho, Lucas Doridio

    2013-08-01

    Glutamate concentration at the synaptic level must be kept low in order to prevent excitotoxicity. Astrocytes play a key role in brain energetics, and also astrocytic glutamate transporters are responsible for the vast majority of glutamate uptake in CNS. Experiments with primary astrocytic cultures suggest that increased influx of glutamate cotransported with sodium at astrocytes favors its flux to the tricarboxylic acid cycle instead of the glutamate-glutamine cycle. Although metabolic coupling can be considered an emergent field of research with important recent discoveries, some basic aspects of glutamate metabolism still have not been characterized in brain tissue. Therefore, the aim of this study was to investigate whether the presence of extracellular glutamate is able to modulate the use of glutamate and glucose as energetic substrates. For this purpose, isolated hippocampi of mice were incubated with radiolabeled substrates, and CO2 radioactivity and extracellular lactate were measured. Our results point to a diminished oxidation of glucose with increasing extracellular glutamate concentration, glutamate presumably being the fuel, and might suggest that oxidation of glutamate could buffer excitotoxic conditions by high glutamate concentrations. In addition, these findings were reversed when glutamate uptake by astrocytes was impaired by the presence of (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-L-aspartic acid (TFB-TBOA). Taken together, our findings argue against the lactate shuttle theory, because glutamate did not cause any detectable increase in extracellular lactate content (or, presumably, in glycolysis), because the glutamate is being used as fuel instead of going to glutamine and back to neurons. Copyright © 2013 Wiley Periodicals, Inc.

  12. Fibroblast-Derived Extracellular Matrices: An Alternative Cell Culture System That Increases Metastatic Cellular Properties.

    Michael T Scherzer

    Full Text Available Poor survival rates from lung cancer can largely be attributed to metastatic cells that invade and spread throughout the body. The tumor microenvironment (TME is composed of multiple cell types, as well as non-cellular components. The TME plays a critical role in the development of metastatic cancers by providing migratory cues and changing the properties of the tumor cells. The Extracellular Matrix (ECM, a main component of the TME, has been shown to change composition during tumor progression, contributing to cancer cell invasion and survival away from the primary cancer site. Although the ECM is well-known to influence the fate of tumor progression, little is known about the molecular mechanisms that are affected by the cancer cell-ECM interactions. It is imperative that these mechanisms are elucidated in order to properly understand and prevent lung cancer dissemination. However, common in vitro studies do not incorporate these interactions into everyday cell culture assays. We have adopted a model that examines decellularized human fibroblast-derived ECM as a 3-dimensional substrate for growth of lung adenocarcinoma cell lines. Here, we have characterized the effect of fibroblast-derived matrices on the properties of various lung-derived epithelial cell lines, including cancerous and non-transformed cells. This work highlights the significance of the cell-ECM interaction and its requirement for incorporation into in vitro experiments. Implementation of a fibroblast-derived ECM as an in vitro technique will provide researchers with an important factor to manipulate to better recreate and study the TME.

  13. Root carbon inputs to the rhizosphere stimulate extracellular enzyme activity and increase nitrogen availability in temperate forest soils

    Brzostek, E. R.; Phillips, R.; Dragoni, D.; Drake, J. E.; Finzi, A. C.

    2011-12-01

    The mobilization of nitrogen (N) from soil organic matter in temperate forest soils is controlled by the microbial production and activity of extracellular enzymes. The exudation of carbon (C) by tree roots into the rhizosphere may subsidize the microbial production of extracellular enzymes in the rhizosphere and increase the access of roots to N. The objective of this research was to investigate whether rates of root exudation and the resulting stimulation of extracellular enzyme activity in the rhizosphere (i.e., rhizosphere effect) differs between tree species that form associations with ectomycorrhizal (ECM) or arbuscular mycorrhizal (AM) fungi. This research was conducted at two temperate forest sites, the Harvard Forest (HF) in Central MA and the Morgan Monroe State Forest (MMSF) in Southern IN. At the HF, we measured rates of root exudation and the rhizosphere effects on enzyme activity, N cycling, and C mineralization in AM and ECM soils. At the MMSF, we recently girdled AM and ECM dominated plots to examine the impact of severing belowground C allocation on rhizosphere processes. At both sites, the rhizosphere effect on proteolytic, chitinolytic and ligninolytic enzyme activities was greater in ECM soils than in AM soils. In particular, higher rates of proteolytic enzyme activity increased the availability of amino acid-N in ECM rhizospheres relative to the bulk soils. Further, this stimulation of enzyme activity was directly correlated with higher rates of C mineralization in the rhizosphere than in the bulk soil. Although not significantly different between species, root exudation of C comprised 3-10% of annual gross primary production at the HF. At the MMSF, experimental girdling led to a larger decline in soil respiration and enzyme activity in ECM plots than in AM plots. In both ECM and AM soils, however, girdling resulted in equivalent rates of enzyme activity in rhizosphere and corresponding bulk soils. The results of this study contribute to the

  14. Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway.

    Kosalková, Katarina; García-Estrada, Carlos; Barreiro, Carlos; Flórez, Martha G; Jami, Mohammad S; Paniagua, Miguel A; Martín, Juan F

    2012-01-10

    The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold) in cells grown with casein or casein phosphopeptides (CPPs). CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs), whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase), which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.

  15. Heme oxygenase-2 gene deletion attenuates oxidative stress in neurons exposed to extracellular hemin

    Benvenisti-Zarom Luna

    2004-09-01

    Full Text Available Abstract Background Hemin, the oxidized form of heme, accumulates in intracranial hematomas and is a potent oxidant. Growing evidence suggests that it contributes to delayed injury to surrounding tissue, and that this process is affected by the heme oxygenase enzymes. In a prior study, heme oxygenase-2 gene deletion increased the vulnerability of cultured cortical astrocytes to hemin. The present study tested the effect of HO-2 gene deletion on protein oxidation, reactive oxygen species formation, and cell viability after mixed cortical neuron/astrocyte cultures were incubated with neurotoxic concentrations of hemin. Results Continuous exposure of wild-type cultures to 1–10 μM hemin for 14 h produced concentration-dependent neuronal death, as detected by both LDH release and fluorescence intensity after propidium iodide staining, with an EC50 of 1–2 μM; astrocytes were not injured by these low hemin concentrations. Cell death was consistently reduced by at least 60% in knockout cultures. Exposure to hemin for 4 hours, a time point that preceded cell lysis, increased protein oxidation in wild-type cultures, as detected by staining of immunoblots for protein carbonyl groups. At 10 μM hemin, carbonylation was increased 2.3-fold compared with control sister cultures subjected to medium exchanges only; this effect was reduced by about two-thirds in knockout cultures. Cellular reactive oxygen species, detected by fluorescence intensity after dihydrorhodamine 123 (DHR staining, was markedly increased by hemin in wild-type cultures and was localized to neuronal cell bodies and processes. In contrast, DHR fluorescence intensity in knockout cultures did not differ from that of sham-washed controls. Neuronal death in wild-type cultures was almost completely prevented by the lipid-soluble iron chelator phenanthroline; deferoxamine had a weaker but significant effect. Conclusions These results suggest that HO-2 gene deletion protects neurons in mixed

  16. Muscle contractures in patients with cerebral palsy and acquired brain injury are associated with extracellular matrix expansion, pro-inflammatory gene expression, and reduced rRNA synthesis.

    von Walden, Ferdinand; Gantelius, Stefan; Liu, Chang; Borgström, Hanna; Björk, Lars; Gremark, Ola; Stål, Per; Nader, Gustavo A; Pontén, Eva

    2018-03-23

    Children with cerebral palsy (CP) and acquired brain injury (ABI) commonly develop muscle contractures with advancing age. An underlying growth defect contributing to skeletal muscle contracture formation in CP/ABI has been suggested. The biceps muscles of children and adolescents with CP/ABI (n=20) and typically developing controls (n=10) were investigated. We used immunohistochemistry, qRT-PCR and western blotting to assess gene expression relevant to growth and size homeostasis. Classical pro-inflammatory cytokines and genes involved in extracellular matrix production were elevated in skeletal muscle of children with CP/ABI. Intramuscular collagen content was increased and satellite cell number decreased and this was associated with reduced levels of RNA polymerase (POL) I transcription factors, 45s pre-rRNA and 28S rRNA. The present study provides novel data suggesting a role for pro-inflammatory cytokines and reduced ribosomal production in the development/maintenance of muscle contractures; possibly underlying stunted growth and perimysial extracellular matrix expansion. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

  17. Comparative Genomic Analysis of Neutrophilic Iron(II Oxidizer Genomes for Candidate Genes in Extracellular Electron Transfer

    Shaomei He

    2017-08-01

    Full Text Available Extracellular electron transfer (EET is recognized as a key biochemical process in circumneutral pH Fe(II-oxidizing bacteria (FeOB. In this study, we searched for candidate EET genes in 73 neutrophilic FeOB genomes, among which 43 genomes are complete or close-to-complete and the rest have estimated genome completeness ranging from 5 to 91%. These neutrophilic FeOB span members of the microaerophilic, anaerobic phototrophic, and anaerobic nitrate-reducing FeOB groups. We found that many microaerophilic and several anaerobic FeOB possess homologs of Cyc2, an outer membrane cytochrome c originally identified in Acidithiobacillus ferrooxidans. The “porin-cytochrome c complex” (PCC gene clusters homologous to MtoAB/PioAB are present in eight FeOB, accounting for 19% of complete and close-to-complete genomes examined, whereas PCC genes homologous to OmbB-OmaB-OmcB in Geobacter sulfurreducens are absent. Further, we discovered gene clusters that may potentially encode two novel PCC types. First, a cluster (tentatively named “PCC3” encodes a porin, an extracellular and a periplasmic cytochrome c with remarkably large numbers of heme-binding motifs. Second, a cluster (tentatively named “PCC4” encodes a porin and three periplasmic multiheme cytochromes c. A conserved inner membrane protein (IMP encoded in PCC3 and PCC4 gene clusters might be responsible for translocating electrons across the inner membrane. Other bacteria possessing PCC3 and PCC4 are mostly Proteobacteria isolated from environments with a potential niche for Fe(II oxidation. In addition to cytochrome c, multicopper oxidase (MCO genes potentially involved in Fe(II oxidation were also identified. Notably, candidate EET genes were not found in some FeOB, especially the anaerobic ones, probably suggesting EET genes or Fe(II oxidation mechanisms are different from the searched models. Overall, based on current EET models, the search extends our understanding of bacterial EET and

  18. Characterization of the Aspergillus niger prtT, a unique regulator of extracellular protease encoding genes

    Punt, P.J.; Schuren, F.H.J.; Lehmbeck, J.; Christensen, T.; Hjort, C.; Hondel, C.A.M.J.J. van den

    2008-01-01

    Expression of several Aspergillus niger genes encoding major secreted, but not vacuolar, protease genes including the major acid protease gene pepA, was shown to be affected in the previously isolated A. niger protease mutant, AB1.13 [Mattern, I.E., van Noort, J.M., van den Berg, P., Archer, D.A.,

  19. A new adsorption-elution technique for the concentration of aquatic extracellular antibiotic resistance genes from large volumes of water.

    Wang, Da-Ning; Liu, Lu; Qiu, Zhi-Gang; Shen, Zhi-Qiang; Guo, Xuan; Yang, Dong; Li, Jing; Liu, Wei-Li; Jin, Min; Li, Jun-Wen

    2016-04-01

    Extracellular antibiotic resistance genes (eARGs) that help in the transmission and spread of antibiotic-resistant bacteria are emerging environmental contaminants in water, and there is therefore a growing need to assess environmental levels and associated risks of eARGs. However, as they are present in low amounts, it is difficult to detect eARGs in water directly with PCR techniques. Here, we prepared a new type of nucleic acid adsorption particle (NAAP) with high capacity and developed an optimal adsorption-elution method to concentrate eARGs from large volumes of water. With this technique, we were able to achieve an eARG recovery rate of above 95% from 10 L of water samples. Moreover, combining this new method with quantitative real-time PCR (qPCR), the sensitivity of the eARG detection was 10(4) times that of single qPCR, with the detection limit lowered to 100 gene copies (GCs)/L. Our analyses showed that the eARG load, virus load and certain water characteristics such as pH, chemical oxygen demand (CODMn), and turbidity affected the eARGs recovery rate. However, high eARGs recovery rates always remained within the standard limits for natural surface water quality, while eARG levels in water were lower than the detection limits of single qPCR assays. The recovery rates were not affected by water temperature and heterotrophic plate counts (HPC). The eARGs whatever located in the plasmids or the short-length linear DNAs can be recovered from the water. Furthermore, the recovery rate was high even in the presence of high concentrations of plasmids in different natural water (Haihe river, well water, raw water for drinking water, Jinhe river, Tuanbo lake and the Yunqiao reservoir). By this technology, eARGs concentrations were found ranging from (2.70 ± 0.73) × 10(2) to (4.58 ± 0.47) × 10(4) GCs/L for the extracellular ampicillin resistance gene and (5.43 ± 0.41) × 10(2) to (2.14 ± 0.23) × 10(4) GCs/L for the extracellular gentamicin

  20. Increases in extracellular serotonin and dopamine metabolite levels in the basal forebrain during sleep deprivation

    Zant, J.C.; Leenaars, C.H.; Kostin, A.; van Someren, E.J.W.; Porrka-Heiskanen, T.

    2011-01-01

    The basal forebrain (BF) is an important mediator of cortical arousal, which is innervated by all ascending arousal systems. During sleep deprivation (SD) a site-specific accumulation of sleep factors in the BF results in increased sleep pressure (Kalinchuk et al., 2006; Porkka-Heiskanen et al.,

  1. Albumin, in the Presence of Calcium, Elicits a Massive Increase in Extracellular Bordetella Adenylate Cyclase Toxin.

    Gonyar, Laura A; Gray, Mary C; Christianson, Gregory J; Mehrad, Borna; Hewlett, Erik L

    2017-06-01

    Pertussis (whooping cough), caused by Bordetella pertussis , is resurging in the United States and worldwide. Adenylate cyclase toxin (ACT) is a critical factor in establishing infection with B. pertussis and acts by specifically inhibiting the response of myeloid leukocytes to the pathogen. We report here that serum components, as discovered during growth in fetal bovine serum (FBS), elicit a robust increase in the amount of ACT, and ≥90% of this ACT is localized to the supernatant, unlike growth without FBS, in which ≥90% is associated with the bacterium. We have found that albumin, in the presence of physiological concentrations of calcium, acts specifically to enhance the amount of ACT and its localization to the supernatant. Respiratory secretions, which contain albumin, promote an increase in amount and localization of active ACT that is comparable to that elicited by serum and albumin. The response to albumin is not mediated through regulation of ACT at the transcriptional level or activation of the Bvg two-component system. As further illustration of the specificity of this phenomenon, serum collected from mice that lack albumin does not stimulate an increase in ACT. These data, demonstrating that albumin and calcium act synergistically in the host environment to increase production and release of ACT, strongly suggest that this phenomenon reflects a novel host-pathogen interaction that is central to infection with B. pertussis and other Bordetella species. Copyright © 2017 American Society for Microbiology.

  2. Dihydrotestostenone increase the gene expression of androgen ...

    HNTEP cells were grown in basal medium and treated with DHT in different conditions. HNTEP cells under treatment with DHT (10-13 M) induced an increase in FHL-2 expression. In turn, high DHT concentrations (10-8 M) induced an increase in the expression SHP-1. The present data suggest that the SHP-1 and FHL-2 ...

  3. Acute hyperammonemia and systemic inflammation is associated with increased extracellular brain adenosine in rats

    Bjerring, Peter Nissen; Dale, Nicholas; Larsen, Fin Stolze

    2015-01-01

    ) and cerebral blood flow (CBF). We measured the adenosine concentration with biosensors in rat brain slices exposed to ammonia and in a rat model with hyperammonemia and systemic inflammation. Exposure to ammonia in concentrations from 0.15-10 mM led to increases in the cortical adenosine concentration up to 18......Acute liver failure (ALF) can lead to brain edema, cerebral hyperperfusion and intracranial hypertension. These complications are thought to be mediated by hyperammonemia and inflammation leading to altered brain metabolism. As increased levels of adenosine degradation products have been found...... in brain tissue of patients with ALF we investigated whether hyperammonemia could induce adenosine release in brain tissue. Since adenosine is a potent vasodilator and modulator of cerebral metabolism we furthermore studied the effect of adenosine receptor ligands on intracranial pressure (ICP...

  4. Myocardial extracellular volume quantified by magnetic resonance is increased in cirrhosis and related to poor outcome

    Wiese, Signe; Hove, Jens; Mo, Silje

    2018-01-01

    .04). Furthermore, ECV increased across the Child Pugh A/B/C classes (26.9±4/ 31.5±5/ 34.4±6%, P=0.02). Four-teen patients experienced the composite end-point of death/LT during follow-up and these patients had higher ECV (33.2±4 vs. 30.4±6%, P=0.04). In a univariate Cox regression analysis ECV was associated...

  5. Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway

    Kosalková Katarina

    2012-01-01

    Full Text Available Abstract Background The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Results Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold in cells grown with casein or casein phosphopeptides (CPPs. CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs, whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase, which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. Conclusions In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.

  6. TOL Plasmid Carriage Enhances Biofilm Formation and Increases Extracellular DNA Content in Pseudomonas Putida KT2440

    Smets, Barth F.; D'Alvise, Paul; Yankelovich, T.

    laser scanning microscopy. The TOL-carrying strains formed pellicles and thick biofilms, whereas the same strains without the plasmid displayed little adherent growth. Microscopy using fluorescent nucleic acid- specific stains (cytox orange, propidium iodide) revealed differences in production...... combined with specific cytostains; release of cytoplasmic material was assayed by a β-glucosidase assay. Enhanced cell lysis due to plasmid carriage was ruled out as the mechanism for eDNA release. We report, for the first time, that carriage of a conjugative plasmid leads to increased biofilm formation...

  7. Enhancement of rat bladder contraction by artificial sweeteners via increased extracellular Ca2+ influx

    Dasgupta, Jaydip; Elliott, Ruth A.; Doshani, Angie; Tincello, Douglas G.

    2006-01-01

    Introduction: Consumption of carbonated soft drinks has been shown to be independently associated with the development of overactive bladder symptoms (OR 1.62, 95% CI 1.18, 2.22) [Dallosso, H.M., McGrother, C.W., Matthews, R.J., Donaldson, M.M.K., 2003. The association of diet and other lifestyle factors with overactive bladder and stress incontinence: a longitudinal study in women. BJU Int. 92, 69-77]. We evaluated the effects of three artificial sweeteners, acesulfame K, aspartame and sodium saccharin, on the contractile response of isolated rat detrusor muscle strips. Methods: Strips of detrusor muscle were placed in an organ bath and stimulated with electrical field stimulation (EFS) in the absence and presence of atropine, and with α,β methylene ATP, potassium, calcium and carbachol. Results: Sweeteners 10 -7 M to 10 -2 M enhanced the contractile response to 10 Hz EFS compared to control (p -6 M, aspartame 10 -7 M and sodium saccharin 10 -7 M. Acesulfame K 10 -6 M increased the maximum contractile response to α,β methylene ATP by 35% (± 9.6%) (p -6 M increased the log EC 5 from -2.79 (± 0.037) to -3.03 (± 0.048, p -7 M from -2.74 (± 0.03) to 2.86 (± 0.031, p +2 channels

  8. Neoplastic and stromal cells contribute to an extracellular matrix gene expression profile defining a breast cancer subtype likely to progress.

    Tiziana Triulzi

    Full Text Available We recently showed that differential expression of extracellular matrix (ECM genes delineates four subgroups of breast carcinomas (ECM1, -2, -3- and -4 with different clinical outcome. To further investigate the characteristics of ECM signature and its impact on tumor progression, we conducted unsupervised clustering analyses in 6 additional independent datasets of invasive breast tumors from different platforms for a total of 643 samples. Use of four different clustering algorithms identified ECM3 tumors as an independent group in all datasets tested. ECM3 showed a homogeneous gene pattern, consisting of 58 genes encoding 43 structural ECM proteins. From 26 to 41% of the cases were ECM3-enriched, and analysis of datasets relevant to gene expression in neoplastic or corresponding stromal cells showed that both stromal and breast carcinoma cells can coordinately express ECM3 genes. In in vitro experiments, β-estradiol induced ECM3 gene production in ER-positive breast carcinoma cell lines, whereas TGFβ induced upregulation of the genes leading to ECM3 gene classification, especially in ER-negative breast carcinoma cells and in fibroblasts. Multivariate analysis of distant metastasis-free survival in untreated breast tumor patients revealed a significant interaction between ECM3 and histological grade (p = 0.001. Cox models, estimated separately in grade I-II and grade III tumors, indicated a highly significant association between ECM3 and worse survival probability only in grade III tumors (HR = 3.0, 95% CI = 1.3-7.0, p = 0.0098. Gene Set Enrichment analysis of ECM3 compared to non-ECM3 tumors revealed significant enrichment of epithelial-mesenchymal transition (EMT genes in both grade I-II and grade III subsets of ECM3 tumors. Thus, ECM3 is a robust cluster that identifies breast carcinomas with EMT features but with accelerated metastatic potential only in the undifferentiated (grade III phenotype. These findings support the

  9. Increased extracellular and intracellular Ca{sup 2+} lead to adipocyte accumulation in bone marrow stromal cells by different mechanisms

    Hashimoto, Ryota, E-mail: hryota@juntendo.ac.jp [Department of Physiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Katoh, Youichi, E-mail: katoyo@juntendo-urayasu.jp [Juntendo University Faculty of International Liberal Arts, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Department of Cardiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Miyamoto, Yuki [Juntendo University Faculty of Health Care and Nursing, Takasu 2-5-1, Urayasu-shi, Chiba 279-0023 (Japan); Itoh, Seigo; Daida, Hiroyuki [Department of Cardiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Nakazato, Yuji [Center for Environmental Research, Department of Cardiology, Juntendo University Faculty of Medicine Urayasu Hospital, Tomioka 2-1-1, Urayasu-shi, Chiba 279-0022 (Japan); Okada, Takao [Department of Physiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan)

    2015-02-20

    Mesenchymal stem cells found in bone marrow stromal cells (BMSCs) are the common progenitors for both adipocyte and osteoblast. An increase in marrow adipogenesis is associated with age-related osteopenia and anemia. Both extracellular and intracellular Ca{sup 2+} ([Ca{sup 2+}]{sub o} and [Ca{sup 2+}]{sub i}) are versatile signaling molecules that are involved in the regulation of cell functions, including proliferation and differentiation. We have recently reported that upon treatment of BMSCs with insulin and dexamethasone, both high [Ca{sup 2+}]{sub o} and high [Ca{sup 2+}]{sub i} enhanced adipocyte accumulation, which suggested that increases in [Ca{sup 2+}]{sub o} caused by bone resorption may accelerate adipocyte accumulation in aging and diabetic patients. In this study, we used primary mouse BMSCs to investigate the mechanisms by which high [Ca{sup 2+}]{sub o} and high [Ca{sup 2+}]{sub i} may enhance adipocyte accumulation. In the process of adipocyte accumulation, two important keys are adipocyte differentiation and the proliferation of BMSCs, which have the potential to differentiate into adipocytes. Use of MTT assay and real-time RT-PCR revealed that high [Ca{sup 2+}]{sub i} (ionomycin)-dependent adipocyte accumulation is caused by enhanced proliferation of BMSCs but not enhanced differentiation into adipocytes. Using fura-2 fluorescence-based approaches, we showed that high [Ca{sup 2+}]{sub o} (addition of CaCl{sub 2}) leads to increases in [Ca{sup 2+}]{sub i}. Flow cytometric methods revealed that high [Ca{sup 2+}]{sub o} suppressed the phosphorylation of ERK independently of intracellular Ca{sup 2+}. The inhibition of ERK by U0126 and PD0325901 enhanced the differentiation of BMSCs into adipocytes. These data suggest that increased extracellular Ca{sup 2+} provides the differentiation of BMSCs into adipocytes by the suppression of ERK activity independently of increased intracellular Ca{sup 2+}, which results in BMSC proliferation. - Highlights:

  10. Inverse dose-rate-effects on the expressions of extra-cellular matrix-related genes in low-dose-rate γ-ray irradiated murine cells

    Sugihara, Takashi; Tanaka, Kimio; Oghiso, Yoichi; Murano, Hayato

    2008-01-01

    Based on the results of previous microarray analyses of murine NIH3T3/PG13Luc cells irradiated with continuous low-dose-rate (LDR) γ-ray or end-high-dose-rate-irradiations (end-HDR) at the end of the LDR-irradiation period, the inverse dose-rate-effects on gene expression levels were observed. To compare differences of the effects between LDR-irradiation and HDR-irradiation, HDR-irradiations at 2 different times, one (ini-HDR) at the same time at the start of LDR-irradiation and the other (end-HDR), were performed. The up-regulated genes were classified into two types, in which one was up-regulated in LDR-, ini-HDR-, and end-HDR irradiation such as Cdkn1a and Ccng1, which were reported as p53-dependent genes, and the other was up-regulated in LDR- and ini-HDR irradiations such as pro-collagen TypeIa2/Colla2, TenascinC/Tnc, and Fibulin5/Fbln5, which were reported as extra-cellular matrix-related (ECM) genes. The time dependent gene expression patterns in LDR-irradiation were also classified into two types, in which one was an early response such as in Cdkn1a and Ccng1 and the other was a delayed response such as the ECM genes which have no linearity to total dose. The protein expression pattern of Cdkn1a increased dose dependently in LDR- and end-HDR-irradiations, but those of p53Ser15/18 and MDM2 in LDR-irradiations were different from end-HDR-irradiations. Furthermore, the gene expression levels of the ECM genes in embryonic fibroblasts from p53-deficient mice were not increased by LDR- and end-HDR-irradiation, so the delayed expressions of the ECM genes seem to be regulated by p53. Consequently, the inverse dose-rate-effects on the expression levels of the ECM genes in LDR- and end-HDR-irradiations may be explained from different time responses by p53 status. (author)

  11. Increased extracellular matrix density decreases MCF10A breast cell acinus formation in 3D culture conditions.

    Lance, Amanda; Yang, Chih-Chao; Swamydas, Muthulekha; Dean, Delphine; Deitch, Sandy; Burg, Karen J L; Dréau, Didier

    2016-01-01

    The extracellular matrix (ECM) contributes to the generation and dynamic of normal breast tissue, in particular to the generation of polarized acinar and ductal structures. In vitro 3D culture conditions, including variations in the composition of the ECM, have been shown to directly influence the formation and organization of acinus-like and duct-like structures. Furthermore, the density of the ECM appears to also play a role in the normal mammary tissue and tumour formation. Here we show that the density of the ECM directly influences the number, organization and function of breast acini. Briefly, non-malignant human breast MCF10A cells were incubated in increasing densities of a Matrigel®-collagen I matrix. Elastic moduli near and distant to the acinus structures were measured by atomic force microscopy, and the number of acinus structures was determined. Immunochemistry was used to investigate the expression levels of E-cadherin, laminin, matrix metalloproteinase-14 and ß-casein in MCF10A cells. The modulus of the ECM was significantly increased near the acinus structures and the number of acinus structures decreased with the increase in Matrigel-collagen I density. As evaluated by the expression of laminin, the organization of the acinus structures present was altered as the density of the ECM increased. Increases in both E-cadherin and MMP14 expression by MCF10A cells as ECM density increased were also observed. In contrast, MCF10A cells expressed lower ß-casein levels as the ECM density increased. Taken together, these observations highlight the key role of ECM density in modulating the number, organization and function of breast acini. Copyright © 2013 John Wiley & Sons, Ltd.

  12. Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

    Medina, D.; Oborn, C.J.; Li, M.L.; Bissell, M.J.

    1987-01-01

    The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of β-casein mRNA in the presence or absence of prolactin. The inducibility of β-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types

  13. Raffinose Induces Biofilm Formation by Streptococcus mutans in Low Concentrations of Sucrose by Increasing Production of Extracellular DNA and Fructan.

    Nagasawa, Ryo; Sato, Tsutomu; Senpuku, Hidenobu

    2017-08-01

    Streptococcus mutans is the primary etiological agent of dental caries and causes tooth decay by forming a firmly attached biofilm on tooth surfaces. Biofilm formation is induced by the presence of sucrose, which is a substrate for the synthesis of extracellular polysaccharides but not in the presence of oligosaccharides. Nonetheless, in this study, we found that raffinose, which is an oligosaccharide with an intestinal regulatory function and antiallergic effect, induced biofilm formation by S. mutans in a mixed culture with sucrose, which was at concentrations less than those required to induce biofilm formation directly. We analyzed the possible mechanism behind the small requirement for sucrose for biofilm formation in the presence of raffinose. Our results suggested that sucrose contributed to an increase in bacterial cell surface hydrophobicity and biofilm formation. Next, we examined how the effects of raffinose interacted with the effects of sucrose for biofilm formation. We showed that the presence of raffinose induced fructan synthesis by fructosyltransferase and aggregated extracellular DNA (eDNA, which is probably genomic DNA released from dead cells) into the biofilm. eDNA seemed to be important for biofilm formation, because the degradation of DNA by DNase I resulted in a significant reduction in biofilm formation. When assessing the role of fructan in biofilm formation, we found that fructan enhanced eDNA-dependent cell aggregation. Therefore, our results show that raffinose and sucrose have cooperative effects and that this induction of biofilm formation depends on supportive elements that mainly consist of eDNA and fructan. IMPORTANCE The sucrose-dependent mechanism of biofilm formation in Streptococcus mutans has been studied extensively. Nonetheless, the effects of carbohydrates other than sucrose are inadequately understood. Our findings concerning raffinose advance the understanding of the mechanism underlying the joint effects of sucrose and

  14. Myocardial extracellular volume fraction quantified by cardiovascular magnetic resonance is increased in hypertension and associated with left ventricular remodeling

    Wang, Shuli; Li, Jinghui; Chen, Xiuyu; Yin, Gang; Lan, Tian; Dai, Linlin; Zhang, Yan; Yin, Xiaorong; Zhao, Shihua; Hu, Hongjie; Lu, Minjie; Sirajuddin, Arlene; Arai, Andrew E.; An, Jing; Song, Lei; Dang, Aimin; Kellman, Peter

    2017-01-01

    To determine whether extracellular volume fraction (ECV) quantification by cardiac magnetic resonance (CMR) can demonstrate left ventricle (LV) abnormalities and relationship between ECV and LV remodeling in hypertension (HTN) patients ECV quantification was prospectively performed in 134 consecutive HTN patients and 97 healthy subjects. Individual and regional ECV were compared to the regions on late gadolinium enhancement (LGE) images. Statistical analysis of the relationship between LV global functional parameters and ECV was carried out using Pearson's correlation, Student's t test and multiple regressions. In the HTN group, 70.1% (94/134) were LGE negative and 29.9% (40/134) LGE positive. The mean ECV after adjusting for age, sex, BMI, diabetes, smoking and dyslipidaemia in healthy controls and LGE-negative patients were 26.9 ± 2.67% and 28.5 ± 2.9% (p < 0.001), respectively. The differences in ECV reached statistical significance among the regions of LGE, LGE-Peri, LGE remote and the normal area between the control and LGE-positive subgroup (all p < 0.05). Global ECV significantly correlated with LVEF (r = -0.466, p < 0.001) and LV hypertrophy (r = 0.667, p < 0.001). ECV can identify LV abnormalities at an early stage in HTN patients without LGE. These abnormalities may reflect an increase in diffuse myocardial fibrosis and are associated with LV remodeling. (orig.)

  15. Myocardial extracellular volume fraction quantified by cardiovascular magnetic resonance is increased in hypertension and associated with left ventricular remodeling

    Wang, Shuli; Li, Jinghui; Chen, Xiuyu; Yin, Gang; Lan, Tian; Dai, Linlin; Zhang, Yan; Yin, Xiaorong; Zhao, Shihua [Chinese Academy of Medical Sciences and Peking Union Medical College, Department of Magnetic Resonance Imaging, Cardiovascular Imaging and Intervention Center, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Beijing (China); Hu, Hongjie [Zhejiang University, Department of Radiology, Sir Run Run Shaw Hospital, School of Medicine, Hangzhou (China); Lu, Minjie [Chinese Academy of Medical Sciences and Peking Union Medical College, Department of Magnetic Resonance Imaging, Cardiovascular Imaging and Intervention Center, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Beijing (China); Laboratory for Advanced Cardiovascular Imaging, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Department of Health and Human Services, Bethesda, MD (United States); Sirajuddin, Arlene; Arai, Andrew E. [Laboratory for Advanced Cardiovascular Imaging, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Department of Health and Human Services, Bethesda, MD (United States); An, Jing [Siemens Shenzhen Magnetic Resonance Ltd., Siemens MRI Center, Shenzhen, Guangdong (China); Song, Lei; Dang, Aimin [Chinese Academy of Medical Sciences and Peking Union Medical College, Department of Cardiology, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Beijing (China); Kellman, Peter [National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health (NIH), US Department of Health and Human Services, Cardiovascular and Pulmonary Branch, Bethesda, MD (United States)

    2017-11-15

    To determine whether extracellular volume fraction (ECV) quantification by cardiac magnetic resonance (CMR) can demonstrate left ventricle (LV) abnormalities and relationship between ECV and LV remodeling in hypertension (HTN) patients ECV quantification was prospectively performed in 134 consecutive HTN patients and 97 healthy subjects. Individual and regional ECV were compared to the regions on late gadolinium enhancement (LGE) images. Statistical analysis of the relationship between LV global functional parameters and ECV was carried out using Pearson's correlation, Student's t test and multiple regressions. In the HTN group, 70.1% (94/134) were LGE negative and 29.9% (40/134) LGE positive. The mean ECV after adjusting for age, sex, BMI, diabetes, smoking and dyslipidaemia in healthy controls and LGE-negative patients were 26.9 ± 2.67% and 28.5 ± 2.9% (p < 0.001), respectively. The differences in ECV reached statistical significance among the regions of LGE, LGE-Peri, LGE remote and the normal area between the control and LGE-positive subgroup (all p < 0.05). Global ECV significantly correlated with LVEF (r = -0.466, p < 0.001) and LV hypertrophy (r = 0.667, p < 0.001). ECV can identify LV abnormalities at an early stage in HTN patients without LGE. These abnormalities may reflect an increase in diffuse myocardial fibrosis and are associated with LV remodeling. (orig.)

  16. Gene Deletions Resulting in Increased Nitrogen Release by Azotobacter vinelandii: Application of a Novel Nitrogen Biosensor

    Eberhart, Lauren J.; Ohlert, Janet M.; Knutson, Carolann M.; Plunkett, Mary H.

    2015-01-01

    Azotobacter vinelandii is a widely studied model diazotrophic (nitrogen-fixing) bacterium and also an obligate aerobe, differentiating it from many other diazotrophs that require environments low in oxygen for the function of the nitrogenase. As a free-living bacterium, A. vinelandii has evolved enzymes and transporters to minimize the loss of fixed nitrogen to the surrounding environment. In this study, we pursued efforts to target specific enzymes and further developed screens to identify individual colonies of A. vinelandii producing elevated levels of extracellular nitrogen. Targeted deletions were done to convert urea into a terminal product by disrupting the urease genes that influence the ability of A. vinelandii to recycle the urea nitrogen within the cell. Construction of a nitrogen biosensor strain was done to rapidly screen several thousand colonies disrupted by transposon insertional mutagenesis to identify strains with increased extracellular nitrogen production. Several disruptions were identified in the ammonium transporter gene amtB that resulted in the production of sufficient levels of extracellular nitrogen to support the growth of the biosensor strain. Further studies substituting the biosensor strain with the green alga Chlorella sorokiniana confirmed that levels of nitrogen produced were sufficient to support the growth of this organism when the medium was supplemented with sufficient sucrose to support the growth of the A. vinelandii in coculture. The nature and quantities of nitrogen released by urease and amtB disruptions were further compared to strains reported in previous efforts that altered the nifLA regulatory system to produce elevated levels of ammonium. These results reveal alternative approaches that can be used in various combinations to yield new strains that might have further application in biofertilizer schemes. PMID:25888177

  17. Abundance and co-occurrence of extracellular capsules increase environmental breadth: Implications for the emergence of pathogens.

    Rendueles, Olaya; Garcia-Garcerà, Marc; Néron, Bertrand; Touchon, Marie; Rocha, Eduardo P C

    2017-07-01

    Extracellular capsules constitute the outermost layer of many bacteria, are major virulence factors, and affect antimicrobial therapies. They have been used as epidemiological markers and recently became vaccination targets. Despite the efforts to biochemically serotype capsules in a few model pathogens, little is known of their taxonomic and environmental distribution. We developed, validated, and made available a computational tool, CapsuleFinder, to identify capsules in genomes. The analysis of over 2500 prokaryotic genomes, accessible in a database, revealed that ca. 50% of them-including Archaea-encode a capsule. The Wzx/Wzy-dependent capsular group was by far the most abundant. Surprisingly, a fifth of the genomes encode more than one capsule system-often from different groups-and their non-random co-occurrence suggests the existence of negative and positive epistatic interactions. To understand the role of multiple capsules, we queried more than 6700 metagenomes for the presence of species encoding capsules and showed that their distribution varied between environmental categories and, within the human microbiome, between body locations. Species encoding capsules, and especially those encoding multiple capsules, had larger environmental breadths than the other species. Accordingly, capsules were more frequent in environmental bacteria than in pathogens and, within the latter, they were more frequent among facultative pathogens. Nevertheless, capsules were frequent in clinical samples, and were usually associated with fast-growing bacteria with high infectious doses. Our results suggest that capsules increase the environmental range of bacteria and make them more resilient to environmental perturbations. Capsules might allow opportunistic pathogens to profit from empty ecological niches or environmental perturbations, such as those resulting from antibiotic therapy, to colonize the host. Capsule-associated virulence might thus be a by-product of environmental

  18. Abundance and co-occurrence of extracellular capsules increase environmental breadth: Implications for the emergence of pathogens.

    Olaya Rendueles

    2017-07-01

    Full Text Available Extracellular capsules constitute the outermost layer of many bacteria, are major virulence factors, and affect antimicrobial therapies. They have been used as epidemiological markers and recently became vaccination targets. Despite the efforts to biochemically serotype capsules in a few model pathogens, little is known of their taxonomic and environmental distribution. We developed, validated, and made available a computational tool, CapsuleFinder, to identify capsules in genomes. The analysis of over 2500 prokaryotic genomes, accessible in a database, revealed that ca. 50% of them-including Archaea-encode a capsule. The Wzx/Wzy-dependent capsular group was by far the most abundant. Surprisingly, a fifth of the genomes encode more than one capsule system-often from different groups-and their non-random co-occurrence suggests the existence of negative and positive epistatic interactions. To understand the role of multiple capsules, we queried more than 6700 metagenomes for the presence of species encoding capsules and showed that their distribution varied between environmental categories and, within the human microbiome, between body locations. Species encoding capsules, and especially those encoding multiple capsules, had larger environmental breadths than the other species. Accordingly, capsules were more frequent in environmental bacteria than in pathogens and, within the latter, they were more frequent among facultative pathogens. Nevertheless, capsules were frequent in clinical samples, and were usually associated with fast-growing bacteria with high infectious doses. Our results suggest that capsules increase the environmental range of bacteria and make them more resilient to environmental perturbations. Capsules might allow opportunistic pathogens to profit from empty ecological niches or environmental perturbations, such as those resulting from antibiotic therapy, to colonize the host. Capsule-associated virulence might thus be a by

  19. Increases in extracellular zinc in the amygdala in acquisition and recall of fear experience and their roles in response to fear.

    Takeda, A; Tamano, H; Imano, S; Oku, N

    2010-07-14

    The amygdala is enriched with histochemically reactive zinc, which is dynamically coupled with neuronal activity and co-released with glutamate. The dynamics of the zinc in the amygdala was analyzed in rats, which were subjected to inescapable stress, to understand the role of the zinc in emotional behavior. In the communication box, two rats were subjected to foot shock stress and anxiety stress experiencing emotional responses of foot-shocked rat under amygdalar perfusion. Extracellular zinc was increased by foot shock stress, while decreased by anxiety stress, suggesting that the differential changes in extracellular zinc are associated with emotional behavior. In rats conditioned with foot shock, furthermore, extracellular zinc was increased again in the recall of fear (foot shock) in the same box without foot shock. When this recall was performed under perfusion with CaEDTA, a membrane-impermeable zinc chelator, to examine the role of the increase in extracellular zinc, the time of freezing behavior was more increased, suggesting that zinc released in the lateral amygdala during the recall of fear participates in freezing behavior. To examine the role of the increase in extracellular zinc during fear conditioning, fear conditioning was also performed under perfusion with CaEDTA. The time of freezing behavior was more increased in the contextual recall, suggesting that zinc released in the lateral nucleus during fear conditioning also participates in freezing behavior in the recall. In brain slice experiment, CaEDTA enhanced presynaptic activity (exocytosis) in the lateral nucleus after activation of the entorhinal cortex. The present paper demonstrates that zinc released in the lateral amygdala may participate in emotional behavior in response to fear. Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

  20. Increased extracellular volume and altered mechanics are associated with LVH in hypertensive heart disease, not hypertension alone.

    Kuruvilla, Sujith; Janardhanan, Rajesh; Antkowiak, Patrick; Keeley, Ellen C; Adenaw, Nebiyu; Brooks, Jeremy; Epstein, Frederick H; Kramer, Christopher M; Salerno, Michael

    2015-02-01

    The goal of this study was to assess the relationship among extracellular volume (ECV), native T1, and systolic strain in hypertensive patients with left ventricular hypertrophy (HTN LVH), hypertensive patients without LVH (HTN non-LVH), and normotensive controls. Diffuse myocardial fibrosis in HTN LVH patients, as reflected by increased ECV and native T1, may be an underlying mechanism contributing to increased cardiovascular risk compared with HTN non-LVH subjects and controls. Furthermore, increased diffuse fibrosis in HTN LVH subjects may be associated with reduced peak systolic and early diastolic strain rate compared with the other 2 groups. T1 mapping was performed in 20 HTN LVH (mean age, 55 ± 11 years), 23 HTN non-LVH (mean age, 61 ± 12 years), and 22 control subjects (mean age, 54 ± 7 years) on a Siemens 1.5-T Avanto (Siemens Healthcare, Erlangen, Germany) using a previously validated modified look-locker inversion-recovery pulse sequence. T1 was measured pre-contrast and 10, 15, and 20 min after injection of 0.15 mmol/kg gadopentetate dimeglumine, and the mean ECV and native T1 were determined for each subject. Measurement of circumferential strain parameters were performed using cine displacement encoding with stimulated echoes. HTN LVH subjects had higher native T1 compared with controls (p < 0.05). HTN LVH subjects had higher ECV compared with HTN non-LVH subjects and controls (p < 0.05). Peak systolic circumferential strain and early diastolic strain rates were reduced in HTN LVH subjects compared with HTN non-LVH subjects and controls (p < 0.05). Increased levels of ECV and native T1 were associated with reduced peak systolic and early diastolic circumferential strain rate across all subjects. HTN LVH patients had higher ECV, longer native T1 and associated reduction in peak systolic circumferential strain, and early diastolic strain rate compared with HTN non-LVH and control subjects. Measurement of ECV and native T1 provide a noninvasive

  1. Carrying photosynthesis genes increases ecological fitness of cyanophage in silico.

    Hellweger, Ferdi L

    2009-06-01

    Several viruses infecting marine cyanobacteria carry photosynthesis genes (e.g. psbA, hli) that are expressed, yield proteins (D1, HLIP) and help maintain the cell's photosynthesis apparatus during the latent period. This increases energy and speeds up virus production, allowing for a reduced latent period (a fitness benefit), but it also increases the DNA size, which slows down new virus production and reduces burst size (a fitness cost). How do these genes affect the net ecological fitness of the virus? Here, this question is explored using a combined systems biology and systems ecology ('systems bioecology') approach. A novel agent-based model simulates individual cyanobacteria cells and virus particles, each with their own genes, transcripts, proteins and other properties. The effect of D1 and HLIP proteins is explicitly considered using a mechanistic photosynthesis component. The model is calibrated to the available database for Prochlorococcus ecotype MED4 and podovirus P-SSP7. Laboratory- and field-scale in silico survival, competition and evolution (gene packaging error) experiments with wild type and genetically engineered viruses are performed to develop vertical survival and fitness profiles, and to determine the optimal gene content. The results suggest that photosynthesis genes are nonessential, increase fitness in a manner correlated with irradiance, and that the wild type has an optimal gene content.

  2. Early alterations in extracellular matrix and transforming growth factor β gene expression in mouse lung indicative of late radiation fibrosis

    Finkelstein, J.N.; Johnston, C.J.; Baggs, R.; Rubin, P.

    1994-01-01

    Fibrosis, characterized by the accumulation of collagen, is a late result of thoracic irradiation. The expression of late radiation injury can be found immediately after irradiation by measuring messenger RNA (mRNA) abundance. To determine if extracellular matrix mRNA and transforming growth factor beta abundance was affected acutely after irradiation, the authors measured mRNA levels of collagen I (CI), collagen III (CIII), collagen IV (CIV), fibronectin (FN), and transforming growth factor β (TGFβ 1,2ampersand3 ) in mouse lungs on day 1 and day 14 after graded doses of radiation. C57BL/6 female mice were irradiated with a single dose to the thorax of 5 or 12.5 Gy. Total lung RNA was prepared and immobilized by Northern and slot blotting and hybridized with radiolabelled cDNA probes for CI, CIII, CIV, FN, TGFβ 1,2ampersand3 and a control probe encoding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Autoradiographic data were quantified by video densitometry and results normalized to GAPDH. Changes in the expression of CI, CIII, CIV, FN and TGFβ 1,2ampersand3 were observed as early as 1 day after exposure. Through 14 days, changes in mRNA up to 5-fold were seen for any one dose. Dose related changes as high as 10-fold were also evident. The CI:CIII ratio increased gradually for the 5 Gy dose at 14 days postirradiation while the CI:CII ratio for the 12.5 Gy dose decreased by approximately 4-fold as compared to the control. These studies suggest that alterations in expression of extracellular matrix and TGFβ mRNA occur very early after radiation injury even at low doses and may play a role in the development of chronic fibrosis. 37 refs., 6 figs

  3. Increased expression of electron transport chain genes in uterine leiomyoma.

    Tuncal, Akile; Aydin, Hikmet Hakan; Askar, Niyazi; Ozkaya, Ali Burak; Ergenoglu, Ahmet Mete; Yeniel, Ahmet Ozgur; Akdemir, Ali; Ak, Handan

    2014-01-01

    The etiology and pathophysiology of uterine leiomyomas, benign smooth muscle tumors of the uterus, are not well understood. To evaluate the role of mitochondria in uterine leiomyoma, we compared electron transport gene expressions of uterine leiomyoma tissue with myometrium tissue in six uterine leiomyoma patients by RT-PCR array. Our results showed an average of 1.562 (±0.445) fold increase in nuclear-encoded electron transport genes. These results might suggest an increase in size, number, or activity of mitochondria in uterine leiomyoma that, to our knowledge, has not been previously reported. © 2014 by the Association of Clinical Scientists, Inc.

  4. Association analysis between mitogen-activated protein/extracellular signal-regulated kinase (MEK) gene polymorphisms and depressive disorder in the Han Chinese population.

    Hu, Yingyan; Hong, Wu; Smith, Alicia; Yu, Shunying; Li, Zezhi; Wang, Dongxiang; Yuan, Chengmei; Cao, Lan; Wu, Zhiguo; Huang, Jia; Fralick, Drew; Phillips, Michael Robert; Fang, Yiru

    2017-11-01

    Recent research findings suggest that BDNF and BDNF signaling pathways participate in the development of major depressive disorder. Mitogen-activated extracellular signal-regulated kinase (MEK) is the most important kinase in the extracellular signal-regulated kinase pathway, and the extracellular signal-regulated kinase pathway is the key signaling pathway of BDNF, so it may play a role in development of depressive disorder. The aim of this study is to investigate the association between polymorphisms of the MAP2K1 (also known as MEK) gene and depressive disorder. Three single nucleotide polymorphisms (SNPs), were significantly associated with depressive disorder: rs1549854 (p = 0.006), rs1432441 (p = 0.025), and rs7182853 (p = 0.039). When subdividing the sample by gender, two of the SNPs remained statistically associated with depressive disorder in females: rs1549854 (p = 0.013) and rs1432441 (p = 0.04). The rs1549854 and rs1432441 polymorphisms of the MAP2K1 gene may be associated with major depressive disorder, especially in females. This study is the first to report that the MAP2K1 gene may be a genetic marker for depressive disorder. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Gene and miRNA expression signature of Lewis lung carcinoma LLC1 cells in extracellular matrix enriched microenvironment

    Stankevicius, Vaidotas; Vasauskas, Gintautas; Bulotiene, Danute; Butkyte, Stase; Jarmalaite, Sonata; Rotomskis, Ricardas; Suziedelis, Kestutis

    2016-01-01

    The extracellular matrix (ECM), one of the key components of tumor microenvironment, has a tremendous impact on cancer development and highly influences tumor cell features. ECM affects vital cellular functions such as cell differentiation, migration, survival and proliferation. Gene and protein expression levels are regulated in cell-ECM interaction dependent manner as well. The rate of unsuccessful clinical trials, based on cell culture research models lacking the ECM microenvironment, indicates the need for alternative models and determines the shift to three-dimensional (3D) laminin rich ECM models, better simulating tissue organization. Recognized advantages of 3D models suggest the development of new anticancer treatment strategies. This is among the most promising directions of 3D cell cultures application. However, detailed analysis at the molecular level of 2D/3D cell cultures and tumors in vivo is still needed to elucidate cellular pathways most promising for the development of targeted therapies. In order to elucidate which biological pathways are altered during microenvironmental shift we have analyzed whole genome mRNA and miRNA expression differences in LLC1 cells cultured in 2D or 3D culture conditions. In our study we used DNA microarrays for whole genome analysis of mRNA and miRNA expression differences in LLC1 cells cultivated in 2D or 3D culture conditions. Next, we indicated the most common enriched functional categories using KEGG pathway enrichment analysis. Finally, we validated the microarray data by quantitative PCR in LLC1 cells cultured under 2D or 3D conditions or LLC1 tumors implanted in experimental animals. Microarray gene expression analysis revealed that 1884 genes and 77 miRNAs were significantly altered in LLC1 cells after 48 h cell growth under 2D and ECM based 3D cell growth conditions. Pathway enrichment results indicated metabolic pathway, MAP kinase, cell adhesion and immune response as the most significantly altered

  6. Proton receptor GPR68 expression in dendritic-cell-like S100β-positive cells of rat anterior pituitary gland: GPR68 induces interleukin-6 gene expression in extracellular acidification.

    Horiguchi, Kotaro; Higuchi, Masashi; Yoshida, Saishu; Nakakura, Takashi; Tateno, Kozue; Hasegawa, Rumi; Takigami, Shu; Ohsako, Shunji; Kato, Takako; Kato, Yukio

    2014-11-01

    S100β-positive cells, which do not express the classical pituitary hormones, appear to possess multifunctional properties and are assumed to be heterogeneous in the anterior pituitary gland. The presence of several protein markers has shown that S100β-positive cells are composed of populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. Recently, we succeeded in separating S100β-positive cells into round-cell (dendritic-cell-like) and process-cell types. We also found the characteristic expression of anti-inflammatory factors (interleukin-6, Il-6) and membrane receptors (integrin β-6) in the round type. Here, we further investigate the function of the subpopulation of S100β-positive cells. Since IL-6 is also a paracrine factor that regulates hormone producing-cells, we examine whether a correlation exists among extracellular acid stress, IL-6 and hormone production by using primary cultures of anterior pituitary cells. Dendritic-cell-like S100β-positive cells notably expressed Gpr68 (proton receptor) and Il-6. Furthermore, the expression of Il-6 and proopiomelanocortin (Pomc) was up-regulated by extracellular acidification. The functional role of IL-6 and GPR68 in the gene expression of Pomc during extracellular acidification was also examined. Small interfering RNA for Il-6 up-regulated Pomc expression and that for Gpr68 reversed the down-regulation of Il-6 and up-regulated Pomc expression by extracellular acidification. Thus, S100β-positive dendritic-like cells can sense an increase in extracellular protons via GPR68 and respond by the production of IL-6 in order to suppress the up-regulation of Pomc expression.

  7. Cardiac-Restricted IGF-1Ea Overexpression Reduces the Early Accumulation of Inflammatory Myeloid Cells and Mediates Expression of Extracellular Matrix Remodelling Genes after Myocardial Infarction

    Enrique Gallego-Colon

    2015-01-01

    Full Text Available Strategies to limit damage and improve repair after myocardial infarct remain a major therapeutic goal in cardiology. Our previous studies have shown that constitutive expression of a locally acting insulin-like growth factor-1 Ea (IGF-1Ea propeptide promotes functional restoration after cardiac injury associated with decreased scar formation. In the current study, we investigated the underlying molecular and cellular mechanisms behind the enhanced functional recovery. We observed improved cardiac function in mice overexpressing cardiac-specific IGF-1Ea as early as day 7 after myocardial infarction. Analysis of gene transcription revealed that supplemental IGF-1Ea regulated expression of key metalloproteinases (MMP-2 and MMP-9, their inhibitors (TIMP-1 and TIMP-2, and collagen types (Col 1α1 and Col 1α3 in the first week after injury. Infiltration of inflammatory cells, which direct the remodelling process, was also altered; in particular there was a notable reduction in inflammatory Ly6C+ monocytes at day 3 and an increase in anti-inflammatory CD206+ macrophages at day 7. Taken together, these results indicate that the IGF-1Ea transgene shifts the balance of innate immune cell populations early after infarction, favouring a reduction in inflammatory myeloid cells. This correlates with reduced extracellular matrix remodelling and changes in collagen composition that may confer enhanced scar elasticity and improved cardiac function.

  8. Patients with HBV-related acute-on-chronic liver failure have increased concentrations of extracellular histones aggravating cellular damage and systemic inflammation.

    Li, X; Gou, C; Yao, L; Lei, Z; Gu, T; Ren, F; Wen, T

    2017-01-01

    Acute-on-chronic liver failure (ACLF) is the most common type of liver failure and associated with grave consequences. Systemic inflammation has been linked to its pathogenesis and outcome, but the identifiable triggers are absent. Recently, extracellular histones, especially H4, have been recognized as important mediators of cell damage in various inflammatory conditions. This study aimed to investigate whether extracellular histones have clinical implications in patients with hepatitis B virus (HBV)-related ACLF. One hundred and twelve patients with HBV-related ACLF, 90 patients with chronic hepatitis B, 88 patients with HBV-related liver cirrhosis and 40 healthy volunteers were entered into this study. Plasma histone H4 levels, cytokine profile and clinical data were obtained. Besides, patient's sera were incubated overnight with human L02 hepatocytes or monocytic U937 cells in the presence or absence of antihistone H4 antibody, and cellular damage and cytokine production were evaluated. We found that plasma histone H4 levels were greatly increased in patients with ACLF as compared with chronic hepatitis B, liver cirrhosis and healthy control subjects and were significantly associated with disease severity, systemic inflammation and outcome. Notably, ACLF patients' sera incubation decreased cultured L02 cell integrity and induced profound cytokine production in the supernatant of U937 cells. Antihistone H4 antibody treatment abrogated these adverse effects, thus confirming a cause-effect relationship between extracellular histones and organ injury/dysfunction. The data support the hypothesis that the increased extracellular histone levels in ACLF patients may aggravate disease severity by inducing cellular injury and systemic inflammation. Histone-targeted therapies may have potentially interventional value in clinical practice. © 2016 John Wiley & Sons Ltd.

  9. The R-enantiomer of citalopram counteracts escitalopram-induced increase in extracellular 5-HT in the frontal cortex of freely moving rats

    Mørk, A; Kreilgaard, Mads; Sánchez, C

    2003-01-01

    The selective serotonin (5-HT) reuptake inhibitor, citalopram, is a racemic mixture of an S(+)- and R(-)-enantiomer, escitalopram and R-citalopram, respectively. The present study compares the effects of escitalopram, R-citalopram and citalopram on extracellular levels of 5-HT in the frontal cortex...... of freely moving rats. In addition, co-injection of escitalopram and R-citalopram (ratios 1:2 and 1:4) were assessed. In some experiments escitalopram and R-citalopram were infused into the frontal cortex by reverse microdialysis. Finally, the extracellular level of escitalopram in the frontal cortex...... was studied after administration of escitalopram alone or in combination with R-citalopram. Escitalopram (1.0-3.9 mg/kg, s.c.) produced a greater maximal increase in extracellular 5-HT than citalopram (2.0-8.0 mg/kg, s.c.). R-citalopram (15.6 mg/kg s.c.) did not affect the 5-HT levels. When co-injected, R...

  10. Direct Involvement of ombB, omaB and omcB Genes in Extracellular Reduction of Fe(III by Geobacter sulfurreducens PCA

    Yimo eLiu

    2015-10-01

    Full Text Available The tandem gene clusters orfR-ombB-omaB-omcB and orfS-ombC-omaC-omcC of the metal-reducing bacterium Geobacter sulfurreducens PCA are responsible for trans-outer membrane electron transfer during extracellular reduction of Fe(III-citrate and ferrihydrite [a poorly crystalline Fe(III oxide]. Each gene cluster encodes a putative transcriptional factor (OrfR/OrfS, a porin-like outer-membrane protein (OmbB/OmbC, a periplasmic c-type cytochrome (c-Cyt, OmaB/OmaC and an outer-membrane c-Cyt (OmcB/OmcC. The individual roles of OmbB, OmaB and OmcB in extracellular reduction of Fe(III, however, have remained either uninvestigated or controversial. Here, we showed that replacements of ombB, omaB, omcB and ombB-omaB with an antibiotic gene in the presence of ombC-omaC-omcC had no impact on reduction of Fe(III-citrate by G. sulfurreducens PCA. Disruption of ombB, omaB, omcB and ombB-omaB in the absence of ombC-omaC-omcC, however, severely impaired the bacterial ability to reduce Fe(III-citrate as well as ferrihydrite. These results unequivocally demonstrate an overlapping role of ombB-omaB-omcB and ombC-omaC-omcC in extracellular Fe(III reduction by G. sulfurreducens PCA. Involvement of both ombB-omaB-omcB and ombC-omaC-omcC in extracellular Fe(III reduction reflects the importance of these trans-outer membrane protein complexes in the physiology of this bacterium. Moreover, the kinetics of Fe(III-citrate and ferrihydrite reduction by these mutants in the absence of ombC-omaC-omcC were nearly identical, which suggests that absence of any protein subunit eliminates function of OmaB/OmbB/OmcB protein complex. Finally, orfS was found to have a negative impact on the extracellular reduction of Fe(III-citrate and ferrihydrite in G. sulfurreducens PCA probably by serving as a transcriptional repressor.

  11. The influence of an in vitro generated bone-like extracellular matrix on osteoblastic gene expression of marrow stromal cells.

    Pham, Q.P.; Kasper, F.K.; Baggett, L.S.; Raphael, R.M.; Jansen, J.A.; Mikos, A.G.

    2008-01-01

    The function and development of cells rely heavily on the signaling interactions with the surrounding extracellular matrix (ECM). Therefore, a tissue engineering scaffold should mimic native ECM to recreate the in vivo environment. Previously, we have shown that an in vitro generated ECM secreted by

  12. Polybrene increases the efficiency of gene transfer by lipofection.

    Abe, A; Miyanohara, A; Friedmann, T

    1998-05-01

    Lipofection involves the introduction of foreign genetic information into mammalian cells through the use of lipophilic reagents that enhance cellular uptake of polynucleotides. Despite the use of currently optimized lipofection conditions, including the use of serum-depleted media, the efficiency of gene transfer is often low. We show here that, in a variety of cell lines, polybrene markedly enhances the efficiency of lipofection under standardized conditions and also compensates the serum-mediated inhibition of lipofection. Although the degree of the polybrene effect depends on the nature of the cell line, these results indicate that individually optimized concentrations of polybrene can be useful for increasing the efficiency of lipofectin-mediated gene transfer in vitro.

  13. Prevalence of genes encoding extracellular virulence factors among meticillin-resistant Staphylococcus aureus isolates from the University Hospital, Olomouc, Czech Republic.

    Sauer, P; Síla, J; Stosová, T; Vecerová, R; Hejnar, P; Vágnerová, I; Kolár, M; Raclavsky, V; Petrzelová, J; Lovecková, Y; Koukalová, D

    2008-04-01

    A rather fast and complicated progression of an infection caused by some strains of Staphylococcus aureus could be associated with the expression and co-action of virulence factor complexes in these strains. This study screened the antibiotic susceptibility and prevalence of virulence markers in isolates of meticillin-resistant S. aureus (MRSA) obtained from patients hospitalized at the University Hospital in Olomouc, Czech Republic. A total of 100 isolates was screened for 13 genes encoding extracellular virulence determinants (tst, pvl, eta, etb, sea, seb, sec, sed, see, seg, seh, sei and sej) and for their distribution in sample types. Eighty-nine isolates were positive for at least one of the genes. Genes for etb, pvl, see and seh were not detected in any of the MRSA isolates. No statistically significant differences in the occurrence of the determinants studied among sample types were found.

  14. Extracellular matrix protein fibulin-1 plasma levels are associated with increased cardiovascular risk in chronic kidney disease

    Scholze, Alexandra

    INTRODUCTION AND AIMS: Fibulin-1 is one of the few extracellular matrix proteins present in blood in high concentrations. We aimed to define the relationship between plasma fibulin-1 levels and risk markers of cardiovascular disease in patients with chronic kidney disease. METHODS: Plasma fibulin-1...... hemodynamic and arterial stiffness indices. RESULTS: We observed a positive correlation of fibulin-1 levels with age (r=0.38; p=0.033), glycated hemoglobin (r=0.80; p=0.003), creatinine (r=0.35; p=0.045), and fibrinogen (r=0.39; p=0.027). Glomerular filtration rate and fibulin-1 were inversely correlated (r......=-0.57; p=0.022). There was a positive correlation between fibulin-1 and central pulse pressure (r=0.44; p=0.011) and central augmentation pressure (r=0.55; p=0.001). In a multivariable regression model, diabetes, creatinine, fibrinogen and central augmentation pressure were independent predictors...

  15. Epigallocatechin-3-gallate increases intracellular [Ca2+] in U87 cells mainly by influx of extracellular Ca2+ and partly by release of intracellular stores.

    Kim, Hee Jung; Yum, Keun Sang; Sung, Jong-Ho; Rhie, Duck-Joo; Kim, Myung-Jun; Min, Do Sik; Hahn, Sang June; Kim, Myung-Suk; Jo, Yang-Hyeok; Yoon, Shin Hee

    2004-02-01

    Green tea has been receiving considerable attention as a possible preventive agent against cancer and cardiovascular disease. Epigallocatechin-3-gallate (EGCG) is a major polyphenol component of green tea. Using digital calcium imaging and an assay for [3H]-inositol phosphates, we determined whether EGCG increases intracellular [Ca2+] ([Ca2+]i) in non-excitable human astrocytoma U87 cells. EGCG induced concentration-dependent increases in [Ca2+]i. The EGCG-induced [Ca2+]i increases were reduced to 20.9% of control by removal of extracellular Ca2+. The increases were also inhibited markedly by treatment with the non-specific Ca2+ channel inhibitors cobalt (3 mM) for 3 min and lanthanum (1 mM) for 5 min. The increases were not significantly inhibited by treatment for 10 min with the L-type Ca2+ channel blocker nifedipine (100 nM). Treatment with the inhibitor of endoplasmic reticulum Ca2+-ATPase thapsigargin (1 micro M) also significantly inhibited the EGCG-induced [Ca2+]i increases. Treatment for 15 min with the phospholipase C (PLC) inhibitor neomycin (300 micro M) attenuated the increases significantly, while the tyrosine kinase inhibitor genistein (30 micro M) had no effect. EGCG increased [3H]-inositol phosphates formation via PLC activation. Treatment for 10 min with mefenamic acid (100 micro M) and flufenamic acid (100 micro M), derivatives of diphenylamine-2-carboxylate, blocked the EGCG-induced [Ca2+]i increase in non-treated and thapsigargin-treated cells but indomethacin (100 micro M) did not affect the increases. Collectively, these data suggest that EGCG increases [Ca2+]i in non-excitable U87 cells mainly by eliciting influx of extracellular Ca2+ and partly by mobilizing intracellular Ca2+ stores by PLC activation. The EGCG-induced [Ca2+]i influx is mediated mainly through channels sensitive to diphenylamine-2-carboxylate derivatives.

  16. Inhibition of Brain Swelling after Ischemia-Reperfusion by β-Adrenergic Antagonists: Correlation with Increased K+ and Decreased Ca2+ Concentrations in Extracellular Fluid

    Dan Song

    2014-01-01

    Full Text Available Infarct size and brain edema following ischemia/reperfusion are reduced by inhibitors of the Na+, K+, 2Cl−, and water cotransporter NKCC1 and by β1-adrenoceptor antagonists. NKCC1 is a secondary active transporter, mainly localized in astrocytes, driven by transmembrane Na+/K+ gradients generated by the Na+,K+-ATPase. The astrocytic Na+,K+-ATPase is stimulated by small increases in extracellular K+ concentration and by the β-adrenergic agonist isoproterenol. Larger K+ increases, as occurring during ischemia, also stimulate NKCC1, creating cell swelling. This study showed no edema after 3 hr medial cerebral artery occlusion but pronounced edema after 8 hr reperfusion. The edema was abolished by inhibitors of specifically β1-adrenergic pathways, indicating failure of K+-mediated, but not β1-adrenoceptor-mediated, stimulation of Na+,K+-ATPase/NKCC1 transport during reoxygenation. Ninety percent reduction of extracellular Ca2+ concentration occurs in ischemia. Ca2+ omission abolished K+ uptake in normoxic cultures of astrocytes after addition of 5 mM KCl. A large decrease in ouabain potency on K+ uptake in cultured astrocytes was also demonstrated in Ca2+-depleted media, and endogenous ouabains are needed for astrocytic K+ uptake. Thus, among the ionic changes induced by ischemia, the decrease in extracellular Ca2+ causes failure of the high-K+-stimulated Na+,K+-ATPase/NKCC1 ion/water uptake, making β1-adrenergic activation the only stimulus and its inhibition effective against edema.

  17. Combinatorial gene therapy renders increased survival in cirrhotic rats

    Armendáriz-Borunda Juan S

    2010-05-01

    Full Text Available Abstract Background Liver fibrosis ranks as the second cause of death in México's productive-age population. This pathology is characterized by acummulation of fibrillar proteins in hepatic parenchyma causing synthetic and metabolic disfunction. Remotion of excessive fibrous proteins might result in benefit for subjects increasing survival index. The goal of this work was to find whether the already known therapeutical effect of human urokinase Plasminogen Activator and human Matrix Metalloprotease 8 extends survival index in cirrhotic animals. Methods Wistar rats (80 g underwent chronic intoxication with CCl4: mineral oil for 8 weeks. Cirrhotic animals were injected with a combined dose of Ad-delta-huPA plus Ad-MMP8 (3 × 1011 and 1.5 × 1011 vp/Kg, respectively or with Ad-beta-Gal (4.5 × 1011 and were killed after 2, 4, 6, 8 and 10 days. Then, liver and serum were collected. An additional set of cirrhotic animals injected with combined gene therapy was also monitored for their probability of survival. Results Only the cirrhotic animals treated with therapeutical genes (Ad-delta-huPA+Ad-MMP-8 showed improvement in liver fibrosis. These results correlated with hydroxyproline determinations. A significant decrement in alpha-SMA and TGF-beta1 gene expression was also observed. Cirrhotic rats treated with Ad-delta-huPA plus Ad-MMP8 had a higher probability of survival at 60 days with respect to Ad-beta-Gal-injected animals. Conclusion A single administration of Ad-delta-huPA plus Ad-MMP-8 is efficient to induce fibrosis regression and increase survival in experimental liver fibrosis.

  18. Oxygen and Glucose Deprivation Induces Bergmann Glia Membrane Depolarization and Ca2+ Rises Mainly Mediated by K+ and ATP Increases in the Extracellular Space

    Romain Helleringer

    2017-11-01

    Full Text Available During brain ischemia, intense energy deficiency induces a complex succession of events including pump failure, acidosis and exacerbated glutamate release. In the cerebellum, glutamate is the principal mediator of Purkinje neuron anoxic depolarization during episodes of oxygen and glucose deprivation (OGD. Here, the impact of OGD is studied in Bergmann glia, specialized astrocytes closely associated to Purkinje neurons. Patch clamp experiments reveal that during OGD Bergmann glial cells develop a large depolarizing current that is not mediated by glutamate and purinergic receptors but is mainly due to the accumulation of K+ in the extracellular space. Furthermore, we also found that increases in the intracellular Ca2+ concentration appear in Bergmann glia processes several minutes following OGD. These elevations require, in an early phase, Ca2+ mobilization from internal stores via P2Y receptor activation, and, over longer periods, Ca2+ entry through store-operated calcium channels. Our results suggest that increases of K+ and ATP concentrations in the extracellular space are primordial mediators of the OGD effects on Bergmann glia. In the cerebellum, glial responses to energy deprivation-triggering events are therefore highly likely to follow largely distinct rules from those of their neuronal counterparts.

  19. Gene polymorphisms and increased DNA damage in morbidly obese women.

    Luperini, B C O; Almeida, D C; Porto, M P; Marcondes, J P C; Prado, R P; Rasera, I; Oliveira, M R M; Salvadori, D M F

    2015-06-01

    Obesity is characterized by increased adipose tissue mass resulting from a chronic imbalance between energy intake and expenditure. Furthermore, there is a clearly defined relationship among fat mass expansion, chronic low-grade systemic inflammation and reactive oxygen species (ROS) generation; leading to ROS-related pathological events. In the past years, genome-wide association studies have generated convincing evidence associating genetic variation at multiple regions of the genome with traits that reflect obesity. Therefore, the present study aimed to evaluate the relationships among the gene polymorphisms ghrelin (GHRL-rs26802), ghrelin receptor (GHSR-rs572169), leptin (LEP-rs7799039), leptin receptor (LEPR-rs1137101) and fat mass and obesity-associated (FTO-rs9939609) and obesity. The relationships among these gene variants and the amount of DNA damage were also investigated. Three hundred Caucasian morbidly obese and 300 eutrophic (controls) women were recruited. In summary, the results demonstrated that the frequencies of the GHRL, GHSR, LEP and LEPR polymorphisms were not different between Brazilian white morbidly obese and eutrophic women. Exceptions were the AA-FTO genotype and allele A, which were significantly more frequent in obese women than in the controls (0.23% vs. 0.10%; 0.46 vs. 0.36, respectively), and the TT-FTO genotype and the T allele, which were less frequent in morbidly obese women (p<0.01). Furthermore, significant differences in the amount of genetic lesions associated with FTO variants were observed only in obese women. In conclusion, this study demonstrated that the analyzed SNPs were not closely associated with morbid obesity, suggesting they are not the major contributors to obesity. Therefore, our data indicated that these gene variants are not good biomarkers for predicting risk susceptibility for obesity, whereas ROS generated by the inflammatory status might be one of the causes of DNA damage in obese women, favoring

  20. Increased spinal prodynorphin gene expression in reinflammation-associated hyperalgesia after neonatal inflammatory insult

    Wen Yeong-Ray

    2010-10-01

    Full Text Available Abstract Background Neuroplasticity induced by neonatal inflammation is the consequence of a combination of activity-dependent changes in neurons. We investigated neuronal sensitivity to a noxious stimulus in a rat model of neonatal hind-paw peripheral inflammation and assessed changes in pain behaviour at the physiological and molecular levels after peripheral reinflammation in adulthood. Results A decrease in paw withdrawal latency (PWL after a heat stimulus was documented in rats that received inflammatory injections in their left hind paws on postnatal day one (P1 and a reinflammation stimulus at postnatal 6-8 weeks of age, compared with normal rats. An increase in the expression of the prodynorphin (proDYN gene was noted after reinflammation in the spinal cord ipsilateral to the afferents of the neonatally treated hind paw. The involvement of the activation of extracellular signal-regulated kinases (ERK in peripheral inflammatory pain hypersensitivity was evidenced evident by the increase in phospho-ERK (pERK activity after reinflammation. Conclusions Our results indicate that peripheral inflammation in neonates can permanently alter the pain processing pathway during the subsequent sensory stimulation of the region. Elucidation of the mechanism underlying the developing pain circuitry will provide new insights into the understanding of the early pain behaviours and the subsequent adaptation to pain.

  1. Association of Protein Translation and Extracellular Matrix Gene Sets with Breast Cancer Metastasis: Findings Uncovered on Analysis of Multiple Publicly Available Datasets Using Individual Patient Data Approach.

    Nilotpal Chowdhury

    Full Text Available Microarray analysis has revolutionized the role of genomic prognostication in breast cancer. However, most studies are single series studies, and suffer from methodological problems. We sought to use a meta-analytic approach in combining multiple publicly available datasets, while correcting for batch effects, to reach a more robust oncogenomic analysis.The aim of the present study was to find gene sets associated with distant metastasis free survival (DMFS in systemically untreated, node-negative breast cancer patients, from publicly available genomic microarray datasets.Four microarray series (having 742 patients were selected after a systematic search and combined. Cox regression for each gene was done for the combined dataset (univariate, as well as multivariate - adjusted for expression of Cell cycle related genes and for the 4 major molecular subtypes. The centre and microarray batch effects were adjusted by including them as random effects variables. The Cox regression coefficients for each analysis were then ranked and subjected to a Gene Set Enrichment Analysis (GSEA.Gene sets representing protein translation were independently negatively associated with metastasis in the Luminal A and Luminal B subtypes, but positively associated with metastasis in Basal tumors. Proteinaceous extracellular matrix (ECM gene set expression was positively associated with metastasis, after adjustment for expression of cell cycle related genes on the combined dataset. Finally, the positive association of the proliferation-related genes with metastases was confirmed.To the best of our knowledge, the results depicting mixed prognostic significance of protein translation in breast cancer subtypes are being reported for the first time. We attribute this to our study combining multiple series and performing a more robust meta-analytic Cox regression modeling on the combined dataset, thus discovering 'hidden' associations. This methodology seems to yield new and

  2. Association of Protein Translation and Extracellular Matrix Gene Sets with Breast Cancer Metastasis: Findings Uncovered on Analysis of Multiple Publicly Available Datasets Using Individual Patient Data Approach.

    Chowdhury, Nilotpal; Sapru, Shantanu

    2015-01-01

    Microarray analysis has revolutionized the role of genomic prognostication in breast cancer. However, most studies are single series studies, and suffer from methodological problems. We sought to use a meta-analytic approach in combining multiple publicly available datasets, while correcting for batch effects, to reach a more robust oncogenomic analysis. The aim of the present study was to find gene sets associated with distant metastasis free survival (DMFS) in systemically untreated, node-negative breast cancer patients, from publicly available genomic microarray datasets. Four microarray series (having 742 patients) were selected after a systematic search and combined. Cox regression for each gene was done for the combined dataset (univariate, as well as multivariate - adjusted for expression of Cell cycle related genes) and for the 4 major molecular subtypes. The centre and microarray batch effects were adjusted by including them as random effects variables. The Cox regression coefficients for each analysis were then ranked and subjected to a Gene Set Enrichment Analysis (GSEA). Gene sets representing protein translation were independently negatively associated with metastasis in the Luminal A and Luminal B subtypes, but positively associated with metastasis in Basal tumors. Proteinaceous extracellular matrix (ECM) gene set expression was positively associated with metastasis, after adjustment for expression of cell cycle related genes on the combined dataset. Finally, the positive association of the proliferation-related genes with metastases was confirmed. To the best of our knowledge, the results depicting mixed prognostic significance of protein translation in breast cancer subtypes are being reported for the first time. We attribute this to our study combining multiple series and performing a more robust meta-analytic Cox regression modeling on the combined dataset, thus discovering 'hidden' associations. This methodology seems to yield new and interesting

  3. Hamstring contractures in children with spastic cerebral palsy result from a stiffer extracellular matrix and increased in vivo sarcomere length.

    Smith, Lucas R; Lee, Ki S; Ward, Samuel R; Chambers, Henry G; Lieber, Richard L

    2011-05-15

    Cerebral palsy (CP) results from an upper motoneuron (UMN)lesion in the developing brain. Secondary to the UMNl esion,which causes spasticity, is a pathological response by muscle - namely, contracture. However, the elements within muscle that increase passive mechanical stiffness, and therefore result in contracture, are unknown. Using hamstring muscle biopsies from pediatric patients with CP (n =33) and control (n =19) patients we investigated passive mechanical properties at the protein, cellular, tissue and architectural levels to identify the elements responsible for contracture. Titin isoform, the major load-bearing protein within muscle cells, was unaltered in CP. Correspondingly, the passive mechanics of individual muscle fibres were not altered. However, CP muscle bundles, which include fibres in their constituent ECM, were stiffer than control bundles. This corresponded to an increase in collagen content of CP muscles measured by hydroxyproline assay and observed using immunohistochemistry. In vivo sarcomere length of CP muscle measured during surgery was significantly longer than that predicted for control muscle. The combination of increased tissue stiffness and increased sarcomere length interact to increase stiffness greatly of the contracture tissue in vivo. These findings provide evidence that contracture formation is not the result of stiffening at the cellular level, but stiffening of the ECM with increased collagen and an increase of in vivo sarcomere length leading to higher passive stresses.

  4. Are specific gene expressions of extracellular matrix and nucleus pulposus affected by primary cell cultures prepared from intact or degenerative intervertebral disc tissues?

    Karaarslan, Numan; Yilmaz, Ibrahim; Ozbek, Hanefi; Sirin Yasar, Duygu; Kaplan, Necati; Akyuva, Yener; Gonultas, Aylin; Ates, Ozkan

    2018-01-22

    In this scientific research project, the researchers aimed to determine the gene expression patterns of nucleus pulposus (NP) in cell cultures obtained from degenerated or intact tissues. Whereas 12 of the cases were diagnosed with lumbar disc hernia and had undergone lumbar microdiscectomy, 12 cases had undergone traumatic intervertebral discectomy and corpectomy, along with discectomy after spinal trauma. NP-specific markers and gene expressions of the reagents of the extracellular matrix in the experimental setup were tested at the 0th, 24th, and 48th hours by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Visual evaluations were simultaneously made in all samples using invert and fluorescence microscopy. Vitality and proliferation analyses were evaluated by UV spectrophotometer. As a method of statistical evaluation, Spearman was used for categorical variants, and the Pearson correlation was used for variants with numerical and plain distribution. No association was found either between the tissue type and times (r=0.000; p=1.000) or between the region that the tissue was obtained from and hypoxia transcription factor-1 alpha (HIF-1α) gene expression (r=0.098; p=0.245). There was no correlation between cell proliferation and chondroadherin (CHAD) expression or between type II collagen (COL2A1) and CHAD gene expressions. It was found that CHAD and HIF-1α gene expressions and HIF-1α and COL2A1 gene expressions affected cell proliferation. Cell culture setups are of paramount importance because they may influence the pattern of changes in the gene expressions of the cells used in these setups.

  5. Fungal Screening on Olive Oil for Extracellular Triacylglycerol Lipases: Selection of a Trichoderma harzianum Strain and Genome Wide Search for the Genes

    Canseco-Pérez, Miguel Angel; Castillo-Avila, Genny Margarita; Islas-Flores, Ignacio; Apolinar-Hernández, Max M.; Rivera-Muñoz, Gerardo; Gamboa-Angulo, Marcela; Couoh-Uicab, Yeny

    2018-01-01

    A lipolytic screening with fungal strains isolated from lignocellulosic waste collected in banana plantation dumps was carried out. A Trichoderma harzianum strain (B13-1) showed good extracellular lipolytic activity (205 UmL−1). Subsequently, functional screening of the lipolytic activity on Rhodamine B enriched with olive oil as the only carbon source was performed. The successful growth of the strain allows us to suggest that a true lipase is responsible for the lipolytic activity in the B13-1 strain. In order to identify the gene(s) encoding the protein responsible for the lipolytic activity, in silico identification and characterization of triacylglycerol lipases from T. harzianum is reported for the first time. A survey in the genome of this fungus retrieved 50 lipases; however, bioinformatic analyses and putative functional descriptions in different databases allowed us to choose seven lipases as candidates. Suitability of the bioinformatic screening to select the candidates was confirmed by reverse transcription polymerase chain reaction (RT-PCR). The gene codifying 526309 was expressed when the fungus grew in a medium with olive oil as carbon source. This protein shares homology with commercial lipases, making it a candidate for further applications. The success in identifying a lipase gene inducible with olive oil and the suitability of the functional screening and bioinformatic survey carried out herein, support the premise that the strategy can be used in other microorganisms with sequenced genomes to search for true lipases, or other enzymes belonging to large protein families. PMID:29370083

  6. Diversity of Two-Domain Laccase-Like Multicopper Oxidase Genes in Streptomyces spp.: Identification of Genes Potentially Involved in Extracellular Activities and Lignocellulose Degradation during Composting of Agricultural Waste

    Lu, Lunhui; Zhang, Jiachao; Chen, Anwei; Chen, Ming; Jiang, Min; Yuan, Yujie; Wu, Haipeng; Lai, Mingyong; He, Yibin

    2014-01-01

    Traditional three-domain fungal and bacterial laccases have been extensively studied for their significance in various biotechnological applications. Growing molecular evidence points to a wide occurrence of more recently recognized two-domain laccase-like multicopper oxidase (LMCO) genes in Streptomyces spp. However, the current knowledge about their ecological role and distribution in natural or artificial ecosystems is insufficient. The aim of this study was to investigate the diversity and composition of Streptomyces two-domain LMCO genes in agricultural waste composting, which will contribute to the understanding of the ecological function of Streptomyces two-domain LMCOs with potential extracellular activity and ligninolytic capacity. A new specific PCR primer pair was designed to target the two conserved copper binding regions of Streptomyces two-domain LMCO genes. The obtained sequences mainly clustered with Streptomyces coelicolor, Streptomyces violaceusniger, and Streptomyces griseus. Gene libraries retrieved from six composting samples revealed high diversity and a rapid succession of Streptomyces two-domain LMCO genes during composting. The obtained sequence types cluster in 8 distinct clades, most of which are homologous with Streptomyces two-domain LMCO genes, but the sequences of clades III and VIII do not match with any reference sequence of known streptomycetes. Both lignocellulose degradation rates and phenol oxidase activity at pH 8.0 in the composting process were found to be positively associated with the abundance of Streptomyces two-domain LMCO genes. These observations provide important clues that Streptomyces two-domain LMCOs are potentially involved in bacterial extracellular phenol oxidase activities and lignocellulose breakdown during agricultural waste composting. PMID:24657870

  7. Increased Obesity-Associated Circulating Levels of the Extracellular Matrix Proteins Osteopontin, Chitinase-3 Like-1 and Tenascin C Are Associated with Colon Cancer.

    Victoria Catalán

    Full Text Available Excess adipose tissue represents a major risk factor for the development of colon cancer with inflammation and extracellular matrix (ECM remodeling being proposed as plausible mechanisms. The aim of this study was to investigate whether obesity can influence circulating levels of inflammation-related extracellular matrix proteins in patients with colon cancer (CC, promoting a microenvironment favorable for tumor growth.Serum samples obtained from 79 subjects [26 lean (LN and 53 obese (OB] were used in the study. Enrolled subjects were further subclassified according to the established diagnostic protocol for CC (44 without CC and 35 with CC. Anthropometric measurements as well as circulating metabolites and hormones were determined. Circulating concentrations of the ECM proteins osteopontin (OPN, chitinase-3-like protein 1 (YKL-40, tenascin C (TNC and lipocalin-2 (LCN-2 were determined by ELISA.Significant differences in circulating OPN, YKL-40 and TNC concentrations between the experimental groups were observed, being significantly increased due to obesity (P<0.01 and colon cancer (P<0.05. LCN-2 levels were affected by obesity (P<0.05, but no differences were detected regarding the presence or not of CC. A positive association (P<0.05 with different inflammatory markers was also detected.To our knowledge, we herein show for the first time that obese patients with CC exhibit increased circulating levels of OPN, YKL-40 and TNC providing further evidence for the influence of obesity on CC development via ECM proteins, representing promising diagnostic biomarkers or target molecules for therapeutics.

  8. Overexpression of KAI1 induces autophagy and increases MiaPaCa-2 cell survival through the phosphorylation of extracellular signal-regulated kinases

    Wu, Chun-Yan; Yan, Jun; Yang, Yue-Feng; Xiao, Feng-Jun; Li, Qing-Fang; Zhang, Qun-Wei; Wang, Li-Sheng; Guo, Xiao-Zhong; Wang, Hua

    2011-01-01

    Research highlights: → We first investigate the effects of KAI1 on autophagy in MiaPaCa-2 cells. → Our findings demonstrate that KAI1 induces autophagy, which in turn inhibits KAI1-induced apoptosis. → This study also supplies a possible novel therapeutic method for the treatment of pancreatic cancer using autophagy inhibitors. -- Abstract: KAI1, a metastasis-suppressor gene belonging to the tetraspanin family, is known to inhibit cancer metastasis without affecting the primary tumorigenicity by inhibiting the epidermal growth factor (EGF) signaling pathway. Recent studies have shown that hypoxic conditions of solid tumors induce high-level autophagy and KAI1 expression. However, the relationship between autophagy and KAI1 remains unclear. By using transmission electron microscopy, confocal microscopy, and Western blotting, we found that KAI1 can induce autophagy in a dose- and time-dependent manner in the human pancreatic cell line MiaPaCa-2. KAI1-induced autophagy was confirmed by the expression of autophagy-related proteins LC3 and Beclin 1. KAI1 induces autophagy through phosphorylation of extracellular signal-related kinases rather than that of AKT. KAI1-induced autophagy protects MiaPaCa-2 cells from apoptosis and proliferation inhibition partially through the downregulation of poly [adenosine diphosphate (ADP)-ribose] polymerase (PARP) cleavage and caspase-3 activation.

  9. Lymphatic Filariasis Increases Tissue Compressibility and Extracellular Fluid in Lower Limbs of Asymptomatic Young People in Central Myanmar

    Janet Douglass

    2017-09-01

    Full Text Available When normal lymphatic function is hampered, imperceptible subcutaneous edema can develop and progress to overt lymphedema. Low-cost reliable devices for objective assessment of lymphedema are well accepted in clinical practice and research on breast-cancer related lymphedema but are untested in populations with lymphatic filariasis (LF. This is a cross-sectional analysis of baseline data in a longitudinal study on asymptomatic, LF antigen-positive and -negative young people in Myanmar. Rapid field screening was used to identify antigen-positive cases and a group of antigen-negative controls of similar age and gender were invited to continue in the study. Tissue compressibility was assessed with three tissue tonometers, and free fluids were assessed using bio-impedance spectroscopy (BIS. Infection status was confirmed by Og4C3 antigen assay. At baseline (n = 98, antigen-positive cases had clinically relevant increases in tissue compressibility at the calf using a digital Indurometer (11.1%, p = 0.021, and in whole-leg free fluid using BIS (9.2%, p = 0.053. Regression analysis for moderating factors (age, gender, hydration reinforced the between-infection group differences. Results demonstrate that sub-clinical changes associated with infection can be detected in asymptomatic cases. Further exploration of these low-cost devices in clinical and research settings on filariasis-related lymphedema are warranted.

  10. Patterns of some extracellular matrix gene expression are similar in cells from cleft lip-palate patients and in human palatal fibroblasts exposed to diazepam in culture

    Marinucci, Lorella; Balloni, Stefania; Bodo, Maria; Carinci, Francesco; Pezzetti, Furio; Stabellini, Giordano; Carmela, Conte; Lumare, Eleonora

    2009-01-01

    Prenatal exposure to diazepam, a prototype sedative drug that belongs to Benzodiazepines, can lead to orofacial clefting in human newborns. By using real-time PCR, in the present study we investigated whether diazepam elicits gene expression alterations in extracellular matrix (ECM) components, growth factors and gamma-aminobutyric acid receptor (GABRB3), implicated in the coordinate regulation of palate development. Palate fibroblasts were treated with diazepam (Dz-N fibroblasts) and compared to cleft lip-palate (CLP) fibroblasts obtained from patients with no known exposure to diazepam or other teratogens. Untreated fibroblasts from non-CLP patients were used as control. The results showed significant convergences in gene expression pattern of collagens, fibromodulin, vitronectin, tenascin C, integrins and metalloprotease MMP13 between Dz-N and CLP fibroblasts. Among the growth factors, constitutive Fibroblast Growth Factor 2 (FGF2) was greatly enhanced in Dz-N and CLP fibroblasts and associated with a higher reduction of FGF receptor. Transforming Growth Factor beta 3 (TGFβ 3 ) resulted up-regulated in CLP fibroblasts and decreased in Dz-N fibroblasts. We found phenotypic differences exhibited by Dz-N and CLP fibroblasts in GABRB3 gene regulation, so further studies are necessary to determine whether GABAergic system could be involved in the development of diazepam mediated CLP phenotype. Taken together the results elucidate the molecular mechanisms underlying possible toxicology effects induced by diazepam. Counselling of women on the safety of diazepam exposure is clinically important, also for the forensic consequences

  11. aguA, the gene encoding an extracellular alpha-glucuronidase from Aspergillus tubingensis, is specifically induced on xylose and not on glucuronic acid.

    de Vries, R P; Poulsen, C H; Madrid, S; Visser, J

    1998-01-01

    An extracellular alpha-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70 degrees C. The alpha-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and beta-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this alpha-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and alpha-glucuronidase. Glucuronic acid did not induce the expression of aguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose.

  12. aguA, the Gene Encoding an Extracellular α-Glucuronidase from Aspergillus tubingensis, Is Specifically Induced on Xylose and Not on Glucuronic Acid

    de Vries, Ronald P.; Poulsen, Charlotte H.; Madrid, Susan; Visser, Jaap

    1998-01-01

    An extracellular α-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70°C. The α-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and β-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this α-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and α-glucuronidase. Glucuronic acid did not induce the expression of aguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose. PMID:9440512

  13. Long-term lithium treatment increases intracellular and extracellular brain-derived neurotrophic factor (BDNF) in cortical and hippocampal neurons at subtherapeutic concentrations.

    De-Paula, Vanessa J; Gattaz, Wagner F; Forlenza, Orestes V

    2016-12-01

    The putative neuroprotective effects of lithium treatment rely on the fact that it modulates several homeostatic mechanisms involved in the neurotrophic response, autophagy, oxidative stress, inflammation, and mitochondrial function. Lithium is a well-established therapeutic option for the acute and long-term management of bipolar disorder and major depression. The aim of this study was to evaluate the effects of subtherapeutic and therapeutic concentrations of chronic lithium treatment on brain-derived neurotrophic factor (BDNF) synthesis and secretion. Primary cultures of cortical and hippocampal neurons were treated with different subtherapeutic (0.02 and 0.2 mM) and therapeutic (2 mM) concentrations of chronic lithium treatment in cortical and hippocampal cell culture. Lithium treatment increased the intracellular protein expression of cortical neurons (10% at 0.02 mM) and hippocampal neurons (28% and 14% at 0.02 mM and 0.2 mM, respectively). Extracellular BDNF of cortical neurons increased 30% and 428% at 0.02 and 0.2 mM, respectively and in hippocampal neurons increased 44% at 0.02 mM. The present study indicates that chronic, low-dose lithium treatment up-regulates BDNF production in primary neuronal cell culture. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Increasing the complexity: new genes and new types of albinism.

    Montoliu, Lluís; Grønskov, Karen; Wei, Ai-Hua; Martínez-García, Mónica; Fernández, Almudena; Arveiler, Benoît; Morice-Picard, Fanny; Riazuddin, Saima; Suzuki, Tamio; Ahmed, Zubair M; Rosenberg, Thomas; Li, Wei

    2014-01-01

    Albinism is a rare genetic condition globally characterized by a number of specific deficits in the visual system, resulting in poor vision, in association with a variable hypopigmentation phenotype. This lack or reduction in pigment might affect the eyes, skin, and hair (oculocutaneous albinism, OCA), or only the eyes (ocular albinism, OA). In addition, there are several syndromic forms of albinism (e.g. Hermansky-Pudlak and Chediak-Higashi syndromes, HPS and CHS, respectively) in which the described hypopigmented and visual phenotypes coexist with more severe pathological alterations. Recently, a locus has been mapped to the 4q24 human chromosomal region and thus represents an additional genetic cause of OCA, termed OCA5, while the gene is eventually identified. In addition, two new genes have been identified as causing OCA when mutated: SLC24A5 and C10orf11, and hence designated as OCA6 and OCA7, respectively. This consensus review, involving all laboratories that have reported these new genes, aims to update and agree upon the current gene nomenclature and types of albinism, while providing additional insights from the function of these new genes in pigment cells. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene.

    Casucci, Monica; Falcone, Laura; Camisa, Barbara; Norelli, Margherita; Porcellini, Simona; Stornaiuolo, Anna; Ciceri, Fabio; Traversari, Catia; Bordignon, Claudio; Bonini, Chiara; Bondanza, Attilio

    2018-01-01

    Chimeric antigen receptor (CAR)-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and safety determinants that should be unveiled in order to advance the field. With the aim of facilitating the isolation and in vivo tracking of CAR-T cells, we here propose the inclusion within the CAR molecule of a novel extracellular spacer based on the low-affinity nerve-growth-factor receptor (NGFR). We screened four different spacer designs using as target antigen the CD44 isoform variant 6 (CD44v6). We successfully generated NGFR-spaced CD44v6 CAR-T cells that could be efficiently enriched with clinical-grade immuno-magnetic beads without negative consequences on subsequent expansion, immuno-phenotype, in vitro antitumor reactivity, and conditional ablation when co-expressing a suicide gene. Most importantly, these cells could be tracked with anti-NGFR monoclonal antibodies in NSG mice, where they expanded, persisted, and exerted potent antitumor effects against both high leukemia and myeloma burdens. Similar results were obtained with NGFR-enriched CAR-T cells specific for CD19 or CEA, suggesting the universality of this strategy. In conclusion, we have demonstrated that the incorporation of the NGFR marker gene within the CAR sequence allows for a single molecule to simultaneously work as a therapeutic and selection/tracking gene. Looking ahead, NGFR spacer enrichment might allow good manufacturing procedures-manufacturing of standardized CAR-T cell products with high therapeutic potential, which could be harmonized in different clinical trials and used in combination with a suicide gene for future application in the allogeneic setting.

  16. Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene

    Monica Casucci

    2018-03-01

    Full Text Available Chimeric antigen receptor (CAR-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and safety determinants that should be unveiled in order to advance the field. With the aim of facilitating the isolation and in vivo tracking of CAR-T cells, we here propose the inclusion within the CAR molecule of a novel extracellular spacer based on the low-affinity nerve-growth-factor receptor (NGFR. We screened four different spacer designs using as target antigen the CD44 isoform variant 6 (CD44v6. We successfully generated NGFR-spaced CD44v6 CAR-T cells that could be efficiently enriched with clinical-grade immuno-magnetic beads without negative consequences on subsequent expansion, immuno-phenotype, in vitro antitumor reactivity, and conditional ablation when co-expressing a suicide gene. Most importantly, these cells could be tracked with anti-NGFR monoclonal antibodies in NSG mice, where they expanded, persisted, and exerted potent antitumor effects against both high leukemia and myeloma burdens. Similar results were obtained with NGFR-enriched CAR-T cells specific for CD19 or CEA, suggesting the universality of this strategy. In conclusion, we have demonstrated that the incorporation of the NGFR marker gene within the CAR sequence allows for a single molecule to simultaneously work as a therapeutic and selection/tracking gene. Looking ahead, NGFR spacer enrichment might allow good manufacturing procedures-manufacturing of standardized CAR-T cell products with high therapeutic potential, which could be harmonized in different clinical trials and used in combination with a suicide gene for future application in the allogeneic setting.

  17. Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene

    Casucci, Monica; Falcone, Laura; Camisa, Barbara; Norelli, Margherita; Porcellini, Simona; Stornaiuolo, Anna; Ciceri, Fabio; Traversari, Catia; Bordignon, Claudio; Bonini, Chiara; Bondanza, Attilio

    2018-01-01

    Chimeric antigen receptor (CAR)-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and safety determinants that should be unveiled in order to advance the field. With the aim of facilitating the isolation and in vivo tracking of CAR-T cells, we here propose the inclusion within the CAR molecule of a novel extracellular spacer based on the low-affinity nerve-growth-factor receptor (NGFR). We screened four different spacer designs using as target antigen the CD44 isoform variant 6 (CD44v6). We successfully generated NGFR-spaced CD44v6 CAR-T cells that could be efficiently enriched with clinical-grade immuno-magnetic beads without negative consequences on subsequent expansion, immuno-phenotype, in vitro antitumor reactivity, and conditional ablation when co-expressing a suicide gene. Most importantly, these cells could be tracked with anti-NGFR monoclonal antibodies in NSG mice, where they expanded, persisted, and exerted potent antitumor effects against both high leukemia and myeloma burdens. Similar results were obtained with NGFR-enriched CAR-T cells specific for CD19 or CEA, suggesting the universality of this strategy. In conclusion, we have demonstrated that the incorporation of the NGFR marker gene within the CAR sequence allows for a single molecule to simultaneously work as a therapeutic and selection/tracking gene. Looking ahead, NGFR spacer enrichment might allow good manufacturing procedures-manufacturing of standardized CAR-T cell products with high therapeutic potential, which could be harmonized in different clinical trials and used in combination with a suicide gene for future application in the allogeneic setting. PMID:29619024

  18. Increased tenascin C and Toll-like receptor 4 levels in visceral adipose tissue as a link between inflammation and extracellular matrix remodeling in obesity.

    Catalán, Victoria; Gómez-Ambrosi, Javier; Rodríguez, Amaia; Ramírez, Beatriz; Rotellar, Fernando; Valentí, Victor; Silva, Camilo; Gil, María J; Salvador, Javier; Frühbeck, Gema

    2012-10-01

    Obesity is associated with an altered inflammatory and extracellular matrix (ECM) profile. Tenascin C (TNC) is an ECM glycoprotein with proinflammatory effects. We aimed to explore the expression levels of TNC in adipose tissue analyzing the contribution of adipocytes and stromovascular fraction cells (SVFC) as well as its impact on inflammation and ECM regulation. We also analyzed the effect of the stimulation with TNF-α and lipopolysaccharide (LPS) on both SVFC and adipocytes. Samples obtained from 75 subjects were used in the study. Expression levels of TNC, TLR4, MMP2, and MMP9 were analyzed in visceral adipose tissue (VAT) as well as in both adipocytes and SVFC. In addition, Tnc expression was measured in two mice models of obesity. We show, for the first time, that VAT expression levels of TNC are increased in normoglycemic and type 2 diabetic obese patients (Pobese patients with nonalcoholic steatohepatitis (Pobesity were significantly increased (Pexogenous TNC induced (Pobesity via visceral adipose tissue inflammation representing a link with ECM remodeling.

  19. Increased extracellular heat shock protein 90α in severe sepsis and SIRS associated with multiple organ failure and related to acute inflammatory-metabolic stress response in children.

    Fitrolaki, Michaela-Diana; Dimitriou, Helen; Venihaki, Maria; Katrinaki, Marianna; Ilia, Stavroula; Briassoulis, George

    2016-08-01

    Mammalian heat-shock-protein (HSP) 90α rapidly responses to environmental insults. We examined the hypothesis that not only serum HSP72 but also HSP90α is increased in the systemic inflammatory response syndrome (SIRS), severe-sepsis (SS), and/or sepsis (S) compared to healthy children (H); we assessed HSP90α relation to (a) multiple organ system failure (MOSF) and (b) inflammatory-metabolic response and severity of illness.A total of 65 children with S, SS, or SIRS and 25 H were included. ELISA was used to evaluate extracellular HSP90α and HSP72, chemiluminescence interleukins (ILs), flow-cytometry neutrophil-CD64 (nCD64)-expression.HSP90α, along with HSP72, were dramatically increased among MOSF patients. Patients in septic groups and SIRS had elevated HSP90α compared to H (P stress, fever, outcome endpoints, and predicted mortality and inversely related to the low-LDL/low-HDL stress metabolic pattern.

  20. Methylselenol, a selenium metabolite, induces cell cycle arrest in G1 phase and apoptosis via the extracellular-regulated kinase 1/2 pathway and other cancer signaling genes.

    Zeng, Huawei; Wu, Min; Botnen, James H

    2009-09-01

    Methylselenol has been hypothesized to be a critical selenium (Se) metabolite for anticancer activity in vivo, and our previous study demonstrated that submicromolar methylselenol generated by incubating methionase with seleno-l-methionine inhibits the migration and invasive potential of HT1080 tumor cells. However, little is known about the association between cancer signal pathways and methylselenol's inhibition of tumor cell invasion. In this study, we demonstrated that methylselenol exposure inhibited cell growth and we used a cancer signal pathway-specific array containing 15 different signal transduction pathways involved in oncogenesis to study the effect of methylselenol on cellular signaling. Using real-time RT-PCR, we confirmed that cellular mRNA levels of cyclin-dependent kinase inhibitor 1C (CDKN1C), heme oxygenase 1, platelet/endothelial cell adhesion molecule, and PPARgamma genes were upregulated to 2.8- to 5.7-fold of the control. BCL2-related protein A1, hedgehog interacting protein, and p53 target zinc finger protein genes were downregulated to 26-52% of the control, because of methylselenol exposure. These genes are directly related to the regulation of cell cycle and apoptosis. Methylselenol increased apoptotic cells up to 3.4-fold of the control and inhibited the extracellular-regulated kinase 1/2 (ERK1/2) signaling and cellular myelocytomatosis oncogene (c-Myc) expression. Taken together, our studies identify 7 novel methylselenol responsive genes and demonstrate that methylselenol inhibits ERK1/2 pathway activation and c-Myc expression. The regulation of these genes is likely to play a key role in G1 cell cycle arrest and apoptosis, which may contribute to the inhibition of tumor cell invasion.

  1. Extracellular Hsp90 serves as a co-factor for MAPK activation and latent viral gene expression during de novo infection by KSHV

    Qin Zhiqiang; DeFee, Michael; Isaacs, Jennifer S.; Parsons, Chris

    2010-01-01

    The Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), an important cause of morbidity and mortality in immunocompromised patients. KSHV interaction with the cell membrane triggers activation of specific intracellular signal transduction pathways to facilitate virus entry, nuclear trafficking, and ultimately viral oncogene expression. Extracellular heat shock protein 90 localizes to the cell surface (csHsp90) and facilitates signal transduction in cancer cell lines, but whether csHsp90 assists in the coordination of KSHV gene expression through these or other mechanisms is unknown. Using a recently characterized non-permeable inhibitor specifically targeting csHsp90 and Hsp90-specific antibodies, we show that csHsp90 inhibition suppresses KSHV gene expression during de novo infection, and that this effect is mediated largely through the inhibition of mitogen-activated protein kinase (MAPK) activation by KSHV. Moreover, we show that targeting csHsp90 reduces constitutive MAPK expression and the release of infectious viral particles by patient-derived, KSHV-infected primary effusion lymphoma cells. These data suggest that csHsp90 serves as an important co-factor for KSHV-initiated MAPK activation and provide proof-of-concept for the potential benefit of targeting csHsp90 for the treatment or prevention of KSHV-associated illnesses.

  2. Characterization of the rcsA Gene from Pantoea sp. Strain PPE7 and Its Influence on Extracellular Polysaccharide Production and Virulence on Pleurotus eryngii

    Min Keun Kim

    2017-06-01

    Full Text Available RcsA is a positive activator of extracellular polysaccharide (EPS synthesis in the Enterobacteriaceae. The rcsA gene of the soft rot pathogen Pantoea sp. strain PPE7 in Pleurotus eryngii was cloned by PCR amplification, and its role in EPS synthesis and virulence was investigated. The RcsA protein contains 3 highly conserved domains, and the C-terminal end of the open reading frame shared significant amino acid homology to the helix-turn-helix DNA binding motif of bacterial activator proteins. The inactivation of rcsA by insertional mutagenesis created mutants that had decreased production of EPS compared to the wild-type strain and abolished the virulence of Pantoea sp. strain PPE7 in P. eryngii. The Pantoea sp. strain PPE7 rcsA gene was shown to strongly affect the formation of the disease symptoms of a mushroom pathogen and to act as the virulence factor to cause soft rot disease in P. eryngii.

  3. The Arabidopsis SKU5 gene encodes an extracellular glycosyl phosphatidylinositol-anchored glycoprotein involved in directional root growth

    Sedbrook, John C.; Carroll, Kathleen L.; Hung, Kai F.; Masson, Patrick H.; Somerville, Chris R.

    2002-01-01

    To investigate how roots respond to directional cues, we characterized a T-DNA-tagged Arabidopsis mutant named sku5 in which the roots skewed and looped away from the normal downward direction of growth on inclined agar surfaces. sku5 roots and etiolated hypocotyls were slightly shorter than normal and exhibited a counterclockwise (left-handed) axial rotation bias. The surface-dependent skewing phenotype disappeared when the roots penetrated the agar surface, but the axial rotation defect persisted, revealing that these two directional growth processes are separable. The SKU5 gene belongs to a 19-member gene family designated SKS (SKU5 Similar) that is related structurally to the multiple-copper oxidases ascorbate oxidase and laccase. However, the SKS proteins lack several of the conserved copper binding motifs characteristic of copper oxidases, and no enzymatic function could be assigned to the SKU5 protein. Analysis of plants expressing SKU5 reporter constructs and protein gel blot analysis showed that SKU5 was expressed most strongly in expanding tissues. SKU5 was glycosylated and modified by glycosyl phosphatidylinositol and localized to both the plasma membrane and the cell wall. Our observations suggest that SKU5 affects two directional growth processes, possibly by participating in cell wall expansion.

  4. Increased expression of matrix extracellular phosphoglycoprotein (MEPE in cortical bone of the rat tibia after mechanical loading: identification by oligonucleotide microarray.

    Christianne M A Reijnders

    Full Text Available Skeletal integrity in humans and animals is maintained by daily mechanical loading. It has been widely accepted that osteocytes function as mechanosensors. Many biochemical signaling molecules are involved in the response of osteocytes to mechanical stimulation. The aim of this study was to identify genes involved in the translation of mechanical stimuli into bone formation. The four-point bending model was used to induce a single period of mechanical loading on the right tibia, while the contra lateral left tibia served as control. Six hours after loading, the effects of mechanical loading on gene-expression were determined with microarray analysis. Protein expression of differentially regulated genes was evaluated with immunohistochemistry. Nine genes were found to exhibit a significant differential gene expression in LOAD compared to control. MEPE, Garnl1, V2R2B, and QFG-TN1 olfactory receptor were up-regulated, and creatine kinase (muscle form, fibrinogen-B beta-polypeptide, monoamine oxidase A, troponin-C and kinesin light chain-C were down-regulated. Validation with real-time RT-PCR analysis confirmed the up-regulation of MEPE and the down-regulation of creatine kinase (muscle form and troponin-C in the loaded tibia. Immunohistochemistry showed that the increase of MEPE protein expression was already detectable six hours after mechanical loading. In conclusion, these genes probably play a role during translation of mechanical stimuli six hours after mechanical loading. The modulation of MEPE expression may indicate a connection between bone mineralization and bone formation after mechanical stimulation.

  5. Extracellular calmodulin regulates growth and cAMP-mediated chemotaxis in Dictyostelium discoideum

    O’Day, Danton H.; Huber, Robert J.; Suarez, Andres

    2012-01-01

    Highlights: ► Extracellular calmodulin is present throughout growth and development in Dictyostelium. ► Extracellular calmodulin localizes within the ECM during development. ► Extracellular calmodulin inhibits cell proliferation and increases chemotaxis. ► Extracellular calmodulin exists in eukaryotic microbes. ► Extracellular calmodulin may be functionally as important as intracellular calmodulin. -- Abstract: The existence of extracellular calmodulin (CaM) has had a long and controversial history. CaM is a ubiquitous calcium-binding protein that has been found in every eukaryotic cell system. Calcium-free apo-CaM and Ca 2+ /CaM exert their effects by binding to and regulating the activity of CaM-binding proteins (CaMBPs). Most of the research done to date on CaM and its CaMBPs has focused on their intracellular functions. The presence of extracellular CaM is well established in a number of plants where it functions in proliferation, cell wall regeneration, gene regulation and germination. While CaM has been detected extracellularly in several animal species, including frog, rat, rabbit and human, its extracellular localization and functions are less well established. In contrast the study of extracellular CaM in eukaryotic microbes remains to be done. Here we show that CaM is constitutively expressed and secreted throughout asexual development in Dictyostelium where the presence of extracellular CaM dose-dependently inhibits cell proliferation but increases cAMP mediated chemotaxis. During development, extracellular CaM localizes within the slime sheath where it coexists with at least one CaMBP, the matricellular CaM-binding protein CyrA. Coupled with previous research, this work provides direct evidence for the existence of extracellular CaM in the Dictyostelium and provides insight into its functions in this model amoebozoan.

  6. Transforming growth factor. beta. sub 1 is present at sites of extracellular matrix gene expression in human pulmonary fibrosis

    Broekelmann, T.J.; Limper, A.H.; McDonald, J.A. (Washington Univ., St. Louis, MO (United States)); Colby, T.V. (Mayo Clinic, Rochester, MN (United States))

    1991-08-01

    Idiopathic pulmonary fibrosis is an inexorably fatal disorder characterized by connective tissue deposition within the terminal air spaces resulting in loss of lung function and eventual respiratory failure. Previously, the authors demonstrated that foci of activated fibroblasts expressing high levels of fibronectin, procollagen, and smooth muscle actin and thus resembling those found in healing wounds are responsible for the connective tissue deposition and scarring in idiopathic pulmonary fibrosis. Using in situ hybridization and immunohistochemistry, they now demonstrate the presence of transforming growth factor {beta}{sub 1} (TGF-{beta}{sub 1}), a potent profibrotic cytokine, in the foci containing these activated fibroblasts. These results suggest that matrix-associated TGF-{beta}{sub 1} may serve as a stimulus for the persistent expression of connective tissue genes. One potential source of the TGF-{beta}{sub 1} is the alveolar macrophage, and they demonstrate the expression of abundant TGF-{beta}{sub 1} mRNA in alveolar macrophages in lung tissue from patients with idiopathic pulmonary fibrosis.

  7. Influence of bone morphogenetic protein-2 on the extracellular matrix, material properties, and gene expression of long-term articular chondrocyte cultures: loss of chondrocyte stability.

    Krawczak, David A; Westendorf, Jennifer J; Carlson, Cathy S; Lewis, Jack L

    2009-06-01

    The aim of this study was to determine the effects of bone morphogenetic protein-2 (BMP-2) on articular chondrocyte tissues grown as monolayers in vitro for up to 8 weeks. Articular chondrocytes were isolated from New Zealand White rabbits and plated in monolayer cultures. The cultures were supplemented with 100 ng/mL of BMP-2 for up to 8 weeks and the extracellular matrix (ECM) composition, material properties, and messenger RNA (mRNA) expression were analyzed. mRNA expression of cartilage-specific genes, type II collagen, and aggrecan showed that BMP-2 enhanced chondrocyte stability for up to 3 weeks. After 3 weeks in culture, there was substantially more type I collagen expression and more osteopontin and runt-related transcription factor 2 expression in 5- and 8-week cultures treated with BMP-2 than in controls. Additionally, matrix metalloproteinase-13 and ADAMTS-5 (A disintegrin-like and metalloproteinase with thrombospondin 5) were upregulated in 5- and 8-week cultures treated with BMP-2, coinciding with a loss of ECM density, collagen, and proteoglycan. Eight-week tissue stimulated with BMP-2 was more fragile and tore more easily when removed from the culture dish as compared to controls, suggesting temporal limitations to the effectiveness of BMP-2 in monolayer systems and perhaps other models to enhance the generation of a cartilage-like tissue for tissue engineering purposes.

  8. Interaction with extracellular matrix proteins influences Lsh/Ity/Bcg (candidate Nramp) gene regulation of macrophage priming/activation for tumour necrosis factor-alpha and nitrite release.

    Formica, S; Roach, T I; Blackwell, J M

    1994-05-01

    The murine resistance gene Lsh/Ity/Bcg regulates activation of macrophages for tumour necrosis factor-alpha (TNF-alpha)-dependent production of nitric oxide mediating antimicrobial activity against Leishmania, Salmonella and Mycobacterium. As Lsh is differentially expressed in macrophages from different tissue sites, experiments were performed to determine whether interaction with extracellular matrix (ECM) proteins would influence the macrophage TNF-alpha response. Plating of bone marrow-derived macrophages onto purified fibrinogen or fibronectin-rich L929 cell-derived matrices, but not onto mannan, was itself sufficient to stimulate TNF-alpha release, with significantly higher levels released from congenic B10.L-Lshr compared to C57BL/10ScSn (Lshs) macrophages. Only macrophages plated onto fibrinogen also released measurable levels of nitrites, again higher in Lshr compared to Lshs macrophages. Addition of interferon-gamma (IFN-gamma), but not bacterial lipopolysaccharide or mycobacterial lipoarabinomannan, as a second signal enhanced the TNF-alpha and nitrite responses of macrophages plated onto fibrinogen, particularly in the Lshr macrophages. Interaction with fibrinogen and fibronectin also primed macrophages for an enhanced TNF-alpha response to leishmanial parasites, but this was only translated into enhanced nitrite responses in the presence of IFN-gamma. In these experiments, Lshr macrophages remained superior in their TNF-alpha responses throughout, but to a degree which reflected the magnitude of the difference observed on ECM alone. Hence, the specificity for the enhanced TNF-alpha responses of Lshr macrophages lay in their interaction with fibrinogen and fibronectin ECM, while a differential nitrite response was only observed with fibrinogen and/or IFN-gamma. The results are discussed in relation to the possible function of the recently cloned candidate gene Nramp, which has structural identity to eukaryote transporters and an N-terminal cytoplasmic

  9. Cutting-edge gene editing techniques for increased vaccine yields ...

    2018-04-09

    Apr 9, 2018 ... Experiments show that reducing the level of IFITMs in chicken cells infected with influenza leads to increased levels of the virus in the ... Accelerating vaccine development for African swine fever virus through synthetic biology.

  10. A potato NOA gene increased salinity tolerance in Arabidopsis ...

    ONOS

    2010-09-06

    Sep 6, 2010 ... in Arabidopsis thaliana salt stress responses and increased its salinity tolerance. Key words: StNOA1 ... (NR)-dependent pathways (Cueto et al., 1996; Delledonne ..... plastome-encoded proteins uncovers a mechanism for the.

  11. The GABA uptake inhibitor beta-alanine reduces pilocarpine-induced tremor and increases extracellular GABA in substantia nigra pars reticulata as measured by microdialysis.

    Ishiwari, Keita; Mingote, Susana; Correa, Merce; Trevitt, Jennifer T; Carlson, Brian B; Salamone, John D

    2004-12-30

    Substantia nigra pars reticulata (SNr) is a major output nucleus of the basal ganglia that receives GABAergic projections from neostriatum and globus pallidus. Previous research has shown that local pharmacological manipulations of GABA in SNr can influence tremulous jaw movements in rats. Tremulous jaw movements are defined as rapid vertical deflections of the lower jaw that resemble chewing but are not directed at a particular stimulus, and evidence indicates that these movements share many characteristics with parkinsonian tremor in humans. In order to investigate the role of GABA in motor functions related to tremor, the present study tested the GABA uptake blocker beta-alanine for its ability to reduce pilocarpine-induced tremulous jaw movements. In a parallel experiment, the effect of an active dose of beta-alanine on dialysate levels of GABA in SNr was assessed using microdialysis methods. GABA levels in dialysis samples were measured using high performance liquid chromatography with electrochemical detection. beta-Alanine (250-500 mg/kg) significantly reduced tremulous jaw movements induced by pilocarpine (4.0 mg/kg). Moreover, systemic administration of beta-alanine at a dose that reduced tremulous jaw movements (500 mg/kg) resulted in a substantial increase in extracellular levels of GABA in SNr compared to the pre-injection baseline. Thus, the present results are consistent with the hypothesis that GABAergic tone in SNr plays a role in the regulation of tremulous jaw movements. This research may lead to a better understanding of how parkinsonian symptoms are modulated by SNr GABA mechanisms.

  12. Increased limbic phosphorylated extracellular-regulated kinase 1 and 2 expression after chronic stress is reduced by cyclic 17 beta-estradiol administration

    Gerrits, M.; Westenbroek, C.; Koch, T.; Grootkarzijn, A.; Ter Horst, G. J.

    2006-01-01

    Chronic stress induced neuronal changes that may have consequences for subsequent stress responses. For example, chronic stress in rats rearranges dendritic branching patterns and disturbs the phosphorylation of extracellular-regulated kinase 1 and 2 (ERK) 1/2 throughout the limbic system.

  13. Increased left ventricular myocardial extracellular volume is associated with longer cardiopulmonary bypass times, biventricular enlargement and reduced exercise tolerance in children after repair of Tetralogy of Fallot.

    Riesenkampff, Eugénie; Luining, Wietske; Seed, Mike; Chungsomprasong, Paweena; Manlhiot, Cedric; Elders, Bernadette; McCrindle, Brian W; Yoo, Shi-Joon; Grosse-Wortmann, Lars

    2016-10-26

    Unfavorable left ventricular (LV) remodelling may be associated with adverse outcomes after Tetralogy of Fallot (TOF) repair. We sought to assess T1 cardiovascular magnetic resonance (CMR) markers of diffuse LV myocardial fibrosis in children after TOF repair, and associated factors. In this prospective, cross-sectional study, native (=non-contrast) T1 times and extracellular volume fraction (ECV) were quantified in the LV myocardium using CMR. Results were related to ventricular volumes and function, degree of pulmonary regurgitation, as well as surgical characteristics, and exercise capacity. There was no difference in native T1 times or ECV between 31 TOF patients (age at CMR 13.9 ± 2.4 years, 19 male) and 15 controls (age at CMR 13.4 ± 2.6 years, 7 male). Female TOF patients had higher ECVs than males (25.2 ± 2.9 % versus 22.7 ± 3.3 %, p < 0.05). In the patient group, higher native T1 and ECV correlated with higher Z-Scores of right and left ventricular end-diastolic volumes, but not with reduced left and right ventricular ejection fraction or higher pulmonary regurgitation fraction. Longer cardiopulmonary bypass and aortic cross clamp times at surgery correlated with increased native T1 times and ECVs (r = 0.48, p < 0.05 and r = 0.65, p < 0.01, respectively). Maximum workload (percent of predicted for normal) correlated inversely with ECV (r = -0.62, p < 0.05). Higher native T1 times correlated with worse LV longitudinal (r = 0.50, p < 0.05) and mid short axis circumferential strain (r = 0.38, p < 0.05). As compared to controls, TOF patients did not express higher markers of diffuse fibrosis. Longer cardiopulmonary bypass and aortic cross clamp times at surgery as well as biventricular enlargement and reduced exercise tolerance are associated with markers of diffuse myocardial fibrosis after TOF repair. Female patients have higher markers of diffuse myocardial fibrosis than males.

  14. Urotensin II Induces ER Stress and EMT and Increase Extracellular Matrix Production in Renal Tubular Epithelial Cell in Early Diabetic Mice

    Xin-Xin Pang

    2016-07-01

    Full Text Available Background/Aims: Urotensin II (UII and its receptor are highly expressed in the kidney tissue of patients with diabetic nephropathy (DN. The aim of this study is to examine the roles of UII in the induction of endoplasmic reticulum stress (ER stress and Epithelial-mesenchymal transition (EMT in DN in vivo and in vitro. Methods: Kidney tissues were collected from patients with DN. C57BL/6 mice and mice with UII receptor knock out were injected with two consecutive doses of streptozotocin to induce diabetes and were sacrificed at 3th week for in vivo study. HK-2 cells in vitro were cultured and treated with UII. Markers of ER stress and EMT, fibronectin and type IV collagen were detected by immunohistochemistry, real time PCR and western blot. Results: We found that the expressions of protein of UII, GRP78, CHOP, ALPHA-SMA, fibronectin and type IV collagen were upregulated while E-cadherin protein was downregulated as shown by immunohistochemistry or western blot analysis in kidney of diabetic mice in comparison to normal control; moreover expressions of GRP78, CHOP, ALPHA-SMA, fibronectin and type IV collagen were inhibited while E-caherin expression was enhanced in kidney in diabetic mice with UII receptor knock out in comparison to C57BL/6 diabetic mice. In HK-2 cells, UII induced upregulation of GRP78, CHOP, ALPHA-SMA, fibroblast-specifc protein 1(FSP-1, fibronectin and type collagen and downregulation of E-cadherin. UII receptor antagonist can block UII-induced ER stress and EMT; moreover, 4-PBA can inhibit the mRNA expression of ALPHA-SMA and FSP1 induced by UII in HK-2 cells. Conclusions: We are the first to verify UII induces ER stress and EMT and increase extracellular matrix production in renal tubular epithelial cell in early diabetic mice. Moreover, UII may induce renal tubular epithelial EMT via triggering ER stress pathway in vitro, which might be the new pathogenic pathway for the development of renal fibrosis in DN.

  15. Signatures derived from increase in SHARPIN gene copy number are associated with poor prognosis in patients with breast cancer

    Diane Ojo

    2017-12-01

    Full Text Available We report three signatures produced from SHARPIN gene copy number increase (GCN-Increase and their effects on patients with breast cancer (BC. In the Metabric dataset (n = 2059, cBioPortal, SHARPIN GCN-Increase occurs preferentially or mutual exclusively with mutations in TP53, PIK3CA, and CDH1. These genomic alterations constitute a signature (SigMut that significantly correlates with reductions in overall survival (OS in BC patients (n = 1980; p = 1.081e−6. Additionally, SHARPIN GCN-Increase is associated with 4220 differentially expressed genes (DEGs. These DEGs are enriched in activation of the pathways regulating cell cycle progression, RNA transport, ribosome biosynthesis, DNA replication, and in downregulation of the pathways related to extracellular matrix. These DEGs are thus likely to facilitate the proliferation and metastasis of BC cells. Additionally, through forward (FWD and backward (BWD stepwise variate selections among the top 160 downregulated and top 200 upregulated DEGs using the Cox regression model, a 6-gene (SigFWD and a 50-gene (SigBWD signature were derived. Both signatures robustly associate with decreases in OS in BC patients within the Curtis (n = 1980; p = 6.16e−11 for SigFWD; p = 1.06e−10, for SigBWD and TCGA cohort (n = 817; p = 4.53e−4 for SigFWD and p = 0.00525 for SigBWD. After adjusting for known clinical factors, SigMut (HR 1.21, p = 0.0297, SigBWD (HR 1.25, p = 0.0263, and likely SigFWD (HR 1.17, p = 0.062 remain independent risk factors of BC deaths. Furthermore, the proportion of patients positive for these signatures is significantly increased in ER−, Her2-enriched, basal-like, and claudin-low BCs compared to ER+ and luminal BCs. Collectively, these SHARPIN GCN-Increase-derived signatures may have clinical applications in management of patients with BC.

  16. Global regulators ExpA (GacA) and KdgR modulate extracellular enzyme gene expression through the RsmA-rsmB system in Erwinia carotovora subsp. carotovora.

    Hyytiäinen, H; Montesano, M; Palva, E T

    2001-08-01

    The production of the main virulence determinants, the extracellular plant cell wall-degrading enzymes, and hence virulence of Erwinia carotovora subsp. carotovora is controlled by a complex regulatory network. One of the global regulators, the response regulator ExpA, a GacA homolog, is required for transcriptional activation of the extracellular enzyme genes of this soft-rot pathogen. To elucidate the mechanism of ExpA control as well as interactions with other regulatory systems, we isolated second-site transposon mutants that would suppress the enzyme-negative phenotype of an expA (gacA) mutant. Inactivation of kdgR resulted in partial restoration of extracellular enzyme production and virulence to the expA mutant, suggesting an interaction between the two regulatory pathways. This interaction was mediated by the RsmA-rsmB system. Northern analysis was used to show that the regulatory rsmB RNA was under positive control of ExpA. Conversely, the expression of rsmA encoding a global repressor was under negative control of ExpA and positive control of KdgR. This study indicates a central role for the RsmA-rsmB regulatory system during pathogenesis, integrating signals from the ExpA (GacA) and KdgR global regulators of extracellular enzyme production in E. carotovora subsp. carotovora.

  17. TGF-β1 Improves Biomechanical Strength by Extracellular Matrix Accumulation Without Increasing the Number of Tenogenic Lineage Cells in a Rat Rotator Cuff Repair Model.

    Arimura, Hitoshi; Shukunami, Chisa; Tokunaga, Takuya; Karasugi, Tatsuki; Okamoto, Nobukazu; Taniwaki, Takuya; Sakamoto, Hidetoshi; Mizuta, Hiroshi; Hiraki, Yuji

    2017-08-01

    Transforming growth factor β1 (TGF-β1) positively regulates the tenogenic marker genes scleraxis ( Scx) and tenomodulin ( Tnmd) in mesenchymal progenitors in vitro. However, little is known about the effect of TGF-β1 on the expression of tenogenic markers during rotator cuff (RC) healing in rats. TGF-β1 improves the biomechanical properties and histological maturity of reparative tissue in a rat RC repair model by stimulating the growth of tenogenic cells. Controlled laboratory study. Adult male Sprague-Dawley rats (N = 180) underwent unilateral supraspinatus tendon-to-bone surgical repair and were randomly treated with a gelatin hydrogel presoaked in TGF-β1 (100 ng) or phosphate-buffered saline. The effects of TGF-β1 on RC healing were investigated at 2, 4, 6, 8, and 12 weeks postoperatively by immunostaining for proliferating cell nuclear antigen, by real-time reverse transcription polymerase chain reaction and in situ hybridization or immunostaining for enthesis-related markers (SRY-box containing gene 9 [ Sox9], Scx, and Tnmd), and by real-time reverse transcription polymerase chain reaction and immunostaining for type I and III collagen. At 6 and 12 weeks postoperatively, biomechanical testing, micro-computed tomography, and biochemical analysis were also performed. At 2 and 4 weeks postoperatively, mesenchymal stem cell-related markers, phospho-Smad2, and matrix metalloproteinase 9 (MMP-9) and MMP-13 were assessed by immunostaining. The TGF-β1-treated group had significantly higher ultimate load to failure and tissue volume at 6 and 12 weeks postoperatively and a higher collagen content at 12 weeks compared with the saline group. Tendon-related gene expression, histological maturity, cell proliferation, and mesenchymal stem cell-related marker immunoreactivity were not affected by exogenously administrated TGF-β1 at all time points. In the TGF-β1-treated group, the percentage of phospho-Smad2-positive cells within the healing tissue increased

  18. Autism and increased paternal age related changes in global levels of gene expression regulation.

    Mark D Alter

    2011-02-01

    Full Text Available A causal role of mutations in multiple general transcription factors in neurodevelopmental disorders including autism suggested that alterations in global levels of gene expression regulation might also relate to disease risk in sporadic cases of autism. This premise can be tested by evaluating for changes in the overall distribution of gene expression levels. For instance, in mice, variability in hippocampal-dependent behaviors was associated with variability in the pattern of the overall distribution of gene expression levels, as assessed by variance in the distribution of gene expression levels in the hippocampus. We hypothesized that a similar change in variance might be found in children with autism. Gene expression microarrays covering greater than 47,000 unique RNA transcripts were done on RNA from peripheral blood lymphocytes (PBL of children with autism (n = 82 and controls (n = 64. Variance in the distribution of gene expression levels from each microarray was compared between groups of children. Also tested was whether a risk factor for autism, increased paternal age, was associated with variance. A decrease in the variance in the distribution of gene expression levels in PBL was associated with the diagnosis of autism and a risk factor for autism, increased paternal age. Traditional approaches to microarray analysis of gene expression suggested a possible mechanism for decreased variance in gene expression. Gene expression pathways involved in transcriptional regulation were down-regulated in the blood of children with autism and children of older fathers. Thus, results from global and gene specific approaches to studying microarray data were complimentary and supported the hypothesis that alterations at the global level of gene expression regulation are related to autism and increased paternal age. Global regulation of transcription, thus, represents a possible point of convergence for multiple etiologies of autism and other

  19. Prolactin 177, prolactin 188, and extracellular osmolality independently regulate the gene expression of ion transport effectors in gill of Mozambique tilapia.

    Inokuchi, Mayu; Breves, Jason P; Moriyama, Shunsuke; Watanabe, Soichi; Kaneko, Toyoji; Lerner, Darren T; Grau, E Gordon; Seale, Andre P

    2015-11-15

    This study characterized the local effects of extracellular osmolality and prolactin (PRL) on branchial ionoregulatory function of a euryhaline teleost, Mozambique tilapia (Oreochromis mossambicus). First, gill filaments were dissected from freshwater (FW)-acclimated tilapia and incubated in four different osmolalities, 280, 330, 380, and 450 mosmol/kg H2O. The mRNA expression of Na(+)/K(+)-ATPase α1a (NKA α1a) and Na(+)/Cl(-) cotransporter (NCC) showed higher expression with decreasing media osmolalities, while Na(+)/K(+)/2Cl(-) cotransporter 1a (NKCC1a) and PRL receptor 2 (PRLR2) mRNA levels were upregulated by increases in media osmolality. We then incubated gill filaments in media containing ovine PRL (oPRL) and native tilapia PRLs (tPRL177 and tPRL188). oPRL and the two native tPRLs showed concentration-dependent effects on NCC, NKAα1a, and PRLR1 expression; Na(+)/H(+) exchanger 3 (NHE3) expression was increased by 24 h of incubation with tPRLs. Immunohistochemical observation showed that oPRL and both tPRLs maintained a high density of NCC- and NKA-immunoreactive ionocytes in cultured filaments. Furthermore, we found that tPRL177 and tPRL188 differentially induce expression of these ion transporters, according to incubation time. Together, these results provide evidence that ionocytes of Mozambique tilapia may function as osmoreceptors, as well as directly respond to PRL to modulate branchial ionoregulatory functions. Copyright © 2015 the American Physiological Society.

  20. FRAS1-related extracellular matrix 3 (FREM3 single-nucleotide polymorphism effects on gene expression, amygdala reactivity and perceptual processing speed

    Yuliya eNikolova

    2015-09-01

    Full Text Available The A allele of the Fras1-related extracellular matrix protein 3 (FREM3 rs7676614 single nucleotide polymorphism (SNP was linked to major depressive disorder (MDD in an early genome-wide association study (GWAS, and to symptoms of psychomotor retardation in a follow-up investigation. In line with significant overlap between age- and depression-related molecular pathways, parallel work has shown that FREM3 expression in postmortem human brain decreases with age. Here we probe the effect of rs7676614 on amygdala reactivity and perceptual processing speed, both of which are altered in depression and aging. Amygdala reactivity was assessed using a face-matching BOLD fMRI paradigm in 365 Caucasian participants in the Duke Neurogenetics Study (192 women, mean age 19.7±1.2. Perceptual processing speed was indexed by reaction times in the same task and the Trails Making Test (TMT. The effect of rs7676614 on FREM3 mRNA brain expression levels was probed in a postmortem cohort of 169 Caucasian individuals (44 women, mean age 50.8±14.9. The A allele of rs7676614 was associated with blunted amygdala reactivity to faces, slower reaction times in the face-matching condition (p<0.04, as well as marginally slower performance on TMT Part B (p=0.056. In the postmortem cohort, the T allele of rs6537170 (proxy for the rs7676614 A allele, was associated with trend-level reductions in gene expression in Brodmann areas 11 and 47 (p=0.066, reminiscent of patterns characteristic of older age. The low-expressing allele of another FREM3 SNP (rs1391187 was similarly associated with reduced amygdala reactivity and slower TMT Part B speed, in addition to reduced BA47 activity and Extraversion (p<0.05. Together, these results suggest common genetic variation associated with reduced FREM3 expression may confer risk for a subtype of depression characterized by reduced reactivity to environmental stimuli and slower perceptual processing speed, possibly suggestive of

  1. Increasing platelet concentrations in leukocyte-reduced platelet-rich plasma decrease collagen gene synthesis in tendons.

    Boswell, Stacie G; Schnabel, Lauren V; Mohammed, Hussni O; Sundman, Emily A; Minas, Tom; Fortier, Lisa A

    2014-01-01

    Platelet-rich plasma (PRP) is used for the treatment of tendinopathy. There are numerous PRP preparations, and the optimal combination of platelets and leukocytes is not known. Within leukocyte-reduced PRP (lrPRP), there is a plateau effect of platelet concentration, with increasing platelet concentrations being detrimental to extracellular matrix synthesis. Controlled laboratory study. Different formulations of lrPRP with respect to the platelet:leukocyte ratio were generated from venous blood of 8 horses. Explants of the superficial digital flexor tendon were cultured in lrPRP products for 96 hours. Platelet-derived growth factor-BB (PDGF-BB), tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), and interleukin-1β (IL-1β) concentrations were determined in the media by enzyme-linked immunosorbent assay. Gene expression in tendon tissue for collagen type I and III (COL1A1 and COL3A1, respectively), matrix metalloproteinase-3 and -13 (MMP-3 and MMP-13, respectively), cartilage oligomeric matrix protein (COMP), and IL-1β was determined. Data were divided into 3 groups of lrPRP based on the ratio of platelets:leukocytes and evaluated to determine the effect of platelet concentration. Complete blood counts verified leukocyte reduction and platelet enrichment in all PRP preparations. In the lrPRP preparation, the anabolic growth factors PDGF-BB and TGF-β1 were increased with increasing platelet concentrations, and the catabolic cytokine IL-1β was decreased with increasing platelet concentrations. Increasing the platelet concentration resulted in a significant reduction in COL1A1 and COL3A1 synthesis in tendons. Increasing the platelet concentration within lrPRP preparations results in the delivery of more anabolic growth factors and less proinflammatory cytokines, but the biological effect on tendons is diminished metabolism as indicated by a decrease in the synthesis of both COL1A1 and COL3A1. Together, this information suggests that

  2. Changes in transcriptional orientation are associated with increases in evolutionary rates of enterobacterial genes

    Hsiung Chao

    2011-10-01

    Full Text Available Abstract Background Changes in transcriptional orientation (“CTOs” occur frequently in prokaryotic genomes. Such changes usually result from genomic inversions, which may cause a conflict between the directions of replication and transcription and an increase in mutation rate. However, CTOs do not always lead to the replication-transcription confrontation. Furthermore, CTOs may cause deleterious disruptions of operon structure and/or gene regulations. The currently existing CTOs may indicate relaxation of selection pressure. Therefore, it is of interest to investigate whether CTOs have an independent effect on the evolutionary rates of the affected genes, and whether these genes are subject to any type of selection pressure in prokaryotes. Methods Three closely related enterbacteria, Escherichia coli, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium, were selected for comparisons of synonymous (dS and nonsynonymous (dN substitution rate between the genes that have experienced changes in transcriptional orientation (changed-orientation genes, “COGs” and those that do not (same-orientation genes, “SOGs”. The dN/dS ratio was also derived to evaluate the selection pressure on the analyzed genes. Confounding factors in the estimation of evolutionary rates, such as gene essentiality, gene expression level, replication-transcription confrontation, and decreased dS at gene terminals were controlled in the COG-SOG comparisons. Results We demonstrate that COGs have significantly higher dN and dS than SOGs when a series of confounding factors are controlled. However, the dN/dS ratios are similar between the two gene groups, suggesting that the increase in dS can sufficiently explain the increase in dN in COGs. Therefore, the increases in evolutionary rates in COGs may be mainly mutation-driven. Conclusions Here we show that CTOs can increase the evolutionary rates of the affected genes. This effect is independent of the

  3. Membrane fusion inducers, chloroquine and spermidine increase lipoplex-mediated gene transfection

    Wong-Baeza, Carlos; Bustos, Israel; Serna, Manuel; Tescucano, Alonso; Alcantara-Farfan, Veronica; Ibanez, Miguel; Montanez, Cecilia; Wong, Carlos; Baeza, Isabel

    2010-01-01

    Gene transfection into mammalian cells can be achieved with viral and non-viral vectors. Non-viral vectors, such as cationic lipids that form lipoplexes with DNA, are safer and more stable than viral vectors, but their transfection efficiencies are lower. Here we describe that the simultaneous treatment with a membrane fusion inducer (chlorpromazine or procainamide) plus the lysosomotropic agent chloroquine increases lipoplex-mediated gene transfection in human (HEK293 and C-33 A) and rat (PC12) cell lines (up to 9.2-fold), as well as in situ in BALB/c mice spleens and livers (up to 6-fold); and that the polyamine spermidine increases lipoplex-mediated gene transfection and expression in cell cultures. The use of these four drugs provides a novel, safe and relatively inexpensive way to considerably increase lipoplex-mediated gene transfection efficiency.

  4. Genetically increased antioxidative protection and decreased chronic obstructive pulmonary disease

    Juul, Klaus; Tybjærg-Hansen, Anne; Marklund, Stefan

    2006-01-01

    RATIONALE: Increased oxidative stress is involved in chronic obstructive pulmonary disease (COPD); however, plasma and bronchial lining fluid contains the antioxidant extracellular superoxide dismutase. Approximately 2% of white individuals carry the R213G polymorphism in the gene encoding extrac...

  5. Nucleases from Prevotella intermedia can degrade neutrophil extracellular traps.

    Doke, M; Fukamachi, H; Morisaki, H; Arimoto, T; Kataoka, H; Kuwata, H

    2017-08-01

    Periodontitis is an inflammatory disease caused by periodontal bacteria in subgingival plaque. These bacteria are able to colonize the periodontal region by evading the host immune response. Neutrophils, the host's first line of defense against infection, use various strategies to kill invading pathogens, including neutrophil extracellular traps (NETs). These are extracellular net-like fibers comprising DNA and antimicrobial components such as histones, LL-37, defensins, myeloperoxidase, and neutrophil elastase from neutrophils that disarm and kill bacteria extracellularly. Bacterial nuclease degrades the NETs to escape NET killing. It has now been shown that extracellular nucleases enable bacteria to evade this host antimicrobial mechanism, leading to increased pathogenicity. Here, we compared the DNA degradation activity of major Gram-negative periodontopathogenic bacteria, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. We found that Pr. intermedia showed the highest DNA degradation activity. A genome search of Pr. intermedia revealed the presence of two genes, nucA and nucD, putatively encoding secreted nucleases, although their enzymatic and biological activities are unknown. We cloned nucA- and nucD-encoding nucleases from Pr. intermedia ATCC 25611 and characterized their gene products. Recombinant NucA and NucD digested DNA and RNA, which required both Mg 2+ and Ca 2+ for optimal activity. In addition, NucA and NucD were able to degrade the DNA matrix comprising NETs. © 2016 The Authors Molecular Oral Microbiology Published by John Wiley & Sons Ltd.

  6. Pycnogenol® effects on skin elasticity and hydration coincide with increased gene expressions of collagen type I and hyaluronic acid synthase in women.

    Marini, A; Grether-Beck, S; Jaenicke, T; Weber, M; Burki, C; Formann, P; Brenden, H; Schönlau, F; Krutmann, J

    2012-01-01

    In recent years there has been an increasing interest in the use of nutritional supplements to benefit human skin. Molecular evidence substantiating such effects, however, is scarce. In the present study we investigated whether nutritional supplementation of women with the standardized pine bark extract Pycnogenol® will improve their cosmetic appearance and relate these effects to expression of corresponding molecular markers of their skin. For this purpose 20 healthy postmenopausal women were supplemented with Pycnogenol for 12 weeks. Before, during and after supplementation, their skin condition was assessed (i) by employing non-invasive, biophysical methods including corneometry, cutometry, visioscan and ultrasound analyses and (ii) by taking biopsies and subsequent PCR for gene expression analyses related to extracellular matrix homeostasis. Pycnogenol supplementation was well tolerated in all volunteers. Pycnogenol significantly improved hydration and elasticity of skin. These effects were most pronounced in women presenting with dry skin conditions prior to the start of supplementation. The skin-physiological improvement was accompanied by a significant increase in the mRNA expression of hyaluronic acid synthase-1 (HAS-1), an enzyme critically involved in the synthesis of hyaluronic acid, and a noticeable increase in gene expression involved in collagen de novo synthesis. This study provides skin-physiological and for the first time molecular evidence that Pycnogenol supplementation benefits human skin by increasing skin hydration and skin elasticity. These effects are most likely due to an increased synthesis of extracellular matrix molecules such as hyaluronic acid and possibly collagen. Pycnogenol supplementation may thus be useful to counteract the clinical signs of skin aging. Copyright © 2012 S. Karger AG, Basel.

  7. Dramatic Increases of Soil Microbial Functional Gene Diversity at the Treeline Ecotone of Changbai Mountain.

    Shen, Congcong; Shi, Yu; Ni, Yingying; Deng, Ye; Van Nostrand, Joy D; He, Zhili; Zhou, Jizhong; Chu, Haiyan

    2016-01-01

    The elevational and latitudinal diversity patterns of microbial taxa have attracted great attention in the past decade. Recently, the distribution of functional attributes has been in the spotlight. Here, we report a study profiling soil microbial communities along an elevation gradient (500-2200 m) on Changbai Mountain. Using a comprehensive functional gene microarray (GeoChip 5.0), we found that microbial functional gene richness exhibited a dramatic increase at the treeline ecotone, but the bacterial taxonomic and phylogenetic diversity based on 16S rRNA gene sequencing did not exhibit such a similar trend. However, the β-diversity (compositional dissimilarity among sites) pattern for both bacterial taxa and functional genes was similar, showing significant elevational distance-decay patterns which presented increased dissimilarity with elevation. The bacterial taxonomic diversity/structure was strongly influenced by soil pH, while the functional gene diversity/structure was significantly correlated with soil dissolved organic carbon (DOC). This finding highlights that soil DOC may be a good predictor in determining the elevational distribution of microbial functional genes. The finding of significant shifts in functional gene diversity at the treeline ecotone could also provide valuable information for predicting the responses of microbial functions to climate change.

  8. Dramatic increases of soil microbial functional gene diversity at the treeline ecotone of Changbai Mountain

    Congcong Shen

    2016-07-01

    Full Text Available The elevational and latitudinal diversity patterns of microbial taxa have attracted great attention in the past decade. Recently, the distribution of functional attributes has been in the spotlight. Here, we report a study profiling soil microbial communities along an elevation gradient (500 to 2200 m on Changbai Mountain. Using a comprehensive functional gene microarray (GeoChip 5.0, we found that microbial functional gene richness exhibited a dramatic increase at the treeline ecotone, but the bacterial taxonomic and phylogenetic diversity based on 16S rRNA gene sequencing did not exhibit such a similar trend. However, the β-diversity (compositional dissimilarity among sites for both bacterial taxa and functional genes was similar, showing significant elevational distance-decay patterns which presented increased dissimilarity with elevation. The bacterial taxonomic diversity/structure was strongly influenced by soil pH, while the functional gene diversity/structure was significantly correlated with soil dissolved organic carbon (DOC. This finding highlights that soil DOC may be a good predictor in determining the elevational distribution of microbial functional genes. The finding of significant shifts in functional gene diversity at the treeline ecotone could also provide valuable information for predicting the responses of microbial functions to climate change.

  9. Identification and manipulation of the pleuromutilin gene cluster from Clitopilus passeckerianus for increased rapid antibiotic production

    Bailey, Andy M.; Alberti, Fabrizio; Kilaru, Sreedhar; Collins, Catherine M.; de Mattos-Shipley, Kate; Hartley, Amanda J.; Hayes, Patrick; Griffin, Alison; Lazarus, Colin M.; Cox, Russell J.; Willis, Christine L.; O'Dwyer, Karen; Spence, David W.; Foster, Gary D.

    2016-05-01

    Semi-synthetic derivatives of the tricyclic diterpene antibiotic pleuromutilin from the basidiomycete Clitopilus passeckerianus are important in combatting bacterial infections in human and veterinary medicine. These compounds belong to the only new class of antibiotics for human applications, with novel mode of action and lack of cross-resistance, representing a class with great potential. Basidiomycete fungi, being dikaryotic, are not generally amenable to strain improvement. We report identification of the seven-gene pleuromutilin gene cluster and verify that using various targeted approaches aimed at increasing antibiotic production in C. passeckerianus, no improvement in yield was achieved. The seven-gene pleuromutilin cluster was reconstructed within Aspergillus oryzae giving production of pleuromutilin in an ascomycete, with a significant increase (2106%) in production. This is the first gene cluster from a basidiomycete to be successfully expressed in an ascomycete, and paves the way for the exploitation of a metabolically rich but traditionally overlooked group of fungi.

  10. Regulatory Network Controlling Extracellular Proteins in Erwinia carotovora subsp. carotovora: FlhDC, the Master Regulator of Flagellar Genes, Activates rsmB Regulatory RNA Production by Affecting gacA and hexA (lrhA) Expression▿

    Cui, Yaya; Chatterjee, Asita; Yang, Hailian; Chatterjee, Arun K.

    2008-01-01

    Erwinia carotovora subsp. carotovora produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). rsmB RNA produc...

  11. Increased fat oxidation and regulation of metabolic genes with ultraendurance exercise

    Helge, Jørn Wulff; Rehrer, N J; Pilegaard, H

    2007-01-01

    AIM: Regular endurance exercise stimulates muscle metabolic capacity, but effects of very prolonged endurance exercise are largely unknown. This study examined muscle substrate availability and utilization during prolonged endurance exercise, and associated metabolic genes. METHODS: Data were...... exercise markedly increases plasma fatty acid availability and fat utilization during exercise. Exercise-induced regulation of genes encoding proteins involved in fatty acid recruitment and oxidation may contribute to these changes....

  12. Transforming growth factor-beta stimulates wound healing and modulates extracellular matrix gene expression in pig skin. I. Excisional wound model.

    Quaglino, D; Nanney, L B; Kennedy, R; Davidson, J M

    1990-09-01

    The effect of transforming growth factor-beta 1 (TGF-beta 1) on matrix gene expression has been investigated during the process of wound repair, where the formation of new connective tissue represents a critical step in restoring tissue integrity. Split-thickness excisional wounds in the pig were studied by in situ hybridization in order to obtain subjective findings on the activity and location of cells involved in matrix gene expression after the administration of recombinant TGF-beta 1. Data focus on the stimulatory role of this growth factor in granulation tissue formation, on the enhanced mRNA content of collagen types I and III, fibronectin, TGF-beta 1 itself, and on the reduction in stromelysin mRNA, suggesting that increased matrix formation measured after treatment with TGF-beta 1 is due to fibroplasia regulated by the abundance of mRNAs for several different structural, matrix proteins as well as inhibition of proteolytic phenomena elicited by metalloproteinases. These studies reveal elastin mRNA early in the repair process, and elastin mRNA expression is enhanced by administration of TGF-beta 1. Moreover, we show that TGF-beta 1 was auto-stimulating in wounds, accounting, at least in part, for the persistent effects of single doses of this multipotential cytokine.

  13. The pde2 gene of Saccharomyces cerevisiae is allelic to rca1 and encodes a phosphodiesterase which protects the cell from extracellular cAMP.

    Wilson, R B; Renault, G; Jacquet, M; Tatchell, K

    1993-07-05

    The high affinity cAMP phosphodiesterase, encoded by PDE2, is an important component of the cAMP-dependent protein kinase signaling system in Saccharomyces cerevisiae. An unexpected phenotype of pde2 mutants is sensitivity to external cAMP. This trait has been found independently for rca1 mutants and has been used to monitor the effects of cAMP on several biological processes. We demonstrate here that RCA1 is identical to PDE2. Further analysis of the phenotype of pde2 deletions reveal that exogenously added cAMP results in an increase in the internal level of cAMP. This increase slows down the rate of cell division by increasing the length of the G1 phase of the cell cycle and leads to increased cell volume. Also, cells with a disrupted PDE2 gene previously arrested by nutrient starvation rapidly lose thermotolerance when incubated with exogenous cAMP. From these observations we propose that a role of the PDE2-encoded phosphodiesterase may be to help insulate the internal cAMP pools from the external environment. This protective role might also be important in other eukaryotic organisms where cAMP is a key second messenger.

  14. The emerging role of skeletal muscle extracellular matrix remodelling in obesity and exercise.

    Martinez-Huenchullan, S; McLennan, S V; Verhoeven, A; Twigg, S M; Tam, C S

    2017-07-01

    Skeletal muscle extracellular matrix remodelling has been proposed as a new feature associated with obesity and metabolic dysfunction. Exercise training improves muscle function in obesity, which may be mediated by regulatory effects on the muscle extracellular matrix. This review examined available literature on skeletal muscle extracellular matrix remodelling during obesity and the effects of exercise. A non-systematic literature review was performed on PubMed of publications from 1970 to 2015. A total of 37 studies from humans and animals were retained. Studies reported overall increases in gene and protein expression of different types of collagen, growth factors and enzymatic regulators of the skeletal muscle extracellular matrix in obesity. Only two studies investigated the effects of exercise on skeletal muscle extracellular matrix during obesity, with both suggesting a regulatory effect of exercise. The effects of exercise on muscle extracellular matrix seem to be influenced by the duration and type of exercise training with variable effects from a single session compared with a longer duration of exercise. More studies are needed to elucidate the mechanisms behind skeletal muscle extracellular matrix remodelling during obesity and the effects of exercise. © 2017 World Obesity Federation.

  15. A positive feedback-based gene circuit to increase the production of a membrane protein

    Gennis Robert B

    2010-05-01

    Full Text Available Abstract Background Membrane proteins are an important class of proteins, playing a key role in many biological processes, and are a promising target in pharmaceutical development. However, membrane proteins are often difficult to produce in large quantities for the purpose of crystallographic or biochemical analyses. Results In this paper, we demonstrate that synthetic gene circuits designed specifically to overexpress certain genes can be applied to manipulate the expression kinetics of a model membrane protein, cytochrome bd quinol oxidase in E. coli, resulting in increased expression rates. The synthetic circuit involved is an engineered, autoinducer-independent variant of the lux operon activator LuxR from V. fischeri in an autoregulatory, positive feedback configuration. Conclusions Our proof-of-concept experiments indicate a statistically significant increase in the rate of production of the bd oxidase membrane protein. Synthetic gene networks provide a feasible solution for the problem of membrane protein production.

  16. The Ser311Cys variation in the paraoxonase 2 gene increases the ...

    Supplementary data: The Ser311Cys variation in the paraoxonase 2 gene increases the risk of type 2 diabetes in northern Chinese. Yanchun Qu, Ze Yang, Feng Jin, Liang Sun, Chuanfang Zhang, Linong Ji, Hong Sun, Binyou Wang and Li Wang. J. Genet. 87, 165–169. Table 1. Clinical characteristics of case and control.

  17. Increased xylose affinity of Hxt2 through gene shuffling of hexose transporters in Saccharomyces cerevisiae

    Nijland, Jeroen G; Shin, Hyun Yong; de Waal, Paul P; Klaassen, Paul; Driessen, Arnold J M

    AIMS: Optimizing D-xylose transport in Saccharomyces cerevisiae is essential for efficient bioethanol production from cellulosic materials. We have used a gene shuffling approach of hexose (Hxt) transporters in order to increase the affinity for D-xylose. METHODS AND RESULTS: Various libraries were

  18. Cholesteryl ester hydroperoxides increase macrophage CD36 gene expression via PPARα

    Jedidi, Iness; Couturier, Martine; Therond, Patrice; Gardes-Albert, Monique; Legrand, Alain; Barouki, Robert; Bonnefont-Rousselot, Dominique; Aggerbeck, Martine

    2006-01-01

    The uptake of oxidized LDL by macrophages is a key event in the development of atherosclerosis. The scavenger receptor CD36 is one major receptor that internalizes oxidized LDL. In differentiated human macrophages, we compared the regulation of CD36 expression by copper-oxidized LDL or their products. Only oxidized derivatives of cholesteryl ester (CEOOH) increased the amount of CD36 mRNA (2.5-fold). Both oxidized LDL and CEOOH treatment increased two to fourfold the transcription of promoters containing peroxisome-proliferator-activated-receptor responsive elements (PPRE) in the presence of PPARα or γ. Electrophoretic-mobility-shift-assays with nuclear extracts prepared from macrophages treated by either oxidized LDL or CEOOH showed increased binding of PPARα to the CD36 gene promoter PPRE. In conclusion, CEOOH present in oxidized LDL increase CD36 gene expression in a pathway involving PPARα

  19. Novel rat Alzheimer's disease models based on AAV-mediated gene transfer to selectively increase hippocampal Aβ levels

    Dicker Bridget L

    2007-06-01

    Full Text Available Abstract Background Alzheimer's disease (AD is characterized by a decline in cognitive function and accumulation of amyloid-β peptide (Aβ in extracellular plaques. Mutations in amyloid precursor protein (APP and presenilins alter APP metabolism resulting in accumulation of Aβ42, a peptide essential for the formation of amyloid deposits and proposed to initiate the cascade leading to AD. However, the role of Aβ40, the more prevalent Aβ peptide secreted by cells and a major component of cerebral Aβ deposits, is less clear. In this study, virally-mediated gene transfer was used to selectively increase hippocampal levels of human Aβ42 and Aβ40 in adult Wistar rats, allowing examination of the contribution of each to the cognitive deficits and pathology seen in AD. Results Adeno-associated viral (AAV vectors encoding BRI-Aβ cDNAs were generated resulting in high-level hippocampal expression and secretion of the specific encoded Aβ peptide. As a comparison the effect of AAV-mediated overexpression of APPsw was also examined. Animals were tested for development of learning and memory deficits (open field, Morris water maze, passive avoidance, novel object recognition three months after infusion of AAV. A range of impairments was found, with the most pronounced deficits observed in animals co-injected with both AAV-BRI-Aβ40 and AAV-BRI-Aβ42. Brain tissue was analyzed by ELISA and immunohistochemistry to quantify levels of detergent soluble and insoluble Aβ peptides. BRI-Aβ42 and the combination of BRI-Aβ40+42 overexpression resulted in elevated levels of detergent-insoluble Aβ. No significant increase in detergent-insoluble Aβ was seen in the rats expressing APPsw or BRI-Aβ40. No pathological features were noted in any rats, except the AAV-BRI-Aβ42 rats which showed focal, amorphous, Thioflavin-negative Aβ42 deposits. Conclusion The results show that AAV-mediated gene transfer is a valuable tool to model aspects of AD pathology in

  20. TIMP-1 gene deficiency increases tumour cell sensitivity to chemotherapy-induced apoptosis

    Davidsen, Marie Louise; Würts, S.Ø.; Rømer, Maria Unni Koefoed

    2006-01-01

    deficiency increases the response to chemotherapy considerably, confirming that TIMP-1 protects the cells from apoptosis. This is to our knowledge the first study investigating TIMP-1 and chemotherapy-induced apoptosis employing a powerful model system comprising TIMP-1 gene-deficient cells...... this hypothesis, we have established TIMP-1 gene-deficient and TIMP-1 wild-type fibrosarcoma cells from mouse lung tissue. We have characterised these cells with regard to TIMP-1 genotype, TIMP-1 expression, malignant transformation and sensitivity to chemotherapy-induced apoptosis. We show that TIMP-1 gene...... and their genetically identical wild-type controls. For future studies, this cell system can be used to uncover the mechanisms and signalling pathways involved in the TIMP-1-mediated inhibition of apoptosis as well as to investigate the possibility of using TIMP-1 inhibitors to optimise the effect of conventional...

  1. Extracellular DNA metabolism in Haloferax volcanii

    Scott eChimileski

    2014-02-01

    Full Text Available Extracellular DNA is found in all environments and is a dynamic component of the micro-bial ecosystem. Microbial cells produce and interact with extracellular DNA through many endogenous mechanisms. Extracellular DNA is processed and internalized for use as genetic information and as a major source of macronutrients, and plays several key roles within prokaryotic biofilms. Hypersaline sites contain some of the highest extracellular DNA con-centrations measured in nature–a potential rich source of carbon, nitrogen and phosphorus for halophilic microorganisms. We conducted DNA growth studies for the halophilic archaeon Haloferax volcanii DS2 and show that this model Halobacteriales strain is capable of using exogenous double-stranded DNA as a nutrient. Further experiments with varying medium composition, DNA concentration and DNA types revealed that DNA is utilized primarily as a phosphorus source, that growth on DNA is concentration-dependent and that DNA isolated from different sources is metabolized selectively, with a bias against highly divergent methylated DNA sources. Additionally, fluorescence microscopy experiments showed that labeled DNA colocalized with Haloferax volcanii cells. The gene Hvo_1477 was also identified using a comparative genomic approach as a factor likely to be involved in extracellular DNA processing at the cell surface, and deletion of Hvo_1477 created an H. volcanii strain deficient in its ability to grow on extracellular DNA. Widespread distribution of Hvo_1477 homologs in archaea suggests metabolism of extracellular DNA may be of broad ecological and physiological relevance in this domain of life.

  2. Placental triglyceride accumulation in maternal type 1 diabetes is associated with increased lipase gene expression

    Lindegaard, Marie Louise Skakkebæk; Damm, Peter; Mathiesen, Elisabeth R

    2006-01-01

    Maternal diabetes can cause fetal macrosomia and increased risk of obesity, diabetes, and cardiovascular disease in adulthood of the offspring. Although increased transplacental lipid transport could be involved, the impact of maternal type 1 diabetes on molecular mechanisms for lipid transport...... in placenta is largely unknown. To examine whether maternal type 1 diabetes affects placental lipid metabolism, we measured lipids and mRNA expression of lipase-encoding genes in placentas from women with type 1 diabetes (n = 27) and a control group (n = 21). The placental triglyceride (TG) concentration....... These results suggest that maternal type 1 diabetes is associated with TG accumulation and increased EL and HSL gene expression in placenta and that optimal metabolic control reduces these effects....

  3. Increased missense mutation burden of Fatty Acid metabolism related genes in nunavik inuit population.

    Zhou, Sirui; Xiong, Lan; Xie, Pingxing; Ambalavanan, Amirthagowri; Bourassa, Cynthia V; Dionne-Laporte, Alexandre; Spiegelman, Dan; Turcotte Gauthier, Maude; Henrion, Edouard; Diallo, Ousmane; Dion, Patrick A; Rouleau, Guy A

    2015-01-01

    Nunavik Inuit (northern Quebec, Canada) reside along the arctic coastline where for generations their daily energy intake has mainly been derived from animal fat. Given this particular diet it has been hypothesized that natural selection would lead to population specific allele frequency differences and unique variants in genes related to fatty acid metabolism. A group of genes, namely CPT1A, CPT1B, CPT1C, CPT2, CRAT and CROT, encode for three carnitine acyltransferases that are important for the oxidation of fatty acids, a critical step in their metabolism. Exome sequencing and SNP array genotyping were used to examine the genetic variations in the six genes encoding for the carnitine acyltransferases in 113 Nunavik Inuit individuals. Altogether ten missense variants were found in genes CPT1A, CPT1B, CPT1C, CPT2 and CRAT, including three novel variants and one Inuit specific variant CPT1A p.P479L (rs80356779). The latter has the highest frequency (0.955) compared to other Inuit populations. We found that by comparison to Asians or Europeans, the Nunavik Inuit have an increased mutation burden in CPT1A, CPT2 and CRAT; there is also a high level of population differentiation based on carnitine acyltransferase gene variations between Nunavik Inuit and Asians. The increased number and frequency of deleterious variants in these fatty acid metabolism genes in Nunavik Inuit may be the result of genetic adaptation to their diet and/or the extremely cold climate. In addition, the identification of these variants may help to understand some of the specific health risks of Nunavik Inuit.

  4. Adaptive evolution of mitochondrial energy metabolism genes associated with increased energy demand in flying insects.

    Yang, Yunxia; Xu, Shixia; Xu, Junxiao; Guo, Yan; Yang, Guang

    2014-01-01

    Insects are unique among invertebrates for their ability to fly, which raises intriguing questions about how energy metabolism in insects evolved and changed along with flight. Although physiological studies indicated that energy consumption differs between flying and non-flying insects, the evolution of molecular energy metabolism mechanisms in insects remains largely unexplored. Considering that about 95% of adenosine triphosphate (ATP) is supplied by mitochondria via oxidative phosphorylation, we examined 13 mitochondrial protein-encoding genes to test whether adaptive evolution of energy metabolism-related genes occurred in insects. The analyses demonstrated that mitochondrial DNA protein-encoding genes are subject to positive selection from the last common ancestor of Pterygota, which evolved primitive flight ability. Positive selection was also found in insects with flight ability, whereas no significant sign of selection was found in flightless insects where the wings had degenerated. In addition, significant positive selection was also identified in the last common ancestor of Neoptera, which changed its flight mode from direct to indirect. Interestingly, detection of more positively selected genes in indirect flight rather than direct flight insects suggested a stronger selective pressure in insects having higher energy consumption. In conclusion, mitochondrial protein-encoding genes involved in energy metabolism were targets of adaptive evolution in response to increased energy demands that arose during the evolution of flight ability in insects.

  5. Increased expression of ID2, PRELP and SMOC2 genes in patients with endometriosis

    F.M. Araujo

    Full Text Available Endometriosis is a benign, estrogen-dependent disease with symptoms such as pelvic pain and infertility, and it is characterized by the ectopic distribution of endometrial tissue. The expression of the ID2, PRELP and SMOC2 genes was compared between the endometrium of women without endometriosis in the proliferative phase of their menstrual cycle and the eutopic and ectopic endometrium of women with endometriosis in the proliferative phase. Paired tissue samples from 20 women were analyzed: 10 from endometrial and peritoneal endometriotic lesions and 10 from endometrial and ovarian endometriotic lesions. As controls, 16 endometrium samples were collected from women without endometriosis in the proliferative phase of menstrual cycle. Analysis was performed by real-time polymerase chain reaction (PCR. There was no significant difference between gene expression in the endometrium of women with and without endometriosis. The ID2 gene expression was increased in the most advanced stage of endometriosis and in ovarian endometriomas, the PRELP was more expressed in peritoneal lesions, and the SMOC2 was highly expressed in both peritoneal and endometrioma lesions. Considering that the genes studied participate either directly or indirectly in cellular processes that can lead to cell migration, angiogenesis, and inappropriate invasion, it is possible that the deregulation of these genes caused the development and maintenance of ectopic tissue.

  6. Testosterone increases renal anti-aging klotho gene expression via the androgen receptor-mediated pathway.

    Hsu, Shih-Che; Huang, Shih-Ming; Lin, Shih-Hua; Ka, Shuk-Man; Chen, Ann; Shih, Meng-Fu; Hsu, Yu-Juei

    2014-12-01

    Gender is known to be associated with longevity and oestrogen administration induced longevity-associated gene expression is one of the potential mechanisms underlying the benefits of oestrogen on lifespan, whereas the role of testosterone in the regulation of longevity-associated gene expressions remains largely unclear. The klotho gene, predominantly expressed in the kidney, has recently been discovered to be an aging suppressor gene. In the present study, we investigated the regulatory effects of testosterone on renal klotho gene expression in vivo and in vitro. In testosterone-administered mouse kidney and NRK-52E cells, increased klotho expression was accompanied by the up-regulation of the nuclear androgen receptor (AR). Overexpression of AR enhanced the expression of klotho mRNA and protein. Conversely, testosterone-induced klotho expression was attenuated in the presence of flutamide, an AR antagonist. A reporter assay and a chromatin immunoprecipitation (ChIP) assay demonstrated that AR directly binds to the klotho promoter via androgen response elements (AREs) which reconfirmed its importance for AR binding via the element mutation. In summary, our study demonstrates that testosterone up-regulates anti-aging klotho together with AR expression in the kidney in vivo and in vitro by recruiting AR on to the AREs of the klotho promoter.

  7. Polymorphisms within the COL5A1 gene and regulators of the extracellular matrix modify the risk of Achilles tendon pathology in a British case-control study.

    Brown, Karryn L; Seale, Kirsten B; El Khoury, Louis Y; Posthumus, Michael; Ribbans, William J; Raleigh, Stuart M; Collins, Malcolm; September, Alison V

    2017-08-01

    Several genetic loci have been associated with risk of Achilles tendon pathology (ATP) within South African and Australian populations. The aim of this study was, therefore, to evaluate eight previously implicated genetic variants in an independent British population. A total of 130 asymptomatic controls (CON) and 112 participants clinically diagnosed with ATP comprising 87 individuals with chronic Achilles tendinopathy (TEN) and 25 with Achilles tendon ruptures (RUP) were included. All participants were genotyped for variants within the COL5A1, MIR608, IL-1β, IL-6 and CASP8 genes. Primary findings implicated COL5A1 and CASP8. Three inferred allele combinations constructed from COL5A1 rs12722, rs3196378 and rs71746744 were identified as risk modifiers. The T-C-D combination was associated with increased risk of ATP (P = 0.023) and RUP (P RUP (P = 0.011) and the C-C-D combination was associated with decreased risk of ATP (P = 0.011) and RUP (P = 0.004). The CASP8 rs3834129 DD genotype was associated with decreased risk of TEN (P = 0.020, odds ratio: 0.45, 95% confidence interval: 0.22-0.90) and the CASP8 I-G (rs3834129-rs1045485) inferred allele combination was associated with increased risk of TEN (P = 0.031). This study further highlights the importance of polymorphisms within COL5A1 and CASP8 in the aetiology of ATP.

  8. Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway

    Kosalková Katarina; García-Estrada Carlos; Barreiro Carlos; Flórez Martha G; Jami Mohammad S; Paniagua Miguel A; Martín Juan F

    2012-01-01

    Abstract Background The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Results Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold) in cells grown with casein or casein phosphopeptides (CPPs). CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% u...

  9. Low-frequency ultrasound increases non-viral gene transfer to the mouse lung.

    Xenariou, Stefania; Liang, Hai-Dong; Griesenbach, Uta; Zhu, Jie; Farley, Raymond; Somerton, Lucinda; Singh, Charanjit; Jeffery, Peter K; Scheule, Ronald K; Cheng, Seng H; Geddes, Duncan M; Blomley, Martin; Alton, Eric W F W

    2010-01-01

    The aim of the study was to assess if low-frequency ultrasound (US), in the range of 30-35 kHz, increases non-viral gene transfer to the mouse lung. US is greatly attenuated in the lung due to large energy losses at the air/tissue interfaces. The advantages of low-frequency US, compared with high-frequency US are: (i) increased cavitation (responsible for the formation of transient pores in the cell membrane) and (ii) reduced energy losses during lung penetration. Cationic lipid GL67/plasmid DNA (pDNA), polyethylenimine (PEI)/pDNA and naked pDNA were delivered via intranasal instillation and the animals were then exposed to US (sonoporation) at 0.07 or 0.1 MPa for 10 min. Under these conditions, US did not enhance GL67 or PEI-mediated transfection. It did, however, increase naked pDNA gene transfer by approximately 4 folds. Importantly, this was achieved in the absence of microbubbles, which are crucial for the commonly used high-frequency (1 MHz) sonoporation but may not be able to withstand nebulization in a clinically relevant setup. Lung hemorrhage was also assessed and shown to increase with US pressure in a dose-dependent manner. We have thus, established that low-frequency US can enhance lung gene transfer with naked pDNA and this enhancement is more effective than the previously reported 1 MHz US.

  10. Production of extracellular fatty acid using engineered Escherichia coli

    Liu Hui

    2012-04-01

    Full Text Available Abstract Background As an alternative for economic biodiesel production, the microbial production of extracellular fatty acid from renewable resources is receiving more concerns recently, since the separation of fatty acid from microorganism cells is normally involved in a series of energy-intensive steps. Many attempts have been made to construct fatty acid producing strains by targeting genes in the fatty acid biosynthetic pathway, while few studies focused on the cultivation process and the mass transfer kinetics. Results In this study, both strain improvements and cultivation process strategies were applied to increase extracellular fatty acid production by engineered Escherichia coli. Our results showed overexpressing ‘TesA and the deletion of fadL in E. coli BL21 (DE3 improved extracellular fatty acid production, while deletion of fadD didn’t strengthen the extracellular fatty acid production for an undetermined mechanism. Moreover, the cultivation process controls contributed greatly to extracellular fatty acid production with respect to titer, cell growth and productivity by adjusting the temperature, adding ampicillin and employing on-line extraction. Under optimal conditions, the E. coli strain (pACY-‘tesA-ΔfadL produced 4.8 g L−1 extracellular fatty acid, with the specific productivity of 0.02 g h−1 g−1dry cell mass, and the yield of 4.4% on glucose, while the ratios of cell-associated fatty acid versus extracellular fatty acid were kept below 0.5 after 15 h of cultivation. The fatty acids included C12:1, C12:0, C14:1, C14:0, C16:1, C16:0, C18:1, C18:0. The composition was dominated by C14 and C16 saturated and unsaturated fatty acids. Using the strain pACY-‘tesA, similar results appeared under the same culture conditions and the titer was also much higher than that ever reported previously, which suggested that the supposedly superior strain did not necessarily perform best for the efficient production of desired

  11. The Gene Expression of Human Endothelial Cells Is Modulated by Subendothelial Extracellular Matrix Proteins: Short-Term Response to Laminar Shear Stress

    Chlupáč, Jaroslav; Filová, Elena; Havlíková, Jana; Matějka, R.; Riedel, Tomáš; Houska, Milan; Brynda, Eduard; Pamula, E.; Rémy, M.; Bareille, R.; Fernandez, P.; Daculsi, R.; Bourget, Ch.; Bačáková, Lucie; Bordenave, L.

    2014-01-01

    Roč. 20, 15-16 (2014), s. 2253-2264 ISSN 2152-4947 R&D Projects: GA ČR(CZ) GAP108/10/1106; GA ČR(CZ) GAP108/11/1857; GA ČR(CZ) GA305/08/0108; GA ČR GAP205/12/1702; GA MŠk(CZ) EE2.3.30.0029; GA MŠk(CZ) ED1.1.00/02.0109; GA MZd(CZ) NT11270 Grant - others:GA MŠk(CZ) Barrande 2005-06-036-1 Institutional support: RVO:67985823 ; RVO:61389013 Keywords : atherosclerosis * endothelial cells * extracellular matrix proteins * tissue engineering Subject RIV: EI - Biotechnology ; Bionics; CD - Macromolecular Chemistry (UMCH-V) Impact factor: 4.448, year: 2014

  12. Increasing cocoa butter-like lipid production of Saccharomyces cerevisiae by expression of selected cocoa genes

    Wei, Yongjun; Gossing, Michael; Bergenholm, David

    2017-01-01

    for CB biosynthesis from the cocoa genome using a phylogenetic analysis approach. By expressing the selected cocoa genes in S. cerevisiae, we successfully increased total fatty acid production, TAG production and CBL production in some S. cerevisiae strains. The relative CBL content in three yeast...... higher level of CBL compared with the control strain. In summary, CBL production by S. cerevisiae were increased through expressing selected cocoa genes potentially involved in CB biosynthesis.......Cocoa butter (CB) extracted from cocoa beans mainly consists of three different kinds of triacylglycerols (TAGs), 1,3-dipalmitoyl-2-oleoyl-glycerol (POP, C16:0-C18:1-C16:0), 1-palmitoyl-3-stearoyl-2-oleoyl-glycerol(POS,C16:0C18:1-C18:0) and 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0-C18:1-C18...

  13. Selective Inhibition of Histone Deacetylation in Melanoma Increases Targeted Gene Delivery by a Bacteriophage Viral Vector

    Samuel Campbell

    2018-04-01

    Full Text Available The previously developed adeno-associated virus/phage (AAVP vector, a hybrid between M13 bacteriophage (phage viruses that infect bacteria only and human Adeno-Associated Virus (AAV, is a promising tool in targeted gene therapy against cancer. AAVP can be administered systemically and made tissue specific through the use of ligand-directed targeting. Cancer cells and tumor-associated blood vessels overexpress the αν integrin receptors, which are involved in tumor angiogenesis and tumor invasion. AAVP is targeted to these integrins via a double cyclic RGD4C ligand displayed on the phage capsid. Nevertheless, there remain significant host-defense hurdles to the use of AAVP in targeted gene delivery and subsequently in gene therapy. We previously reported that histone deacetylation in cancer constitutes a barrier to AAVP. Herein, to improve AAVP-mediated gene delivery to cancer cells, we combined the vector with selective adjuvant chemicals that inhibit specific histone deacetylases (HDAC. We examined the effects of the HDAC inhibitor C1A that mainly targets HDAC6 and compared this to sodium butyrate, a pan-HDAC inhibitor with broad spectrum HDAC inhibition. We tested the effects on melanoma, known for HDAC6 up-regulation, and compared this side by side with a normal human kidney HEK293 cell line. Varying concentrations were tested to determine cytotoxic levels as well as effects on AAVP gene delivery. We report that the HDAC inhibitor C1A increased AAVP-mediated transgene expression by up to ~9-fold. These findings indicate that selective HDAC inhibition is a promising adjuvant treatment for increasing the therapeutic value of AAVP.

  14. Typical and Atypical Antipsychotic Drugs Increase Extracellular Histamine Levels in the Rat Medial Prefrontal Cortex: Contribution of Histamine H1 Receptor Blockade

    Kjell A Svensson

    2012-05-01

    Full Text Available Atypical antipsychotics such as clozapine and olanzapine have been shown to enhance histamine turnover and this effect has been hypothesized to contribute to their improved therapeutic profile compared to typical antipsychotics. In the present study, we examined the effects of antipsychotic drugs on histamine (HA efflux in the mPFC of the rat by means of in vivo microdialysis and sought to differentiate the receptor mechanisms which underlie such effects. Olanzapine and clozapine increased mPFC HA efflux in a dose related manner. Increased HA efflux was also observed after quetiapine, chlorpromazine and perphenazine treatment. We found no effect of the selective 5-HT2A antagonist MDL100907, 5-HT2c antagonist SB242084 or the 5-HT6 antagonist Ro 04-6790 on mPFC HA efflux. HA efflux was increased following treatment with selective H1 receptor antagonists pyrilamine, diphenhydramine and triprolidine, the H3 receptor antagonist ciproxifan and the mixed 5HT2A/H1 receptor antagonist ketanserin. The potential novel antipsychotic drug FMPD, which has a lower affinity at H1 receptors than olanzapine, did not affect HA efflux. Similarly, other antipsychotics with lower H1 receptor affinity (risperidone, aripiprazole and haloperidol were also without effect on HA efflux. Perfusion of clozapine and pyrilamine into the TMN, but not the mPFC, increased local HA efflux. Finally, HA efflux after antipsychotic treatment was significantly correlated with affinity at H1 receptors whereas 9 other receptors, including 5-HT2A, were not. These results demonstrate that both typical and atypical antipsychotics increase mPFC histamine efflux and this effect may be mediated via antagonism of histamine H1 receptors.

  15. Oral Administration of Recombinant Lactococcus lactis Expressing the Cellulase Gene Increases Digestibility of Fiber in Geese.

    Zhou, Haizhu; Gao, Yunhang; Gao, Guang; Lou, Yujie

    2015-12-01

    Enhancing cellulose digestibility in animals is important for improving the utilization of forage, which can decrease the amount of food used in animal production. The aim of the present study was to achieve recombinant expression of the cellulase gene in Lactococcus lactis and evaluate the effects of oral administration of the recombinant L. lactis on fiber digestibility in geese. Cellulase (Cell) and green fluorescent protein (GFP) genes were cloned into a L. lactis expression vector (pNZ8149) to construct the recombinant expression plasmid (pNZ8149-GFP-Cell). Then, the recombinant expression plasmid was transformed into L. lactis (NZ3900) competent cells by electroporation to obtain recombinant L. lactis (pNZ8149-GFP-Cell/NZ3900) in which protein expression was induced by Nisin. Expression of GFP and Cell by the recombinant L. lactis was confirmed using SDS-PAGE, fluorescence detection, and Congo red assays. A feeding experiment showed that oral administration of pNZ8149-GFP-Cell/NZ3900 significantly increased the digestibility of dietary fiber in geese fed either a maize stalk diet or a rice chaff diet. Therefore, oral administration of recombinant L. lactis cells expressing the cellulase gene increases fiber digestibility in geese, offering a way to increase the utilization of dietary fiber in geese.

  16. Fatness-Associated FTO Gene Variant Increases Mortality Independent of Fatness - in Cohorts of Danish Men

    Zimmermann, E; Kring, SI; Berentzen, TL

    2009-01-01

    The A-allele of the single nucleotide polymorphism (SNP), rs9939609, in the FTO gene is associated with increased fatness. We hypothesized that the SNP is associated with morbidity and mortality through the effect on fatness. METHODOLOGY/PRINCIPAL FINDINGS: In a population of 362,200 Danish young...... prior to death suggested a general protective effect of the TT genotype, whereas there were only weak associations with disease incidence, except for diseases of the nervous system. CONCLUSION: Independent of fatness, the A-allele of the FTO SNP appears to increase mortality of a magnitude similar...

  17. Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number.

    Price, Ric N; Uhlemann, Anne-Catrin; Brockman, Alan; McGready, Rose; Ashley, Elizabeth; Phaipun, Lucy; Patel, Rina; Laing, Kenneth; Looareesuwan, Sornchai; White, Nicholas J; Nosten, François; Krishna, Sanjeev

    The borders of Thailand harbour the world's most multidrug resistant Plasmodium falciparum parasites. In 1984 mefloquine was introduced as treatment for uncomplicated falciparum malaria, but substantial resistance developed within 6 years. A combination of artesunate with mefloquine now cures more than 95% of acute infections. For both treatment regimens, the underlying mechanisms of resistance are not known. The relation between polymorphisms in the P falciparum multidrug resistant gene 1 (pfmdr1) and the in-vitro and in-vivo responses to mefloquine were assessed in 618 samples from patients with falciparum malaria studied prospectively over 12 years. pfmdr1 copy number was assessed by a robust real-time PCR assay. Single nucleotide polymorphisms of pfmdr1, P falciparum chloroquine resistance transporter gene (pfcrt) and P falciparum Ca2+ ATPase gene (pfATP6) were assessed by PCR-restriction fragment length polymorphism. Increased copy number of pfmdr1 was the most important determinant of in-vitro and in-vivo resistance to mefloquine, and also to reduced artesunate sensitivity in vitro. In a Cox regression model with control for known confounders, increased pfmdr1 copy number was associated with an attributable hazard ratio (AHR) for treatment failure of 6.3 (95% CI 2.9-13.8, p<0.001) after mefloquine monotherapy and 5.4 (2.0-14.6, p=0.001) after artesunate-mefloquine therapy. Single nucleotide polymorphisms in pfmdr1 were associated with increased mefloquine susceptibility in vitro, but not in vivo. Amplification in pfmdr1 is the main cause of resistance to mefloquine in falciparum malaria. Multidrug resistant P falciparum malaria is common in southeast Asia, but difficult to identify and treat. Genes that encode parasite transport proteins maybe involved in export of drugs and so cause resistance. In this study we show that increase in copy number of pfmdr1, a gene encoding a parasite transport protein, is the best overall predictor of treatment failure with

  18. Simulated physiological stretch increases expression of extracellular matrix proteins in human bladder smooth muscle cells via integrin α4/αv-FAK-ERK1/2 signaling pathway.

    Chen, Shulian; Peng, Chuandu; Wei, Xin; Luo, Deyi; Lin, Yifei; Yang, Tongxin; Jin, Xi; Gong, Lina; Li, Hong; Wang, Kunjie

    2017-08-01

    To investigate the effect of simulated physiological stretch on the expression of extracellular matrix (ECM) proteins and the role of integrin α4/αv, focal adhesion kinase (FAK), extracellular regulated protein kinases 1/2 (ERK1/2) in the stretch-induced ECM protein expression of human bladder smooth muscle cells (HBSMCs). HBSMCs were seeded onto silicone membrane and subjected to simulated physiological stretch at the range of 5, 10, and 15% elongation. Expression of primary ECM proteins in HBSMCs was analyzed by real-time polymerase chain reaction and Western blot. Specificity of the FAK and ERK1/2 was determined by Western blot with FAK inhibitor and ERK1/2 inhibitor (PD98059). Specificity of integrin α4 and integrin αv was determined with small interfering ribonucleic acid (siRNA) transfection. The expression of collagen I (Col1), collagen III (Col3), and fibronectin (Fn) was increased significantly under the simulated physiological stretch of 10 and 15%. Integrin α4 and αv, FAK, ERK1/2 were activated by 10% simulated physiological stretch compared with the static condition. Pretreatment of ERK1/2 inhibitor, FAK inhibitor, integrin α4 siRNA, or integrin αv siRNA reduced the stretch-induced expression of ECM proteins. And FAK inhibitor decreased the stretch-induced ERK1/2 activity and ECM protein expression. Integrin α4 siRNA or integrin αv siRNA inhibited the stretch-induced activity of FAK. Simulated physiological stretch increases the expression of ECM proteins in HBSMCs, and integrin α4/αv-FAK-ERK1/2 signaling pathway partly modulates the mechano-transducing process.

  19. Identification of a receptor for extracellular renalase.

    Ling Wang

    Full Text Available An increased risk for developing essential hypertension, stroke and diabetes is associated with single nucleotide gene polymorphisms in renalase, a newly described secreted flavoprotein with oxidoreductase activity. Gene deletion causes hypertension, and aggravates acute ischemic kidney (AKI and cardiac injury. Independent of its intrinsic enzymatic activities, extracellular renalase activates MAPK signaling and prevents acute kidney injury (AKI in wild type (WT mice. Therefore, we sought to identity the receptor for extracellular renalase.RP-220 is a previously identified, 20 amino acids long renalase peptide that is devoid of any intrinsic enzymatic activity, but it is equally effective as full-length recombinant renalase at protecting against toxic and ischemic injury. Using biotin transfer studies with RP-220 in the human proximal tubular cell line HK-2 and protein identification by mass spectrometry, we identified PMCA4b as a renalase binding protein. This previously characterized plasma membrane ATPase is involved in cell signaling and cardiac hypertrophy. Co-immunoprecipitation and co-immunolocalization confirmed protein-protein interaction between endogenous renalase and PMCA4b. Down-regulation of endogenous PMCA4b expression by siRNA transfection, or inhibition of its enzymatic activity by the specific peptide inhibitor caloxin1b each abrogated RP-220 dependent MAPK signaling and cytoprotection. In control studies, these maneuvers had no effect on epidermal growth factor mediated signaling, confirming specificity of the interaction between PMCA4b and renalase.PMCA4b functions as a renalase receptor, and a key mediator of renalase dependent MAPK signaling.

  20. Suicide Gene Therapy to Increase the Safety of Chimeric Antigen Receptor-Redirected T Lymphocytes

    Monica Casucci, Attilio Bondanza

    2011-01-01

    Full Text Available Chimeric antigen receptors (CARs are generated by fusing the antigen-binding motif of a monoclonal antibody (mAb with the signal transduction machinery of the T-cell receptor (TCR. The genetic modification of T lymphocytes with chimeric receptors specific for tumor-associated antigens (TAAs allows for the redirection towards tumor cells. Clinical experience with CAR-redirected T cells suggests that antitumor efficacy associates with some degree of toxicity, especially when TAA expression is shared with healthy tissues. This situation closely resembles the case of allogeneic hematopoietic stem cell transplantation (HSCT, wherein allorecognition causes both the graft-versus-leukemia (GVL effect and graft-versus-host disease (GVHD. Suicide gene therapy, i.e. the genetic induction of a conditional suicide phenotype into donor T cells, enables dissociating the GVL effect from GVHD. Applying suicide gene modification to CAR-redirected T cells may therefore greatly increase their safety profile and facilitate their clinical development.

  1. Increasing the amylose content of durum wheat through silencing of the SBEIIa genes

    Masci Stefania

    2010-07-01

    Full Text Available Abstract Background High amylose starch has attracted particular interest because of its correlation with the amount of Resistant Starch (RS in food. RS plays a role similar to fibre with beneficial effects for human health, providing protection from several diseases such as colon cancer, diabetes, obesity, osteoporosis and cardiovascular diseases. Amylose content can be modified by a targeted manipulation of the starch biosynthetic pathway. In particular, the inactivation of the enzymes involved in amylopectin synthesis can lead to the increase of amylose content. In this work, genes encoding starch branching enzymes of class II (SBEIIa were silenced using the RNA interference (RNAi technique in two cultivars of durum wheat, using two different methods of transformation (biolistic and Agrobacterium. Expression of RNAi transcripts was targeted to the seed endosperm using a tissue-specific promoter. Results Amylose content was markedly increased in the durum wheat transgenic lines exhibiting SBEIIa gene silencing. Moreover the starch granules in these lines were deformed, possessing an irregular and deflated shape and being smaller than those present in the untransformed controls. Two novel granule bound proteins, identified by SDS-PAGE in SBEIIa RNAi lines, were investigated by mass spectrometry and shown to have strong homologies to the waxy proteins. RVA analysis showed new pasting properties associated with high amylose lines in comparison with untransformed controls. Finally, pleiotropic effects on other starch genes were found by semi-quantitative and Real-Time reverse transcription-polymerase chain reaction (RT-PCR. Conclusion We have found that the silencing of SBEIIa genes in durum wheat causes obvious alterations in granule morphology and starch composition, leading to high amylose wheat. Results obtained with two different methods of transformation and in two durum wheat cultivars were comparable.

  2. Involvement of extracellular matrix constituents in breast cancer

    Lochter, Andre; Bissell, Mina J

    1995-06-01

    It has recently been established that the extracellular matrix is required for normal functional differentiation of mammary epithelia not only in culture, but also in vivo. The mechanisms by which extracellular matrix affects differentiation, as well as the nature of extracellular matrix constituents which have major impacts on mammary gland function, have only now begun to be dissected. The intricate variety of extracellular matrix-mediated events and the remarkable degree of plasticity of extracellular matrix structure and composition at virtually all times during ontogeny, make such studies difficult. Similarly, during carcinogenesis, the extracellular matrix undergoes gross alterations, the consequences of which are not yet precisely understood. Nevertheless, an increasing amount of data suggests that the extracellular matrix and extracellular matrix-receptors might participate in the control of most, if not all, of the successive stages of breast tumors, from appearance to progression and metastasis.

  3. Inactivation of genes encoding extracellular proteases in bacillus halodurans BhFC01 and the impact on its modified flagellin type III secretion pathway towards improving peptide expression

    Berger, E

    2009-01-01

    Full Text Available The flagellin type III secretion pathway of Bacillus halodurans BhFC01 (-hag) was modified by the inactivation of fliD. An in-frame flagellin gene fusion polypeptide construct was expressed, and the heterologous peptides were secreted as flagellin...

  4. Effect of the deletion of genes encoding proteins of the extracellular virion form of vaccinia virus on vaccine immunogenicity and protective effectiveness in the mouse model.

    Clement A Meseda

    Full Text Available Antibodies to both infectious forms of vaccinia virus, the mature virion (MV and the enveloped virion (EV, as well as cell-mediated immune response appear to be important for protection against smallpox. EV virus particles, although more labile and less numerous than MV, are important for dissemination and spread of virus in infected hosts and thus important in virus pathogenesis. The importance of the EV A33 and B5 proteins for vaccine induced immunity and protection in a murine intranasal challenge model was evaluated by deletion of both the A33R and B5R genes in a vaccine-derived strain of vaccinia virus. Deletion of either A33R or B5R resulted in viruses with a small plaque phenotype and reduced virus yields, as reported previously, whereas deletion of both EV protein-encoding genes resulted in a virus that formed small infection foci that were detectable and quantifiable only by immunostaining and an even more dramatic decrease in total virus yield in cell culture. Deletion of B5R, either as a single gene knockout or in the double EV gene knockout virus, resulted in a loss of EV neutralizing activity, but all EV gene knockout viruses still induced a robust neutralizing activity against the vaccinia MV form of the virus. The effect of elimination of A33 and/or B5 on the protection afforded by vaccination was evaluated by intranasal challenge with a lethal dose of either vaccinia virus WR or IHD-J, a strain of vaccinia virus that produces relatively higher amounts of EV virus. The results from multiple experiments, using a range of vaccination doses and virus challenge doses, and using mortality, morbidity, and virus dissemination as endpoints, indicate that the absence of A33 and B5 have little effect on the ability of a vaccinia vaccine virus to provide protection against a lethal intranasal challenge in a mouse model.

  5. Childhood and later life stressors and increased inflammatory gene expression at older ages.

    Levine, M E; Cole, S W; Weir, D R; Crimmins, E M

    2015-04-01

    Adverse experiences in early life have the ability to "get under the skin" and affect future health. This study examined the relative influence of adversities during childhood and adulthood in accounting for individual differences in pro-inflammatory gene expression in late life. Using a pilot-sample from the Health and Retirement Study (N = 114) aged from 51 to 95, OLS regression models were run to determine the association between a composite score from three proinflammatory gene expression levels (PTGS2, ILIB, and IL8) and 1) childhood trauma, 2) childhood SES, 3) childhood health, 4) adult traumas, and 5) low SES in adulthood. Our results showed that only childhood trauma was found to be associated with increased inflammatory transcription in late life. Furthermore, examination of interaction effects showed that childhood trauma exacerbated the influence of low SES in adulthood on elevated levels of inflammatory gene expression-signifying that having low SES in adulthood was most damaging for persons who had experienced traumatic events during their childhood. Overall our study suggests that traumas experienced during childhood may alter the stress response, leading to more sensitive reactivity throughout the lifespan. As a result, individuals who experienced greater adversity in early life may be at higher risk of late life health outcomes, particularly if adulthood adversity related to SES persists. Copyright © 2015. Published by Elsevier Ltd.

  6. Increased MCP-1 gene expression in monocytes of severe OSA patients and under intermittent hypoxia.

    Chuang, Li-Pang; Chen, Ning-Hung; Lin, Yuling; Ko, Wen-Shan; Pang, Jong-Hwei S

    2016-03-01

    Obstructive sleep apnea (OSA) is known to be a risk factor of coronary artery disease. Monocyte chemoattractant protein-1 (MCP-1), as a critical factor for monocyte infiltration, is known to play a role in the development of atherosclerosis. This study aimed to investigate the effect of intermittent hypoxia, the hallmark of OSA, on the MCP-1 expression of monocytes. Peripheral blood was sampled from 61 adults enrolled for suspected OSA. RNA was prepared from the isolated monocytes for the analysis of MCP-1. The effect of in vitro intermittent hypoxia on the regulation and function of MCP-1 was investigated on THP-1 monocytic cells and human monocytes. The mRNA and secreted protein levels were investigated by RT/real-time PCR and enzyme-linked immunosorbent assay, respectively. Monocytic MCP-1 gene expression was found to be increased significantly in severe OSA patients. In vitro intermittent hypoxia was demonstrated to increase the mRNA and protein expression levels of MCP-1 dose- and time-dependently in THP-1 monocytic cells. The MCP-1 mRNA expression in monocytes isolated from OSA patient was induced to a much higher level compared to that from normal control. Pre-treatment with inhibitor for p42/44 MAPK or p38 MAPK suppressed the activation of MCP-1 expression by intermittent hypoxia. This is the first study to demonstrate the increase of MCP-1 gene expression in monocytes of severe OSA patients. In addition, monocytic MCP-1 gene expression can be induced under intermittent hypoxia.

  7. Mice lacking the kf-1 gene exhibit increased anxiety- but not despair-like behavior

    Atsushi Tsujimura

    2008-09-01

    Full Text Available KF-1 was originally identified as a protein encoded by human gene with increased expression in the cerebral cortex of a patient with Alzheimer’s disease. In mouse brain, kf-1 mRNA is detected predominantly in the hippocampus and cerebellum, and kf-1 gene expression is elevated also in the frontal cortex of rats after chronic antidepressant treatments. KF-1 mediates E2-dependent ubiquitination and may modulate cellular protein levels as an E3 ubiquitin ligase, though its target proteins are not yet identified. To elucidate the role of kf-1 in the central nervous system, we generated kf-1 knockout mice by gene targeting, using Cre-lox recombination. The resulting kf-1−/− mice were normal and healthy in appearance. Behavioral analyses revealed that kf-1−/− mice showed significantly increased anxiety-like behavior compared with kf-1+/+ littermates in the light/dark transition and elevated plus maze tests; however, no significant differences were observed in exploratory locomotion using the open field test or in behavioral despair using the forced swim and tail suspension tests. These observations suggest that KF-1 suppresses selectively anxiety under physiological conditions probably through modulating protein levels of its unknown target(s. Interestingly, kf-1−/− mice exhibited significantly increased prepulse inhibition, which is usually reduced in human schizophrenic patients. Thus, the kf-1−/− mice provide a novel animal model for elucidating molecular mechanisms of psychiatric diseases such as anxiety/depression, and may be useful for screening novel anxiolytic/antidepressant compounds.

  8. Restoration using Azolla imbricata increases nitrogen functional bacterial groups and genes in soil.

    Lu, Xiao-Ming; Lu, Peng-Zhen; Yang, Ke

    2017-05-01

    Microbial groups are major factors that influence soil function. Currently, there is a lack of studies on microbial functional groups. Although soil microorganisms play an important role in the nitrogen cycle, systematic studies of the effects of environmental factors on microbial populations in relation to key metabolic processes in the nitrogen cycle are seldom reported. In this study, we conducted a systematic analysis of the changes in nitrogen functional groups in mandarin orange garden soil treated with Azolla imbricata. The structures of the major functional bacterial groups and the functional gene abundances involved in key processes of the soil nitrogen cycle were analyzed using high-throughput sequencing (HTS) and quantitative real-time PCR, respectively. The results indicated that returning A. imbricata had an important influence on the composition of soil nitrogen functional bacterial communities. Treatment with A. imbricata increased the diversity of the nitrogen functional bacteria. The abundances of nitrogen functional genes were significantly higher in the treated soil compared with the control soil. Both the diversity of the major nitrogen functional bacteria (nifH bacteria, nirK bacteria, and narG bacteria) and the abundances of nitrogen functional genes in the soil showed significant positive correlations with the soil pH, the organic carbon content, available nitrogen, available phosphorus, and NH 4 + -N and NO 3 - -N contents. Treatment with 12.5 kg fresh A. imbricata per mandarin orange tree was effective to improve the quality of the mandarin orange garden soil. This study analyzed the mechanism of the changes in functional bacterial groups and genes involved in key metabolic processes of the nitrogen cycle in soil treated by A. imbricata.

  9. Extracellular Alkalinization as a Defense Response in Potato Cells.

    Moroz, Natalia; Fritch, Karen R; Marcec, Matthew J; Tripathi, Diwaker; Smertenko, Andrei; Tanaka, Kiwamu

    2017-01-01

    A quantitative and robust bioassay to assess plant defense response is important for studies of disease resistance and also for the early identification of disease during pre- or non-symptomatic phases. An increase in extracellular pH is known to be an early defense response in plants. In this study, we demonstrate extracellular alkalinization as a defense response in potatoes. Using potato suspension cell cultures, we observed an alkalinization response against various pathogen- and plant-derived elicitors in a dose- and time-dependent manner. We also assessed the defense response against a variety of potato pathogens, such as protists ( Phytophthora infestans and Spongospora subterranea ) and fungi ( Verticillium dahliae and Colletotrichum coccodes ). Our results show that extracellular pH increases within 30 min in proportion to the number of pathogen spores added. Consistently with the alkalinization effect, the higher transcription level of several defense-related genes and production of reactive oxygen species was observed. Our results demonstrate that the alkalinization response is an effective marker to study early stages of defense response in potatoes.

  10. Neutrophil Extracellular Traps in Ulcerative Colitis

    Bjerg Bennike, Tue; Carlsen, Thomas Gelsing; Ellingsen, Torkell

    2015-01-01

    microscopy and confocal microscopy. RESULTS: We identified and quantified 5711 different proteins with proteomics. The abundance of the proteins calprotectin and lactotransferrin in the tissue correlated with the degree of tissue inflammation as determined by histology. However, fecal calprotectin did...... not correlate. Forty-six proteins were measured with a statistically significant differences in abundances between the UC colon tissue and controls. Eleven of the proteins with increased abundances in the UC biopsies were associated with neutrophils and neutrophil extracellular traps. The findings were...... validated by microscopy, where an increased abundance of neutrophils and the presence of neutrophil extracellular traps by extracellular DNA present in the UC colon tissue were confirmed. CONCLUSIONS: Neutrophils, induced neutrophil extracellular traps, and several proteins that play a part in innate...

  11. Impaired barrier function by dietary fructo-oligosaccharides (FOS in rats is accompanied by increased colonic mitochondrial gene expression

    Kramer Evelien

    2008-03-01

    Full Text Available Abstract Background Dietary non-digestible carbohydrates stimulate the gut microflora and are therefore presumed to improve host resistance to intestinal infections. However, several strictly controlled rat infection studies showed that non-digestible fructo-oligosaccharides (FOS increase, rather than decrease, translocation of Salmonella towards extra-intestinal sites. In addition, it was shown that FOS increases intestinal permeability already before infection. The mechanism responsible for this adverse effect of FOS is unclear. Possible explanations are altered mucosal integrity due to changes in tight junctions or changes in expression of defense molecules such as antimicrobials and mucins. To examine the mechanisms underlying weakening of the intestinal barrier by FOS, a controlled dietary intervention study was performed. Two groups of 12 rats were adapted to a diet with or without FOS. mRNA was collected from colonic mucosa and changes in gene expression were assessed for each individual rat using Agilent rat whole genome microarrays. Results Among the 997 FOS induced genes we observed less mucosal integrity related genes than expected with the clear permeability changes. FOS did not induce changes in tight junction genes and only 8 genes related to mucosal defense were induced by FOS. These small effects are unlikely the cause for the clear increase in intestinal permeability that is observed. FOS significantly increased expression of 177 mitochondria-related genes. More specifically, induced expression of genes involved in all five OXPHOS complexes and the TCA cycle was observed. These results indicate that dietary FOS influences intestinal mucosal energy metabolism. Furthermore, increased expression of 113 genes related to protein turnover, including proteasome genes, ribosomal genes and protein maturation related genes, was seen. FOS upregulated expression of the peptide hormone proglucagon gene, in agreement with previous studies, as

  12. Extracellular Matrix, Nuclear and Chromatin Structure and GeneExpression in Normal Tissues and Malignant Tumors: A Work inProgress

    Spencer, Virginia A.; Xu, Ren; Bissell, Mina J.

    2006-08-01

    Almost three decades ago, we presented a model where theextracellular matrix (ECM) was postulated to influence gene expressionand tissue-specificity through the action of ECM receptors and thecytoskeleton. This hypothesis implied that ECM molecules could signal tothe nucleus and that the unit of function in higher organisms was not thecell alone, but the cell plus its microenvironment. We now know that ECMinvokes changes in tissue and organ architecture and that tissue, cell,nuclear, and chromatin structure are changed profoundly as a result ofand during malignant progression. Whereas some evidence has beengenerated for a link between ECM-induced alterations in tissuearchitecture and changes in both nuclear and chromatin organization, themanner by which these changes actively induce or repress gene expressionin normal and malignant cells is a topic in need of further attention.Here, we will discuss some key findings that may provide insights intomechanisms through which ECM could influence gene transcription and howtumor cells acquire the ability to overcome these levels ofcontrol.

  13. The novel virulence-related gene nlxA in the lipopolysaccharide cluster of Xanthomonas citri ssp. citri is involved in the production of lipopolysaccharide and extracellular polysaccharide, motility, biofilm formation and stress resistance.

    Yan, Qing; Hu, Xiufang; Wang, Nian

    2012-10-01

    Lipopolysaccharide (LPS) is an important virulence factor of Xanthomonas citri ssp. citri, the causative agent of citrus canker disease. In this research, a novel gene, designated as nlxA (novel LPS cluster gene of X. citri ssp. citri), in the LPS cluster of X. citri ssp. citri 306, was characterized. Our results indicate that nlxA is required for O-polysaccharide biosynthesis by encoding a putative rhamnosyltransferase. This is supported by several lines of evidence: (i) NlxA shares 40.14% identity with WsaF, which acts as a rhamnosyltransferase; (ii) sodium dodecylsulphate-polyacrylamide gel electrophoresis analysis showed that four bands of the O-antigen part of LPS were missing in the LPS production of the nlxA mutant; this is also consistent with a previous report that the O-antigen moiety of LPS of X. citri ssp. citri is composed of a rhamnose homo-oligosaccharide; (iii) mutation of nlxA resulted in a significant reduction in the resistance of X. citri ssp. citri to different stresses, including sodium dodecylsulphate, polymyxin B, H(2)O(2), phenol, CuSO(4) and ZnSO(4). In addition, our results indicate that nlxA plays an important role in extracellular polysaccharide production, biofilm formation, stress resistance, motility on semi-solid plates, virulence and in planta growth in the host plant grapefruit. © 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

  14. Expression of jasmonic ethylene responsive factor gene in transgenic poplar tree leads to increased salt tolerance.

    Li, Yiliang; Su, Xiaohua; Zhang, Bingyu; Huang, Qinjun; Zhang, Xianghua; Huang, Rongfeng

    2009-02-01

    The stress resistance of plants can be enhanced by regulating the expression of multiple downstream genes associated with stress resistance. We used the Agrobacterium method to transfer the tomato jasmonic ethylene responsive factors (JERFs) gene that encodes the ethylene response factor (ERF) like transcription factor to the genome of a hybrid poplar (Populus alba x Populus berolinensis). Eighteen resistant plants were obtained, of which 13 were identified by polymerase chain reaction (PCR), reverse transcriptase PCR and Southern blot analyses as having incorporated the JERFs gene and able to express it at the transcriptional level. Salinity tests were conducted in a greenhouse with 0, 100, 200 and 300 mM NaCl. In the absence of NaCl, the transgenic plants were significantly taller than the control plants, but no statistically significant differences in the concentrations of proline and chlorophyll were observed. With increasing salinity, the extent of damage was significantly less in transgenic plants than that in control plants, and the reductions in height, basal diameter and biomass were less in transgenic plants than those in control plants. At 200 and 300 mM NaCl concentrations, transgenic plants were 128.9% and 98.8% taller, respectively, and had 199.8% and 113.0% more dry biomass, respectively, than control plants. The saline-induced reduction in leaf water content and increase in root/crown ratio were less in transgenic plants than in control plants. Foliar proline concentration increased more in response to salt treatment in transgenic plants than in control plants. Foliar Na(+) concentration was higher in transgenic plants than in control plants. In the coastal area in Panjin of Liaoning where the total soil salt concentration is 0.3%, a salt tolerance trial of transgenic plants indicated that 3-year-old transgenic plants were 14.5% and 33.6% taller than the control plants at two field sites. The transgenic plants at the two field sites were growing

  15. Reproductive gene expression in a coral reef fish exposed to increasing temperature across generations

    Veilleux, Heather D; Donelson, Jennifer M; Munday, Philip L

    2017-01-01

    Reproduction in marine fish is generally tightly linked with water temperature. Consequently, when adults are exposed to projected future ocean temperatures, reproductive output of many species declines precipitously. Recent research has shown that in the common reef fish, Acanthochromis polyacanthus, step-wise exposure to higher temperatures over two generations (parents: +1.5°C, offspring: +3.0°C) can improve reproductive output in the F2 generation compared to F2 fish that have experienced the same high temperatures over two generations (F1 parents: +3.0°C, F2 offspring: +3.0°C). To investigate how a step-wise increase in temperature between generations improved reproductive capacity, we tested the expression of well-known teleost reproductive genes in the brain and gonads of F2 fish using quantitative reverse transcription PCR and compared it among control (+0.0°C for two generations), developmental (+3.0°C in second generation only), step (+1.5°C in first generation and +3.0°C in second generation), and transgenerational (+3.0°C for two generations) treatments. We found that levels of gonadotropin receptor gene expression (Fshr and Lhcgr) in the testes were reduced in developmental and transgenerational temperature treatments, but were similar to control levels in the step treatment. This suggests Fshr and Lhcgr may be involved in regulating male reproductive capacity in A. polyacanthus. In addition, lower Fshb expression in the brain of females in all temperature treatments compared to control, suggests that Fshb expression, which is involved in vitellogenesis, is sensitive to high temperatures. Our results help elucidate key genes that facilitate successful reproduction in reef fishes when they experience a gradual increase in temperature across generations consistent with the trajectory of climate change.

  16. Reproductive gene expression in a coral reef fish exposed to increasing temperature across generations.

    Veilleux, Heather D; Donelson, Jennifer M; Munday, Philip L

    2018-01-01

    Reproduction in marine fish is generally tightly linked with water temperature. Consequently, when adults are exposed to projected future ocean temperatures, reproductive output of many species declines precipitously. Recent research has shown that in the common reef fish, Acanthochromis polyacanthus , step-wise exposure to higher temperatures over two generations (parents: +1.5°C, offspring: +3.0°C) can improve reproductive output in the F2 generation compared to F2 fish that have experienced the same high temperatures over two generations (F1 parents: +3.0°C, F2 offspring: +3.0°C). To investigate how a step-wise increase in temperature between generations improved reproductive capacity, we tested the expression of well-known teleost reproductive genes in the brain and gonads of F2 fish using quantitative reverse transcription PCR and compared it among control (+0.0°C for two generations), developmental (+3.0°C in second generation only), step (+1.5°C in first generation and +3.0°C in second generation), and transgenerational (+3.0°C for two generations) treatments. We found that levels of gonadotropin receptor gene expression ( Fshr and Lhcgr ) in the testes were reduced in developmental and transgenerational temperature treatments, but were similar to control levels in the step treatment. This suggests Fshr and Lhcgr may be involved in regulating male reproductive capacity in A. polyacanthus . In addition, lower Fshb expression in the brain of females in all temperature treatments compared to control, suggests that Fshb expression, which is involved in vitellogenesis, is sensitive to high temperatures. Our results help elucidate key genes that facilitate successful reproduction in reef fishes when they experience a gradual increase in temperature across generations consistent with the trajectory of climate change.

  17. Kaempferol increases apoptosis in human acute promyelocytic leukemia cells and inhibits multidrug resistance genes.

    Moradzadeh, Maliheh; Tabarraei, Alijan; Sadeghnia, Hamid Reza; Ghorbani, Ahmad; Mohamadkhani, Ashraf; Erfanian, Saiedeh; Sahebkar, Amirhossein

    2018-02-01

    Acute promyelocytic leukemia (APL) is one of the most life-threatening hematological malignancies. Defects in the cell growth and apoptotic pathways are responsible for both disease pathogenesis and treatment resistance. Therefore, pro-apoptotic agents are potential candidates for APL treatment. Kaempferol is a flavonoid with antioxidant and anti-tumor properties. This study was designed to investigate the cytotoxic, pro-apoptotic, and differentiation-inducing effects of kaempferol on HL-60 and NB4 leukemia cells. Resazurin assay was used to determine cell viability following treatment with kaempferol (12.5-100 μM) and all-trans retinoic acid (ATRA; 10 μM; used as a positive control). Apoptosis and differentiation were also detected using propidium iodide and NBT staining techniques, respectively. Furthermore, the expression levels of genes involved in apoptosis (PI3 K, AKT, BCL2, BAX, p53, p21, PTEN, CASP3, CASP8, and CASP9), differentiation (PML-RAR and HDAC1), and multi-drug resistance (ABCB1 and ABCC1) were determined using quantitative real-time PCR. The protein expressions of Bax/Bcl2 and casp3 were confirmed using Western blot. The results showed that kaempferol decreased cell viability and increased subG1 population in the tested leukemic cells. This effect was associated with decreased expression of Akt, BCL2, ABCB1, and ABCC1 genes, while the expression of CASP3 and BAX/BCL-2 ratio were significantly increased at both gene and protein levels. Kaempferol promoted apoptosis and inhibited multidrug resistance in a concentration-dependent manner, without any differential effect on leukemic cells. In conclusion, this study suggested that kaempferol may be utilized as an appropriate alternative for ATRA in APL patients. © 2017 Wiley Periodicals, Inc.

  18. Reproductive gene expression in a coral reef fish exposed to increasing temperature across generations

    Veilleux, Heather D

    2017-12-07

    Reproduction in marine fish is generally tightly linked with water temperature. Consequently, when adults are exposed to projected future ocean temperatures, reproductive output of many species declines precipitously. Recent research has shown that in the common reef fish, Acanthochromis polyacanthus, step-wise exposure to higher temperatures over two generations (parents: +1.5°C, offspring: +3.0°C) can improve reproductive output in the F2 generation compared to F2 fish that have experienced the same high temperatures over two generations (F1 parents: +3.0°C, F2 offspring: +3.0°C). To investigate how a step-wise increase in temperature between generations improved reproductive capacity, we tested the expression of well-known teleost reproductive genes in the brain and gonads of F2 fish using quantitative reverse transcription PCR and compared it among control (+0.0°C for two generations), developmental (+3.0°C in second generation only), step (+1.5°C in first generation and +3.0°C in second generation), and transgenerational (+3.0°C for two generations) treatments. We found that levels of gonadotropin receptor gene expression (Fshr and Lhcgr) in the testes were reduced in developmental and transgenerational temperature treatments, but were similar to control levels in the step treatment. This suggests Fshr and Lhcgr may be involved in regulating male reproductive capacity in A. polyacanthus. In addition, lower Fshb expression in the brain of females in all temperature treatments compared to control, suggests that Fshb expression, which is involved in vitellogenesis, is sensitive to high temperatures. Our results help elucidate key genes that facilitate successful reproduction in reef fishes when they experience a gradual increase in temperature across generations consistent with the trajectory of climate change.

  19. Reduced methylation of the thromboxane synthase gene is correlated with its increased vascular expression in preeclampsia.

    Mousa, Ahmad A; Strauss, Jerome F; Walsh, Scott W

    2012-06-01

    Preeclampsia is characterized by increased thromboxane and decreased prostacyclin levels, which predate symptoms, and can explain some of the clinical manifestations of preeclampsia, including hypertension and thrombosis. In this study, we examined DNA methylation of the promoter region of the thromboxane synthase gene (TBXAS1) and the expression of thromboxane synthase in systemic blood vessels of normal pregnant and preeclamptic women. Thromboxane synthase is responsible for the synthesis of thromboxane A(2), a potent vasoconstrictor and activator of platelets. We also examined the effect of experimentally induced DNA hypomethylation on the expression of thromboxane synthase in a neutrophil-like cell line (HL-60 cells) and in cultured vascular smooth muscle and endothelial cells. We found that DNA methylation of the TBXAS1 promoter was decreased and thromboxane synthase expression was increased in omental arteries of preeclamptic women as compared with normal pregnant women. Increased thromboxane synthase expression was observed in vascular smooth muscles cells, endothelial cells, and infiltrating neutrophils. Experimentally induced DNA hypomethylation only increased expression of thromboxane synthase in the neutrophil-like cell line, whereas tumor necrosis factor-α, a neutrophil product, increased its expression in cultured vascular smooth muscle cells. Our study suggests that epigenetic mechanisms and release of tumor necrosis factor-α by infiltrating neutrophils could contribute to the increased expression of thromboxane synthase in maternal systemic blood vessels, contributing to the hypertension and coagulation abnormalities associated with preeclampsia.

  20. Placental triglyceride accumulation in maternal type 1 diabetes is associated with increased lipase gene expression

    Lindegaard, Marie Louise Skakkebæk; Damm, Peter; Mathiesen, Elisabeth R

    2006-01-01

    Maternal diabetes can cause fetal macrosomia and increased risk of obesity, diabetes, and cardiovascular disease in adulthood of the offspring. Although increased transplacental lipid transport could be involved, the impact of maternal type 1 diabetes on molecular mechanisms for lipid transport...... in placenta is largely unknown. To examine whether maternal type 1 diabetes affects placental lipid metabolism, we measured lipids and mRNA expression of lipase-encoding genes in placentas from women with type 1 diabetes (n = 27) and a control group (n = 21). The placental triglyceride (TG) concentration......RNA expression of lipoprotein lipase and lysosomal lipase were similar in women with diabetes and the control group. Immunohistochemistry showed EL protein in syncytiotrophoblasts facing the maternal blood and endothelial cells facing the fetal blood in placentas from both normal women and women with diabetes...

  1. Receptor-like protein-tyrosine phosphatase alpha specifically inhibits insulin-increased prolactin gene expression

    Jacob, K K; Sap, J; Stanley, F M

    1998-01-01

    A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin...... is specific by two criteria. A number of potential RPTPalpha targets were ruled out by finding (a) that they are not affected or (b) that they are not on the pathway to insulin-increased prolactin-CAT activity. The negative effect of RPTPalpha on insulin activation of the prolactin promoter is not due...... to reduced phosphorylation or kinase activity of the insulin receptor or to reduced phosphorylation of insulin receptor substrate-1 or Shc. Inhibitor studies suggest that insulin-increased prolactin gene expression is mediated by a Ras-like GTPase but is not mitogen-activated protein kinase dependent...

  2. Genetically increased antioxidative protection and decreased chronic obstructive pulmonary disease

    Juul, Klaus; Tybjærg-Hansen, Anne; Marklund, Stefan

    2006-01-01

    RATIONALE: Increased oxidative stress is involved in chronic obstructive pulmonary disease (COPD); however, plasma and bronchial lining fluid contains the antioxidant extracellular superoxide dismutase. Approximately 2% of white individuals carry the R213G polymorphism in the gene encoding......-sectionally and prospectively (during 24 yr) 9,258 individuals from the Danish general population genotyped for R213G. MEASUREMENTS: We determined plasma extracellular superoxide dismutase concentration, pulmonary function and COPD diagnosed by means of spirometry or through national hospitalization and death registers. MAIN...... extracellular superoxide dismutase, which increases plasma extracellular superoxide dismutase 10-fold and presumably also renders bronchial lining fluid high in extracellular superoxide dismutase. OBJECTIVE: We tested the hypothesis that R213G reduces the risk of COPD. METHODS: We studied cross...

  3. Sources of extracellular tau and its signaling.

    Avila, Jesús; Simón, Diana; Díaz-Hernández, Miguel; Pintor, Jesús; Hernández, Félix

    2014-01-01

    The pathology associated with tau protein, tauopathy, has been recently analyzed in different disorders, leading to the suggestion that intracellular and extracellular tau may itself be the principal agent in the transmission and spreading of tauopathies. Tau pathology is based on an increase in the amount of tau, an increase in phosphorylated tau, and/or an increase in aggregated tau. Indeed, phosphorylated tau protein is the main component of tau aggregates, such as the neurofibrillary tangles present in the brain of Alzheimer's disease patients. It has been suggested that intracellular tau could be toxic to neurons in its phosphorylated and/or aggregated form. However, extracellular tau could also damage neurons and since neuronal death is widespread in Alzheimer's disease, mainly among cholinergic neurons, these cells may represent a possible source of extracellular tau. However, other sources of extracellular tau have been proposed that are independent of cell death. In addition, several ways have been proposed for cells to interact with, transmit, and spread extracellular tau, and to transduce signals mediated by this tau. In this work, we will discuss the role of extracellular tau in the spreading of the tau pathology.

  4. Carrier of Wingless (Cow), a Secreted Heparan Sulfate Proteoglycan, Promotes Extracellular Transport of Wingless

    Chang, Yung-Heng; Sun, Yi Henry

    2014-01-01

    Morphogens are signaling molecules that regulate growth and patterning during development by forming a gradient and activating different target genes at different concentrations. The extracellular distribution of morphogens is tightly regulated, with the Drosophila morphogen Wingless (Wg) relying on Dally-like (Dlp) and transcytosis for its distribution. However, in the absence of Dlp or endocytic activity, Wg can still move across cells along the apical (Ap) surface. We identified a novel secreted heparan sulfate proteoglycan (HSPG) that binds to Wg and promotes its extracellular distribution by increasing Wg mobility, which was thus named Carrier of Wg (Cow). Cow promotes the Ap transport of Wg, independent of Dlp and endocytosis, and this function addresses a previous gap in the understanding of Wg movement. This is the first example of a diffusible HSPG acting as a carrier to promote the extracellular movement of a morphogen. PMID:25360738

  5. Cannabidivarin (CBDV suppresses pentylenetetrazole (PTZ-induced increases in epilepsy-related gene expression

    Naoki Amada

    2013-11-01

    Full Text Available To date, anticonvulsant effects of the plant cannabinoid, cannabidivarin (CBDV, have been reported in several animal models of seizure. However, these behaviourally observed anticonvulsant effects have not been confirmed at the molecular level. To examine changes to epilepsy-related gene expression following chemical convulsant treatment and their subsequent control by phytocannabinoid administration, we behaviourally evaluated effects of CBDV (400 mg/kg, p.o. on acute, pentylenetetrazole (PTZ: 95 mg/kg, i.p.-induced seizures, quantified expression levels of several epilepsy-related genes (Fos, Casp 3, Ccl3, Ccl4, Npy, Arc, Penk, Camk2a, Bdnf and Egr1 by qPCR using hippocampal, neocortical and prefrontal cortical tissue samples before examining correlations between expression changes and seizure severity. PTZ treatment alone produced generalised seizures (median: 5.00 and significantly increased expression of Fos, Egr1, Arc, Ccl4 and Bdnf. Consistent with previous findings, CBDV significantly decreased PTZ-induced seizure severity (median: 3.25 and increased latency to the first sign of seizure. Furthermore, there were correlations between reductions of seizure severity and mRNA expression of Fos, Egr1, Arc, Ccl4 and Bdnf in the majority of brain regions in the CBDV+PTZ treated group. When CBDV treated animals were grouped into CBDV responders (criterion: seizure severity ≤3.25 and non-responders (criterion: seizure severity >3.25, PTZ-induced increases of Fos, Egr1, Arc, Ccl4 and Bdnf expression were suppressed in CBDV responders. These results provide the first molecular confirmation of behaviourally observed effects of the non-psychoactive, anticonvulsant cannabinoid, CBDV, upon chemically-induced seizures and serve to underscore its suitability for clinical development.

  6. The BTNL2 G16071A gene polymorphism increases granulomatous disease susceptibility

    Tong, Xiang; Ma, Yao; Niu, Xundong; Yan, Zhipeng; Liu, Sitong; Peng, Bo; Peng, Shifeng; Fan, Hong

    2016-01-01

    Abstract Objective: The butyrophilin-like 2 (BTNL2) G16071A gene polymorphism has been implicated in the susceptibility to granulomatous diseases, but the results were inconclusive. The objective of the current study was to precisely explore the relationship between BTNL2 G16071A gene polymorphism and granulomatous disease susceptibility by the meta-analysis including false-positive report probability (FPRP) test. Methods: A systematic literature search in the PubMed, Embase, and Wanfang databases, China National Knowledge Internet, and commercial Internet search engines was conducted to identify studies published up to April 1, 2016. The odds ratio (OR) with 95% confidence interval (CI) was used to assess the effect size. Statistical analysis was conducted using the STATA 12.0 software and FPRP test sheet. Results: In total, all 4324 cases and 4386 controls from 14 eligible studies were included in the current meta-analysis. By the overall meta-analysis, we found a significant association between BTNL2 G16071A gene polymorphism and granulomatous disease susceptibility (A vs G: OR = 1.25, 95% CI = 1.07–1.45, P = 0.005). The meta-regression analyses showed that a large proportion of the between-study heterogeneity was significantly attributed to the ethnicity (A vs G, P = 0.013) and the types of granulomatous diseases (A vs G, P = 0.002). By the subgroup meta-analysis, the BTNL2 G16071A gene polymorphism was associated with granulomatous disease susceptibility in Caucasians (A vs G: OR = 1.37, 95% CI = 1.18–1.58, P susceptibility (A vs G: OR = 1.52, 95% CI = 1.39–1.66, P susceptibility (A vs G, FPRP susceptibility among Caucasians (A vs G, FPRP susceptibility, especially increasing the sarcoidosis susceptibility. In addition, the polymorphism may be greatly associated with likelihood of granulomatous diseases among Caucasians. PMID:27472712

  7. Cloning of a Gene Whose Expression is Increased in Scrapie and in Senile Plaques in Human Brain

    Wietgrefe, S.; Zupancic, M.; Haase, A.; Chesebro, B.; Race, R.; Frey, W.; Rustan, T.; Friedman, R. L.

    1985-12-01

    A complementary DNA library was constructed from messenger RNA's extracted from the brains of mice infected with the scrapie agent. The library was differentially screened with the objectives of finding clones that might be used as markers of infection and finding clones of genes whose increased expression might be correlated with the pathological changes common to scrapie and Alzheimer's disease. A gene was identified whose expression is increased in scrapie. The complementary DNA corresponding to this gene hybridized preferentially and focally to cells in the brains of scrapie-infected animals. The cloned DNA also hybridized to the neuritic plaques found with increased frequency in brains of patients with Alzheimer's disease.

  8. Low AMY1 Gene Copy Number Is Associated with Increased Body Mass Index in Prepubertal Boys.

    M Loredana Marcovecchio

    Full Text Available Genome-wide association studies have identified more than 60 single nucleotide polymorphisms associated with Body Mass Index (BMI. Additional genetic variants, such as copy number variations (CNV, have also been investigated in relation to BMI. Recently, the highly polymorphic CNV in the salivary amylase (AMY1 gene, encoding an enzyme implicated in the first step of starch digestion, has been associated with obesity in adults and children. We assessed the potential association between AMY1 copy number and a wide range of BMI in a population of Italian school-children.744 children (354 boys, 390 girls, mean age (±SD: 8.4±1.4years underwent anthropometric assessments (height, weight and collection of saliva samples for DNA extraction. AMY1 copies were evaluated by quantitative PCR.A significant increase of BMI z-score by decreasing AMY1 copy number was observed in boys (β: -0.117, p = 0.033, but not in girls. Similarly, waist circumference (β: -0.155, p = 0.003, adjusted for age was negatively influenced by AMY1 copy number in boys. Boys with 8 or more AMY1 copy numbers presented a significant lower BMI z-score (p = 0.04 and waist circumference (p = 0.01 when compared to boys with less than 8 copy numbers.In this pediatric-only, population-based study, a lower AMY1 copy number emerged to be associated with increased BMI in boys. These data confirm previous findings from adult studies and support a potential role of a higher copy number of the salivary AMY1 gene in protecting from excess weight gain.

  9. MicroRNA gene polymorphisms and environmental factors increase patient susceptibility to hepatocellular carcinoma.

    Yin-Hung Chu

    Full Text Available BACKGROUND: Micro RNAs (miRNAs are small RNA fragments that naturally exist in the human body. Through various physiological mechanisms, miRNAs can generate different functions for regulating RNA protein levels and balancing abnormalities. Abnormal miRNA expression has been reported to be highly related to several diseases and cancers. Single-nucleotide polymorphisms (SNPs in miRNAs have been reported to increase patient susceptibility and affect patient prognosis and survival. We adopted a case-control research design to verify the relationship between miRNAs and hepatocellular carcinoma. METHODOLOGY/PRINCIPAL FINDINGS: A total of 525 subjects, including 377 controls and 188 hepatocellular carcinoma patients, were selected. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP and real-time PCR were used to analyze miRNA146a (rs2910164, miRNA149 (rs2292832, miRNA196 (rs11614913, and miRNA499 (rs3746444 genetic polymorphisms between the control group and the case group. The results indicate that people who carry the rs3746444 CT or CC genotypes may have a significantly increased susceptibility to hepatocellular carcinoma (adjusted odds ratio [AOR] = 2.84, 95% confidence interval [CI] = 1.88-4.30. In addition, when combined with environmental risk factors, such as smoking and alcohol consumption, interaction effects were observed between gene polymorphisms and environmental factors (odds ratio [OR] = 4.69, 95% CI = 2.52-8.70; AOR = 3.38, 95% CI = 1.68-6.80. CONCLUSIONS: These results suggest that a significant association exists between miRNA499 SNPs and hepatocellular carcinoma. Gene-environment interactions of miRNA499 polymorphisms, smoking, and alcohol consumption might alter hepatocellular carcinoma susceptibility.

  10. Pseudogene PHBP1 promotes esophageal squamous cell carcinoma proliferation by increasing its cognate gene PHB expression.

    Feng, Feiyue; Qiu, Bin; Zang, Ruochuan; Song, Peng; Gao, Shugeng

    2017-04-25

    Natural antisense transcripts (NATs) as one of the most diverse classes of long noncoding RNAs (lncRNAs), have been demonstrated involved in fundamental biological processes in human. Here, we reported that human prohibitin gene pseudogene 1 (PHBP1) was upregulated in ESCC, and increased PHBP1 expression in ESCC was associated with clinical advanced stage. Functional experiments showed that PHBP1 knockdown inhibited ESCC cells proliferation, colony formation and xenograft tumor growth in vitro and in vivo by causing cell-cycle arrest at the G1-G0 phase. Mechanisms analysis revealed that PHBP1 transcript as an antisense transcript of PHB is partially complementary to PHB mRNA and formed an RNA-RNA hybrid with PHB, consequently inducing an increase of PHB expression at both the mRNA and protein levels. Furthermore, PHBP1 expression is strongly correlated with PHB expression in ESCC tissues. Collectively, this study elucidates an important role of PHBP1 in promoting ESCC partly via increasing PHB expression.

  11. Regulatory network controlling extracellular proteins in Erwinia carotovora subsp. carotovora: FlhDC, the master regulator of flagellar genes, activates rsmB regulatory RNA production by affecting gacA and hexA (lrhA) expression.

    Cui, Yaya; Chatterjee, Asita; Yang, Hailian; Chatterjee, Arun K

    2008-07-01

    Erwinia carotovora subsp. carotovora produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (PecT in Erwinia chrysanthemi; LrhA [LysR homolog A] in Escherichia coli) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to the flhDC operon of E. coli also controls extracellular enzyme production. A transposon insertion FlhDC(-) mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, and E. carotovora subsp. carotovora Harpin (Harpin(Ecc)) and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhDC(+) plasmid. Sequence analysis and transcript assays disclosed that the flhD operon of E. carotovora subsp. carotovora, like those of other enterobacteria, consists of flhD and flhC. Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. The data presented here show that FlhDC positively regulates gacA, rsmC, and fliA and negatively regulates hexA (lrhA). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA. Reduced levels of GacA and elevated levels of HexA in the FlhDC(-) mutant are responsible for the inhibition of rsmB RNA

  12. Regulatory Network Controlling Extracellular Proteins in Erwinia carotovora subsp. carotovora: FlhDC, the Master Regulator of Flagellar Genes, Activates rsmB Regulatory RNA Production by Affecting gacA and hexA (lrhA) Expression▿

    Cui, Yaya; Chatterjee, Asita; Yang, Hailian; Chatterjee, Arun K.

    2008-01-01

    Erwinia carotovora subsp. carotovora produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (PecT in Erwinia chrysanthemi; LrhA [LysR homolog A] in Escherichia coli) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to the flhDC operon of E. coli also controls extracellular enzyme production. A transposon insertion FlhDC− mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, and E. carotovora subsp. carotovora Harpin (HarpinEcc) and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhDC+ plasmid. Sequence analysis and transcript assays disclosed that the flhD operon of E. carotovora subsp. carotovora, like those of other enterobacteria, consists of flhD and flhC. Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. The data presented here show that FlhDC positively regulates gacA, rsmC, and fliA and negatively regulates hexA (lrhA). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA. Reduced levels of GacA and elevated levels of HexA in the FlhDC− mutant are responsible for the inhibition of rsmB RNA production

  13. Fabp1 gene ablation inhibits high-fat diet-induced increase in brain endocannabinoids.

    Martin, Gregory G; Landrock, Danilo; Chung, Sarah; Dangott, Lawrence J; Seeger, Drew R; Murphy, Eric J; Golovko, Mikhail Y; Kier, Ann B; Schroeder, Friedhelm

    2017-01-01

    The endocannabinoid system shifts energy balance toward storage and fat accumulation, especially in the context of diet-induced obesity. Relatively little is known about factors outside the central nervous system that may mediate the effect of high-fat diet (HFD) on brain endocannabinoid levels. One candidate is the liver fatty acid binding protein (FABP1), a cytosolic protein highly prevalent in liver, but not detected in brain, which facilitates hepatic clearance of fatty acids. The impact of Fabp1 gene ablation (LKO) on the effect of high-fat diet (HFD) on brain and plasma endocannabinoid levels was examined and data expressed for each parameter as the ratio of high-fat diet/control diet. In male wild-type mice, HFD markedly increased brain N-acylethanolamides, but not 2-monoacylglycerols. LKO blocked these effects of HFD in male mice. In female wild-type mice, HFD slightly decreased or did not alter these endocannabinoids as compared with male wild type. LKO did not block the HFD effects in female mice. The HFD-induced increase in brain arachidonic acid-derived arachidonoylethanolamide in males correlated with increased brain-free and total arachidonic acid. The ability of LKO to block the HFD-induced increase in brain arachidonoylethanolamide correlated with reduced ability of HFD to increase brain-free and total arachidonic acid in males. In females, brain-free and total arachidonic acid levels were much less affected by either HFD or LKO in the context of HFD. These data showed that LKO markedly diminished the impact of HFD on brain endocannabinoid levels, especially in male mice. © 2016 International Society for Neurochemistry.

  14. Polymorphisms in the prion protein gene and in the doppel gene increase susceptibility for Creutzfeldt-Jakob disease

    E.A. Croes (Esther); B.Z. Alizadeh (Behrooz); A.M. Bertoli Avella (Aida); T.A.M. Rademaker (Tessa); J. Vergeer-Drop (Jeannette); B. Dermaut (Bart); J.J. Houwing-Duistermaat (Jeanine); D.P.W.M. Wientjens (Dorothee); A. Hofman (Albert); C. van Broeckhoven (Christine); C.M. van Duijn (Cornelia)

    2004-01-01

    textabstractThe prion protein gene (PRNP) plays a central role in the origin of Creutzfeldt-Jakob disease (CJD), but there is growing interest in other polymorphisms that may be involved in CJD. Polymorphisms upstream of PRNP that may modulate the prion protein production as well as polymorphisms in

  15. Horizontal gene transfer contributed to the evolution of extracellular surface structures: the freshwater polyp Hydra is covered by a complex fibrous cuticle containing glycosaminoglycans and proteins of the PPOD and SWT (sweet tooth families.

    Angelika Böttger

    Full Text Available The single-cell layered ectoderm of the fresh water polyp Hydra fulfills the function of an epidermis by protecting the animals from the surrounding medium. Its outer surface is covered by a fibrous structure termed the cuticle layer, with similarity to the extracellular surface coats of mammalian epithelia. In this paper we have identified molecular components of the cuticle. We show that its outermost layer contains glycoproteins and glycosaminoglycans and we have identified chondroitin and chondroitin-6-sulfate chains. In a search for proteins that could be involved in organising this structure we found PPOD proteins and several members of a protein family containing only SWT (sweet tooth domains. Structural analyses indicate that PPODs consist of two tandem β-trefoil domains with similarity to carbohydrate-binding sites found in lectins. Experimental evidence confirmed that PPODs can bind sulfated glycans and are secreted into the cuticle layer from granules localized under the apical surface of the ectodermal epithelial cells. PPODs are taxon-specific proteins which appear to have entered the Hydra genome by horizontal gene transfer from bacteria. Their acquisition at the time Hydra evolved from a marine ancestor may have been critical for the transition to the freshwater environment.

  16. Integrated analysis of genetic variation and gene expression reveals novel variant for increased warfarin dose requirement in African Americans

    Hernandez, W.; Gamazon, E. R.; Aquino-Michaels, K.; Smithberger, E.; O'Brien, T. J.; Harralson, A. F.; Tuck, M.; Barbour, A.; Cavallari, L. H.; Perera, M. A.

    2017-01-01

    Essentials Genetic variants controlling gene regulation have not been explored in pharmacogenomics. We tested liver expression quantitative trait loci for association with warfarin dose response. A novel predictor for increased warfarin dose response in African Americans was identified. Precision

  17. Significantly increased risk of carotid atherosclerosis with arsenic exposure and polymorphisms in arsenic metabolism genes

    Hsieh, Yi-Chen; Lien, Li-Ming; Chung, Wen-Ting; Hsieh, Fang-I; Hsieh, Pei-Fan; Wu, Meei-Maan; Tseng, Hung-Pin; Chiou, Hung-Yi; Chen, Chien-Jen

    2011-01-01

    Individual susceptibility to arsenic-induced carotid atherosclerosis might be associated with genetic variations in arsenic metabolism. The purpose of this study is to explore the interaction effect on risk of carotid atherosclerosis between arsenic exposure and risk genotypes of purine nucleoside phosphorylase (PNP), arsenic (+3) methyltransferase (As3MT), and glutathione S-transferase omega 1 (GSTO1) and omega 2 (GSTO2). A community-based case-control study was conducted in northeastern Taiwan to investigate the arsenic metabolic-related genetic susceptibility to carotid atherosclerosis. In total, 863 subjects, who had been genotyped and for whom the severity of carotid atherosclerosis had been determined, were included in the present study. Individual well water was collected and arsenic concentration determined using hydride generation combined with flame atomic absorption spectrometry. The result showed that a significant dose-response trend (P=0.04) of carotid atherosclerosis risk associated with increasing arsenic concentration. Non-significant association between genetic polymorphisms of PNP Gly51Ser, Pro57Pro, As3MT Met287Thr, GSTO1 Ala140Asp, and GSTO2 A-183G and the risk for development of carotid atherosclerosis were observed. However, the significant interaction effect on carotid atherosclerosis risk was found for arsenic exposure (>50 μg/l) and the haplotypes of PNP (p=0.0115). A marked elevated risk of carotid atherosclerosis was observed in subjects with arsenic exposure of >50 μg/l in drinking water and those who carried the PNP A-T haplotype and at least either of the As3MT risk polymorphism or GSTO risk haplotypes (OR, 6.43; 95% CI, 1.79-23.19). In conclusion, arsenic metabolic genes, PNP, As3MT, and GSTO, may exacerbate the formation of atherosclerosis in individuals with high levels of arsenic concentration in well water (>50 μg/l). - Highlights: →Arsenic metabolic genes might be associated with carotid atherosclerosis. → A case

  18. The Increasing Complexity of the Oncofetal H19 Gene Locus: Functional Dissection and Therapeutic Intervention

    Abraham Hochberg

    2013-02-01

    Full Text Available The field of the long non-coding RNA (lncRNA is advancing rapidly. Currently, it is one of the most popular fields in the biological and medical sciences. It is becoming increasingly obvious that the majority of the human transcriptome has little or no-protein coding capacity. Historically, H19 was the first imprinted non-coding RNA (ncRNA transcript identified, and the H19/IGF2 locus has served as a paradigm for the study of genomic imprinting since its discovery. In recent years, we have extensively investigated the expression of the H19 gene in a number of human cancers and explored the role of H19 RNA in tumor development. Here, we discuss recently published data from our group and others that provide further support for a central role of H19 RNA in the process of tumorigenesis. Furthermore, we focus on major transcriptional modulators of the H19 gene and discuss them in the context of the tumor-promoting activity of the H19 RNA. Based on the pivotal role of the H19 gene in human cancers, we have developed a DNA-based therapeutic approach for the treatment of cancers that have upregulated levels of H19 expression. This approach uses a diphtheria toxin A (DTA protein expressed under the regulation of the H19 promoter to treat tumors with significant expression of H19 RNA. In this review, we discuss the treatment of four cancer indications in human subjects using this approach, which is currently under development. This represents perhaps one of the very few examples of an existing DNA-based therapy centered on an lncRNA system. Apart from cancer, H19 expression has been reported also in other conditions, syndromes and diseases, where deregulated imprinting at the H19 locus was obvious in some cases and will be summarized below. Moreover, the H19 locus proved to be much more complicated than initially thought. It houses a genomic sequence that can transcribe, yielding various transcriptional outputs, both in sense and antisense directions. The

  19. Significantly increased risk of carotid atherosclerosis with arsenic exposure and polymorphisms in arsenic metabolism genes

    Hsieh, Yi-Chen [School of Public Health, College of Public Health and Nutrition, Taipei Medical University, 250 Wusing St., Taipei 11031, Taiwan (China); Lien, Li-Ming [Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); School of Medicine, Taipei Medical University, Taipei, Taiwan (China); Department of Neurology, Shin Kong WHS Memorial Hospital, Taipei, Taiwan (China); Chung, Wen-Ting [Department of Neurology, Wanfang Hospital, Taipei Medical University, Taipei, Taiwan (China); Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei, Taiwan (China); Hsieh, Fang-I; Hsieh, Pei-Fan [School of Public Health, College of Public Health and Nutrition, Taipei Medical University, 250 Wusing St., Taipei 11031, Taiwan (China); Wu, Meei-Maan [School of Public Health, College of Public Health and Nutrition, Taipei Medical University, 250 Wusing St., Taipei 11031, Taiwan (China); Graduate Institute of Basic Medicine, College of Medicine, Fu-Jen Catholic University, Taipei, Taiwan (China); Tseng, Hung-Pin [Department of Neurology, Lotung Poh-Ai Hospital, I-Lan, Taiwan (China); Chiou, Hung-Yi, E-mail: hychiou@tmu.edu.tw [School of Public Health, College of Public Health and Nutrition, Taipei Medical University, 250 Wusing St., Taipei 11031, Taiwan (China); Chen, Chien-Jen [Genomics Research Center, Academia Sinica, Taipei, Taiwan (China)

    2011-08-15

    Individual susceptibility to arsenic-induced carotid atherosclerosis might be associated with genetic variations in arsenic metabolism. The purpose of this study is to explore the interaction effect on risk of carotid atherosclerosis between arsenic exposure and risk genotypes of purine nucleoside phosphorylase (PNP), arsenic (+3) methyltransferase (As3MT), and glutathione S-transferase omega 1 (GSTO1) and omega 2 (GSTO2). A community-based case-control study was conducted in northeastern Taiwan to investigate the arsenic metabolic-related genetic susceptibility to carotid atherosclerosis. In total, 863 subjects, who had been genotyped and for whom the severity of carotid atherosclerosis had been determined, were included in the present study. Individual well water was collected and arsenic concentration determined using hydride generation combined with flame atomic absorption spectrometry. The result showed that a significant dose-response trend (P=0.04) of carotid atherosclerosis risk associated with increasing arsenic concentration. Non-significant association between genetic polymorphisms of PNP Gly51Ser, Pro57Pro, As3MT Met287Thr, GSTO1 Ala140Asp, and GSTO2 A-183G and the risk for development of carotid atherosclerosis were observed. However, the significant interaction effect on carotid atherosclerosis risk was found for arsenic exposure (>50 {mu}g/l) and the haplotypes of PNP (p=0.0115). A marked elevated risk of carotid atherosclerosis was observed in subjects with arsenic exposure of >50 {mu}g/l in drinking water and those who carried the PNP A-T haplotype and at least either of the As3MT risk polymorphism or GSTO risk haplotypes (OR, 6.43; 95% CI, 1.79-23.19). In conclusion, arsenic metabolic genes, PNP, As3MT, and GSTO, may exacerbate the formation of atherosclerosis in individuals with high levels of arsenic concentration in well water (>50 {mu}g/l). - Highlights: {yields}Arsenic metabolic genes might be associated with carotid atherosclerosis. {yields

  20. miR-200–containing extracellular vesicles promote breast cancer cell metastasis

    Le, Minh T.N.; Hamar, Peter; Guo, Changying; Basar, Emre; Perdigão-Henriques, Ricardo; Balaj, Leonora; Lieberman, Judy

    2014-01-01

    Metastasis is associated with poor prognosis in breast cancer patients. Not all cancer cells within a tumor are capable of metastasizing. The microRNA-200 (miR-200) family, which regulates the mesenchymal-to-epithelial transition, is enriched in the serum of patients with metastatic cancers. Ectopic expression of miR-200 can confer metastatic ability to poorly metastatic tumor cells in some settings. Here, we investigated whether metastatic capability could be transferred between metastatic and nonmetastatic cancer cells via extracellular vesicles. miR-200 was secreted in extracellular vesicles from metastatic murine and human breast cancer cell lines, and miR-200 levels were increased in sera of mice bearing metastatic tumors. In culture, murine and human metastatic breast cancer cell extracellular vesicles transferred miR-200 microRNAs to nonmetastatic cells, altering gene expression and promoting mesenchymal-to-epithelial transition. In murine cancer and human xenograft models, miR-200–expressing tumors and extracellular vesicles from these tumors promoted metastasis of otherwise weakly metastatic cells either nearby or at distant sites and conferred to these cells the ability to colonize distant tissues in a miR-200–dependent manner. Together, our results demonstrate that metastatic capability can be transferred by the uptake of extracellular vesicles. PMID:25401471

  1. Increased isobutanol production in Saccharomyces cerevisiae by overexpression of genes in valine metabolism

    Karhumaa Kaisa

    2011-07-01

    Full Text Available Abstract Background Isobutanol can be a better biofuel than ethanol due to its higher energy density and lower hygroscopicity. Furthermore, the branched-chain structure of isobutanol gives a higher octane number than the isomeric n-butanol. Saccharomyces cerevisiae was chosen as the production host because of its relative tolerance to alcohols, robustness in industrial fermentations, and the possibility for future combination of isobutanol production with fermentation of lignocellulosic materials. Results The yield of isobutanol was improved from 0.16 to 0.97 mg per g glucose by simultaneous overexpression of biosynthetic genes ILV2, ILV3, and ILV5 in valine metabolism in anaerobic fermentation of glucose in mineral medium in S. cerevisiae. Isobutanol yield was further improved by twofold by the additional overexpression of BAT2, encoding the cytoplasmic branched-chain amino-acid aminotransferase. Overexpression of ILV6, encoding the regulatory subunit of Ilv2, in the ILV2 ILV3 ILV5 overexpression strain decreased isobutanol production yield by threefold. In aerobic cultivations in shake flasks in mineral medium, the isobutanol yield of the ILV2 ILV3 ILV5 overexpression strain and the reference strain were 3.86 and 0.28 mg per g glucose, respectively. They increased to 4.12 and 2.4 mg per g glucose in yeast extract/peptone/dextrose (YPD complex medium under aerobic conditions, respectively. Conclusions Overexpression of genes ILV2, ILV3, ILV5, and BAT2 in valine metabolism led to an increase in isobutanol production in S. cerevisiae. Additional overexpression of ILV6 in the ILV2 ILV3 ILV5 overexpression strain had a negative effect, presumably by increasing the sensitivity of Ilv2 to valine inhibition, thus weakening the positive impact of overexpression of ILV2, ILV3, and ILV5 on isobutanol production. Aerobic cultivations of the ILV2 ILV3 ILV5 overexpression strain and the reference strain showed that supplying amino acids in cultivation media

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

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

    2015-01-15

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

  3. Increased burden of deleterious variants in essential genes in autism spectrum disorder.

    Ji, Xiao; Kember, Rachel L; Brown, Christopher D; Bućan, Maja

    2016-12-27

    Autism spectrum disorder (ASD) is a heterogeneous, highly heritable neurodevelopmental syndrome characterized by impaired social interaction, communication, and repetitive behavior. It is estimated that hundreds of genes contribute to ASD. We asked if genes with a strong effect on survival and fitness contribute to ASD risk. Human orthologs of genes with an essential role in pre- and postnatal development in the mouse [essential genes (EGs)] are enriched for disease genes and under strong purifying selection relative to human orthologs of mouse genes with a known nonlethal phenotype [nonessential genes (NEGs)]. This intolerance to deleterious mutations, commonly observed haploinsufficiency, and the importance of EGs in development suggest a possible cumulative effect of deleterious variants in EGs on complex neurodevelopmental disorders. With a comprehensive catalog of 3,915 mammalian EGs, we provide compelling evidence for a stronger contribution of EGs to ASD risk compared with NEGs. By examining the exonic de novo and inherited variants from 1,781 ASD quartet families, we show a significantly higher burden of damaging mutations in EGs in ASD probands compared with their non-ASD siblings. The analysis of EGs in the developing brain identified clusters of coexpressed EGs implicated in ASD. Finally, we suggest a high-priority list of 29 EGs with potential ASD risk as targets for future functional and behavioral studies. Overall, we show that large-scale studies of gene function in model organisms provide a powerful approach for prioritization of genes and pathogenic variants identified by sequencing studies of human disease.

  4. Extracellular Matrix Proteins Expression Profiling in Chemoresistant Variants of the A2780 Ovarian Cancer Cell Line

    Radosław Januchowski

    2014-01-01

    Full Text Available Ovarian cancer is the leading cause of death among gynaecological malignancies. Extracellular matrix (ECM can affect drug resistance by preventing the penetration of the drug into cancer cells and increased resistance to apoptosis. This study demonstrates alterations in the expression levels of ECM components and related genes in cisplatin-, doxorubicin-, topotecan-, and paclitaxel-resistant variants of the A2780 ovarian cancer cell line. Affymetrix Gene Chip Human Genome Array Strips were used for hybridisations. The genes that had altered expression levels in drug-resistant sublines were selected and filtered by scatter plots. The genes that were up- or downregulated more than fivefold were selected and listed. Among the investigated genes, 28 genes were upregulated, 10 genes were downregulated, and two genes were down- or upregulated depending on the cell line. Between upregulated genes 12 were upregulated very significantly—over 20-fold. These genes included COL1A2, COL12A1, COL21A1, LOX, TGFBI, LAMB1, EFEMP1, GPC3, SDC2, MGP, MMP3, and TIMP3. Four genes were very significantly downregulated: COL11A1, LAMA2, GPC6, and LUM. The expression profiles of investigated genes provide a preliminary insight into the relationship between drug resistance and the expression of ECM components. Identifying correlations between investigated genes and drug resistance will require further analysis.

  5. Fluvastatin increases insulin-like growth factor-1 gene expression in rat model of metabolic syndrome

    Mansy, Wael H.; Sourour, Doaa A.; Shaker, Olfat G.; Mahfouz, Mahmoud M.

    2008-01-01

    Insulin-like growth factor-1 (IGF-1) was found to have a role in both glucose homeostasis and cardiovascular diseases. The present study was designed to compare the effects of fluvastatin and metformin on IGF-1 mRNA expression within the liver and other individual components of the metabolic syndrome induced in rats by high fructose feeding. Rats fed 60% fructose in diet for 6 weeks were treated daily with fluvastatin (3.75 mg/kg/day) during the last two weeks and were compared with untreated fructose fed group. Fasting levels of plasma cholesterol, triglyceride, glucose, insulin, nitric oxide products, IGF-1 mRNA within the liver as well as systolic blood pressure and body weight were determined. Compared to control rats, the fructose fed group developed hypertension, hyperlipidemia, hyperinsulinemia, hyperglycemia and endothelial dysfunction as well as decreased levels of plasma IGF-1 and its mRNA within the liver. Fructose fed rats treated with fluvastatin or metformin for 2 weeks showed significant decrease in plasma cholesterol, triglyceride, insulin and glucose levels compared to untreated fructose fed group. Also, both drugs increased significantly plasma levels of nitric oxide products and IGF-1 together with significant increase in IGF-1 mRNA within the liver. However, only metformin treated rats showed significant decrease in systolic blood pressure compared to fructose fed group. This study showed that in a rat model of insulin resistance, fluvastatin improves the metabolic profile and increases plasma level of IGF-1 and its gene expression as effective as metformin. (author)

  6. Increased expression of clp genes in Lactobacillus delbrueckii UFV H2b20 exposed to acid stress and bile salts.

    Ferreira, A B; De Oliveira, M N V; Freitas, F S; Alfenas-Zerbini, P; Da Silva, D F; De Queiroz, M V; Borges, A C; De Moraes, C A

    2013-12-01

    The ability to survive in harsh environments is an important criterion to select potential probiotics strains. The objective of this study was to identify and carry out phylogenetic and expression analysis by quantitative real-time PCR of the clpP, clpE, clpL and clpX genes in the probiotic strain Lactobacillus delbrueckii UFV H2b20 exposed to the conditions prevailing in the gastrointestinal tract (GIT). Phylogenetic trees reconstructed by Bayesian inference showed that the L. delbrueckii UFV H2b20 clpP, clpL and clpE genes and the ones from L. delbrueckii ATCC 11842 were grouped. The exposure of cells to MRS broth of pH 3.5 for 30 and 60 min resulted in an increased expression of the four genes. Exposure of the L. delbrueckii UFV H2b20 cells for 30 and 60 min to MRS broth containing 0.1% bile salts increased the expression of the clpP and clpE genes, while the expression level of the clpL and clpX genes increased only after 30 min of exposure. The involvement of the studied genes in the responses to acid stress and bile salts suggests a possible central role of these genes in the survival of L. delbrueckii UFV H2b20 during the passage through the GIT, a characteristic necessary for probiotic strains.

  7. Arsenic exposure from drinking water is associated with decreased gene expression and increased DNA methylation in peripheral blood

    Ameer, Syeda Shegufta [Department of Laboratory Medicine, Division of Occupational and Environmental Medicine, Lund University, Lund (Sweden); Engström, Karin [Department of Laboratory Medicine, Division of Occupational and Environmental Medicine, Lund University, Lund (Sweden); Institute of Environmental Medicine, Unit of Metals & Health, Karolinska Institutet, Stockholm (Sweden); Hossain, Mohammad Bakhtiar [Department of Laboratory Medicine, Division of Occupational and Environmental Medicine, Lund University, Lund (Sweden); Concha, Gabriela [Science Department, Risk Benefit Assessment Unit, National Food Agency, Uppsala (Sweden); Vahter, Marie [Institute of Environmental Medicine, Unit of Metals & Health, Karolinska Institutet, Stockholm (Sweden); Broberg, Karin, E-mail: Karin.broberg@ki.se [Institute of Environmental Medicine, Unit of Metals & Health, Karolinska Institutet, Stockholm (Sweden)

    2017-04-15

    Background: Exposure to inorganic arsenic increases the risk of cancer and non-malignant diseases. Inefficient arsenic metabolism is a marker for susceptibility to arsenic toxicity. Arsenic may alter gene expression, possibly by altering DNA methylation. Objectives: To elucidate the associations between arsenic exposure, gene expression, and DNA methylation in peripheral blood, and the modifying effects of arsenic metabolism. Methods: The study participants, women from the Andes, Argentina, were exposed to arsenic via drinking water. Arsenic exposure was assessed as the sum of arsenic metabolites in urine (U-As), using high performance liquid-chromatography hydride-generation inductively-coupled-plasma-mass-spectrometry, and arsenic metabolism efficiency was assessed by the urinary fractions (%) of the individual metabolites. Genome-wide gene expression (N = 80 women) and DNA methylation (N = 93; 80 overlapping with gene expression) in peripheral blood were measured using Illumina DirectHyb HumanHT-12 v4.0 and Infinium Human-Methylation 450K BeadChip, respectively. Results: U-As concentrations, ranging 10–1251 μg/L, was associated with decreased gene expression: 64% of the top 1000 differentially expressed genes were down-regulated with increasing U-As. U-As was also associated with hypermethylation: 87% of the top 1000 CpGs were hypermethylated with increasing U-As. The expression of six genes and six individual CpG sites were significantly associated with increased U-As concentration. Pathway analyses revealed enrichment of genes related to cell death and cancer. The pathways differed somewhat depending on arsenic metabolism efficiency. We found no overlap between arsenic-related gene expression and DNA methylation for individual genes. Conclusions: Increased arsenic exposure was associated with lower gene expression and hypermethylation in peripheral blood, but with no evident overlap. - Highlights: • Women exposed to inorganic arsenic were studied for

  8. Germline heterozygous variants in genes associated with familial hemophagocytic lymphohistiocytosis as a cause of increased bleeding

    Fager Ferrari, Marcus; Leinoe, Eva; Rossing, Maria

    2018-01-01

    Familial hemophagocytic lymphohistiocytosis (FHL) is caused by biallelic variants in genes regulating granule secretion in cytotoxic lymphocytes. In FHL3-5, the affected genes UNC13D, STX11 and STXBP2 have further been shown to regulate the secretion of platelet granules, giving rise to compromised...

  9. TOP1 gene copy numbers are increased in cancers of the bile duct and pancreas

    Grunnet, Mie; Calatayud, Dan; Schultz, Nicolai Aa.

    2015-01-01

    ) poison. Top1 protein, TOP1 gene copy number and mRNA expression, respectively, have been proposed as predictive biomarkers of response to irinotecan in other cancers. Here we investigate the occurrence of TOP1 gene aberrations in cancers of the bile ducts and pancreas. Material and methods. TOP1...

  10. Antisense pre-treatment increases gene therapy efficacy in dystrophic muscles.

    Peccate, Cécile; Mollard, Amédée; Le Hir, Maëva; Julien, Laura; McClorey, Graham; Jarmin, Susan; Le Heron, Anita; Dickson, George; Benkhelifa-Ziyyat, Sofia; Piétri-Rouxel, France; Wood, Matthew J; Voit, Thomas; Lorain, Stéphanie

    2016-08-15

    In preclinical models for Duchenne muscular dystrophy, dystrophin restoration during adeno-associated virus (AAV)-U7-mediated exon-skipping therapy was shown to decrease drastically after six months in treated muscles. This decline in efficacy is strongly correlated with the loss of the therapeutic AAV genomes, probably due to alterations of the dystrophic myofiber membranes. To improve the membrane integrity of the dystrophic myofibers at the time of AAV-U7 injection, mdx muscles were pre-treated with a single dose of the peptide-phosphorodiamidate morpholino (PPMO) antisense oligonucleotides that induced temporary dystrophin expression at the sarcolemma. The PPMO pre-treatment allowed efficient maintenance of AAV genomes in mdx muscles and enhanced the AAV-U7 therapy effect with a ten-fold increase of the protein level after 6 months. PPMO pre-treatment was also beneficial to AAV-mediated gene therapy with transfer of micro-dystrophin cDNA into muscles. Therefore, avoiding vector genome loss after AAV injection by PPMO pre-treatment would allow efficient long-term restoration of dystrophin and the use of lower and thus safer vector doses for Duchenne patients. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput.

    Uwase, Grace; Enrico, Taylor P; Chelimo, David S; Keyser, Benjamin R; Johnson, Russell R

    2018-03-30

    The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (β-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.

  12. Linkage of the VNTR/insulin-gene and type I diabetes mellitus: Increased gene sharing in affected sibling pairs

    Owerbach, D.; Gabbay, K.H. (Baylor College of Medicine, Houston, TX (United States))

    1994-05-01

    Ninety-six multiplex type I diabetic families were typed at the 5' flanking region of the insulin gene by using a PCR assay that better resolves the VNTR into multiple alleles. Affected sibling pairs shared 2, 1, and 0 VNTR alleles - identical by descent - at a frequency of .47, .45, and .08, respectively, a ratio that deviated from the expected 1:2:1 ratio (P<.001). These results confirm linkage of the chromosome 11p15.5 region with type I diabetes mellitus susceptibility. 20 refs., 2 tabs.

  13. High-level inducible Smad4-reexpression in the cervical cancer cell line C4-II is associated with a gene expression profile that predicts a preferential role of Smad4 in extracellular matrix composition

    Klein-Scory, Susanne; Zapatka, Marc; Eilert-Micus, Christina; Hoppe, Sabine; Schwarz, Elisabeth; Schmiegel, Wolff; Hahn, Stephan A; Schwarte-Waldhoff, Irmgard

    2007-01-01

    Smad4 is a tumour suppressor frequently inactivated in pancreatic and colorectal cancers. We have recently reported loss of Smad4 in every fourth carcinoma of the uterine cervix. Smad4 transmits signals from the TGF-β superfamily of cytokines and functions as a versatile transcriptional co-modulator. The prevailing view suggests that the tumour suppressor function of Smad4 primarily resides in its capability to mediate TGF-β growth inhibitory responses. However, accumulating evidence indicates, that the acquisition of TGF-β resistance and loss of Smad4 may be independent events in the carcinogenic process. Through inducible reexpression of Smad4 in cervical cancer cells we wished to shed more light on this issue and to identify target genes implicated in Smad4 dependent tumor suppression. Smad4-deficient human C4-II cervical carcinoma cells were used to establish inducible Smad4 reexpression using the commercial Tet-on™ system (Clontech). The impact of Smad4 reexpression on cell growth was analysed in vitro and in vivo. Transcriptional responses were assessed through profiling on cDNA macroarrays (Clontech) and validated through Northern blotting. Clones were obtained that express Smad4 at widely varying levels from approximately physiological to 50-fold overexpression. Smad4-mediated tumour suppression in vivo was apparent at physiological expression levels as well as in Smad4 overexpressing clones. Smad4 reexpression in a dose-dependent manner was associated with transcriptional induction of the extracellular matrix-associated genes, BigH3, fibronectin and PAI-1, in response to TGF-β. Smad4-dependent regulation of these secreted Smad4 targets is not restricted to cervical carcinoma cells and was confirmed in pancreatic carcinoma cells reexpressing Smad4 after retroviral transduction and in a stable Smad4 knockdown model. On the other hand, the classical cell cycle-associated TGF-β target genes, c-myc, p21 and p15, remained unaltered. Our results show that

  14. Increased prevalence of VNTR III of the insulin gene in women with gestational diabetes mellitus (GDM).

    Litou, Hariklia; Anastasiou, Eleni; Thalassinou, Louminitsa; Sarika, Helen-Leda; Philippou, George; Alevizaki, Maria

    2007-05-01

    The VNTR polymorphism in the promoter region of the insulin gene (INS-VNTR) affects transcription rate and has been associated with insulin resistance and DM2. Gestational diabetes mellitus (GDM) is a multifactorial disorder, where both impaired insulin secretion and action may be involved. The aim of the study was to examine the distribution of the INS-VNTRs in women with GDM and to investigate possible associations with features of beta cell function and glycaemic control in this population. One hundred and sixty-one women with GDM and 111 normal pregnant women (n) were genotyped for INS-VNTR during the 24th-32nd pregnancy week. Glucose and insulin levels were determined during the diagnostic OGTT. The majority of the previous GDM women were also examined at 3-6 months post-partum. VNTR class III/III genotype was significantly more frequent in the GDM group 8.7% versus 2.7%, p=0.02 giving an OR of 3.97 (1.1-14.29). An increased frequency of the VNTR class III allele was found in those GDM women who required insulin for treatment compared to those controlled with diet alone (12.4% versus 4%, pwomen homozygous for the class III allele without reaching statistical significance (p=0.09). The INS-VNTR class III is more frequent in women who develop GDM, and may be associated with decreased ability of the beta cell to meet the increased insulin requirements as reflected by the need for insulin supplementation for adequate glycaemic control.

  15. Prophage Rs551 and Its Repressor Gene orf14 Reduce Virulence and Increase Competitive Fitness of Its Ralstonia solanacearum Carrier Strain UW551.

    Ahmad, Abdelmonim Ali; Stulberg, Michael J; Huang, Qi

    2017-01-01

    We previously characterized a filamentous lysogenic bacteriophage, ϕRs551, isolated directly from the race 3 biovar 2 phylotype IIB sequevar 1 strain UW551 of Ralstonia solanacearum grown under normal culture conditions. The genome of ϕRs551 was identified with 100% identity in the deposited genomes of 11 race 3 biovar 2 phylotype IIB sequevar 1 strains of R. solanacearum , indicating evolutionary and biological importance, and ORF14 of ϕRs551 was annotated as a putative type-2 repressor. In this study, we determined the effect of the prophage and its ORF14 on the virulence and competitive fitness of its carrier strain UW551 by deleting the orf14 gene only (the UW551 orf14 mutant), and nine of the prophage's 14 genes including orf14 and six out of seven structural genes (the UW551 prophage mutant), respectively, from the genome of UW551. The two mutants were increased in extracellular polysaccharide production, twitching motility, expression of targeted virulence and virulence regulatory genes ( pilT, egl, pehC, hrPB, and phcA ), and virulence, suggesting that the virulence of UW551 was negatively regulated by ϕRs551, at least partially through ORF14. Interestingly, we found that the wt ϕRs551-carrying strain UW551 of R. solanacearum significantly outcompeted the wt strain RUN302 which lacks the prophage in tomato plants co-inoculated with the two strains. When each of the two mutant strains was co-inoculated with RUN302, however, the mutants were significantly out-competed by RUN302 for the same colonization site. Our results suggest that ecologically, ϕRs551 may play an important role by regulating the virulence of and offering a competitive fitness advantage to its carrier bacterial strain for persistence of the bacterium in the environment, which in turn prolongs the symbiotic relationship between the phage ϕRs551 and the R. solanacearum strain UW551. Our study is the first toward a better understanding of the co-existence between a lysogenic phage and

  16. Prophage Rs551 and Its Repressor Gene orf14 Reduce Virulence and Increase Competitive Fitness of Its Ralstonia solanacearum Carrier Strain UW551

    Abdelmonim Ali Ahmad

    2017-12-01

    Full Text Available We previously characterized a filamentous lysogenic bacteriophage, ϕRs551, isolated directly from the race 3 biovar 2 phylotype IIB sequevar 1 strain UW551 of Ralstonia solanacearum grown under normal culture conditions. The genome of ϕRs551 was identified with 100% identity in the deposited genomes of 11 race 3 biovar 2 phylotype IIB sequevar 1 strains of R. solanacearum, indicating evolutionary and biological importance, and ORF14 of ϕRs551 was annotated as a putative type-2 repressor. In this study, we determined the effect of the prophage and its ORF14 on the virulence and competitive fitness of its carrier strain UW551 by deleting the orf14 gene only (the UW551 orf14 mutant, and nine of the prophage’s 14 genes including orf14 and six out of seven structural genes (the UW551 prophage mutant, respectively, from the genome of UW551. The two mutants were increased in extracellular polysaccharide production, twitching motility, expression of targeted virulence and virulence regulatory genes (pilT, egl, pehC, hrPB, and phcA, and virulence, suggesting that the virulence of UW551 was negatively regulated by ϕRs551, at least partially through ORF14. Interestingly, we found that the wt ϕRs551-carrying strain UW551 of R. solanacearum significantly outcompeted the wt strain RUN302 which lacks the prophage in tomato plants co-inoculated with the two strains. When each of the two mutant strains was co-inoculated with RUN302, however, the mutants were significantly out-competed by RUN302 for the same colonization site. Our results suggest that ecologically, ϕRs551 may play an important role by regulating the virulence of and offering a competitive fitness advantage to its carrier bacterial strain for persistence of the bacterium in the environment, which in turn prolongs the symbiotic relationship between the phage ϕRs551 and the R. solanacearum strain UW551. Our study is the first toward a better understanding of the co-existence between a

  17. Prophage Rs551 and Its Repressor Gene orf14 Reduce Virulence and Increase Competitive Fitness of Its Ralstonia solanacearum Carrier Strain UW551

    Ahmad, Abdelmonim Ali; Stulberg, Michael J.; Huang, Qi

    2017-01-01

    We previously characterized a filamentous lysogenic bacteriophage, ϕRs551, isolated directly from the race 3 biovar 2 phylotype IIB sequevar 1 strain UW551 of Ralstonia solanacearum grown under normal culture conditions. The genome of ϕRs551 was identified with 100% identity in the deposited genomes of 11 race 3 biovar 2 phylotype IIB sequevar 1 strains of R. solanacearum, indicating evolutionary and biological importance, and ORF14 of ϕRs551 was annotated as a putative type-2 repressor. In this study, we determined the effect of the prophage and its ORF14 on the virulence and competitive fitness of its carrier strain UW551 by deleting the orf14 gene only (the UW551 orf14 mutant), and nine of the prophage’s 14 genes including orf14 and six out of seven structural genes (the UW551 prophage mutant), respectively, from the genome of UW551. The two mutants were increased in extracellular polysaccharide production, twitching motility, expression of targeted virulence and virulence regulatory genes (pilT, egl, pehC, hrPB, and phcA), and virulence, suggesting that the virulence of UW551 was negatively regulated by ϕRs551, at least partially through ORF14. Interestingly, we found that the wt ϕRs551-carrying strain UW551 of R. solanacearum significantly outcompeted the wt strain RUN302 which lacks the prophage in tomato plants co-inoculated with the two strains. When each of the two mutant strains was co-inoculated with RUN302, however, the mutants were significantly out-competed by RUN302 for the same colonization site. Our results suggest that ecologically, ϕRs551 may play an important role by regulating the virulence of and offering a competitive fitness advantage to its carrier bacterial strain for persistence of the bacterium in the environment, which in turn prolongs the symbiotic relationship between the phage ϕRs551 and the R. solanacearum strain UW551. Our study is the first toward a better understanding of the co-existence between a lysogenic phage and

  18. Elevated CO2 increases R gene-dependent resistance of Medicago truncatula against the pea aphid by up-regulating a heat shock gene.

    Sun, Yucheng; Guo, Huijuan; Yuan, Erliang; Ge, Feng

    2018-03-01

    Resistance against pathogens and herbivorous insects in many plant results from the expression of resistance (R) genes. Few reports, however, have considered the effects of elevated CO 2 on R gene-based resistance in plants. The current study determined the responses of two near isogenic Medicago truncatula genotypes (Jester has an R gene and A17 does not) to the pea aphid and elevated CO 2 in open-top chambers in the field. Aphid abundance, mean relative growth rate and feeding efficiency were increased by elevated CO 2 on A17 plants but were reduced on Jester plants. According to proteomic and gene expression data, elevated CO 2 enhanced pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) but decreased the effector-triggered immunity (ETI) in aphid-infested A17 plants. For aphid-infested Jester plants, by contrast, elevated CO 2 enhanced the ETI-related heat shock protein (HSP) 90 and its co-chaperones, the jasmonic acid (JA) signaling pathway, and ubiquitin-mediated proteolysis. In a loss-of-function experiment, silencing of the HSP90 gene in Jester plants impaired the JA signaling pathway and ubiquitin-mediated proteolysis against the aphid under ambient CO 2 , and negated the increased resistance against the aphid under elevated CO 2 . Our results suggest that increases in expression of HSP90 are responsible for the enhanced resistance against the aphid under elevated CO 2 . © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  19. Extensive innate immune gene activation accompanies brain aging, increasing vulnerability to cognitive decline and neurodegeneration: a microarray study

    2012-01-01

    Background This study undertakes a systematic and comprehensive analysis of brain gene expression profiles of immune/inflammation-related genes in aging and Alzheimer’s disease (AD). Methods In a well-powered microarray study of young (20 to 59 years), aged (60 to 99 years), and AD (74 to 95 years) cases, gene responses were assessed in the hippocampus, entorhinal cortex, superior frontal gyrus, and post-central gyrus. Results Several novel concepts emerge. First, immune/inflammation-related genes showed major changes in gene expression over the course of cognitively normal aging, with the extent of gene response far greater in aging than in AD. Of the 759 immune-related probesets interrogated on the microarray, approximately 40% were significantly altered in the SFG, PCG and HC with increasing age, with the majority upregulated (64 to 86%). In contrast, far fewer immune/inflammation genes were significantly changed in the transition to AD (approximately 6% of immune-related probesets), with gene responses primarily restricted to the SFG and HC. Second, relatively few significant changes in immune/inflammation genes were detected in the EC either in aging or AD, although many genes in the EC showed similar trends in responses as in the other brain regions. Third, immune/inflammation genes undergo gender-specific patterns of response in aging and AD, with the most pronounced differences emerging in aging. Finally, there was widespread upregulation of genes reflecting activation of microglia and perivascular macrophages in the aging brain, coupled with a downregulation of select factors (TOLLIP, fractalkine) that when present curtail microglial/macrophage activation. Notably, essentially all pathways of the innate immune system were upregulated in aging, including numerous complement components, genes involved in toll-like receptor signaling and inflammasome signaling, as well as genes coding for immunoglobulin (Fc) receptors and human leukocyte antigens I

  20. Extensive innate immune gene activation accompanies brain aging, increasing vulnerability to cognitive decline and neurodegeneration: a microarray study

    Cribbs David H

    2012-07-01

    Full Text Available Abstract Background This study undertakes a systematic and comprehensive analysis of brain gene expression profiles of immune/inflammation-related genes in aging and Alzheimer’s disease (AD. Methods In a well-powered microarray study of young (20 to 59 years, aged (60 to 99 years, and AD (74 to 95 years cases, gene responses were assessed in the hippocampus, entorhinal cortex, superior frontal gyrus, and post-central gyrus. Results Several novel concepts emerge. First, immune/inflammation-related genes showed major changes in gene expression over the course of cognitively normal aging, with the extent of gene response far greater in aging than in AD. Of the 759 immune-related probesets interrogated on the microarray, approximately 40% were significantly altered in the SFG, PCG and HC with increasing age, with the majority upregulated (64 to 86%. In contrast, far fewer immune/inflammation genes were significantly changed in the transition to AD (approximately 6% of immune-related probesets, with gene responses primarily restricted to the SFG and HC. Second, relatively few significant changes in immune/inflammation genes were detected in the EC either in aging or AD, although many genes in the EC showed similar trends in responses as in the other brain regions. Third, immune/inflammation genes undergo gender-specific patterns of response in aging and AD, with the most pronounced differences emerging in aging. Finally, there was widespread upregulation of genes reflecting activation of microglia and perivascular macrophages in the aging brain, coupled with a downregulation of select factors (TOLLIP, fractalkine that when present curtail microglial/macrophage activation. Notably, essentially all pathways of the innate immune system were upregulated in aging, including numerous complement components, genes involved in toll-like receptor signaling and inflammasome signaling, as well as genes coding for immunoglobulin (Fc receptors and human

  1. Osmotic regulation of expression of two extracellular matrix-binding proteins and a haemolysin of Leptospira interrogans: differential effects on LigA and Sph2 extracellular release.

    Matsunaga, James; Medeiros, Marco A; Sanchez, Yolanda; Werneid, Kristian F; Ko, Albert I

    2007-10-01

    The life cycle of the pathogen Leptospira interrogans involves stages outside and inside the host. Entry of L. interrogans from moist environments into the host is likely to be accompanied by the induction of genes encoding virulence determinants and the concomitant repression of genes encoding products required for survival outside of the host. The expression of the adhesin LigA, the haemolysin Sph2 (Lk73.5) and the outer-membrane lipoprotein LipL36 of pathogenic Leptospira species have been reported to be regulated by mammalian host signals. A previous study demonstrated that raising the osmolarity of the leptospiral growth medium to physiological levels encountered in the host by addition of various salts enhanced the levels of cell-associated LigA and LigB and extracellular LigA. In this study, we systematically examined the effects of osmotic upshift with ionic and non-ionic solutes on expression of the known mammalian host-regulated leptospiral genes. The levels of cell-associated LigA, LigB and Sph2 increased at physiological osmolarity, whereas LipL36 levels decreased, corresponding to changes in specific transcript levels. These changes in expression occurred irrespective of whether sodium chloride or sucrose was used as the solute. The increase of cellular LigA, LigB and Sph2 protein levels occurred within hours of adding sodium chloride. Extracellular Sph2 levels increased when either sodium chloride or sucrose was added to achieve physiological osmolarity. In contrast, enhanced levels of extracellular LigA were observed only with an increase in ionic strength. These results indicate that the mechanisms for release of LigA and Sph2 differ during host infection. Thus, osmolarity not only affects leptospiral gene expression by affecting transcript levels of putative virulence determinants but also affects the release of such proteins into the surroundings.

  2. Functional transferred DNA within extracellular vesicles

    Cai, Jin; Wu, Gengze; Jose, Pedro A.; Zeng, Chunyu

    2016-01-01

    Extracellular vesicles (EVs) are small membrane vesicles including exosomes and shedding vesicles that mediated a cell-to-cell communication. EVs are released from almost all cell types under both physiological and pathological conditions and incorporate nuclear and cytoplasmic molecules for intercellular delivery. Besides protein, mRNA, and microRNA of these molecules, as recent studies show, specific DNA are prominently packaged into EVs. It appears likely that some of exosomes or shedding vesicles, bearing nuclear molecules are released upon bubble-like blebs. Specific interaction of EVs with susceptible recipients performs the uptake of EVs into the target cells, discharging their cargo including nuclear and cytoplasmic macromolecules into the cytosol. These findings expand the nucleic acid content of EVs to include increased levels of specific DNA. Thus, EVs contain a repertoire of genetic information available for horizontal gene transfer and potential use as blood biomarkers for cancer and atherosclerosis. In this review, the focus is on the characteristics, biological functions, and roles in diseases of DNA within EVs. - Highlights: • This review is focused on the DNA within EVs including its characteristics, biological functions, and roles in diseases. • It is clear that DNA within EVs might have important physiological and pathological roles in various diseases. • Knowledge in this area may provides us alternative methods for disease diagnosis or therapy in the future.

  3. Functional transferred DNA within extracellular vesicles

    Cai, Jin [Department of Cardiology, Daping Hospital, The Third Military Medical University, Chongqing 400042 (China); Department of Neurology, Jinling Hospital, Nanjing University School of Medicine, Jiangsu Province (China); Wu, Gengze [Department of Cardiology, Daping Hospital, The Third Military Medical University, Chongqing 400042 (China); Jose, Pedro A. [Division of Nephrology, Department of Medicine and Physiology, University of Maryland, School of Medicine, Baltimore, MD 21201 (United States); Zeng, Chunyu, E-mail: Chunyuzeng01@163.com [Department of Cardiology, Daping Hospital, The Third Military Medical University, Chongqing 400042 (China)

    2016-11-15

    Extracellular vesicles (EVs) are small membrane vesicles including exosomes and shedding vesicles that mediated a cell-to-cell communication. EVs are released from almost all cell types under both physiological and pathological conditions and incorporate nuclear and cytoplasmic molecules for intercellular delivery. Besides protein, mRNA, and microRNA of these molecules, as recent studies show, specific DNA are prominently packaged into EVs. It appears likely that some of exosomes or shedding vesicles, bearing nuclear molecules are released upon bubble-like blebs. Specific interaction of EVs with susceptible recipients performs the uptake of EVs into the target cells, discharging their cargo including nuclear and cytoplasmic macromolecules into the cytosol. These findings expand the nucleic acid content of EVs to include increased levels of specific DNA. Thus, EVs contain a repertoire of genetic information available for horizontal gene transfer and potential use as blood biomarkers for cancer and atherosclerosis. In this review, the focus is on the characteristics, biological functions, and roles in diseases of DNA within EVs. - Highlights: • This review is focused on the DNA within EVs including its characteristics, biological functions, and roles in diseases. • It is clear that DNA within EVs might have important physiological and pathological roles in various diseases. • Knowledge in this area may provides us alternative methods for disease diagnosis or therapy in the future.

  4. Extracellular vesicles: fundamentals and clinical relevance

    Wael Nassar

    2015-01-01

    Full Text Available All types of cells of eukaryotic organisms produce and release small nanovesicles into their extracellular environment. Early studies have described these vesicles as ′garbage bags′ only to remove obsolete cellular molecules. Valadi and colleagues, in 2007, were the first to discover the capability of circulating extracellular vesicles (EVs to horizontally transfer functioning gene information between cells. These extracellular vesicles express components responsible for angiogenesis promotion, stromal remodeling, chemoresistance, genetic exchange, and signaling pathway activation through growth factor/receptor transfer. EVs represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, signaling proteins, and RNAs. They contribute to physiology and pathology, and they have a myriad of potential clinical applications in health and disease. Moreover, vesicles can pass the blood-brain barrier and may perhaps even be considered as naturally occurring liposomes. These cell-derived EVs not only represent a central mediator of the disease microenvironment, but their presence in the peripheral circulation may serve as a surrogate for disease biopsies, enabling real-time diagnosis and disease monitoring. In this review, we′ll be addressing the characteristics of different types of extracellular EVs, as well as their clinical relevance and potential as diagnostic markers, and also define therapeutic options.

  5. Extracellular Gd-CA

    Thomsen, Henrik S; Marckmann, Peter

    2008-01-01

    Until recently it was believed that extracellular gadolinium-based contrast agents were safe for both the kidneys and all other organs within the dose range up to 0.3 mmol/kg body weight. However, in 2006, it was demonstrated that some gadolinium-based contrast agents may trig the development...... gadolinium-based agent (3-7% versus 0-1% per injection) in patients with reduced renal function. Prevalence after exposure to two gadodiamide injections is as high as 36% in patients with chronic kidney disease (CKD) stage 5. No report of NSF after the most stable agents has been reported in the peer...

  6. Knock down of the myostatin gene by RNA interference increased body weight in chicken.

    Bhattacharya, T K; Shukla, R; Chatterjee, R N; Dushyanth, K

    2017-01-10

    Myostatin is a negative regulator of muscular growth in poultry and other animals. Of several approaches, knocking down the negative regulator is an important aspect to augment muscular growth in chicken. Knock down of myostatin gene has been performed by shRNA acting against the expression of gene in animals. Two methods of knock down of gene in chicken such as embryo manipulation and sperm mediated method have been performed. The hatching percentage in embryo manipulation and sperm mediated method of knock down was 58.0 and 41.5%, respectively. The shRNA in knock down chicken enhanced body weight at 6 weeks by 26.9%. The dressing percentage and serum biochemical parameters such as SGPT and alkaline phosphatase differed significantly (Pknock down and control birds. It is concluded that knocking down the myostatin gene successfully augmented growth in chicken. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. The interaction of BDNF and NTRK2 gene increases the susceptibility of paranoid schizophrenia.

    Zheng Lin

    Full Text Available The association between BDNF gene functional Val66Met polymorphism rs6265 and the schizophrenia is far from being consistent. In addition to the heterogeneous in schizophrenia per se leading to the inconsistent results, the interaction among multi-genes is probably playing the main role in the pathogenesis of schizophrenia, but not a single gene. Neurotrophic tyrosine kinase receptor 2 (NTRK2 is the high-affinity receptor of BDNF, and was reported to be associated with mood disorders, though no literature reported the association with schizophrenia. Thus, in the present study, total 402 patients with paranoid schizophrenia (the most common subtype of schizophrenia and matched 406 healthy controls were recruited to investigate the role of rs6265 in BDNF, three polymorphisms in NTRK2 gene (rs1387923, rs2769605 and rs1565445 and their interaction in the susceptibility to paranoid schizophrenia in a Chinese Han population. We did not observe significant differences in allele and genotype frequencies between patients and healthy controls for all four polymorphisms separately. The haplotype analysis also showed no association between haplotype of NTRK2 genes (rs1387923, rs2769605, and rs1565445 and paranoid schizophrenia. However, we found the association between the interaction of BDNF and NTRK2 with paranoid schizophrenia by using the MDR method followed by conventional statistical analysis. The best gene-gene interaction model was a three-locus model (BDNF rs6265, NTRK2 rs1387923 and NTRK2 rs2769605, in which one low-risk and three high-risk four-locus genotype combinations were identified. Our findings implied that single polymorphism of rs6265 rs1387923, rs2769605, and rs1565445 in BDNF and NTRK2 were not associated with the development of paranoid schizophrenia in a Han population, however, the interaction of BDNF and NTRK2 genes polymorphisms (BDNF-rs6265, NTRK2-rs1387923 and NTRK2-rs2769605 may be involved in the susceptibility to paranoid

  8. The interaction of BDNF and NTRK2 gene increases the susceptibility of paranoid schizophrenia.

    Lin, Zheng; Su, Yousong; Zhang, Chengfang; Xing, Mengjuan; Ding, Wenhua; Liao, Liwei; Guan, Yangtai; Li, Zezhi; Cui, Donghong

    2013-01-01

    The association between BDNF gene functional Val66Met polymorphism rs6265 and the schizophrenia is far from being consistent. In addition to the heterogeneous in schizophrenia per se leading to the inconsistent results, the interaction among multi-genes is probably playing the main role in the pathogenesis of schizophrenia, but not a single gene. Neurotrophic tyrosine kinase receptor 2 (NTRK2) is the high-affinity receptor of BDNF, and was reported to be associated with mood disorders, though no literature reported the association with schizophrenia. Thus, in the present study, total 402 patients with paranoid schizophrenia (the most common subtype of schizophrenia) and matched 406 healthy controls were recruited to investigate the role of rs6265 in BDNF, three polymorphisms in NTRK2 gene (rs1387923, rs2769605 and rs1565445) and their interaction in the susceptibility to paranoid schizophrenia in a Chinese Han population. We did not observe significant differences in allele and genotype frequencies between patients and healthy controls for all four polymorphisms separately. The haplotype analysis also showed no association between haplotype of NTRK2 genes (rs1387923, rs2769605, and rs1565445) and paranoid schizophrenia. However, we found the association between the interaction of BDNF and NTRK2 with paranoid schizophrenia by using the MDR method followed by conventional statistical analysis. The best gene-gene interaction model was a three-locus model (BDNF rs6265, NTRK2 rs1387923 and NTRK2 rs2769605), in which one low-risk and three high-risk four-locus genotype combinations were identified. Our findings implied that single polymorphism of rs6265 rs1387923, rs2769605, and rs1565445 in BDNF and NTRK2 were not associated with the development of paranoid schizophrenia in a Han population, however, the interaction of BDNF and NTRK2 genes polymorphisms (BDNF-rs6265, NTRK2-rs1387923 and NTRK2-rs2769605) may be involved in the susceptibility to paranoid schizophrenia.

  9. Intracerebroventricular Injection of Lipopolysaccharide Increases Gene Expression of Connexin32 Gap Junction in Rat Hippocampus

    Mohammad Abbasian

    2013-11-01

    Full Text Available Introduction: Gap junctions are intercellular membrane channels that provide direct cytoplasmic continuity between adjacent cells. This communication can be affected by changes in expression of gap junctional subunits called Connexins (Cx. Changes in the expression and function of connexins are associated with number of brain neurodegenerative diseases. Neuroinflammation is a hallmark of various central nervous system (CNS diseases, like multiple sclerosis, Alzheimer's disease and epilepsy. Neuroinflammation causes change in Connexins expression. Hippocampus, one of the main brain regions with a wide network of Gap junctions between different neural cell types, has particular vulnerability to damage and consequent inflammation. Cx32 – among Connexins– is expressed in hippocampal Olygodandrocytes and some neural subpopulations. Although multiple lines of evidence indicate that there is an association between neuroinflammation and the expression of connexin, the direct effect of neuroinflammation on the expression of connexins has not been well studied. In the present study, the effect of neuroinflammation induced by the Lipopolysaccharide (LPS on Cx32 gene and protein expressions in rat hippocampus is evaluated. Methods: LPS (2.5μg/rat was infused into the rat cerebral ventricles for 14 days. Cx32 mRNA and protein levels were measured by Real Time PCR and Western Blot after 1st, 7th and 14th injection of LPS in the hippocampus. Results: Significant increase in Cx32 mRNA expression was observed after 7th injection of LPS (P<0.001. However, no significant change was observed in Cx32 protein level. Conclusion: LPS seems to modify Cx32 GJ communication in the hippocampus at transcription level but not at translation or post-translation level. In order to have a full view concerning modification of Cx32 GJ communication, effect of LPS on Cx32 channel gating should also be determined.

  10. Genes Whose Gain or Loss-Of-Function Increases Skeletal Muscle Mass in Mice: A Systematic Literature Review

    Sander A. J. Verbrugge

    2018-05-01

    Full Text Available Skeletal muscle mass differs greatly in mice and humans and this is partially inherited. To identify muscle hypertrophy candidate genes we conducted a systematic review to identify genes whose experimental loss or gain-of-function results in significant skeletal muscle hypertrophy in mice. We found 47 genes that meet our search criteria and cause muscle hypertrophy after gene manipulation. They are from high to small effect size: Ski, Fst, Acvr2b, Akt1, Mstn, Klf10, Rheb, Igf1, Pappa, Ppard, Ikbkb, Fstl3, Atgr1a, Ucn3, Mcu, Junb, Ncor1, Gprasp1, Grb10, Mmp9, Dgkz, Ppargc1a (specifically the Ppargc1a4 isoform, Smad4, Ltbp4, Bmpr1a, Crtc2, Xiap, Dgat1, Thra, Adrb2, Asb15, Cast, Eif2b5, Bdkrb2, Tpt1, Nr3c1, Nr4a1, Gnas, Pld1, Crym, Camkk1, Yap1, Inhba, Tp53inp2, Inhbb, Nol3, Esr1. Knock out, knock down, overexpression or a higher activity of these genes causes overall muscle hypertrophy as measured by an increased muscle weight or cross sectional area. The mean effect sizes range from 5 to 345% depending on the manipulated gene as well as the muscle size variable and muscle investigated. Bioinformatical analyses reveal that Asb15, Klf10, Tpt1 are most highly expressed hypertrophy genes in human skeletal muscle when compared to other tissues. Many of the muscle hypertrophy-regulating genes are involved in transcription and ubiquitination. Especially genes belonging to three signaling pathways are able to induce hypertrophy: (a Igf1-Akt-mTOR pathway, (b myostatin-Smad signaling, and (c the angiotensin-bradykinin signaling pathway. The expression of several muscle hypertrophy-inducing genes and the phosphorylation of their protein products changes after human resistance and high intensity exercise, in maximally stimulated mouse muscle or in overloaded mouse plantaris.

  11. Cytokinins and Expression of SWEET, SUT, CWINV and AAP Genes Increase as Pea Seeds Germinate

    Paula E. Jameson

    2016-12-01

    Full Text Available Transporter genes and cytokinins are key targets for crop improvement. These genes are active during the development of the seed and its establishment as a strong sink. However, during germination, the seed transitions to being a source for the developing root and shoot. To determine if the sucrose transporter (SUT, amino acid permease (AAP, Sugar Will Eventually be Exported Transporter (SWEET, cell wall invertase (CWINV, cytokinin biosynthesis (IPT, activation (LOG and degradation (CKX gene family members are involved in both the sink and source activities of seeds, we used RT-qPCR to determine the expression of multiple gene family members, and LC-MS/MS to ascertain endogenous cytokinin levels in germinating Pisum sativum L. We show that genes that are actively expressed when the seed is a strong sink during its development, are also expressed when the seed is in the reverse role of being an active source during germination and early seedling growth. Cytokinins were detected in the imbibing seeds and were actively biosynthesised during germination. We conclude that, when the above gene family members are targeted for seed yield improvement, a downstream effect on subsequent seed germination or seedling vigour must be taken into consideration.

  12. Impact of Maspin Polymorphism rs2289520 G/C and Its Interaction with Gene to Gene, Alcohol Consumption Increase Susceptibility to Oral Cancer Occurrence.

    Yang, Po-Yu; Miao, Nae-Fang; Lin, Chiao-Wen; Chou, Ying-Erh; Yang, Shun-Fa; Huang, Hui-Chuan; Chang, Hsiu-Ju; Tsai, Hsiu-Ting

    2016-01-01

    The purpose of this study was to identify gene polymorphisms of mammary serine protease inhibitor (Maspin) specific to patients with oral cancer susceptibility and clinicopathological status. Three single-nucleotide polymorphisms (SNPs) of the Maspin gene from 741 patients with oral cancer and 601 non-cancer controls were analyzed by real-time PCR. The participants with G/G homozygotes or with G/C heterozygotes of Maspin rs2289520 polymorphism had a 2.07-fold (p = 0.01) and a 2.01-fold (p = 0.02) risk of developing oral cancer compared to those with C/C homozygotes. Moreover, gene-gene interaction increased the risk of oral cancer susceptibility among subjects expose to oral cancer related risk factors, including areca, alcohol, and tobacco consumption. G allele of Maspin rs2289520 polymorphism may be a factor that increases the susceptibility to oral cancer. The interactions of gene to oral cancer-related environmental risk factors have a synergetic effect that can further enhance oral cancer development.

  13. Transgenic Suppression of AGAMOUS Genes in Apple Reduces Fertility and Increases Floral Attractiveness

    Klocko, Amy L.; Borejsza-Wysocka, Ewa; Brunner, Amy M.; Shevchenko, Olga; Aldwinckle, Herb; Strauss, Steven H.

    2016-01-01

    We investigated the ability of RNA interference (RNAi) directed against two co-orthologs of AGAMOUS (AG) from Malus domestica (domestic apple, MdAG) to reduce the risks of invasiveness and provide genetic containment of transgenes, while also promoting the attractiveness of flowers for ornamental usage. Suppression of two MdAG-like genes, MdMADS15 and MdMADS22, led to the production of trees with highly showy, polypetalous flowers. These “double-flowers” had strongly reduced expression of both MdAG-like genes. Members of the two other clades within in the MdAG subfamily showed mild to moderate differences in gene expression, or were unchanged, with the level of suppression approximately proportional to the level of sequence identity between the gene analyzed and the RNAi fragment. The double-flowers also exhibited reduced male and female fertility, had few viable pollen grains, a decreased number of stigmas, and produced few viable seeds after cross-pollination. Despite these floral alterations, RNAi-AG trees with double-flowers set full-sized fruit. Suppression or mutation of apple AG-like genes appears to be a promising method for combining genetic containment with improved floral attractiveness. PMID:27500731

  14. Acute and repeated ECS treatment increases CRF, POMC and PENK gene expression in selected regions of the rat hypothalamus.

    Garcia-Garcia, L; Llewellyn-Jones, V; Fernandez Fernandez, I; Fuentes, J A; Manzanares, J

    1998-01-05

    The purpose of this study was to investigate the effects of acute and repeated electroconvulsive shock (ECS) on corticotropin releasing factor (CRF), proopiomelanocortin (POMC) and proenkephalin (PENK) gene expression in selected regions of the brain and pituitary of the rat. Acute ECS increased CRF gene expression in the paraventricular nucleus (PVN) by 20%, an effect that was further enhanced to 38% when rats received repeated ECS treatment. Acute and repeated ECS increased POMC gene expression in the arcuate nucleus (ARC) by 49-59% but failed to alter these mRNA levels in the anterior lobe (AL) of the pituitary gland. PENK gene expression was increased by 35% in the nucleus accumbens (NA) and by 180% the ventromedial nucleus (VMN) after acute or repeated ECS treatment but no significant changes were found in the PVN or striatum (ST). Taken together, these results indicate a differential CRF and opioid gene expression regulation after acute or repeated ECS treatment that may be relevant to their therapeutic or side effects in depression.

  15. Gene expression profiling of preovulatory follicle in the buffalo cow: effects of increased IGF-I concentration on periovulatory events.

    Jyotsna U Rao

    Full Text Available The preovulatory follicle in response to gonadotropin surge undergoes dramatic biochemical, and morphological changes orchestrated by expression changes in hundreds of genes. Employing well characterized bovine preovulatory follicle model, granulosa cells (GCs and follicle wall were collected from the preovulatory follicle before, 1, 10 and 22 h post peak LH surge. Microarray analysis performed on GCs revealed that 450 and 111 genes were differentially expressed at 1 and 22 h post peak LH surge, respectively. For validation, qPCR and immunocytochemistry analyses were carried out for some of the differentially expressed genes. Expression analysis of many of these genes showed distinct expression patterns in GCs and the follicle wall. To study molecular functions and genetic networks, microarray data was analyzed using Ingenuity Pathway Analysis which revealed majority of the differentially expressed genes to cluster within processes like steroidogenesis, cell survival and cell differentiation. In the ovarian follicle, IGF-I is established to be an important regulator of the above mentioned molecular functions. Thus, further experiments were conducted to verify the effects of increased intrafollicular IGF-I levels on the expression of genes associated with the above mentioned processes. For this purpose, buffalo cows were administered with exogenous bGH to transiently increase circulating and intrafollicular concentrations of IGF-I. The results indicated that increased intrafollicular concentrations of IGF-I caused changes in expression of genes associated with steroidogenesis (StAR, SRF and apoptosis (BCL-2, FKHR, PAWR. These results taken together suggest that onset of gonadotropin surge triggers activation of various biological pathways and that the effects of growth factors and peptides on gonadotropin actions could be examined during preovulatory follicle development.

  16. Increased production of free fatty acids in Aspergillus oryzae by disruption of a predicted acyl-CoA synthetase gene.

    Tamano, Koichi; Bruno, Kenneth S; Koike, Hideaki; Ishii, Tomoko; Miura, Ai; Umemura, Myco; Culley, David E; Baker, Scott E; Machida, Masayuki

    2015-04-01

    Fatty acids are attractive molecules as source materials for the production of biodiesel fuel. Previously, we attained a 2.4-fold increase in fatty acid production by increasing the expression of fatty acid synthesis-related genes in Aspergillus oryzae. In this study, we achieved an additional increase in the production of fatty acids by disrupting a predicted acyl-CoA synthetase gene in A. oryzae. The A. oryzae genome is predicted to encode six acyl-CoA synthetase genes and disruption of AO090011000642, one of the six genes, resulted in a 9.2-fold higher accumulation (corresponding to an increased production of 0.23 mmol/g dry cell weight) of intracellular fatty acid in comparison to the wild-type strain. Furthermore, by introducing a niaD marker from Aspergillus nidulans to the disruptant, as well as changing the concentration of nitrogen in the culture medium from 10 to 350 mM, fatty acid productivity reached 0.54 mmol/g dry cell weight. Analysis of the relative composition of the major intracellular free fatty acids caused by disruption of AO090011000642 in comparison to the wild-type strain showed an increase in stearic acid (7 to 26 %), decrease in linoleic acid (50 to 27 %), and no significant changes in palmitic or oleic acid (each around 20-25 %).

  17. Maternal low protein diet and postnatal high fat diet increases adipose imprinted gene expression

    Maternal and postnatal diet can alter Igf2 gene expression and DNA methylation. To test whether maternal low protein and postnatal high fat (HF) diet result in alteration in Igf2 expression and obesity, we fed obese-prone Sprague-Dawley rats 8% (LP) or 20% (NP) protein for 3 wk prior to breeding and...

  18. Two Alzheimer’s disease risk genes increase entorhinal cortex volume in young adults

    Amanda Marie Dibattista

    2014-10-01

    Full Text Available Alzheimer’s disease (AD risk genes alter brain structure and function decades before disease onset. Apolipoprotein E (APOE is the strongest known genetic risk factor for Alzheimer’s disease, and a related gene, apolipoprotein J (APOJ, also affects disease risk. However, the extent to which these genes affect brain structure in young adults remains unclear. Here, we report that AD risk alleles of these two genes, APOE-ε4 and APOJ-C, cumulatively alter brain volume in young adults. Using voxel-based morphometry in 57 individuals, we examined the entorhinal cortex, one of the earliest brain regions affected in AD pathogenesis. APOE-ε4 carriers exhibited higher right entorhinal cortex volume compared to non-carriers. Interestingly, APOJ-C risk genotype was associated with higher bilateral entorhinal cortex volume in non-APOE-ε4 carriers. To determine the combined disease risk of APOE and APOJ status per subject, we used cumulative odds ratios as regressors for volumetric measurements. Higher disease risk corresponded to greater right entorhinal cortex volume. These results suggest that, years before disease onset, two key AD genetic risk factors may exert influence on the structure of a brain region where AD pathogenesis takes root.

  19. Two Alzheimer’s disease risk genes increase entorhinal cortex volume in young adults

    DiBattista, Amanda Marie; Stevens, Benson W.; Rebeck, G. William; Green, Adam E.

    2014-01-01

    Alzheimer’s disease (AD) risk genes alter brain structure and function decades before disease onset. Apolipoprotein E (APOE) is the strongest known genetic risk factor for AD, and a related gene, apolipoprotein J (APOJ), also affects disease risk. However, the extent to which these genes affect brain structure in young adults remains unclear. Here, we report that AD risk alleles of these two genes, APOE-ε4 and APOJ-C, cumulatively alter brain volume in young adults. Using voxel-based morphometry (VBM) in 57 individuals, we examined the entorhinal cortex, one of the earliest brain regions affected in AD pathogenesis. Apolipoprotein E-ε4 carriers exhibited higher right entorhinal cortex volume compared to non-carriers. Interestingly, APOJ-C risk genotype was associated with higher bilateral entorhinal cortex volume in non-APOE-ε4 carriers. To determine the combined disease risk of APOE and APOJ status per subject, we used cumulative odds ratios as regressors for volumetric measurements. Higher disease risk corresponded to greater right entorhinal cortex volume. These results suggest that, years before disease onset, two key AD genetic risk factors may exert influence on the structure of a brain region where AD pathogenesis takes root. PMID:25339884

  20. Disruption of the GH Receptor Gene in Adult Mice Increases Maximal Lifespan in Females

    Junnila, Riia K.; Duran-Ortiz, Silvana; Suer, Ozan

    2016-01-01

    GH and IGF-1 are important for a variety of physiological processes including growth, development, and aging. Mice with reduced levels of GH and IGF-1 have been shown to live longer than wild-type controls. Our laboratory has previously found that mice with a GH receptor gene knockout (GHRKO) fro...

  1. Transcription factor 7-like 2 gene links increased in vivo insulin synthesis to type 2 diabetes

    S. Jainandunsing (Sjaam); Koole, H.R. (H. Rita); van Miert, J.N.I. (Joram N.I.); T. Rietveld (Trinet); J.L.D. Wattimena (Josias); E.J.G. Sijbrands (Eric); F.W.M. de Rooij (Felix)

    2018-01-01

    textabstractTranscription factor 7-like 2 (TCF7L2) is the main susceptibility gene for type 2 diabetes, primarily through impairing the insulin secretion by pancreatic β cells. However, the exact in vivo mechanisms remain poorly understood. We performed a family study and determined if the T risk

  2. A common haplotype in the G-protein-coupled receptor gene GPR74 is associated with leanness and increased lipolysis

    Dahlman, Ingrid; Dicker, Andrea; Jiao, Hong

    2007-01-01

    0.36; P=.036) among those selected for obese or lean phenotypes. The ATAG haplotype was associated with increased adipocyte lipid mobilization (lipolysis) in vivo and in vitro. In human fat cells, GPR74 receptor stimulation and inhibition caused a significant and marked decrease and increase......, respectively, of lipolysis, which could be linked to catecholamine stimulation of adipocytes through beta -adrenergic receptors. These findings suggest that a common haplotype in the GPR74 gene protects against obesity, which, at least in part, is caused by a relief of inhibition of lipid mobilization from......The G-protein-coupled receptor GPR74 is a novel candidate gene for body weight regulation. In humans, it is predominantly expressed in brain, heart, and adipose tissue. We report a haplotype in the GPR74 gene, ATAG, with allele frequency ~4% in Scandinavian cohorts, which was associated...

  3. The Interaction of TXNIP and AFq1 Genes Increases the Susceptibility of Schizophrenia.

    Su, Yousong; Ding, Wenhua; Xing, Mengjuan; Qi, Dake; Li, Zezhi; Cui, Donghong

    2017-08-01

    Although previous studies showed the reduced risk of cancer in patients with schizophrenia, whether patients with schizophrenia possess genetic factors that also contribute to tumor suppressor is still unknown. In the present study, based on our previous microarray data, we focused on the tumor suppressor genes TXNIP and AF1q, which differentially expressed in patients with schizophrenia. A total of 413 patients and 578 healthy controls were recruited. We found no significant differences in genotype, allele, or haplotype frequencies at the selected five single nucleotide polymorphisms (SNPs) (rs2236566 and rs7211 in TXNIP gene; rs10749659, rs2140709, and rs3738481 in AF1q gene) between patients with schizophrenia and controls. However, we found the association between the interaction of TXNIP and AF1q with schizophrenia by using the MDR method followed by traditional statistical analysis. The best gene-gene interaction model identified was a three-locus model TXNIP (rs2236566, rs7211)-AF1q (rs2140709). After traditional statistical analysis, we found the high-risk genotype combination was rs2236566 (GG)-rs7211(CC)-rs2140709(CC) (OR = 1.35 [1.03-1.76]). The low-risk genotype combination was rs2236566 (GT)-rs7211(CC)-rs2140709(CC) (OR = 0.67 [0.49-0.91]). Our finding suggested statistically significant role of interaction of TXNIP and AF1q polymorphisms (TXNIP-rs2236566, TXNIP-rs7211, and AF1q-rs2769605) in schizophrenia susceptibility.

  4. Identification of adaptive mutations in the influenza A virus non-structural 1 gene that increase cytoplasmic localization and differentially regulate host gene expression.

    Nicole Forbes

    Full Text Available The NS1 protein of influenza A virus (IAV is a multifunctional virulence factor. We have previously characterized gain-of-function mutations in the NS1 protein arising from the experimental adaptation of the human isolate A/Hong Kong/1/1968(H3N2 (HK to the mouse. The majority of these mouse adapted NS1 mutations were demonstrated to increase virulence, viral fitness, and interferon antagonism, but differ in binding to the post-transcriptional processing factor cleavage and polyadenylation specificity factor 30 (CPSF30. Because nuclear trafficking is a major genetic determinant of influenza virus host adaptation, we assessed subcellular localization and host gene expression of NS1 adaptive mutations. Recombinant HK viruses with adaptive mutations in the NS1 gene were assessed for NS1 protein subcellular localization in mouse and human cells using confocal microscopy and cellular fractionation. In human cells the HK wild-type (HK-wt virus NS1 protein partitioned equivalently between the cytoplasm and nucleus but was defective in cytoplasmic localization in mouse cells. Several adaptive mutations increased the proportion of NS1 in the cytoplasm of mouse cells with the greatest effects for mutations M106I and D125G. The host gene expression profile of the adaptive mutants was determined by microarray analysis of infected mouse cells to show either high or low extents of host-gene regulation (HGR or LGR phenotypes. While host genes were predominantly down regulated for the HGR group of mutants (D2N, V23A, F103L, M106I+L98S, L98S, M106V, and M106V+M124I, the LGR phenotype mutants (D125G, M106I, V180A, V226I, and R227K were characterized by a predominant up regulation of host genes. CPSF30 binding affinity of NS1 mutants did not predict effects on host gene expression. To our knowledge this is the first report of roles of adaptive NS1 mutations that impact intracellular localization and regulation of host gene expression.

  5. Sulfamethoxazole and COD increase abundance of sulfonamide resistance genes and change bacterial community structures within sequencing batch reactors.

    Guo, Xueping; Pang, Weihai; Dou, Chunling; Yin, Daqiang

    2017-05-01

    The abundant microbial community in biological treatment processes in wastewater treatment plants (WWTPs) may potentially enhance the horizontal gene transfer of antibiotic resistance genes with the presence of antibiotics. A lab-scale sequencing batch reactor was designed to investigate response of sulfonamide resistance genes (sulI, sulII) and bacterial communities to various concentrations of sulfamethoxazole (SMX) and chemical oxygen demand (COD) of wastewater. The SMX concentrations (0.001 mg/L, 0.1 mg/L and 10 mg/L) decreased with treatment time and higher SMX level was more difficult to remove. The presence of SMX also significantly reduced the removal efficiency of ammonia nitrogen, affecting the normal function of WWTPs. All three concentrations of SMX raised both sulI and sulII genes with higher concentrations exhibiting greater increases. The abundance of sul genes was positive correlated with treatment time and followed the second-order reaction kinetic model. Interestingly, these two genes have rather similar activity. SulI and sulII gene abundance also performed similar response to COD. Simpson index and Shannon-Weiner index did not show changes in the microbial community diversity. However, the 16S rRNA gene cloning and sequencing results showed the bacterial community structures varied during different stages. The results demonstrated that influent antibiotics into WWTPs may facilitate selection of ARGs and affect the wastewater conventional treatment as well as the bacteria community structures. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Interaction between CRHR1 and BDNF genes increases the risk of recurrent major depressive disorder in Chinese population.

    Zheman Xiao

    Full Text Available BACKGROUND: An important etiological hypothesis about depression is stress has neurotoxic effects that damage the hippocampal cells. Corticotropin-releasing hormone (CRH regulates brain-derived neurotrophic factor (BDNF expression through influencing cAMP and Ca2+ signaling pathways during the course. The aim of this study is to examine the single and combined effects of CRH receptor 1 (CRHR1 and BDNF genes in recurrent major depressive disorder (MDD. METHODOLOGY/PRINCIPAL FINDING: The sample consists of 181 patients with recurrent MDD and 186 healthy controls. Whether genetic variations interaction between CRHR1 and BDNF genes might be associated with increased susceptibility to recurrent MDD was studied by using a gene-based association analysis of single-nucleotide polymorphisms (SNPs. CRHR1 gene (rs1876828, rs242939 and rs242941 and BDNF gene (rs6265 were identified in the samples of patients diagnosed with recurrent MDD and matched controls. Allelic association between CRHR1 rs242939 and recurrent MDD was found in our sample (allelic: p = 0.018, genotypic: p = 0.022 with an Odds Ratio 0.454 (95% CI 0.266-0.775. A global test of these four haplotypes showed a significant difference between recurrent MDD group and control group (chi-2 = 13.117, df = 3, P = 0.016. Furthermore, BDNF and CRHR1 interactions were found in the significant 2-locus, gene-gene interaction models (p = 0.05 using a generalized multifactor dimensionality reduction (GMDR method. CONCLUSION: Our results suggest that an interaction between CRHR1 and BDNF genes constitutes susceptibility to recurrent MDD.

  7. Meta-analysis of Gene Expression in the Mouse Liver Reveals Biomarkers Associated with Inflammation Increased Early During Aging

    Aging is associated with a predictable loss of cellular homeostasis, a decline in physiological function and an increase in various diseases. We hypothesized that similar age-related gene expression profiles would be observed in mice across independent studies. Employing a metaan...

  8. Insulin increases transcription of rat gene 33 through cis-acting elements in 5[prime]-flanking DNA

    Cadilla, C.; Isham, K.R.; Lee, K.L.; Ch' ang, L.Y.; Kenney, F.T. (Oak Ridge National Lab., TN (United States)); Johnson, A.C. (National Cancer Institute, Bethesda, MD (United States). Lab. of Molecular Biology)

    1992-01-01

    Gene 33 is a multihormonally-regulated rat gene whose transcription is rapidly and markedly enhanced by insulin in liver and cultured hepatoma cells. To examine the mechanism by which insulin regulates transcription, the authors have constructed chimeric plasmids in which expression of the bacterial cat gene, encoding chloramphenicol acetyltransferase (CAT), is governed by gene 33 promoter elements and contiguous sequence in DNA flanking the transcription start point (tsp). When transfected into H4IIE hepatoma cells, these constructs gave rise to stably transformed cell lines producing the bacterial CAT enzyme. This expression was increased by insulin treatment in a fashion resembling the effect of this hormone on transcription of the native gene. In vitro transcription assays in nuclear extracts also revealed increased transcription of the chimeric plasmids when the extracts were prepared from insulin-treated rat hepatoma cells. The results demonstrate that induction by insulin is mediated by cis-acting nucleotide sequences located between bp [minus]480 to +27 relative to the tsp.

  9. Differential expression patterns of housekeeping genes increase diagnostic and prognostic value in lung cancer

    Yu-Chun Chang

    2018-05-01

    Full Text Available Background Using DNA microarrays, we previously identified 451 genes expressed in 19 different human tissues. Although ubiquitously expressed, the variable expression patterns of these “housekeeping genes” (HKGs could separate one normal human tissue type from another. Current focus on identifying “specific disease markers” is problematic as single gene expression in a given sample represents the specific cellular states of the sample at the time of collection. In this study, we examine the diagnostic and prognostic potential of the variable expressions of HKGs in lung cancers. Methods Microarray and RNA-seq data for normal lungs, lung adenocarcinomas (AD, squamous cell carcinomas of the lung (SQCLC, and small cell carcinomas of the lung (SCLC were collected from online databases. Using 374 of 451 HKGs, differentially expressed genes between pairs of sample types were determined via two-sided, homoscedastic t-test. Principal component analysis and hierarchical clustering classified normal lung and lung cancers subtypes according to relative gene expression variations. We used uni- and multi-variate cox-regressions to identify significant predictors of overall survival in AD patients. Classifying genes were selected using a set of training samples and then validated using an independent test set. Gene Ontology was examined by PANTHER. Results This study showed that the differential expression patterns of 242, 245, and 99 HKGs were able to distinguish normal lung from AD, SCLC, and SQCLC, respectively. From these, 70 HKGs were common across the three lung cancer subtypes. These HKGs have low expression variation compared to current lung cancer markers (e.g., EGFR, KRAS and were involved in the most common biological processes (e.g., metabolism, stress response. In addition, the expression pattern of 106 HKGs alone was a significant classifier of AD versus SQCLC. We further highlighted that a panel of 13 HKGs was an independent predictor of

  10. Intrahepatic injection of adenovirus reduces inflammation and increases gene transfer and therapeutic effect in mice

    Crettaz, J. (Julien); Berraondo, P. (Pedro); Mauleon, I. (Itsaso); Ochoa, L. (Laura); Shankar, V. (Vijay); Barajas, M. (Miguel); Rooijen, N. (Nico) van; Kochanek, S. (Stefan); Qian, C. (Cheng); Prieto, J. (Jesús); Hernandez-Alcoceba, R. (Rubén); Gonzalez-Aseguinolaza, G. (Gloria)

    2006-01-01

    Recombinant adenoviruses (Ad) are among the most extensively used vectors for liver gene transfer. One of the major limitations for the clinical application of these vectors is the inflammatory immune response associated with systemic administration of high dose of virus. We evaluated the effect of Ad administration route on the inflammatory immune response and liver transgene expression. We compared direct intrahepatic injection (IH) with the systemic administration via tail vein (IV). IH in...

  11. Gemfibrozil, a lipid-lowering drug, increases myelin genes in human oligodendrocytes via peroxisome proliferator-activated receptor-β.

    Jana, Malabendu; Mondal, Susanta; Gonzalez, Frank J; Pahan, Kalipada

    2012-10-05

    An increase in CNS remyelination and a decrease in CNS inflammation are important steps to halt the progression of multiple sclerosis. Earlier studies have shown that gemfibrozil, a lipid-lowering drug, has anti-inflammatory properties. The current study identified another novel property of gemfibrozil in stimulating the expression of myelin-specific genes (myelin basic protein, myelin oligodendrocyte glycoprotein, 2',3'-cyclic-nucleotide 3'-phosphodiesterase, and proteolipid protein (PLP)) in primary human oligodendrocytes, mixed glial cells, and spinal cord organotypic cultures. Although gemfibrozil is a known activator of peroxisome proliferator-activated receptor-α (PPAR-α), we were unable to detect PPAR-α in either gemfibrozil-treated or untreated human oligodendrocytes, and gemfibrozil increased the expression of myelin genes in oligodendrocytes isolated from both wild type and PPAR-α(-/-) mice. On the other hand, gemfibrozil markedly increased the expression of PPAR-β but not PPAR-γ. Consistently, antisense knockdown of PPAR-β, but not PPAR-γ, abrogated the stimulatory effect of gemfibrozil on myelin genes in human oligodendrocytes. Gemfibrozil also did not up-regulate myelin genes in oligodendroglia isolated from PPAR-β(-/-) mice. Chromatin immunoprecipitation analysis showed that gemfibrozil induced the recruitment of PPAR-β to the promoter of PLP and myelin oligodendrocyte glycoprotein genes in human oligodendrocytes. Furthermore, gemfibrozil treatment also led to the recruitment of PPAR-β to the PLP promoter in vivo in the spinal cord of experimental autoimmune encephalomyelitis mice and suppression of experimental autoimmune encephalomyelitis symptoms in PLP-T cell receptor transgenic mice. These results suggest that gemfibrozil stimulates the expression of myelin genes via PPAR-β and that gemfibrozil, a prescribed drug for humans, may find further therapeutic use in demyelinating diseases.

  12. Gemfibrozil, a Lipid-lowering Drug, Increases Myelin Genes in Human Oligodendrocytes via Peroxisome Proliferator-activated Receptor-β*

    Jana, Malabendu; Mondal, Susanta; Gonzalez, Frank J.; Pahan, Kalipada

    2012-01-01

    An increase in CNS remyelination and a decrease in CNS inflammation are important steps to halt the progression of multiple sclerosis. Earlier studies have shown that gemfibrozil, a lipid-lowering drug, has anti-inflammatory properties. The current study identified another novel property of gemfibrozil in stimulating the expression of myelin-specific genes (myelin basic protein, myelin oligodendrocyte glycoprotein, 2′,3′-cyclic-nucleotide 3′-phosphodiesterase, and proteolipid protein (PLP)) in primary human oligodendrocytes, mixed glial cells, and spinal cord organotypic cultures. Although gemfibrozil is a known activator of peroxisome proliferator-activated receptor-α (PPAR-α), we were unable to detect PPAR-α in either gemfibrozil-treated or untreated human oligodendrocytes, and gemfibrozil increased the expression of myelin genes in oligodendrocytes isolated from both wild type and PPAR-α(−/−) mice. On the other hand, gemfibrozil markedly increased the expression of PPAR-β but not PPAR-γ. Consistently, antisense knockdown of PPAR-β, but not PPAR-γ, abrogated the stimulatory effect of gemfibrozil on myelin genes in human oligodendrocytes. Gemfibrozil also did not up-regulate myelin genes in oligodendroglia isolated from PPAR-β(−/−) mice. Chromatin immunoprecipitation analysis showed that gemfibrozil induced the recruitment of PPAR-β to the promoter of PLP and myelin oligodendrocyte glycoprotein genes in human oligodendrocytes. Furthermore, gemfibrozil treatment also led to the recruitment of PPAR-β to the PLP promoter in vivo in the spinal cord of experimental autoimmune encephalomyelitis mice and suppression of experimental autoimmune encephalomyelitis symptoms in PLP-T cell receptor transgenic mice. These results suggest that gemfibrozil stimulates the expression of myelin genes via PPAR-β and that gemfibrozil, a prescribed drug for humans, may find further therapeutic use in demyelinating diseases. PMID:22879602

  13. Overexpression of D-Xylose Reductase (xyl1 Gene and Antisense Inhibition of D-Xylulokinase (xyiH Gene Increase Xylitol Production in Trichoderma reesei

    Yuanyuan Hong

    2014-01-01

    Full Text Available T. reesei is an efficient cellulase producer and biomass degrader. To improve xylitol production in Trichoderma reesei strains by genetic engineering, two approaches were used in this study. First, the presumptive D-xylulokinase gene in T. reesei (xyiH, which has high homology to known fungi D-xylulokinase genes, was silenced by transformation of T. reesei QM9414 strain with an antisense construct to create strain S6-2-2. The expression of the xyiH gene in the transformed strain S6-2-2 decreased at the mRNA level, and D-xylulokinase activity decreased after 48 h of incubation. This led to an increase in xylitol production from undetectable levels in wild-type T. reesei QM9414 to 8.6 mM in S6-2-2. The T. reesei Δxdh is a xylose dehydrogenase knockout strain with increased xylitol production compared to the wild-type T. reesei QM9414 (22.8 mM versus undetectable. The copy number of the xylose reductase gene (xyl1 in T. reesei Δxdh strain was increased by genetic engineering to create a new strain Δ9-5-1. The Δ9-5-1 strain showed a higher xyl1 expression and a higher yield of xylose reductase, and xylitol production was increased from 22.8 mM to 24.8 mM. Two novel strains S6-2-2 and Δ9-5-1 are capable of producing higher yields of xylitol. T. reesei has great potential in the industrial production of xylitol.

  14. Overexpression of D-Xylose Reductase (xyl1) Gene and Antisense Inhibition of D-Xylulokinase (xyiH) Gene Increase Xylitol Production in Trichoderma reesei

    Hong, Yuanyuan; Dashtban, Mehdi; Kepka, Greg; Chen, Sanfeng; Qin, Wensheng

    2014-01-01

    T. reesei is an efficient cellulase producer and biomass degrader. To improve xylitol production in Trichoderma reesei strains by genetic engineering, two approaches were used in this study. First, the presumptive D-xylulokinase gene in T. reesei (xyiH), which has high homology to known fungi D-xylulokinase genes, was silenced by transformation of T. reesei QM9414 strain with an antisense construct to create strain S6-2-2. The expression of the xyiH gene in the transformed strain S6-2-2 decreased at the mRNA level, and D-xylulokinase activity decreased after 48 h of incubation. This led to an increase in xylitol production from undetectable levels in wild-type T. reesei QM9414 to 8.6 mM in S6-2-2. The T. reesei Δxdh is a xylose dehydrogenase knockout strain with increased xylitol production compared to the wild-type T. reesei QM9414 (22.8 mM versus undetectable). The copy number of the xylose reductase gene (xyl1) in T. reesei Δxdh strain was increased by genetic engineering to create a new strain Δ9-5-1. The Δ9-5-1 strain showed a higher xyl1 expression and a higher yield of xylose reductase, and xylitol production was increased from 22.8 mM to 24.8 mM. Two novel strains S6-2-2 and Δ9-5-1 are capable of producing higher yields of xylitol. T. reesei has great potential in the industrial production of xylitol. PMID:25013760

  15. Differential gene expression in liver and small intestine from lactating rats compared to age-matched virgin controls detects increased mRNA of cholesterol biosynthetic genes

    Jungsuwadee Paiboon

    2011-02-01

    Full Text Available Abstract Background Lactation increases energy demands four- to five-fold, leading to a two- to three-fold increase in food consumption, requiring a proportional adjustment in the ability of the lactating dam to absorb nutrients and to synthesize critical biomolecules, such as cholesterol, to meet the dietary needs of both the offspring and the dam. The size and hydrophobicity of the bile acid pool increases during lactation, implying an increased absorption and disposition of lipids, sterols, nutrients, and xenobiotics. In order to investigate changes at the transcriptomics level, we utilized an exon array and calculated expression levels to investigate changes in gene expression in the liver, duodenum, jejunum, and ileum of lactating dams when compared against age-matched virgin controls. Results A two-way mixed models ANOVA was applied to detect differentially expressed genes. Significance calls were defined as a p Cyp7a1, which catalyzes the rate limiting step in the bile acid biosynthetic pathway, was also significantly increased in liver. In addition, decreased levels of mRNA associated with T-cell signaling were found in the jejunum and ileum. Several members of the Solute Carrier (SLC and Adenosine Triphosphate Binding Cassette (ABC superfamilies of membrane transporters were found to be differentially expressed; these genes may play a role in differences in nutrient and xenobiotic absorption and disposition. mRNA expression of SLC39a4_predicted, a zinc transporter, was increased in all tissues, suggesting that it is involved in increased zinc uptake during lactation. Microarray data are available through GEO under GSE19175. Conclusions We detected differential expression of mRNA from several pathways in lactating dams, including upregulation of the cholesterol biosynthetic pathway in liver and intestine, consistent with Srebp activation. Differential T-Cell signaling in the two most distal regions of the small intestine (ileum and

  16. Wounding stimulates ALLENE OXIDE SYNTHASE gene and increases the level of jasmonic acid in Ipomoea nil cotyledons

    Emilia Wilmowicz

    2016-03-01

    Full Text Available Allene oxide synthase (AOS encodes the first enzyme in the lipoxygenase pathway, which is responsible for jasmonic acid (JA formation. In this study we report the molecular cloning and characterization of InAOS from Ipomoea nil. The full-length gene is composed of 1662 bp and encodes for 519 amino acids. The predicted InAOS contains PLN02648 motif, which is evolutionarily conserved and characteristic for functional enzymatic proteins. We have shown that wounding led to a strong stimulation of the examined gene activity in cotyledons and an increase in JA level, which suggest that this compound may be a modulator of stress responses in I. nil.

  17. Increased natriuretic peptide receptor A and C gene expression in rats with pressure-overload cardiac hypertrophy

    Christoffersen, Tue E.H.; Aplin, Mark; Strom, Claes C.

    2006-01-01

    also affects cardiac hypertrophy and fibrosis. In this study we examined the expression of genes for the NPRs in rats with pressure-overload cardiac hypertrophy. The ANG II type 1 receptor was blocked with losartan (10 mg.kg(-1).day(-1)) to investigate a possible role of the renin-angiotensin system......RNAs for the natriuretic peptides or their receptors. Although increased gene expression does not necessarily convey a higher concentration of the protein, the data suggest that pressure overload is accompanied by upregulation of not only ANP and BNP but also their receptors NPR-A and NPR-C in the left ventricle....

  18. FRAS1-related extracellular matrix 3 (FREM3) single-nucleotide polymorphism effects on gene expression, amygdala reactivity and perceptual processing speed: An accelerated aging pathway of depression risk

    Nikolova, Yuliya S.; Iruku, Swetha P.; Lin, Chien-Wei; Conley, Emily Drabant; Puralewski, Rachel; French, Beverly; Hariri, Ahmad R.; Sibille, Etienne

    2015-01-01

    The A allele of the FRAS1-related extracellular matrix protein 3 (FREM3) rs7676614 single nucleotide polymorphism (SNP) was linked to major depressive disorder (MDD) in an early genome-wide association study (GWAS), and to symptoms of psychomotor retardation in a follow-up investigation. In line with significant overlap between age- and depression-related molecular pathways, parallel work has shown that FREM3 expression in postmortem human brain decreases with age. Here, we probe the effect of rs7676614 on amygdala reactivity and perceptual processing speed, both of which are altered in depression and aging. Amygdala reactivity was assessed using a face-matching BOLD fMRI paradigm in 365 Caucasian participants in the Duke Neurogenetics Study (DNS) (192 women, mean age 19.7 ± 1.2). Perceptual processing speed was indexed by reaction times in the same task and the Trail Making Test (TMT). The effect of rs7676614 on FREM3 mRNA brain expression levels was probed in a postmortem cohort of 169 Caucasian individuals (44 women, mean age 50.8 ± 14.9). The A allele of rs7676614 was associated with blunted amygdala reactivity to faces, slower reaction times in the face-matching condition (p < 0.04), as well as marginally slower performance on TMT Part B (p = 0.056). In the postmortem cohort, the T allele of rs6537170 (proxy for the rs7676614 A allele), was associated with trend-level reductions in gene expression in Brodmann areas 11 and 47 (p = 0.066), reminiscent of patterns characteristic of older age. The low-expressing allele of another FREM3 SNP (rs1391187) was similarly associated with reduced amygdala reactivity and slower TMT Part B speed, in addition to reduced BA47 activity and extraversion (p < 0.05). Together, these results suggest common genetic variation associated with reduced FREM3 expression may confer risk for a subtype of depression characterized by reduced reactivity to environmental stimuli and slower perceptual processing speed, possibly suggestive of

  19. Cell stiffness, contractile stress and the role of extracellular matrix

    An, Steven S.; Kim, Jina; Ahn, Kwangmi; Trepat, Xavier; Drake, Kenneth J.; Kumar, Sarvesh; Ling, Guoyu; Purington, Carolyn; Rangasamy, Tirumalai; Kensler, Thomas W.; Mitzner, Wayne; Fredberg, Jeffrey J.; Biswal, Shyam

    2009-01-01

    Here we have assessed the effects of extracellular matrix (ECM) composition and rigidity on mechanical properties of the human airway smooth muscle (ASM) cell. Cell stiffness and contractile stress showed appreciable changes from the most relaxed state to the most contracted state: we refer to the maximal range of these changes as the cell contractile scope. The contractile scope was least when the cell was adherent upon collagen V, followed by collagen IV, laminin, and collagen I, and greatest for fibronectin. Regardless of ECM composition, upon adherence to increasingly rigid substrates, the ASM cell positively regulated expression of antioxidant genes in the glutathione pathway and heme oxygenase, and disruption of a redox-sensitive transcription factor, nuclear erythroid 2 p45-related factor (Nrf2), culminated in greater contractile scope. These findings provide biophysical evidence that ECM differentially modulates muscle contractility and, for the first time, demonstrate a link between muscle contractility and Nrf2-directed responses.

  20. Cell stiffness, contractile stress and the role of extracellular matrix

    An, Steven S., E-mail: san@jhsph.edu [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E-7616, Baltimore, MD 21205 (United States); Kim, Jina [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E-7616, Baltimore, MD 21205 (United States); Ahn, Kwangmi [Division of Biostatistics, Penn State College of Medicine, Hershey, PA 17033 (United States); Trepat, Xavier [CIBER, Enfermedades Respiratorias, 07110 Bunyola (Spain); Drake, Kenneth J. [Division of Molecular and Integrative Physiological Sciences, Harvard School of Public Health, Boston, MA 02115 (United States); Kumar, Sarvesh; Ling, Guoyu; Purington, Carolyn; Rangasamy, Tirumalai; Kensler, Thomas W.; Mitzner, Wayne [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E-7616, Baltimore, MD 21205 (United States); Fredberg, Jeffrey J. [Division of Molecular and Integrative Physiological Sciences, Harvard School of Public Health, Boston, MA 02115 (United States); Biswal, Shyam [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E-7616, Baltimore, MD 21205 (United States); Division of Pulmonary and Critical Care Medicine, Johns Hopkins School of Medicine, Baltimore, MD 21205 (United States)

    2009-05-15

    Here we have assessed the effects of extracellular matrix (ECM) composition and rigidity on mechanical properties of the human airway smooth muscle (ASM) cell. Cell stiffness and contractile stress showed appreciable changes from the most relaxed state to the most contracted state: we refer to the maximal range of these changes as the cell contractile scope. The contractile scope was least when the cell was adherent upon collagen V, followed by collagen IV, laminin, and collagen I, and greatest for fibronectin. Regardless of ECM composition, upon adherence to increasingly rigid substrates, the ASM cell positively regulated expression of antioxidant genes in the glutathione pathway and heme oxygenase, and disruption of a redox-sensitive transcription factor, nuclear erythroid 2 p45-related factor (Nrf2), culminated in greater contractile scope. These findings provide biophysical evidence that ECM differentially modulates muscle contractility and, for the first time, demonstrate a link between muscle contractility and Nrf2-directed responses.

  1. Dietary selenomethionine increases exon-specific DNA methylation of the p53 gene in rat liver and colon mucosa.

    Zeng, Huawei; Yan, Lin; Cheng, Wen-Hsing; Uthus, Eric O

    2011-08-01

    The regulation of site-specific DNA methylation of tumor suppressor genes has been considered as a leading mechanism by which certain nutrients exert their anticancer property. This study was to investigate whether selenium (Se) affects the methylation of globe genomic DNA and the exon-specific p53 gene. Three groups of rats (n = 6-7/group) were fed the AIN-93G basal diet supplemented with 0 [Se deficient (D)], 0.15 [Se adequate (A)], or 4 mg [Se supranutritional (S)] (Se as l-selenomethionine)/kg diet for 104 d, respectively. Rats fed the A or S diet had greater plasma and liver glutathione peroxidase activity, liver thioredoxin reductase activity, and plasma homocysteine concentration than those fed the D diet. However, compared with the A diet, rats fed the S diet did not further increase these Se-dependent enzyme activities or homocysteine concentration. In contrast, Se concentrations in kidney, liver, gastrocnemius muscle, and plasma were increased in a Se-dose-dependent manner. Interestingly, rats fed the S diet had significantly less global liver genomic DNA methylation than those fed the D diet. However, the S diet significantly increased the methylation of the p53 gene (exons 5-8) but not the β-actin gene (exons 2-3) DNA in liver and colon mucosa compared with those fed the D diet. Taken together, long-term Se consumption not only affects selenoprotein enzyme activities, homocysteine, tissue Se concentrations, and global genomic DNA methylation but also increases exon-specific DNA methylation of the p53 gene in a Se-dose-dependent manner in rat liver and colon mucosa.

  2. Osteoblast-specific transcription factor Osterix increases vitamin D receptor gene expression in osteoblasts.

    Chi Zhang

    Full Text Available Osterix (Osx is an osteoblast-specific transcription factor required for osteoblast differentiation from mesenchymal stem cells. In Osx knock-out mice, no bone formation occurs. The vitamin D receptor (VDR is a member of the nuclear hormone receptor superfamily that regulates target gene transcription to ensure appropriate control of calcium homeostasis and bone development. Here, we provide several lines of evidence that show that the VDR gene is a target for transcriptional regulation by Osx in osteoblasts. For example, calvaria obtained from Osx-null embryos displayed dramatic reductions in VDR expression compared to wild-type calvaria. Stable overexpression of Osx stimulated VDR expression in C2C12 mesenchymal cells. Inhibition of Osx expression by siRNA led to downregulation of VDR. In contrast, Osx levels remained unchanged in osteoblasts in VDR-null mice. Mechanistic approaches using transient transfection assays showed that Osx directly activated a 1 kb fragment of the VDR promoter in a dose-dependent manner. To define the region of the VDR promoter that was responsive to Osx, a series of VDR promoter deletion mutants were examined and the minimal Osx-responsive region was refined to the proximal 120 bp of the VDR promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VDR promoter activation by Osx. Chromatin immunoprecipitation assays demonstrated that endogenous Osx was associated with the native VDR promoter in primary osteoblasts in vivo. Cumulatively, these data strongly support a direct regulatory role for Osx in VDR gene expression. They further provide new insight into potential mechanisms and pathways that Osx controls in osteoblasts and during the process of osteoblastic cell differentiation.

  3. Quinapril treatment increases insulin-stimulated endothelial function and adiponectin gene expression in patients with type 2 diabetes

    Hermann, Thomas S; Li, Weijie; Dominguez, Helena

    2005-01-01

    OBJECTIVE: Angiotensin-converting enzyme inhibitors reduce cardiovascular mortality and improve endothelial function in type 2 diabetic patients. We hypothesized that 2 months of quinapril treatment would improve insulin-stimulated endothelial function and glucose uptake in type 2 diabetic subjects...... and simultaneously increase the expression of genes that are pertinent for endothelial function and metabolism. METHODS: Twenty-four type 2 diabetic subjects were randomized to receive 2 months of quinapril 20 mg daily or no treatment in an open parallel study. Endothelium-dependent and -independent vasodilation...... occlusion plethysmography. Gene expression was measured by real-time PCR. RESULTS: Quinapril treatment increased insulin-stimulated endothelial function in the type 2 diabetic subjects (P = 0.005), whereas forearm glucose uptake was unchanged. Endothelial function was also increased by quinapril (P = 0...

  4. Fluconazole Resistance Associated with Drug Efflux and Increased Transcription of a Drug Transporter Gene, PDH1, in Candida glabrata

    Miyazaki, Haruko; Miyazaki, Yoshitsugu; Geber, Antonia; Parkinson, Tanya; Hitchcock, Christopher; Falconer, Derek J.; Ward, Douglas J.; Marsden, Katherine; Bennett, John E.

    1998-01-01

    Sequential Candida glabrata isolates were obtained from the mouth of a patient infected with human immunodeficiency virus type 1 who was receiving high doses of fluconazole for oropharyngeal thrush. Fluconazole-susceptible colonies were replaced by resistant colonies that exhibited both increased fluconazole efflux and increased transcripts of a gene which codes for a protein with 72.5% identity to Pdr5p, an ABC multidrug transporter in Saccharomyces cerevisiae. The deduced protein had a molecular mass of 175 kDa and was composed of two homologous halves, each with six putative transmembrane domains and highly conserved sequences of ATP-binding domains. When the earliest and most azole-susceptible isolate of C. glabrata from this patient was exposed to fluconazole, increased transcripts of the PDR5 homolog appeared, linking azole exposure to regulation of this gene. PMID:9661006

  5. Extracellular matrix structure.

    Theocharis, Achilleas D; Skandalis, Spyros S; Gialeli, Chrysostomi; Karamanos, Nikos K

    2016-02-01

    Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network composed of collagens, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminins, and several other glycoproteins. Matrix components bind each other as well as cell adhesion receptors forming a complex network into which cells reside in all tissues and organs. Cell surface receptors transduce signals into cells from ECM, which regulate diverse cellular functions, such as survival, growth, migration, and differentiation, and are vital for maintaining normal homeostasis. ECM is a highly dynamic structural network that continuously undergoes remodeling mediated by several matrix-degrading enzymes during normal and pathological conditions. Deregulation of ECM composition and structure is associated with the development and progression of several pathologic conditions. This article emphasizes in the complex ECM structure as to provide a better understanding of its dynamic structural and functional multipotency. Where relevant, the implication of the various families of ECM macromolecules in health and disease is also presented. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Tacrolimus increases Nox4 expression in human renal fibroblasts and induces fibrosis-related genes by aberrant TGF-beta receptor signalling.

    Georg Kern

    Full Text Available Chronic nephrotoxicity of immunosuppressives is one of the main limiting factors in the long-term outcome of kidney transplants, leading to tissue fibrosis and ultimate organ failure. The cytokine TGF-β is considered a key factor in this process. In the human renal fibroblast cell line TK-173, the macrolide calcineurin inhibitor tacrolimus (FK-506 induced TGF-β-like effects, manifested by increased expression of NAD(PH-oxidase 4 (Nox4, transgelin, tropomyosin 1, and procollagen α1(V mRNA after three days. The macrolide mTOR inhibitor rapamycin had similar effects, while cyclosporine A did not induce fibrose-related genes. Concentration dependence curves were sigmoid, where mRNA expression was induced already at low nanomolar levels of tacrolimus, and reached saturation at 100-300 nM. The effects were independent of extracellular TGF-β as confirmed by the use of neutralizing antibodies, and thus most likely caused by aberrant TGF-β receptor signaling, where binding of tacrolimus to the regulatory FKBP12 protein results in a "leaky" TGF-β receptor. The myofibroblast marker α-smooth muscle actin was neither induced by tacrolimus nor by TGF-β1, indicating an incomplete activation of TK-173 fibroblasts under culture conditions. Tacrolimus- and TGF-β1-induced Nox4 protein upregulation was confirmed by Western blotting, and was accompanied by a rise in intracellular H2O2 concentration. Si-RNA mediated knock-down of Nox4 expression prevented up-regulation of procollagen α1(V mRNA in tacrolimus-treated cells, but induced procollagen α1(V expression in control cells. Nox4 knock-down had no significant effect on the other genes tested. TGF-β is a key molecule in fibrosis, and the constant activation of aberrant receptor signaling by tacrolimus might contribute to the long-term development of interstitial kidney fibrosis in immunosuppressed patients. Nox4 levels possibly play a regulatory role in these processes.

  7. Planting increases the abundance and structure complexity of soil core functional genes relevant to carbon and nitrogen cycling.

    Wang, Feng; Liang, Yuting; Jiang, Yuji; Yang, Yunfeng; Xue, Kai; Xiong, Jinbo; Zhou, Jizhong; Sun, Bo

    2015-09-23

    Plants have an important impact on soil microbial communities and their functions. However, how plants determine the microbial composition and network interactions is still poorly understood. During a four-year field experiment, we investigated the functional gene composition of three types of soils (Phaeozem, Cambisols and Acrisol) under maize planting and bare fallow regimes located in cold temperate, warm temperate and subtropical regions, respectively. The core genes were identified using high-throughput functional gene microarray (GeoChip 3.0), and functional molecular ecological networks (fMENs) were subsequently developed with the random matrix theory (RMT)-based conceptual framework. Our results demonstrated that planting significantly (P soils and 83.5% of microbial alpha-diversity can be explained by the plant factor. Moreover, planting had significant impacts on the microbial community structure and the network interactions of the microbial communities. The calculated network complexity was higher under maize planting than under bare fallow regimes. The increase of the functional genes led to an increase in both soil respiration and nitrification potential with maize planting, indicating that changes in the soil microbial communities and network interactions influenced ecological functioning.

  8. Extracellular proteases of Trichoderma species. A review.

    Kredics, L; Antal, Zsuzsanna; Szekeres, A; Hatvani, L; Manczinger, L; Vágvölgyi, Cs; Nagy, Erzsébet

    2005-01-01

    Cellulolytic, xylanolytic, chitinolytic and beta-1,3-glucanolytic enzyme systems of species belonging to the filamentous fungal genus Trichoderma have been investigated in details and are well characterised. The ability of Trichoderma strains to produce extracellular proteases has also been known for a long time, however, the proteolytic enzyme system is relatively unknown in this genus. Fortunately, in the recent years more and more attention is focused on the research in this field. The role of Trichoderma proteases in the biological control of plant pathogenic fungi and nematodes has been demonstrated, and it is also suspected that they may be important for the competitive saprophytic ability of green mould isolates and may represent potential virulence factors of Trichoderma strains as emerging fungal pathogens of clinical importance. The aim of this review is to summarize the information available about the extracellular proteases of Trichoderma. Numerous studies are available about the extracellular proteolytic enzyme profiles of Trichoderma strains and about the effect of abiotic environmental factors on protease activities. A number of protease enzymes have been purified to homogeneity and some protease encoding genes have been cloned and characterized. These results will be reviewed and the role of Trichoderma proteases in biological control as well as their advantages and disadvantages in biotechnology will be discussed.

  9. Overexpression of SAMDC1 gene in Arabidopsis thaliana increases expression of defense-related genes as well as resistance to Pseudomonas syringae and Hyaloperonospora arabidopsidis

    Francisco eMarco

    2014-03-01

    Full Text Available It has been previously described that elevation of endogenous spermine levels in Arabidopsis could be achieved by transgenic overexpression of S-Adenosylmethionine decarboxylase (SAMDC or Spermine synthase (SPMS. In both cases, spermine accumulation had an impact on the plant transcriptome, with up-regulation of a set of genes enriched in functional categories involved in defense-related processes against both biotic and abiotic stresses. In this work, the response of SAMDC1-overexpressing plants against bacterial and oomycete pathogens has been tested. The expression of several pathogen defense-related genes was induced in these plants as well as in wild type plants exposed to an exogenous supply of spermine. SAMDC1-overexpressing plants showed an increased tolerance to infection by Pseudomonas syringae and by Hyaloperonospora arabidopsidis. Both results add more evidence to the hypothesis that spermine plays a key role in plant resistance to biotic stress.

  10. Deletion of the Wolfram syndrome-related gene Wfs1 results in increased sensitivity to ethanol in female mice.

    Raud, Sirli; Reimets, Riin; Loomets, Maarja; Sütt, Silva; Altpere, Alina; Visnapuu, Tanel; Innos, Jürgen; Luuk, Hendrik; Plaas, Mario; Volke, Vallo; Vasar, Eero

    2015-08-01

    Wolfram syndrome, induced by mutation in WFS1 gene, increases risk of developing mood disorders in humans. In mice, Wfs1 deficiency cause higher anxiety-like behaviour and increased response to anxiolytic-like effect of diazepam, a GABAA receptor agonist. As GABAergic system is also target for ethanol, we analysed its anxiolytic-like and sedative properties in Wfs1-deficient mice using elevated plus-maze test and tests measuring locomotor activity and coordination, respectively. Additionally loss of righting reflex test was conducted to study sedative/hypnotic properties of ethanol, ketamine and pentobarbital. To evaluate pharmacokinetics of ethanol in mice enzymatic colour test was used. Finally, gene expression of alpha subunits of GABAA receptors following ethanol treatment was studied by real-time-PCR. Compared to wild-types, Wfs1-deficient mice were more sensitive to ethanol-induced anxiolytic-like effect, but less responsive to impairment of motor coordination. Ethanol and pentobarbital, but not ketamine, caused longer duration of hypnosis in Wfs1-deficient mice. The expression of Gabra2 subunit at 30 minutes after ethanol injection was significantly increased in the frontal cortex of Wfs1-deficient mice as compared to respective vehicle-treated mice. For the temporal lobe, similar change in Gabra2 mRNA occurred at 60 minutes after ethanol treatment in Wfs1-deficient mice. No changes were detected in Gabra1 and Gabra3 mRNA following ethanol treatment. Taken together, increased anxiolytic-like effect of ethanol in Wfs1-deficient mice is probably related to altered Gabra2 gene expression. Increased anti-anxiety effect of GABAA receptor agonists in the present work and earlier studies (Luuk et al., 2009) further suggests importance of Wfs1 gene in the regulation of emotional behaviour. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Growth arrest- and DNA-damage-inducible 45beta gene inhibits c-Jun N-terminal kinase and extracellular signal-regulated kinase and decreases IL-1beta-induced apoptosis in insulin-producing INS-1E cells

    Larsen, Claus Morten; Døssing, M G; Papa, S

    2006-01-01

    IL-1beta is a candidate mediator of apoptotic beta cell destruction, a process that leads to type 1 diabetes and progression of type 2 diabetes. IL-1beta activates beta cell c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38, all of which are members of the mitogen...

  12. GENE-07. MOLECULAR NEUROPATHOLOGY 2.0 - INCREASING DIAGNOSTIC ACCURACY IN PEDIATRIC NEUROONCOLOGY

    Sturm, Dominik; Jones, David T.W.; Capper, David; Sahm, Felix; von Deimling, Andreas; Rutkoswki, Stefan; Warmuth-Metz, Monika; Bison, Brigitte; Gessi, Marco; Pietsch, Torsten; Pfister, Stefan M.

    2017-01-01

    Abstract The classification of central nervous system (CNS) tumors into clinically and biologically distinct entities and subgroups is challenging. Children and adolescents can be affected by >100 histological variants with very variable outcomes, some of which are exceedingly rare. The current WHO classification has introduced a number of novel molecular markers to aid routine neuropathological diagnostics, and DNA methylation profiling is emerging as a powerful tool to distinguish CNS tumor classes. The Molecular Neuropathology 2.0 study aims to integrate genome wide (epi-)genetic diagnostics with reference neuropathological assessment for all newly-diagnosed pediatric brain tumors in Germany. To date, >350 patients have been enrolled. A molecular diagnosis is established by epigenetic tumor classification through DNA methylation profiling and targeted panel sequencing of >130 genes to detect diagnostically and/or therapeutically useful DNA mutations, structural alterations, and fusion events. Results are aligned with the reference neuropathological diagnosis, and discrepant findings are discussed in a multi-disciplinary tumor board including reference neuroradiological evaluation. Ten FFPE sections as input material are sufficient to establish a molecular diagnosis in >95% of tumors. Alignment with reference pathology results in four broad categories: a) concordant classification (~77%), b) discrepant classification resolvable by tumor board discussion and/or additional data (~5%), c) discrepant classification without currently available options to resolve (~8%), and d) cases currently unclassifiable by molecular diagnostics (~10%). Discrepancies are enriched in certain histopathological entities, such as histological high grade gliomas with a molecularly low grade profile. Gene panel sequencing reveals predisposing germline events in ~10% of patients. Genome wide (epi-)genetic analyses add a valuable layer of information to routine neuropathological

  13. Association of the STAT4 gene with increased susceptibility for some immune-mediated diseases.

    Martínez, A; Varadé, J; Márquez, A; Cénit, M C; Espino, L; Perdigones, N; Santiago, J L; Fernández-Arquero, M; de la Calle, H; Arroyo, R; Mendoza, J L; Fernández-Gutiérrez, B; de la Concha, E G; Urcelay, E

    2008-09-01

    The STAT4 gene encodes a transcription factor involved in the signaling pathways of several cytokines, including interleukin-12 (IL-12), the type I interferons, and IL-23. Recently, the association of a STAT4 haplotype marked by rs7574865 with rheumatoid arthritis (RA) and systemic lupus erythematosus was reported. The aim of this study was to investigate the role of this STAT4 tagging polymorphism in other immune-mediated diseases. The study group comprised 2,776 consecutively recruited Spanish individuals: 575 with RA, 440 with multiple sclerosis, 700 with inflammatory bowel disease, 311 with type 1 diabetes, and 723 ethnically matched healthy control subjects. The STAT4 polymorphism rs7574865 was genotyped using a predesigned TaqMan assay. Allele and genotype frequencies in patients and control subjects were compared by chi-square test. The association of STAT4 polymorphism rs7574865 with RA was validated in patients of Spanish origin (for T versus G, P = 1.2 x 10(-6), odds ratio [OR] 1.59, 95% confidence interval [95% CI] 1.31-1.92), and the association was described for the first time in both clinical forms of inflammatory bowel disease, Crohn's disease and ulcerative colitis (for T versus G, P = 0.006, OR 1.29, 95% CI 1.07-1.55), and in type 1 diabetes mellitus (for T versus G, P = 0.008, OR 1.36, 95% CI 1.07-1.71). In contrast, the genotypic distribution of this polymorphism showed no difference between patients with multiple sclerosis and healthy control subjects (for T versus G, P = 0.83, OR 1.02, 95% CI 0.82-1.28). The STAT4 gene is emerging as a novel common risk factor for diverse complex diseases.

  14. Over-expression of a novel JAZ family gene from Glycine soja, increases salt and alkali stress tolerance

    Zhu, Dan; Cai, Hua; Luo, Xiao; Bai, Xi; Deyholos, Michael K.; Chen, Qin; Chen, Chao; Ji, Wei; Zhu, Yanming

    2012-01-01

    Highlights: ► We isolated and characterized a novel JAZ family gene, GsJAZ2, from Glycine soja. ► Overexpression of GsJAZ2 enhanced plant tolerance to salt and alkali stress. ► The transcriptions of stress marker genes were higher in GsJAZ2 overexpression lines. ► GsJAZ2 was localized to nucleus. -- Abstract: Salt and alkali stress are two of the main environmental factors limiting crop production. Recent discoveries show that the JAZ family encodes plant-specific genes involved in jasmonate signaling. However, there is only limited information about this gene family in abiotic stress response, and in wild soybean (Glycine soja), which is a species noted for its tolerance to alkali and salinity. Here, we isolated and characterized a novel JAZ family gene, GsJAZ2, from G. soja. Transcript abundance of GsJAZ2 increased following exposure to salt, alkali, cold and drought. Over-expression of GsJAZ2 in Arabidopsis resulted in enhanced plant tolerance to salt and alkali stress. The expression levels of some alkali stress response and stress-inducible marker genes were significantly higher in the GsJAZ2 overexpression lines as compared to wild-type plants. Subcellular localization studies using a GFP fusion protein showed that GsJAZ2 was localized to the nucleus. These results suggest that the newly isolated wild soybean GsJAZ2 is a positive regulator of plant salt and alkali stress tolerance.

  15. Enhanced lysosomal acidification leads to increased chloroquine accumulation in CHO cells expressing the pfmdr1 gene

    van Es, H. H.; Renkema, H.; Aerts, H.; Schurr, E.

    1994-01-01

    Expression of the pfmdr1-encoded Pgh1 protein of Plasmodium falciparum in CHO cells confers a phenotype of increased sensitivity to chloroquine due to an increased Pgh1-mediated accumulation of this antimalarial. Pgh1 carrying amino acid substitutions associated with chloroquine resistance in P.

  16. The Peanut (Arachis hypogaea L. Gene AhLPAT2 Increases the Lipid Content of Transgenic Arabidopsis Seeds.

    Silong Chen

    Full Text Available Lysophosphatidic acid acyltransferase (LPAT, which converts lysophosphatidic acid (LPA to phosphatidic acid (PA, catalyzes the addition of fatty acyl moieties to the sn-2 position of the LPA glycerol backbone in triacylglycerol (TAG biosynthesis. We recently reported the cloning and temporal-spatial expression of a peanut (Arachis hypogaea AhLPAT2gene, showing that an increase in AhLPAT2 transcript levels was closely correlated with an increase in seed oil levels. However, the function of the enzyme encoded by the AhLPAT2 gene remains unclear. Here, we report that AhLPAT2 transcript levels were consistently higher in the seeds of a high-oil cultivar than in those of a low-oil cultivar across different seed developmental stages. Seed-specific overexpression of AhLPAT2 in Arabidopsis results in a higher percentage of oil in the seeds and greater-than-average seed weight in the transgenic plants compared with the wild-type plants, leading to a significant increase in total oil yield per plant. The total fatty acid (FA content and the proportion of unsaturated FAs also increased. In the developing siliques of AhLPAT2-overexpressing plants, the expression levels of genes encoding crucial enzymes involved in de novo FA synthesis, acetyl-CoA subunit (AtBCCP2 and acyl carrier protein 1 (AtACP1 were elevated. AhLPAT2 overexpression also promoted the expression of several key genes related to TAG assembly, sucrose metabolism, and glycolysis. These results demonstrate that the expression of AhLPAT2 plays an important role in glycerolipid production in peanuts.

  17. High dietary zinc supplementation increases the occurrence of tetracycline and sulfonamide resistance genes in the intestine of weaned pigs

    Vahjen, Wilfried; Pietruszy?ska, Dominika; Starke, Ingo C.; Zentek, J?rgen

    2015-01-01

    Background Dietary zinc oxide is used in pig nutrition to combat post weaning diarrhoea. Recent data suggests that high doses (2.5?g/kg feed) increase the bacterial antibiotic resistance development in weaned pigs. Therefore, the aim of this study was to investigate the development of enterobacterial antibiotic resistance genes in the intestinal tract of weaned pigs. Findings Weaned pigs were fed diets for 4?weeks containing 57 (low), 164 (intermediate) or 2425 (high) mg?kg?1 analytical grade...

  18. Increased Abundance and Transferability of Resistance Genes after Field Application of Manure from Sulfadiazine-Treated Pigs

    Jechalke, Sven; Kopmann, Christoph; Rosendahl, Ingrid; Groeneweg, Joost; Weichelt, Viola; Krögerrecklenfort, Ellen; Brandes, Nikola; Nordwig, Mathias; Ding, Guo-Chun; Siemens, Jan; Heuer, Holger

    2013-01-01

    Spreading manure containing antibiotics in agriculture is assumed to stimulate the dissemination of antibiotic resistance in soil bacterial populations. Plant roots influencing the soil environment and its microflora by exudation of growth substrates might considerably increase this effect. In this study, the effects of manure from pigs treated with sulfadiazine (SDZ), here called SDZ manure, on the abundance and transferability of sulfonamide resistance genes sul1 and sul2 in the rhizosphere of maize and grass were compared to the effects in bulk soil in a field experiment. In plots that repeatedly received SDZ manure, a significantly higher abundance of both sul genes was detected compared to that in plots where manure from untreated pigs was applied. Significantly lower abundances of sul genes relative to bacterial ribosomal genes were encountered in the rhizosphere than in bulk soil. However, in contrast to results for bulk soil, the sul gene abundance in the SDZ manure-treated rhizosphere constantly deviated from control treatments over a period of 6 weeks after manuring, suggesting ongoing antibiotic selection over this period. Transferability of sulfonamide resistance was analyzed by capturing resistance plasmids from soil communities into Escherichia coli. Increased rates of plasmid capture were observed in samples from SDZ manure-treated bulk soil and the rhizosphere of maize and grass. More than 97% of the captured plasmids belonged to the LowGC type (having low G+C content), giving further evidence for their important contribution to the environmental spread of antibiotic resistance. In conclusion, differences between bulk soil and rhizosphere need to be considered when assessing the risks associated with the spreading of antibiotic resistance. PMID:23315733

  19. Molecular control of copper homeostasis in filamentous fungi: increased expression of a metallothionein gene during aging of Podospora anserina.

    Averbeck, N B; Borghouts, C; Hamann, A; Specke, V; Osiewacz, H D

    2001-01-01

    The lifespan of the ascomycete Podospora anserina was previously demonstrated to be significantly increased in a copper-uptake mutant, suggesting that copper is a potential stressor involved in degenerative processes. In order to determine whether changes in copper stress occur in the cells during normal aging of cultures, we cloned and characterized a gene coding for a component of the molecular machinery involved in the control of copper homeostasis. This gene, PaMt1, is a single-copy gene that encodes a metallothionein of 26 amino acids. The coding sequence of PaMt1 is interrupted by a single intron. The deduced amino acid sequence shows a high degree of sequence identity to metallothioneins of the filamentous ascomycete Neurospora crassa and the basidiomycete Agaricus bisporus, and to the N-terminal portion of mammalian metallothioneins. Levels of PaMt1 transcript increase in response to elevated amounts of copper in the growth medium and during aging of wild-type cultures. In contrast, in the long-lived mutant grisea, transcript levels first increase but then decrease again. The ability of wild-type cultures to respond to exogenous copper stress via the induction of PaMt1 transcription is not affected as they grow older.

  20. Expression of an Arabidopsis molybdenum cofactor sulphurase gene in soybean enhances drought tolerance and increases yield under field conditions.

    Li, Yajun; Zhang, Jiachang; Zhang, Juan; Hao, Ling; Hua, Jinping; Duan, Liusheng; Zhang, Mingcai; Li, Zhaohu

    2013-08-01

    LOS5/ABA3 gene encoding molybdenum cofactor sulphurase is involved in aldehyde oxidase (AO) activity in Arabidopsis, which indirectly regulates ABA biosynthesis and increased stress tolerance. Here, we used a constitutive super promoter to drive LOS5/ABA3 overexpression in soybean (Glycine max L.) to enhance drought tolerance in growth chamber and field conditions. Expression of LOS5/ABA3 was up-regulated by drought stress, which led to increasing AO activity and then a notable increase in ABA accumulation. Transgenic soybean under drought stress had reduced water loss by decreased stomatal aperture size and transpiration rate, which alleviated leaf wilting and maintained higher relative water content. Exposed to drought stress, transgenic soybean exhibited reduced cell membrane damage by reducing electrolyte leakage and production of malondialdehyde and promoting proline accumulation and antioxidant enzyme activities. Also, overexpression of LOS5/ABA3 enhanced expression of stress-up-regulated genes. Furthermore, the seed yield of transgenic plants is at least 21% higher than that of wide-type plants under drought stress conditions in the field. These data suggest that overexpression of LOS5/ABA3 could improve drought tolerance in transgenic soybean via enhanced ABA accumulation, which could activate expression of stress-up-regulated genes and cause a series of physiological and biochemical resistant responses. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  1. Sterol regulatory element binding protein-1 (SREBP1) gene expression is similarly increased in polycystic ovary syndrome and endometrial cancer.

    Shafiee, Mohamad N; Mongan, Nigel; Seedhouse, Claire; Chapman, Caroline; Deen, Suha; Abu, Jafaru; Atiomo, William

    2017-05-01

    Women with polycystic ovary syndrome have a three-fold higher risk of endometrial cancer. Insulin resistance and hyperlipidemia may be pertinent factors in the pathogenesis of both conditions. The aim of this study was to investigate endometrial sterol regulatory element binding protein-1 gene expression in polycystic ovary syndrome and endometrial cancer endometrium, and to correlate endometrial sterol regulatory element binding protein-1 gene expression with serum lipid profiles. A cross-sectional study was performed at Nottingham University Hospital, UK. A total of 102 women (polycystic ovary syndrome, endometrial cancer and controls; 34 participants in each group) were recruited. Clinical and biochemical assessments were performed before endometrial biopsies were obtained from all participants. Taqman real-time polymerase chain reaction for endometrial sterol regulatory element binding protein-1 gene and its systemic protein expression were analyzed. The body mass indices of women with polycystic ovary syndrome (29.28 ± 2.91 kg/m 2 ) and controls (28.58 ± 2.62 kg/m 2 ) were not significantly different. Women with endometrial cancer had a higher mean body mass index (32.22 ± 5.70 kg/m 2 ). Sterol regulatory element binding protein-1 gene expression was significantly increased in polycystic ovary syndrome and endometrial cancer endometrium compared with controls (p ovary syndrome, but this was not statistically significant. Similarly, statistically insignificant positive correlations were found between endometrial sterol regulatory element binding protein-1 gene expression and body mass index in endometrial cancer (r = 0.643, p = 0.06) and waist-hip ratio (r = 0.096, p = 0.073). Sterol regulatory element binding protein-1 gene expression was significantly positively correlated with triglyceride in both polycystic ovary syndrome and endometrial cancer (p = 0.028 and p = 0.027, respectively). Quantitative serum sterol regulatory element

  2. Genome Plasticity and Polymorphisms in Critical Genes Correlate with Increased Virulence of Dutch Outbreak-Related Coxiella burnetii Strains

    Runa Kuley

    2017-08-01

    Full Text Available Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever. During 2007–2010 the largest Q fever outbreak ever reported occurred in The Netherlands. It is anticipated that strains from this outbreak demonstrated an increased zoonotic potential as more than 40,000 individuals were assumed to be infected. The acquisition of novel genetic factors by these C. burnetii outbreak strains, such as virulence-related genes, has frequently been proposed and discussed, but is not proved yet. In the present study, the whole genome sequence of several Dutch strains (CbNL01 and CbNL12 genotypes, a few additionally selected strains from different geographical locations and publicly available genome sequences were used for a comparative bioinformatics approach. The study focuses on the identification of specific genetic differences in the outbreak related CbNL01 strains compared to other C. burnetii strains. In this approach we investigated the phylogenetic relationship and genomic aspects of virulence and host-specificity. Phylogenetic clustering of whole genome sequences showed a genotype-specific clustering that correlated with the clustering observed using Multiple Locus Variable-number Tandem Repeat Analysis (MLVA. Ortholog analysis on predicted genes and single nucleotide polymorphism (SNP analysis of complete genome sequences demonstrated the presence of genotype-specific gene contents and SNP variations in C. burnetii strains. It also demonstrated that the currently used MLVA genotyping methods are highly discriminatory for the investigated outbreak strains. In the fully reconstructed genome sequence of the Dutch outbreak NL3262 strain of the CbNL01 genotype, a relatively large number of transposon-linked genes were identified as compared to the other published complete genome sequences of C. burnetii. Additionally, large numbers of SNPs in its membrane proteins and predicted virulence-associated genes were identified

  3. Mucin 4 Gene Silencing Reduces Oxidative Stress and Calcium Oxalate Crystal Formation in Renal Tubular Epithelial Cells Through the Extracellular Signal-Regulated Kinase Signaling Pathway in Nephrolithiasis Rat Model

    Ling Sun

    2018-05-01

    Full Text Available Background/Aims: Nephrolithiasis plagues a great number of patients all over the world. Increasing evidence shows that the extracellular signal-regulated kinase (ERK signaling pathway and renal tubular epithelial cell (RTEC dysfunction and attrition are central to the pathogenesis of kidney diseases. Mucin 4 (MUC4 is reported as an activator of ERK signaling pathway in epithelial cells. In this study, using rat models of calcium oxalate (CaOx nephrolithiasis, the present study aims to define the roles of MUC4 and ERK signaling pathway as contributors to oxidative stress and CaOx crystal formation in RTEC. Methods: Data sets of nephrolithiasis were searched using GEO database and a heat flow map was drawn. Then MUC4 function was predicted. Wistar rats were prepared for the purpose of model establishment of ethylene glycol and ammonium chloride induced CaOx nephrolithiasis. In order to assess the detailed regulatory mechanism of MUC4 silencing on the ERK signaling pathway and RTEC, we used recombinant plasmid to downregulate MUC4 expression in Wistar rat-based models. Samples from rat urine, serum and kidney tissues were reviewed to identify oxalic acid and calcium contents, BUN, Cr, Ca2+ and P3+ levels, calcium crystal formation in renal tubules and MUC4 positive expression rate. Finally, RT-qPCR, Western blot analysis, and ELISA were employed to access oxidative stress state and CaOx crystal formation in RTEC. Results: Initially, MUC4 was found to have an influence on the process of nephrolithiasis. MUC4 was upregulated in the CaOx nephrolithiasis model rats. We proved that the silencing of MUC4 triggered the inactivation of ERK signaling pathway. Following the silencing of MUC4 or the inhibition of ERK signaling pathway, the oxalic acid and calcium contents in rat urine, BUN, Cr, Ca2+ and P3+ levels in rat serum, p-ERK1/2, MCP-1 and OPN expressions in RTEC and H2O2 and MDA levels in the cultured supernatant were downregulated, but the GSH

  4. Management with willow short rotation coppice increase the functional gene diversity and functional activity of a heavy metal polluted soil.

    Xue, K; van Nostrand, J D; Vangronsveld, J; Witters, N; Janssen, J O; Kumpiene, J; Siebielec, G; Galazka, R; Giagnoni, L; Arenella, M; Zhou, J-Z; Renella, G

    2015-11-01

    We studied the microbial functional diversity, biochemical activity, heavy metals (HM) availability and soil toxicity of Cd, Pb and Zn contaminated soils, kept under grassland or short rotation coppice (SRC) to attenuate the risks associated with HM contamination and restore the soil ecological functions. Soil microbial functional diversity was analyzed by the GeoChip, a functional gene microarray containing probes for genes involved in nutrient cycling, metal resistance and stress response. Soil under SRC showed a higher abundance of microbial genes involved in C, N, P and S cycles and resistance to various HM, higher microbial biomass, respiration and enzyme activity rates, and lower HM availability than the grassland soil. The linkages between functional genes of soil microbial communities and soil chemical properties, HM availability and biochemical activity were also investigated. Soil toxicity and N, P and Pb availability were important factors in shaping the microbial functional diversity, as determined by CCA. We concluded that in HM contaminated soils the microbial functional diversity was positively influenced by SRC management through the reduction of HM availability and soil toxicity increase of nutrient cycling. The presented results can be important in predicting the long term environmental sustainability of plant-based soil remediation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Mutations in the NOT Genes or in the Translation Machinery Similarly Display Increased Resistance to Histidine Starvation

    Martine A. Collart

    2017-05-01

    Full Text Available The NOT genes encode subunits of the conserved Ccr4-Not complex, a global regulator of gene expression, and in particular of mRNA metabolism. They were originally identified in a selection for increased resistance to histidine starvation in the yeast S. cerevisiae. Recent work indicated that the Not5 subunit, ortholog of mammalian CNOT3, determines global translation levels by defining binding of the Ccr4-Not scaffold protein Not1 to ribosomal mRNAs during transcription. This is needed for optimal translation of ribosomal proteins. In this work we searched for mutations in budding yeast that were resistant to histidine starvation using the same selection that originally led to the isolation of the NOT genes. We thereby isolated mutations in ribosome-related genes. This common phenotype of ribosome mutants and not mutants is in good agreement with the positive role of the Not proteins for translation. In this regard, it is interesting that frequent mutations in RPL5 and RPL10 or in CNOT3 have been observed to accumulate in adult T-cell acute lymphoblastic leukemia (T-ALL. This suggests that in metazoans a common function implicating ribosome subunits and CNOT3 plays a role in the development of cancer. In this perspective we suggest that the Ccr4-Not complex, according to translation levels and fidelity, could itself be involved in the regulation of amino acid biosynthesis levels. We discuss how this could explain why mutations have been identified in many cancers.

  6. Over-expression of a novel JAZ family gene from Glycine soja, increases salt and alkali stress tolerance.

    Zhu, Dan; Cai, Hua; Luo, Xiao; Bai, Xi; Deyholos, Michael K; Chen, Qin; Chen, Chao; Ji, Wei; Zhu, Yanming

    2012-09-21

    Salt and alkali stress are two of the main environmental factors limiting crop production. Recent discoveries show that the JAZ family encodes plant-specific genes involved in jasmonate signaling. However, there is only limited information about this gene family in abiotic stress response, and in wild soybean (Glycine soja), which is a species noted for its tolerance to alkali and salinity. Here, we isolated and characterized a novel JAZ family gene, GsJAZ2, from G. soja. Transcript abundance of GsJAZ2 increased following exposure to salt, alkali, cold and drought. Over-expression of GsJAZ2 in Arabidopsis resulted in enhanced plant tolerance to salt and alkali stress. The expression levels of some alkali stress response and stress-inducible marker genes were significantly higher in the GsJAZ2 overexpression lines as compared to wild-type plants. Subcellular localization studies using a GFP fusion protein showed that GsJAZ2 was localized to the nucleus. These results suggest that the newly isolated wild soybean GsJAZ2 is a positive regulator of plant salt and alkali stress tolerance. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  7. Increasing galactose consumption by Saccharomyces cerevisiae through metabolic engineering of the GAL gene regulatory network

    Østergaard, Simon; Olsson, Lisbeth; Johnston, M.

    2000-01-01

    Increasing the flux through central carbon metabolism is difficult because of rigidity in regulatory structures, at both the genetic and the enzymatic levels. Here we describe metabolic engineering of a regulatory network to obtain a balanced increase in the activity of all the enzymes in the pat...... media. The improved galactose consumption of the gal mutants did not favor biomass formation, but rather caused excessive respiro-fermentative metabolism, with the ethanol production rate increasing linearly with glycolytic flux....... by eliminating three known negative regulators of the GAL system: Gale, Gal80, and Mig1. This led to a 41% increase in flux through the galactose utilization pathway compared with the wild-type strain. This is of significant interest within the field of biotechnology since galactose is present in many industrial...

  8. A functional brain-derived neurotrophic factor (BDNF) gene variant increases the risk of moderate-to-severe allergic rhinitis.

    Jin, Peng; Andiappan, Anand Kumar; Quek, Jia Min; Lee, Bernett; Au, Bijin; Sio, Yang Yie; Irwanto, Astrid; Schurmann, Claudia; Grabe, Hans Jörgen; Suri, Bani Kaur; Matta, Sri Anusha; Westra, Harm-Jan; Franke, Lude; Esko, Tonu; Sun, Liangdan; Zhang, Xuejun; Liu, Hong; Zhang, Furen; Larbi, Anis; Xu, Xin; Poidinger, Michael; Liu, Jianjun; Chew, Fook Tim; Rotzschke, Olaf; Shi, Li; Wang, De Yun

    2015-06-01

    Brain-derived neurotrophic factor (BDNF) is a secretory protein that has been implicated in the pathogenesis of allergic rhinitis (AR), atopic asthma, and eczema, but it is currently unknown whether BDNF polymorphisms influence susceptibility to moderate-to-severe AR. We sought to identify disease associations and the functional effect of BDNF genetic variants in patients with moderate-to-severe AR. Tagging single nucleotide polymorphisms (SNPs) spanning the BDNF gene were selected from the human HapMap Han Chinese from Beijing (CHB) data set, and associations with moderate-to-severe AR were assessed in 2 independent cohorts of Chinese patients (2216 from Shandong province and 1239 living in Singapore). The functional effects of the BDNF genetic variants were determined by using both in vitro and ex vivo assays. The tagging SNP rs10767664 was significantly associated with the risk of moderate-to-severe AR in both Singapore Chinese (P = .0017; odds ratio, 1.324) and Shandong Chinese populations (P = .039; odds ratio, 1.180). The coding nonsynonymous SNP rs6265 was in perfect linkage with rs10767664 and conferred increased BDNF protein secretion by a human cell line in vitro. Subjects bearing the AA genotype of rs10767664 exhibited increased risk of moderate-to-severe AR and displayed increased BDNF protein and total IgE levels in plasma. Using a large-scale expression quantitative trait locus study, we demonstrated that BDNF SNPs are significantly associated with altered BDNF concentrations in peripheral blood. A common genetic variant of the BDNF gene is associated with increased risk of moderate-to-severe AR, and the AA genotype is associated with increased BDNF mRNA levels in peripheral blood. Together, these data indicate that functional BDNF gene variants increase the risk of moderate-to-severe AR. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  9. Cells involved in extracellular matrix remodeling after acute myocardial infarction

    Garcia, Larissa Ferraz [Faculdade de Medicina do ABC, Santo André, SP (Brazil); Mataveli, Fábio D’Aguiar [Universidade Federal de São Paulo, São Paulo, SP (Brazil); Mader, Ana Maria Amaral Antônio; Theodoro, Thérèse Rachell [Faculdade de Medicina do ABC, Santo André, SP (Brazil); Justo, Giselle Zenker; Pinhal, Maria Aparecida da Silva [Universidade Federal de São Paulo, São Paulo, SP (Brazil)

    2015-07-01

    Evaluate the effects of VEGF{sub 165} gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF{sub 165} treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF{sub 165}. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF{sub 165}, suggesting greater tissue differentiation. The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF{sub 165} seems to provide a protective effect in the treatment of acute myocardial infarct.

  10. Cells involved in extracellular matrix remodeling after acute myocardial infarction

    Garcia, Larissa Ferraz; Mataveli, Fábio D’Aguiar; Mader, Ana Maria Amaral Antônio; Theodoro, Thérèse Rachell; Justo, Giselle Zenker; Pinhal, Maria Aparecida da Silva

    2015-01-01

    Evaluate the effects of VEGF_1_6_5 gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF_1_6_5 treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF_1_6_5. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF_1_6_5, suggesting greater tissue differentiation. The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF_1_6_5 seems to provide a protective effect in the treatment of acute myocardial infarct

  11. Dermal extracellular lipid in birds.

    Stromberg, M W; Hinsman, E J; Hullinger, R L

    1990-01-01

    A light and electron microscopic study of the skin of domestic chickens, seagulls, and antarctic penguins revealed abundant extracellular dermal lipid and intracellular epidermal lipid. Dermal lipid appeared ultrastructurally as extracellular droplets varying from less than 1 micron to more than 25 microns in diameter. The droplets were often irregularly contoured, sometimes round, and of relatively low electron density. Processes of fibrocytes were often seen in contact with extracellular lipid droplets. Sometimes a portion of such a droplet was missing, and this missing part appeared to have been "digested away" by the cell process. In places where cells or cell processes are in contact with fact droplets, there are sometimes extracellular membranous whorls or fragments which have been associated with the presence of fatty acids. Occasionally (in the comb) free fat particles were seen in intimate contact with extravasated erythrocytes. Fat droplets were seen in the lumen of small dermal blood and lymph vessels. We suggest that the dermal extracellular lipid originates in the adipocyte layer and following hydrolysis the free fatty acids diffuse into the epidermis. Here they become the raw material for forming the abundant neutral lipid contained in many of the epidermal cells of both birds and dolphins. The heretofore unreported presence and apparently normal utilization of abundant extracellular lipid in birds, as well as the presence of relatively large droplets of neutral lipid in dermal vessels, pose questions which require a thorough reappraisal of present concepts of the ways in which fat is distributed and utilized in the body.

  12. Hematopoietically expressed homeobox (HHEX) gene polymorphism (rs5015480) is associated with increased risk of gestational diabetes mellitus.

    Tarnowski, M; Malinowski, D; Safranow, K; Dziedziejko, V; Czerewaty, M; Pawlik, A

    2017-06-01

    Gestational diabetes mellitus (GDM) is a metabolic disorder that occurs during pregnancy. HHEX and PROX1 are genetic loci associated with diabetes mellitus type 2. HHEX and PROX1 play significant roles in carbohydrate intolerance and diabetes because these transcription factors may be involved in the regulation of insulin secretion and in glucose and lipid metabolism. The aim of this study was to examine the association between HHEX (rs5015480) and PROX1 (rs340874) gene polymorphisms and GDM. This study included 204 pregnant women with GDM and 207 pregnant women with the normal glucose tolerance (NGT). The diagnosis of GDM was based on a 75-g oral glucose tolerance test at 24-28 weeks' gestation. There was a statistically significant prevalence of the HHEX rs5015480 CC genotype and C allele among women with GDM (C vs T allele, p = 0.021, odds ratio OR = 1.40, 95% CI: 1.05-1.87). Statistically significant higher increase of body mass and BMI during pregnancy was found in women with the HHEX rs5015480 CC genotype. The results of our study suggest an association between the HHEX gene rs5015480 polymorphism and risk of GDM. The HHEX gene rs5015480 C allele may be a risk allele of GDM that is associated with increased BMI during pregnancy. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Over expression of Zmda1-1 gene increases seed mass of corn ...

    Genetic engineering of seed size and increasing biomass in crop plants has an important significant contribution to the world. Arabidopsis DA1 is one of the key factors that negatively control seed and organ size by restricting the period of cell proliferation, and the mutant of Arabidopsis DA1, da1-1 (DA1R358K) can ...

  14. Increased Rrm2 gene dosage reduces fragile site breakage and prolongs survival of ATR mutant mice

    Lopez-Contreras, Andres J; Specks, Julia; Barlow, Jacqueline H

    2015-01-01

    In Saccharomyces cerevisiae, absence of the checkpoint kinase Mec1 (ATR) is viable upon mutations that increase the activity of the ribonucleotide reductase (RNR) complex. Whether this pathway is conserved in mammals remains unknown. Here we show that cells from mice carrying extra alleles of the...

  15. Influenza A virus NS1 gene mutations F103L and M106I increase replication and virulence

    Ping Jihui

    2011-01-01

    Full Text Available Abstract Background To understand the evolutionary steps required for a virus to become virulent in a new host, a human influenza A virus (IAV, A/Hong Kong/1/68(H3N2 (HK-wt, was adapted to increased virulence in the mouse. Among eleven mutations selected in the NS1 gene, two mutations F103L and M106I had been previously detected in the highly virulent human H5N1 isolate, A/HK/156/97, suggesting a role for these mutations in virulence in mice and humans. Results To determine the selective advantage of these mutations, reverse genetics was used to rescue viruses containing each of the NS1 mouse adapted mutations into viruses possessing the HK-wt NS1 gene on the A/PR/8/34 genetic backbone. Both F103L and M106I NS1 mutations significantly enhanced growth in vitro (mouse and canine cells and in vivo (BALB/c mouse lungs as well as enhanced virulence in the mouse. Only the M106I NS1 mutation enhanced growth in human cells. Furthermore, these NS1 mutations enhanced early viral protein synthesis in MDCK cells and showed an increased ability to replicate in mouse interferon β (IFN-β pre-treated mouse cells relative to rPR8-HK-NS-wt NS1. The double mutant, rPR8-HK-NS-F103L + M106I, demonstrated growth attenuation late in infection due to increased IFN-β induction in mouse cells. We then generated a rPR8 virus possessing the A/HK/156/97 NS gene that possesses 103L + 106I, and then rescued the L103F + I106M mutant. The 103L + 106I mutations increased virulence by >10 fold in BALB/c mice. We also inserted the avian A/Ck/Beijing/1/95 NS1 gene (the source lineage of the A/HK/156/97 NS1 gene that possesses 103L + 106I, onto the A/WSN/33 backbone and then generated the L103F + I106M mutant. None of the H5N1 and H9N2 NS containing viruses resulted in increased IFN-β induction. The rWSN-A/Ck/Beijing/1/95-NS1 gene possessing 103L and 106I demonstrated 100 fold enhanced growth and >10 fold enhanced virulence that was associated with increased tropism for lung

  16. Reduced intracellular c-di-GMP content increases expression of quorum sensing-regulated genes in Pseudomonas aeruginosa

    Chua, Song Lin; Liu, Yang; Li, Yingying

    2017-01-01

    Cyclic-di-GMP (c-di-GMP) is an intracellular secondary messenger which controls the biofilm life cycle in many bacterial species. High intracellular c-di-GMP content enhances biofilm formation via the reduction of motility and production of biofilm matrix, while low c-di-GMP content in biofilm...... cells leads to increased motility and biofilm dispersal. While the effect of high c-di-GMP levels on bacterial lifestyles is well studied, the physiology of cells at low c-di-GMP levels remains unclear. Here, we showed that Pseudomonas aeruginosa cells with high and low intracellular c-di-GMP contents...... possessed distinct transcriptome profiles. There were 535 genes being upregulated and 432 genes downregulated in cells with low c-di-GMP, as compared to cells with high c-di-GMP. Interestingly, both rhl and pqs quorum-sensing (QS) operons were expressed at higher levels in cells with low intracellular c-di-GMP...

  17. Silencing of Soybean Raffinose Synthase Gene Reduced Raffinose Family Oligosaccharides and Increased True Metabolizable Energy of Poultry Feed

    Michelle F. Valentine

    2017-05-01

    Full Text Available Soybean [Glycine max (L. Merr.] is the number one oil and protein crop in the United States, but the seed contains several anti-nutritional factors that are toxic to both humans and livestock. RNA interference technology has become an increasingly popular technique in gene silencing because it allows for both temporal and spatial targeting of specific genes. The objective of this research is to use RNA-mediated gene silencing to down-regulate the soybean gene raffinose synthase 2 (RS2, to reduce total raffinose content in mature seed. Raffinose is a trisaccharide that is indigestible to humans and monogastric animals, and as monogastric animals are the largest consumers of soy products, reducing raffinose would improve the nutritional quality of soybean. An RNAi construct targeting RS2 was designed, cloned, and transformed to the soybean genome via Agrobacterium-mediated transformation. Resulting plants were analyzed for the presence and number of copies of the transgene by PCR and Southern blot. The efficiency of mRNA silencing was confirmed by real-time quantitative PCR. Total raffinose content was determined by HPLC analysis. Transgenic plant lines were recovered that exhibited dramatically reduced levels of raffinose in mature seed, and these lines were further analyzed for other phenotypes such as development and yield. Additionally, a precision-fed rooster assay was conducted to measure the true metabolizable energy (TME in full-fat soybean meal made from the wild-type or transgenic low-raffinose soybean lines. Transgenic low-raffinose soy had a measured TME of 2,703 kcal/kg, an increase as compared with 2,411 kcal/kg for wild-type. As low digestible energy is a major limiting factor in the percent of soybean meal that can be used in poultry diets, these results may substantiate the use of higher concentrations of low-raffinose, full-fat soy in formulated livestock diets.

  18. Silencing of Soybean Raffinose Synthase Gene Reduced Raffinose Family Oligosaccharides and Increased True Metabolizable Energy of Poultry Feed

    Valentine, Michelle F.; De Tar, Joann R.; Mookkan, Muruganantham; Firman, Jeffre D.; Zhang, Zhanyuan J.

    2017-01-01

    Soybean [Glycine max (L.) Merr.] is the number one oil and protein crop in the United States, but the seed contains several anti-nutritional factors that are toxic to both humans and livestock. RNA interference technology has become an increasingly popular technique in gene silencing because it allows for both temporal and spatial targeting of specific genes. The objective of this research is to use RNA-mediated gene silencing to down-regulate the soybean gene raffinose synthase 2 (RS2), to reduce total raffinose content in mature seed. Raffinose is a trisaccharide that is indigestible to humans and monogastric animals, and as monogastric animals are the largest consumers of soy products, reducing raffinose would improve the nutritional quality of soybean. An RNAi construct targeting RS2 was designed, cloned, and transformed to the soybean genome via Agrobacterium-mediated transformation. Resulting plants were analyzed for the presence and number of copies of the transgene by PCR and Southern blot. The efficiency of mRNA silencing was confirmed by real-time quantitative PCR. Total raffinose content was determined by HPLC analysis. Transgenic plant lines were recovered that exhibited dramatically reduced levels of raffinose in mature seed, and these lines were further analyzed for other phenotypes such as development and yield. Additionally, a precision-fed rooster assay was conducted to measure the true metabolizable energy (TME) in full-fat soybean meal made from the wild-type or transgenic low-raffinose soybean lines. Transgenic low-raffinose soy had a measured TME of 2,703 kcal/kg, an increase as compared with 2,411 kcal/kg for wild-type. As low digestible energy is a major limiting factor in the percent of soybean meal that can be used in poultry diets, these results may substantiate the use of higher concentrations of low-raffinose, full-fat soy in formulated livestock diets. PMID:28559898

  19. Western environment/lifestyle is associated with increased genome methylation and decreased gene expression in Chinese immigrants living in Australia.

    Zhang, Guicheng; Wang, Kui; Schultz, Ennee; Khoo, Siew-Kim; Zhang, Xiaopeng; Annamalay, Alicia; Laing, Ingrid A; Hales, Belinda J; Goldblatt, Jack; Le Souëf, Peter N

    2016-01-01

    Several human diseases and conditions are disproportionally distributed in the world with a significant "Western-developed" vs. "Eastern-developing" gradient. We compared genome-wide DNA methylation of peripheral blood mononuclear cells in 25 newly arrived Chinese immigrants living in a Western environment for less than 6 months ("Newly arrived") with 23 Chinese immigrants living in the Western environment for more than two years ("Long-term") with a mean of 8.7 years, using the Infinium HumanMethylation450 BeadChip. In a sub-group of both subject groups (n = 12 each) we also investigated genome-wide gene expression using a Human HT-12 v4 expression beadChip. There were 62.5% probes among the total number of 382,250 valid CpG sites with greater mean Beta (β) in "Long-term" than in "Newly arrived". In the regions of CpG islands and gene promoters, compared with the CpG sites in all other regions, lower percentages of CpG sites with mean methylation levels in "Long-term" greater than "Newly arrived" were observed, but still >50%. The increase of methylation was associated with a general decrease of gene expression in Chinese immigrants living in the Western environment for a longer period of time. After adjusting for age, gender and other confounding factors the findings remained. Chinese immigrants living in Australia for a longer period of time have increased overall genome methylation and decreased overall gene expression compared with newly arrived immigrants. © 2015 Wiley Periodicals, Inc.

  20. Increased PRPP synthetase activity in cultured rat hepatoma cells containing mutations in the hypoxanthine-guanine phosphoribosyltransferase gene.

    Graf, L H; McRoberts, J A; Harrison, T M; Martin, D W

    1976-07-01

    Nine independently derived clones of mutagenized rat hepatoma cells selected for resistance to 6-mercaptopurine (6-MP) or 6-thioguanine (6-ThioG) have been isolated. Each has severely reduced catalytic activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and seven of them possess significantly increased activities of phosphoribosylpyrophosphate (PRPP) synthetase. The degrees of elevations of PRPP synthetase activities do not correlate with the degrees of deficiencies of HPRT activities. The cells from one of these clones, 1020/12, posses 40% of the normal HPRT catalytic activity and overproduce purines. We have extensively examined the cells from this clone. Immunotration studies of 1020/12 cells indicate that there is a mutation in the structural gene for HPRT. Although they possess increased specific catalytic activities of the enzyme. PRPP synthetase, the catalytic parameters, heat stability, and isoelectric pH of PRPP synthetase from 1020/12 cells are indistinguishable from those of the enzyme from wild-type cells. The cause of purine overproduction by 1020/12 cells appears to be the elevated PRPP synthetase activity, rather than a PRPP "sparing" effect stemming from reduced HPRT activity. Support for this idea is provided by the observation that the complete loss of HPRT activity in a clone derived from 1020/12 cells does not further enhance the levels of PRPP synthetase or purine overproduction. We propose that the elevated levels of PRPP synthetase activity in these HPRT deficient cells result from a mutational event in the structural gene for HPRT, and that this causes the disruption of a previously undescribed regulatory function of this gene on the expression of the PRPP synthetase gene.

  1. Acute resistance exercise reduces increased gene expression in muscle atrophy of ovariectomised arthritic rats

    Roberto Furlanetto Jr

    2017-02-01

    Full Text Available Objective: We studied the effect of resistance exercise (RE on mRNA levels of atrogin-1, MuRF-1, and myostatin in the gastrocnemius muscle of arthritic rats after loss of ovarian function (LOF. Material and methods : Thirty female Wistar rats (nine weeks old, 195.3 ±17.4 grams were randomly allocated into five groups: control group (CT-Sham; n = 6; group with rheumatoid arthritis (RA; n = 6; group with rheumatoid arthritis subjected to RE (RAEX; n = 6; ovariectomy group with rheumatoid arthritis (RAOV; n = 6; and an ovariectomy group with rheumatoid arthritis subjected to RE (RAOVEX; n = 6. After 15 days of intra-articular injections with Met-BSA the animals were subjected to RE and six hours after workout were euthanised. Results : The rheumatoid arthritis provoked reduction in the cross-sectional area (CSA of muscle fibres, but the CSA was lower in the RAOV when compared to the RA groups. Skeletal muscle atrogin-1 mRNA level was increased in arthritic rats (RA and RAOV, but the atrogin-1 level was higher in RAOV group when compared to other arthritic groups. The Muscle MuRF-1 mRNA level was also increased in the RAOV group. The increased atrogin-1 and MuRF-1 mRNA levels were lower in the RAOVEX group than in the RAOV group. The myostatin mRNA level was similar in all groups, except for the RAOVEX group, in which it was lower than the other groups. Conclusions : LOF results in increased loss of skeletal muscle-related ubiquitin ligases (atrogin-1 and MuRF-1. However, the RE reduces the atrogin-1, MuRF-1, and myostatin mRNA levels in muscle of arthritic rats affected by LOF.

  2. Lipid lowering and HDL raising gene transfer increase endothelial progenitor cells, enhance myocardial vascularity, and improve diastolic function.

    Stephanie C Gordts

    Full Text Available BACKGROUND: Hypercholesterolemia and low high density lipoprotein (HDL cholesterol contribute to coronary heart disease but little is known about their direct effects on myocardial function. Low HDL and raised non-HDL cholesterol levels carried increased risk for heart failure development in the Framingham study, independent of any association with myocardial infarction. The objective of this study was to test the hypothesis that increased endothelial progenitor cell (EPC number and function after lipid lowering or HDL raising gene transfer in C57BL/6 low density lipoprotein receptor deficient (LDLr(-/- mice may be associated with an enhanced relative vascularity in the myocardium and an improved cardiac function. METHODOLOGY/PRINCIPAL FINDINGS: Lipid lowering and HDL raising gene transfer were performed using the E1E3E4-deleted LDLr expressing adenoviral vector AdLDLr and the human apolipoprotein A-I expressing vector AdA-I, respectively. AdLDLr transfer in C57BL/6 LDLr(-/- mice resulted in a 2.0-fold (p<0.05 increase of the circulating number of EPCs and in an improvement of EPC function as assessed by ex vivo EPC migration and EPC adhesion. Capillary density and relative vascularity in the myocardium were 28% (p<0.01 and 22% (p<0.05 higher, respectively, in AdLDLr mice compared to control mice. The peak rate of isovolumetric relaxation was increased by 12% (p<0.05 and the time constant of isovolumetric relaxation was decreased by 14% (p<0.05 after AdLDLr transfer. Similarly, HDL raising gene transfer increased EPC number and function and raised both capillary density and relative vascularity in the myocardium by 24% (p<0.05. The peak rate of isovolumetric relaxation was increased by 16% (p<0.05 in AdA-I mice compared to control mice. CONCLUSIONS/SIGNIFICANCE: Both lipid lowering and HDL raising gene transfer have beneficial effects on EPC biology, relative myocardial vascularity, and diastolic function. These findings raise concerns over the

  3. The role of extracellular vesicles in phenotypic cancer transformation:

    Kralj-Iglič, Veronika; Ogorevc, Eva; Veranič, Peter

    2013-01-01

    Background. Cancer has traditionally been considered as a disease resulting from gene mutations. New findings in biology are challenging gene-centered explanations of cancer progression and redirecting them to the non-genetic origins of tumorigenicity. It has become clear that intercellular communication plays a crucial role in cancer progression. Among the most intriguing ways of intercellular communication is that via extracellular vesicles (EVs). EVs are membrane structures released from v...

  4. Recent urbanization in China is correlated with a Westernized microbiome encoding increased virulence and antibiotic resistance genes.

    Winglee, Kathryn; Howard, Annie Green; Sha, Wei; Gharaibeh, Raad Z; Liu, Jiawu; Jin, Donghui; Fodor, Anthony A; Gordon-Larsen, Penny

    2017-09-15

    Urbanization is associated with an increased risk for a number of diseases, including obesity, diabetes, and cancer, which all also show associations with the microbiome. While microbial community composition has been shown to vary across continents and in traditional versus Westernized societies, few studies have examined urban-rural differences in neighboring communities within a single country undergoing rapid urbanization. In this study, we compared the gut microbiome, plasma metabolome, dietary habits, and health biomarkers of rural and urban people from a single Chinese province. We identified significant differences in the microbiota and microbiota-related plasma metabolites in rural versus recently urban subjects from the Hunan province of China. Microbes with higher relative abundance in Chinese urban samples have been associated with disease in other studies and were substantially more prevalent in the Human Microbiome Project cohort of American subjects. Furthermore, using whole metagenome sequencing, we found that urbanization was associated with a loss of microbial diversity and changes in the relative abundances of Viruses, Archaea, and Bacteria. Gene diversity, however, increased with urbanization, along with the proportion of reads associated with antibiotic resistance and virulence, which were strongly correlated with the presence of Escherichia and Shigella. Our data suggest that urbanization has produced convergent evolution of the gut microbial composition in American and urban Chinese populations, resulting in similar compositional patterns of abundant microbes through similar lifestyles on different continents, including a loss of potentially beneficial bacteria and an increase in potentially harmful genes via increased relative abundance of Escherichia and Shigella.

  5. Chronic REM-sleep deprivation of rats elevates metabolic rate and increases UCP1 gene expression in brown adipose tissue.

    Koban, Michael; Swinson, Kevin L

    2005-07-01

    A cluster of unique pathologies progressively develops during chronic total- or rapid eye movement-sleep deprivation (REM-SD) of rats. Two prominent and readily observed symptoms are hyperphagia and decline in body weight. For body weight to be lost despite a severalfold increase in food consumption suggests that SD elevates metabolism as the subject enters a state of negative energy balance. To test the hypothesis that mediation of this hypermetabolism involves increased gene expression of uncoupling protein-1 (UCP1), which dissipates the thermodynamic energy of the mitochondrial proton-motive force as heat instead of ATP formation in brown adipose tissue (BAT), we 1) established the time course and magnitude of change in metabolism by measuring oxygen consumption, 2) estimated change in UCP1 gene expression in BAT by RT-PCR and Western blot, and 3) assayed serum leptin because of its role in regulating energy balance and food intake. REM-SD of male Sprague-Dawley rats was enforced for 20 days with the platform (flowerpot) method, wherein muscle atonia during REM sleep causes contact with surrounding water and awakens it. By day 20, rats more than doubled food consumption while losing approximately 11% of body weight; metabolism rose to 166% of baseline with substantial increases in UCP1 mRNA and immunoreactive UCP1 over controls; serum leptin decreased and remained suppressed. The decline in leptin is consistent with the hyperphagic response, and we conclude that one of the mediators of elevated metabolism during prolonged REM-SD is increased gene expression of UCP1 in BAT.

  6. Heme oxygenase-1 gene expression modulates angiotensin II-induced increase in blood pressure.

    Yang, Liming; Quan, Shuo; Nasjletti, Alberto; Laniado-Schwartzman, Michal; Abraham, Nader G

    2004-06-01

    The heme-heme oxygenase (HO) system has been implicated in the regulation of vascular reactivity and blood pressure. This study examines the notion that overexpression of HO decreases pressor responsiveness to angiotensin II (Ang II). Five-day-old Sprague-Dawley rats received an intraleft ventricular injection of approximately 5x10(9) cfu/mL of retroviruses containing human HO-1 sense (LSN-HHO-1), rat HO-1 antisense (LSN-RHO-1-AS), or control retrovirus (LXSN). Three months later, rats were instrumented with femoral arterial and venous catheters for mean arterial pressure (MAP) determination and Ang II administration, respectively. Rats injected with LSN-HHO-1, but not with LXSN, expressed human HO-1 mRNA and protein in several tissues. BP increased with administration of Ang II in rats expressing and not expressing human HO-1. However, the Ang II-induced pressor response (mm Hg) in LSN-HHO-1 rats (16+/-3, 27+/-3, and 38+/-3 at 0.5, 2, and 10 ng) was surpassed (PHHO-1 rats with the HO inhibitor tin mesoporphyrin (SnMP) enhanced (P<0.05) the Ang II-induced pressor response to a level not different from that observed in LXSN rats. Rats injected with LSN-RHO-1-AS showed a decrease in renal HO-1 protein expression and HO activity relative to control LXSN rats. Administration of Ang II (0.1 to 2 ng) caused small (4 to 5 mm Hg) but significant increases in MAP in rats injected with LSN-RHO-1-AS (P<0.05) compared with rats injected with LXSN. These data demonstrate that overexpression of HO-1 brings about a reduction in pressor responsiveness to Ang II, which is most likely due to increased generation of an HO-1 product, presumably CO, with the ability to inhibit vascular reactivity to constrictor stimuli.

  7. It Remains Unknown Whether Filaggrin Gene Mutations Evolved to Increase Cutaneous Synthesis of Vitamin D

    Thyssen, Jacob P; Elias, Peter M

    2017-01-01

    encountered in Northern Europeans. Importantly, FLG mutation carriers have 10% increased serum vitamin D concentrations compared to controls. Based on these observations, we have proposed that this latitude-dependent gradient of FLG mutations across Europe, Asia and Africa could have provided an evolutionary...... would have provided an evolutionary advantage for modern humans, living in the far North of Europe, where little UV-B penetrates the atomosphere. In a recent article, it was concluded not only that the UVB-Vitamin D3 hypothesis is invalid, but also that FLG genetic variations, including loss...

  8. Sequence-Specific Targeting of Bacterial Resistance Genes Increases Antibiotic Efficacy

    Wong, Michael; Daly, Seth M.; Greenberg, David E.; Toprak, Erdal

    2016-01-01

    The lack of effective and well-tolerated therapies against antibiotic-resistant bacteria is a global public health problem leading to prolonged treatment and increased mortality. To improve the efficacy of existing antibiotic compounds, we introduce a new method for strategically inducing antibiotic hypersensitivity in pathogenic bacteria. Following the systematic verification that the AcrAB-TolC efflux system is one of the major determinants of the intrinsic antibiotic resistance levels in Escherichia coli, we have developed a short antisense oligomer designed to inhibit the expression of acrA and increase antibiotic susceptibility in E. coli. By employing this strategy, we can inhibit E. coli growth using 2- to 40-fold lower antibiotic doses, depending on the antibiotic compound utilized. The sensitizing effect of the antisense oligomer is highly specific to the targeted gene’s sequence, which is conserved in several bacterial genera, and the oligomer does not have any detectable toxicity against human cells. Finally, we demonstrate that antisense oligomers improve the efficacy of antibiotic combinations, allowing the combined use of even antagonistic antibiotic pairs that are typically not favored due to their reduced activities. PMID:27631336

  9. Long term repeated prescribed burning increases evenness in the basidiomycete laccase gene pool in forest soils.

    Artz, Rebekka R E; Reid, Eileen; Anderson, Ian C; Campbell, Colin D; Cairney, John W G

    2009-03-01

    Repeated prescribed burning alters the biologically labile fraction of nutrients and carbon of soil organic matter (SOM). Using a long-term (30 years) repeated burning experiment where burning has been carried out at a 2- or 4-year frequency, we analysed the effect of prescribed burning on gross potential C turnover rates and phenol oxidase activity in relation to shifts in SOM composition as observed using Fourier-transform infrared spectroscopy. In tandem, we assessed the genetic diversity of basidiomycete laccases. While the overall effect of burning was a decline in phenol oxidase activity, Shannon diversity and evenness of laccases was significantly higher in burned sites. Co-correspondence analysis of SOM composition and laccase operational taxonomic unit frequency data also suggested a strong correlation. While this correlation could indicate that the observed increase in laccase genetic diversity due to burning is due to increased resource diversity, a temporal replacement of the most abundant members of the assembly by an otherwise dormant pool of fungi cannot be excluded. As such, our results fit the intermediate disturbance hypothesis. Effects were stronger in plots burned in 2-year rotations, suggesting that the 4-year burn frequency may be a more sustainable practice to ensure the long-term stability of C cycling in such ecosystems.

  10. Tibial loading increases osteogenic gene expression and cortical bone volume in mature and middle-aged mice.

    Matthew J Silva

    Full Text Available There are conflicting data on whether age reduces the response of the skeleton to mechanical stimuli. We examined this question in female BALB/c mice of different ages, ranging from young to middle-aged (2, 4, 7, 12 months. We first assessed markers of bone turnover in control (non-loaded mice. Serum osteocalcin and CTX declined significantly from 2 to 4 months (p<0.001. There were similar age-related declines in tibial mRNA expression of osteoblast- and osteoclast-related genes, most notably in late osteoblast/matrix genes. For example, Col1a1 expression declined 90% from 2 to 7 months (p<0.001. We then assessed tibial responses to mechanical loading using age-specific forces to produce similar peak strains (-1300 µε endocortical; -2350 µε periosteal. Axial tibial compression was applied to the right leg for 60 cycles/day on alternate days for 1 or 6 weeks. qPCR after 1 week revealed no effect of loading in young (2-month mice, but significant increases in osteoblast/matrix genes in older mice. For example, in 12-month old mice Col1a1 was increased 6-fold in loaded tibias vs. controls (p = 0.001. In vivo microCT after 6 weeks revealed that loaded tibias in each age group had greater cortical bone volume (BV than contralateral control tibias (p<0.05, due to relative periosteal expansion. The loading-induced increase in cortical BV was greatest in 4-month old mice (+13%; p<0.05 vs. other ages. In summary, non-loaded female BALB/c mice exhibit an age-related decline in measures related to bone formation. Yet when subjected to tibial compression, mice from 2-12 months have an increase in cortical bone volume. Older mice respond with an upregulation of osteoblast/matrix genes, which increase to levels comparable to young mice. We conclude that mechanical loading of the tibia is anabolic for cortical bone in young and middle-aged female BALB/c mice.

  11. Alpha-synuclein gene ablation increases docosahexaenoic acid incorporation and turnover in brain phospholipids

    Golovko, Mikhail Y; Rosenberger, Thad A; Feddersen, Søren

    2007-01-01

    Previously, we demonstrated that ablation of alpha-synuclein (Snca) reduces arachidonate (20:4n-6) turnover in brain phospholipids through modulation of an endoplasmic reticulum-localized acyl-CoA synthetase (Acsl). The effect of Snca ablation on docosahexaenoic acid (22:6n-3) metabolism is unknown...... and turnover in ethanolamine glycerophospholipid, phosphatidylserine, and phosphatidylinositol pools. Increased 22:6n-3-CoA mass was not the result of altered Acsl activity, which was unaffected by the absence of Snca. While Snca bound 22:6n-3, Kd = 1.0 +/- 0.5 micromol/L, it did not bind 22:6n-3-Co...

  12. A Novel FOXE1 Mutation (R73S) in Bamforth–Lazarus Syndrome Causing Increased Thyroidal Gene Expression

    Carré, Aurore; Hamza, Rasha T.; Kariyawasam, Dulanjalee; Guillot, Loïc; Teissier, Raphaël; Tron, Elodie; Castanet, Mireille; Dupuy, Corinne; El Kholy, Mohamed; Polak, Michel

    2014-01-01

    Background: Homozygous loss-of-function mutations in the FOXE1 gene have been reported in several patients with partial or complete Bamforth–Lazarus syndrome: congenital hypothyroidism (CH) with thyroid dysgenesis (usually athyreosis), cleft palate, spiky hair, with or without choanal atresia, and bifid epiglottis. Here, our objective was to evaluate potential functional consequences of a FOXE1 mutation in a patient with a similar clinical phenotype. Methods: FOXE1 was sequenced in eight patients with thyroid dysgenesis and cleft palate. Transient transfection was performed in HEK293 cells using the thyroglobulin (TG) and thyroid peroxidase (TPO) promoters in luciferase reporter plasmids to assess the functional impact of the FOXE1 mutations. Primary human thyrocytes transfected with wild type and mutant FOXE1 served to assess the impact of the mutation on endogenous TG and TPO expression. Results: We identified and characterized the function of a new homozygous FOXE1 missense mutation (p.R73S) in a boy with a typical phenotype (athyreosis, cleft palate, and partial choanal atresia). This new mutation located within the forkhead domain was inherited from the heterozygous healthy consanguineous parents. In vitro functional studies in HEK293 cells showed that this mutant gene enhanced the activity of the TG and TPO gene promoters (1.5-fold and 1.7-fold respectively vs. wild type FOXE1; p<0.05), unlike the five mutations previously reported in Bamforth–Lazarus syndrome. The gain-of-function effect of the FOXE1-p.R73S mutant gene was confirmed by an increase in endogenous TG production in primary human thyrocytes. Conclusion: We identified a new homozygous FOXE1 mutation responsible for enhanced expression of the TG and TPO genes in a boy whose phenotype is similar to that reported previously in patients with loss-of-function FOXE1 mutations. This finding further delineates the role for FOXE1 in both thyroid and palate development, and shows that enhanced gene

  13. Endocannabinoid receptor 1 gene variations increase risk for obesity and modulate body mass index in European populations

    Benzinou, Michael; Chèvre, Jean-Claude; Ward, Kirsten J

    2008-01-01

    The therapeutic effects of cannabinoid receptor blockade on obesity-associated phenotypes underline the importance of the endocannabinoid pathway on the energy balance. Using a staged-approach, we examined the contribution of the endocannabinoid receptor 1 gene (CNR1) on obesity and body mass ind...... variations increase the risk for obesity and modulate BMI in our European population. As CB1 is a drug target for obesity, a pharmacogenetic analysis of the endocannabinoid blockade obesity treatment may be of interest to identify best responders....

  14. Perfusion of veins at arterial pressure increases the expression of KLF5 and cell cycle genes in smooth muscle cells

    Amirak, Emre; Zakkar, Mustafa; Evans, Paul C.; Kemp, Paul R.

    2010-01-01

    Vascular smooth muscle cell (VSMC) proliferation remains a major cause of veno-arterial graft failure. We hypothesised that exposure of venous SMCs to arterial pressure would increase KLF5 expression and that of cell cycle genes. Porcine jugular veins were perfused at arterial or venous pressure in the absence of growth factors. The KLF5, c-myc, cyclin-D and cyclin-E expression were elevated within 24 h of perfusion at arterial pressure but not at venous pressure. Arterial pressure also reduced the decline in SM-myosin heavy chain expression. These data suggest a role for KLF5 in initiating venous SMCs proliferation in response to arterial pressure.

  15. RNAi-mediated knockdown of serine protease inhibitor genes increases the mortality of Plutella xylostella challenged by destruxin A.

    Han, Pengfei; Fan, Jiqiao; Liu, Yu; Cuthbertson, Andrew G S; Yan, Shaoqiao; Qiu, Bao-Li; Ren, Shunxiang

    2014-01-01

    Destruxin A is a mycotoxin that is secreted by entomopathogenic fungi which has a broad-spectrum insecticidal effect. Previous transcript and protein profiling analysis showed that destruxin A has significant effects on the expression of serine protease inhibitor genes (serpin-2, 4, 5) in the larvae of Plutella xylostella. In the current study, we aimed to understand the role of serpins under application of destruxin A. We obtained two full-length cDNA sequences of P. xylostella serpins, named serpin-4 and serpin-5, and cloned the serpin-2 gene whose full-length has already been published. Phylogenetic analysis indicated that these two serpin genes were highly clustered with other serpins associated with the immune response in other insects. The temporal and spatial expression of serpin-2, serpin-4 and serpin-5 were determined to be the highest in the fat body and hemolymph of 4th larval stage using qRT-PCR and western blot detection techniques. RNA interference (RNAi) mediated knockdown of P. xylostella serpin genes was carried out by microinjection of double-stranded RNA (dsRNA). The expression levels of serpins decreased significantly after RNAi. Results showed that the depletion of serpins induced cecropins expression, increased phenoloxidase (PO) activity, body melanization and mortality in the larvae of P. xylostella under the same lethal concentration of destruxin A. The superimposed effects of serpins RNAi were similar with the destruxin A treatment upon mortality of P. xylostella larvae. We discovered for the first time that serpins play indispensable role in P. xylostella when challenged by destruxin A and deduced the possible function mechanism of destruxin A. Our findings are conducive to fully understanding the potential insecticidal mechanism of destruxin A and constitute a well-defined potential molecular target for novel insecticides.

  16. RNAi-Mediated Knockdown of Serine Protease Inhibitor Genes Increases the Mortality of Plutella xylostella Challenged by Destruxin A

    Han, Pengfei; Fan, Jiqiao; Liu, Yu; Cuthbertson, Andrew G. S.; Yan, Shaoqiao; Qiu, Bao-Li; Ren, Shunxiang

    2014-01-01

    Destruxin A is a mycotoxin that is secreted by entomopathogenic fungi which has a broad-spectrum insecticidal effect. Previous transcript and protein profiling analysis showed that destruxin A has significant effects on the expression of serine protease inhibitor genes (serpin-2, 4, 5) in the larvae of Plutella xylostella. In the current study, we aimed to understand the role of serpins under application of destruxin A. We obtained two full-length cDNA sequences of P. xylostella serpins, named serpin-4 and serpin-5, and cloned the serpin-2 gene whose full-length has already been published. Phylogenetic analysis indicated that these two serpin genes were highly clustered with other serpins associated with the immune response in other insects. The temporal and spatial expression of serpin-2, serpin-4 and serpin-5 were determined to be the highest in the fat body and hemolymph of 4th larval stage using qRT-PCR and western blot detection techniques. RNA interference (RNAi) mediated knockdown of P. xylostella serpin genes was carried out by microinjection of double-stranded RNA (dsRNA). The expression levels of serpins decreased significantly after RNAi. Results showed that the depletion of serpins induced cecropins expression, increased phenoloxidase (PO) activity, body melanization and mortality in the larvae of P. xylostella under the same lethal concentration of destruxin A. The superimposed effects of serpins RNAi were similar with the destruxin A treatment upon mortality of P. xylostella larvae. We discovered for the first time that serpins play indispensable role in P. xylostella when challenged by destruxin A and deduced the possible function mechanism of destruxin A. Our findings are conducive to fully understanding the potential insecticidal mechanism of destruxin A and constitute a well-defined potential molecular target for novel insecticides. PMID:24837592

  17. Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations

    Li, Yongchao [ORNL; Tschaplinski, Timothy J [ORNL; Engle, Nancy L [ORNL; Hamilton, Choo Yieng [ORNL; Rodriguez, Jr., Miguel [ORNL; Liao, James C [ORNL; Schadt, Christopher Warren [ORNL; Guss, Adam M [ORNL; Yang, Yunfeng [ORNL; Graham, David E [ORNL

    2012-01-01

    Background: The model bacterium Clostridium cellulolyticum efficiently hydrolyzes crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H2 and CO2, the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels. Therefore genetic engineering will likely be required to improve the ethanol yield. Random mutagenesis, plasmid transformation, and heterologous expression systems have previously been developed for C. cellulolyticum, but targeted mutagenesis has not been reported for this organism. Results: The first targeted gene inactivation system was developed for C. cellulolyticum, based on a mobile group II intron originating from the Lactococcus lactis L1.LtrB intron. This markerless mutagenesis system was used to disrupt both the paralogous L-lactate dehydrogenase (Ccel_2485; ldh) and L-malate dehydrogenase (Ccel_0137; mdh) genes, distinguishing the overlapping substrate specificities of these enzymes. Both mutations were then combined in a single strain. This double mutant produced 8.5-times more ethanol than wild-type cells growing on crystalline cellulose. Ethanol constituted 93% of the major fermentation products (by molarity), corresponding to a molar ratio of ethanol to organic acids of 15, versus 0.18 in wild-type cells. During growth on acid-pretreated switchgrass, the double mutant also produced four-times as much ethanol as wild-type cells. Detailed metabolomic analyses identified increased flux through the oxidative branch of the mutant s TCA pathway. Conclusions: The efficient intron-based gene inactivation system produced the first gene-targeted mutations in C. cellulolyticum. As a key component of the genetic toolbox for this bacterium, markerless targeted mutagenesis enables functional genomic research in C. cellulolyticum and rapid genetic engineering to

  18. Differential gene expression in Rhododendron fortunei roots colonized by an ericoid mycorrhizal fungus and increased nitrogen absorption and plant growth

    Xiangying Wei

    2016-10-01

    Full Text Available Ericoid mycorrhizal (ERM fungi are specifically symbiotic with plants in the family Ericaceae. Little is known thus far about their symbiotic establishment and subsequent nitrogen (N uptake at the molecular level. The present study devised a system for establishing a symbiotic relationship between Rhododendron fortunei Lindl. and an ERM fungus (Oidiodendron maius var. maius strain Om19, quantified seedling growth and N uptake, and compared transcriptome profiling between colonized and uncolonized roots using RNA-Seq. The Om19 colonization induced 16,892 genes that were differentially expressed in plant roots, of which 14,364 were upregulated and 2,528 were downregulated. These genes included those homologous to ATP-binding cassette transporters, calcium/calmodulin-dependent kinases, and symbiosis receptor-like kinases. N metabolism was particularly active in Om19-colonized roots, and 51 genes were upregulated, such as nitrate transporters, nitrate reductase, nitrite reductase, ammonium transporters, glutamine synthetase, and glutamate synthase. Transcriptome analysis also identified a series of genes involving endocytosis, Fc-gamma R-mediated phagocytosis, glycerophospholipid metabolism, and GnRH signal pathway that have not been reported previously. Their roles in the symbiosis require further investigation. The Om19 colonization significantly increased N uptake and seedling growth. Total N content and dry weight of colonized seedlings were 36.6% and 46.6% greater than control seedlings. This is the first transcriptome analysis of a species from the family Ericaceae colonized by an ERM fungus. The findings from this study will shed light on the mechanisms underlying symbiotic relationships of ericaceous species with ERM fungi and the symbiosis-resultant N uptake and plant growth.

  19. 'Cold shock' increases the frequency of homology directed repair gene editing in induced pluripotent stem cells.

    Guo, Q; Mintier, G; Ma-Edmonds, M; Storton, D; Wang, X; Xiao, X; Kienzle, B; Zhao, D; Feder, John N

    2018-02-01

    Using CRISPR/Cas9 delivered as a RNA modality in conjunction with a lipid specifically formulated for large RNA molecules, we demonstrate that homology directed repair (HDR) rates between 20-40% can be achieved in induced pluripotent stem cells (iPSC). Furthermore, low HDR rates (between 1-20%) can be enhanced two- to ten-fold in both iPSCs and HEK293 cells by 'cold shocking' cells at 32 °C for 24-48 hours following transfection. This method can also increases the proportion of loci that have undergone complete sequence conversion across the donor sequence, or 'perfect HDR', as opposed to partial sequence conversion where nucleotides more distal to the CRISPR cut site are less efficiently incorporated ('partial HDR'). We demonstrate that the structure of the single-stranded DNA oligo donor can influence the fidelity of HDR, with oligos symmetric with respect to the CRISPR cleavage site and complementary to the target strand being more efficient at directing 'perfect HDR' compared to asymmetric non-target strand complementary oligos. Our protocol represents an efficient method for making CRISPR-mediated, specific DNA sequence changes within the genome that will facilitate the rapid generation of genetic models of human disease in iPSCs as well as other genome engineered cell lines.

  20. Inactivation of the Autolysis-Related Genes lrgB and yycI in Staphylococcus aureus Increases Cell Lysis-Dependent eDNA Release and Enhances Biofilm Development In Vitro and In Vivo.

    Cristiana Ossaille Beltrame

    Full Text Available Staphylococcus aureus ica-independent biofilms are multifactorial in nature, and various bacterial proteins have been associated with biofilm development, including fibronectin-binding proteins A and B, protein A, surface protein SasG, proteases, and some autolysins. The role of extracellular DNA (eDNA has also been demonstrated in some S. aureus biofilms. Here, we constructed a Tn551 library, and the screening identified two genes that affected biofilm formation, lrgB and yycI. The repressive effect of both genes on the development of biofilm was also confirmed in knockout strains constructed by allelic recombination. In contrast, the superexpression of either lrgB or yycI by a cadmium-inducible promoter led to a decrease in biofilm accumulation. Indeed, a significant increase in the cell-lysis dependent eDNA release was detected when lrgB or yycI were inactivated, explaining the enhanced biofilm formed by these mutants. In fact, lrgB and yycI genes belong to distinct operons that repress bacterial autolysis through very different mechanisms. LrgB is associated with the synthesis of phage holin/anti-holin analogues, while YycI participates in the activation/repression of the two-component system YycGF (WalKR. Our in vivo data suggest that autolysins activation lead to increased bacterial virulence in the foreign body animal model since a higher number of attached cells was recovered from the implanted catheters inoculated with lrgB or yycI knockout mutants.

  1. Cloning and characterization of Bacillus subtilis iep, which has positive and negative effects on production of extracellular proteases.

    Tanaka, T; Kawata, M

    1988-08-01

    We have isolated a DNA fragment from Bacillus subtilis 168 which, when present in a high-copy plasmid, inhibited production of extracellular alkaline and neutral proteases. The gene responsible for this activity was referred to as iep. The open reading frame of iep was found to be incomplete in the cloned DNA fragment. When the intact iep gene was reconstructed after the missing part of the iep gene had been cloned, it showed an enhancing effect on the production of the extracellular proteases. The open reading frame encodes a polypeptide of 229 amino acids with a molecular weight of ca. 25,866. Deletion of two amino acids from the N-terminal half of the putative iep protein resulted in dual effects, i.e., a decrease in the inhibitory activity shown by the incomplete iep gene and a slight increase in the enhancing activity shown by the complete iep gene. These results show that the iep gene product is a bifunctional protein, containing inhibitory and enhancing activities for the exoprotease production in the N-terminal and C-terminal regions, respectively. It was found by genetic and functional analyses that iep lies very close to sacU.

  2. Common variants in mismatch repair genes associated with increased risk of sperm DNA damage and male infertility

    Ji Guixiang

    2012-05-01

    Full Text Available Abstract Background The mismatch repair (MMR pathway plays an important role in the maintenance of the genome integrity, meiotic recombination and gametogenesis. This study investigated whether genetic variations in MMR genes are associated with an increased risk of sperm DNA damage and male infertility. Methods We selected and genotyped 21 tagging single nucleotide polymorphisms (SNPs in five MMR genes (MLH1, MLH3, PMS2, MSH4 and MSH5 using the SNPstream 12-plex platform in a case-control study of 1,292 idiopathic infertility patients and 480 fertile controls in a Chinese population. Sperm DNA damage levels were detected with the Tdt-mediated dUTP nick end labelling (TUNEL assay in 450 cases. Fluorescence resonance energy transfer (FRET and co-immunoprecipitation techniques were employed to determine the effects of functional variants. Results One intronic SNP in MLH1 (rs4647269 and two non-synonymous SNPs in PMS2 (rs1059060, Ser775Asn and MSH5 (rs2075789, Pro29Ser seem to be risk factors for the development of azoospermia or oligozoospermia. Meanwhile, we also identified a possible contribution of PMS2 rs1059060 to the risk of male infertility with normal sperm count. Among patients with normal sperm count, MLH1 rs4647269 and PMS2 rs1059060 were associated with increased sperm DNA damage. Functional analysis revealed that the PMS2 rs1059060 can affect the interactions between MLH1 and PMS2. Conclusions Our results provide evidence supporting the involvement of genetic polymorphisms in MMR genes in the aetiology of male infertility.

  3. Intermittent Fasting Promotes Fat Loss With Lean Mass Retention, Increased Hypothalamic Norepinephrine Content, and Increased Neuropeptide Y Gene Expression in Diet-Induced Obese Male Mice.

    Gotthardt, Juliet D; Verpeut, Jessica L; Yeomans, Bryn L; Yang, Jennifer A; Yasrebi, Ali; Roepke, Troy A; Bello, Nicholas T

    2016-02-01

    Clinical studies indicate alternate-day, intermittent fasting (IMF) protocols result in meaningful weight loss in obese individuals. To further understand the mechanisms sustaining weight loss by IMF, we investigated the metabolic and neural alterations of IMF in obese mice. Male C57/BL6 mice were fed a high-fat diet (HFD; 45% fat) ad libitum for 8 weeks to promote an obese phenotype. Mice were divided into four groups and either maintained on ad libitum HFD, received alternate-day access to HFD (IMF-HFD), and switched to ad libitum low-fat diet (LFD; 10% fat) or received IMF of LFD (IMF-LFD). After 4 weeks, IMF-HFD (∼13%) and IMF-LFD (∼18%) had significantly lower body weights than the HFD. Body fat was also lower (∼40%-52%) in all diet interventions. Lean mass was increased in the IMF-LFD (∼12%-13%) compared with the HFD and IMF-HFD groups. Oral glucose tolerance area under the curve was lower in the IMF-HFD (∼50%), whereas the insulin tolerance area under the curve was reduced in all diet interventions (∼22%-42%). HPLC measurements of hypothalamic tissue homogenates indicated higher (∼55%-60%) norepinephrine (NE) content in the anterior regions of the medial hypothalamus of IMF compared with the ad libitum-fed groups, whereas NE content was higher (∼19%-32%) in posterior regions in the IMF-LFD group only. Relative gene expression of Npy in the arcuate nucleus was increased (∼65%-75%) in IMF groups. Our novel findings indicate that intermittent fasting produces alterations in hypothalamic NE and neuropeptide Y, suggesting the counterregulatory processes of short-term weight loss are associated with an IMF dietary strategy.

  4. Gene

    U.S. Department of Health & Human Services — Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes,...

  5. Adenosine A(2A) receptor gene (ADORA2A) variants may increase autistic symptoms and anxiety in autism spectrum disorder.

    Freitag, Christine M; Agelopoulos, Konstantin; Huy, Ellen; Rothermundt, Matthias; Krakowitzky, Petra; Meyer, Jobst; Deckert, Jürgen; von Gontard, Alexander; Hohoff, Christa

    2010-01-01

    Autism spectrum disorders (ASDs) are heterogeneous disorders presenting with increased rates of anxiety. The adenosine A(2A) receptor gene (ADORA2A) is associated with panic disorder and is located on chromosome 22q11.23. Its gene product, the adenosine A(2A) receptor, is strongly expressed in the caudate nucleus, which also is involved in ASD. As autistic symptoms are increased in individuals with 22q11.2 deletion syndrome, and large 22q11.2 deletions and duplications have been observed in ASD individuals, in this study, 98 individuals with ASD and 234 control individuals were genotyped for eight single-nucleotide polymorphisms in ADORA2A. Nominal association with the disorder was observed for rs2236624-CC, and phenotypic variability in ASD symptoms was influenced by rs3761422, rs5751876 and rs35320474. In addition, association of ADORA2A variants with anxiety was replicated for individuals with ASD. Findings point toward a possible mediating role of ADORA2A variants on phenotypic expression in ASD that need to be replicated in a larger sample.

  6. Preharvest Ultraviolet C Irradiation Increased the Level of Polyphenol Accumulation and Flavonoid Pathway Gene Expression in Strawberry Fruit.

    Xu, Yanqun; Charles, Marie Thérèse; Luo, Zisheng; Mimee, Benjamin; Veronneau, Pierre-Yves; Rolland, Daniel; Roussel, Dominique

    2017-11-22

    Preharvest ultraviolet C (UV-C) irradiation is an innovative approach for increasing the bioactive phytochemical content of strawberries to increase the disease resistance and nutritional value. This study investigated the changes in individual flavonoids in strawberry developed with three different cumulative doses of preharvest UV-C treatment (low, 9.6 kJ m -2 ; middle, 15 kJ m -2 ; and high , 29.4 kJ m -2 ). Significant accumulation (p radiation. The expression of the flavonoid pathway structural genes, i.e., FaCHS1, FaCHI, FaFHT, FaDFR, FaFLS, and FaFGT, was upregulated in the low- and middle-dose groups, while the early stage genes were not affected by the high dose. FaMYB1 was also relatively enhanced in the low- and middle-dose groups, while FaASR was upregulated in only the low-dose group. Hormetic preharvest UV-C dose ranges for enhancing the polyphenol content of strawberries were established for the first time.

  7. Increased asthma and adipose tissue inflammatory gene expression with obesity and Inuit migration to a western country.

    Backer, Vibeke; Baines, Katherine J; Powell, Heather; Porsbjerg, Celeste; Gibson, Peter G

    2016-02-01

    An overlap between obesity and asthma exists, and inflammatory cells in adipose tissue could drive the development of asthma. Comparison of adipose tissue gene expression among Inuit living in Greenland to those in Denmark provides an opportunity to assess how changes in adipose tissue inflammation can be modified by migration and diet. To examine mast cell and inflammatory markers in adipose tissue and the association with asthma. Two Inuit populations were recruited, one living in Greenland and another in Denmark. All underwent adipose subcutaneous biopsy, followed by clinical assessment of asthma, and measurement of AHR. Adipose tissue biopsies were homogenised, RNA extracted, and PCR was performed to determine the relative gene expression of mast cell (tryptase, chymase, CPA3) and inflammatory markers (IL-6, IL-1β, and CD163). Of the 1059 Greenlandic Inuit participants, 556 were living in Greenland and 6.4% had asthma. Asthma was increased in Denmark (9%) compared to Greenland (3.6%, p Inuit (p Inuit, adipose tissue inflammation is also increased in those who migrate to Denmark, possibly as a result of dietary changes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Ortho-aminoazotoluene activates mouse constitutive androstane receptor (mCAR) and increases expression of mCAR target genes

    Smetanina, Mariya A.; Pakharukova, Mariya Y.; Kurinna, Svitlana M.; Dong, Bingning; Hernandez, Juan P.; Moore, David D.; Merkulova, Tatyana I.

    2011-01-01

    2'-3-dimethyl-4-aminoazobenzene (ortho-aminoazotoluene, OAT) is an azo dye and a rodent carcinogen that has been evaluated by the International Agency for Research on Cancer (IARC) as a possible (class 2B) human carcinogen. Its mechanism of action remains unclear. We examined the role of the xenobiotic receptor Constitutive Androstane Receptor (CAR, NR1I3) as a mediator of the effects of OAT. We found that OAT increases mouse CAR (mCAR) transactivation in a dose-dependent manner. This effect is specific because another closely related azo dye, 3'-methyl-4-dimethyl-aminoazobenzene (3'MeDAB), did not activate mCAR. Real-time Q-PCR analysis in wild-type C57BL/6 mice revealed that OAT induces the hepatic mRNA expression of the following CAR target genes: Cyp2b10, Cyp2c29, Cyp3a11, Ugt1a1, Mrp4, Mrp2 and c-Myc. CAR-null (Car -/- ) mice showed no increased expression of these genes following OAT treatment, demonstrating that CAR is required for their OAT dependent induction. The OAT-induced CAR-dependent increase of Cyp2b10 and c-Myc expression was confirmed by Western blotting. Immunohistochemistry analysis of wild-type and Car -/- livers showed that OAT did not acutely induce hepatocyte proliferation, but at much later time points showed an unexpected CAR-dependent proliferative response. These studies demonstrate that mCAR is an OAT xenosensor, and indicate that at least some of the biological effects of this compound are mediated by this nuclear receptor. - Highlights: → The azo dye and mouse carcinogen OAT is a very effective mCAR activator. → OAT increases mCAR transactivation in a dose-dependent manner. → OAT CAR-dependently increases the expression of a specific subset of CAR target genes. → OAT induces an unexpectedly deferred, but CAR-dependent hepatocyte proliferation.

  9. Myostatin-deficiency in mice increases global gene expression at the Dlk1-Dio3 locus in the skeletal muscle.

    Hitachi, Keisuke; Tsuchida, Kunihiro

    2017-01-24

    Myostatin, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle growth and development. Myostatin inhibition leads to increased skeletal muscle mass in mammals; hence, myostatin is considered a potential therapeutic target for skeletal muscle wasting. However, downstream molecules of myostatin in the skeletal muscle have not been fully elucidated. Here, we identified the Dlk1-Dio3 locus at the mouse chromosome 12qF1, also called as the callipyge locus in sheep, as a novel downstream target of myostatin. In skeletal muscle of myostatin knockout mice, the expression of mature miRNAs at the Dlk1-Dio3 locus was significantly increased. The increased miRNA levels are caused by the transcriptional activation of the Dlk1-Dio3 locus, because a significant increase in the primary miRNA transcript was observed in myostatin knockout mice. In addition, we found increased expression of coding and non-coding genes (Dlk1, Gtl2, Rtl1/Rtl1as, and Rian) at the Dlk1-Dio3 locus in myostatin-deficient skeletal muscle. Moreover, epigenetic changes, associated with the regulation of the Dlk1-Dio3 locus, were observed in myostatin knockout mice. Taken together, this is the first report demonstrating the role of myostatin in regulating the Dlk1-Dio3 (the callipyge) locus in the skeletal muscle.

  10. When stress predicts a shrinking gene pool, trading early reproduction for longevity can increase fitness, even with lower fecundity.

    William C Ratcliff

    2009-06-01

    Full Text Available Stresses like dietary restriction or various toxins increase lifespan in taxa as diverse as yeast, Caenorhabditis elegans, Drosophila and rats, by triggering physiological responses that also tend to delay reproduction. Food odors can reverse the effects of dietary restriction, showing that key mechanisms respond to information, not just resources. Such environmental cues can predict population trends, not just individual prospects for survival and reproduction. When population size is increasing, each offspring produced earlier makes a larger proportional contribution to the gene pool, but the reverse is true when population size is declining.We show mathematically that natural selection can favor facultative delay in reproduction when environmental cues predict a decrease in total population size, even if lifetime fecundity decreases with delay. We also show that increased reproduction from waiting for better conditions does not increase fitness (proportional representation when the whole population benefits similarly.We conclude that the beneficial effects of stress on longevity (hormesis in diverse taxa are a side-effect of delaying reproduction in response to environmental cues that population size is likely to decrease. The reversal by food odors of the effects of dietary restriction can be explained as a response to information that population size is less likely to decrease, reducing the chance that delaying reproduction will increase fitness.

  11. The role of extracellular histones in haematological disorders.

    Alhamdi, Yasir; Toh, Cheng-Hock

    2016-06-01

    Over the past decades, chromosomal alterations have been extensively investigated for their pathophysiological relevance in haematological malignancies. In particular, epigenetic modifications of intra-nuclear histones are now known as key regulators of healthy cell cycles that have also evolved into novel therapeutic targets for certain blood cancers. Thus, for most haematologists, histones are DNA-chained proteins that are buried deep within chromatin. However, the plot has deepened with recent revelations on the function of histones when unchained and released extracellularly upon cell death or from activated neutrophils as part of neutrophil extracellular traps (NETs). Extracellular histones and NETs are increasingly recognized for profound cytotoxicity and pro-coagulant effects. This article highlights the importance of recognizing this new paradigm of extracellular histones as a key player in host defence through its damage-associated molecular patterns, which could translate into novel diagnostic and therapeutic biomarkers in various haematological and critical disorders. © 2016 John Wiley & Sons Ltd.

  12. Targeted transfection increases siRNA uptake and gene silencing of primary endothelial cells in vitro--a quantitative study.

    Asgeirsdóttir, Sigridur A; Talman, Eduard G; de Graaf, Inge A; Kamps, Jan A A M; Satchell, Simon C; Mathieson, Peter W; Ruiters, Marcel H J; Molema, Grietje

    2010-01-25

    Applications of small-interfering RNA (siRNA) call for specific and efficient delivery of siRNA into particular cell types. We developed a novel, non-viral targeting system to deliver siRNA specifically into inflammation-activated endothelial cells. This was achieved by conjugating the cationic amphiphilic lipid SAINT to antibodies recognizing the inflammatory cell adhesion molecule E-selectin. These anti-E-selectin-SAINT lipoplexes (SAINTarg) maintained antigen recognition capacity of the parental antibody in vitro, and ex vivo in human kidney tissue slices subjected to inflammatory conditions. Regular SAINT mediated transfection resulted in efficient gene silencing in human microvascular endothelial cells (HMEC-1) and conditionally immortalized glomerular endothelial cells (ciGEnC). However, primary human umbilical vein endothelial cells (HUVEC) transfected poorly, a phenomenon that we could quantitatively correlate with a cell-type specific capacity to facilitate siRNA uptake. Importantly, SAINTarg increased siRNA uptake and transfection specificity for activated endothelial cells. Transfection with SAINTarg delivered significantly more siRNA into activated HUVEC, compared to transfection with non-targeted SAINT. The enhanced uptake of siRNA was corroborated by improved silencing of both gene- and protein expression of VE-cadherin in activated HUVEC, indicating that SAINTarg delivered functionally active siRNA into endothelial cells. The obtained results demonstrate a successful design of a small nucleotide carrier system with improved and specific siRNA delivery into otherwise difficult-to-transfect primary endothelial cells, which in addition reduced considerably the amount of siRNA needed for gene silencing. Copyright 2009 Elsevier B.V. All rights reserved.

  13. Specific extracellular matrix remodeling signature of colon hepatic metastases.

    Maguy Del Rio

    Full Text Available To identify genes implicated in metastatic colonization of the liver in colorectal cancer, we collected pairs of primary tumors and hepatic metastases before chemotherapy in 13 patients. We compared mRNA expression in the pairs of patients to identify genes deregulated during metastatic evolution. We then validated the identified genes using data obtained by different groups. The 33-gene signature was able to classify 87% of hepatic metastases, 98% of primary tumors, 97% of normal colon mucosa, and 95% of normal liver tissues in six datasets obtained using five different microarray platforms. The identified genes are specific to colon cancer and hepatic metastases since other metastatic locations and hepatic metastases originating from breast cancer were not classified by the signature. Gene Ontology term analysis showed that 50% of the genes are implicated in extracellular matrix remodeling, and more precisely in cell adhesion, extracellular matrix organization and angiogenesis. Because of the high efficiency of the signature to classify colon hepatic metastases, the identified genes represent promising targets to develop new therapies that will specifically affect hepatic metastasis microenvironment.

  14. An Engineered Survival-Selection Assay for Extracellular Protein Expression Uncovers Hypersecretory Phenotypes in Escherichia coli.

    Natarajan, Aravind; Haitjema, Charles H; Lee, Robert; Boock, Jason T; DeLisa, Matthew P

    2017-05-19

    The extracellular expression of recombinant proteins using laboratory strains of Escherichia coli is now routinely achieved using naturally secreted substrates, such as YebF or the osmotically inducible protein Y (OsmY), as carrier molecules. However, secretion efficiency through these pathways needs to be improved for most synthetic biology and metabolic engineering applications. To address this challenge, we developed a generalizable survival-based selection strategy that effectively couples extracellular protein secretion to antibiotic resistance and enables facile isolation of rare mutants from very large populations (i.e., 10 10-12 clones) based simply on cell growth. Using this strategy in the context of the YebF pathway, a comprehensive library of E. coli single-gene knockout mutants was screened and several gain-of-function mutations were isolated that increased the efficiency of extracellular expression without compromising the integrity of the outer membrane. We anticipate that this user-friendly strategy could be leveraged to better understand the YebF pathway and other secretory mechanisms-enabling the exploration of protein secretion in pathogenesis as well as the creation of designer E. coli strains with greatly expanded secretomes-all without the need for expensive exogenous reagents, assay instruments, or robotic automation.

  15. Collagen VI disorders: Insights on form and function in the extracellular matrix and beyond.

    Lamandé, Shireen R; Bateman, John F

    2017-12-22

    Mutations in the three canonical collagen VI genes, COL6A1, COL6A2 and COL6A3, cause a spectrum of muscle disease from Bethlem myopathy at the mild end to the severe Ullrich congenital muscular dystrophy. Mutations can be either dominant or recessive and the resulting clinical severity is influenced by the way mutations impact the complex collagen VI assembly process. Most mutations are found towards the N-terminus of the triple helical collagenous domain and compromise extracellular microfibril assembly. Outside the triple helix collagen VI is highly polymorphic and discriminating mutations from rare benign changes remains a major diagnostic challenge. Collagen VI deficiency alters extracellular matrix structure and biomechanical properties and leads to increased apoptosis and oxidative stress, decreased autophagy, and impaired muscle regeneration. Therapies that target these downstream consequences have been tested in a collagen VI null mouse and also in small human trials where they show modest clinical efficacy. An important role for collagen VI in obesity, cancer and diabetes is emerging. A major barrier to developing effective therapies is the paucity of information about how collagen VI deficiency in the extracellular matrix signals the final downstream consequences - the receptors involved and the intracellular messengers await further characterization. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

  16. Leukemia inhibitory factor increases the proliferation of human endometrial stromal cells and expression of genes related to pluripotency

    Mojdeh Salehnia

    2017-08-01

    Full Text Available Background: Concerning the low population of human endometrial mesenchymal cells within the tissue and their potential application in the clinic and tissue engineering, some researches have been focused on their in vitro expansion. Objective: The aim of this study was to evaluate the effect of leukemia inhibitory factor (LIF as a proliferative factor on the expansion and proliferation of human endometrial stromal cells. Materials and Methods: In this experimental study, the isolated and cultured human endometrial stromal cells from women at ovulatory phase aged 20-35 years, after fourth passage were divided into control and LIF-treated groups. In the experimental group, the endometrial cells were treated by 10 ng/ml LIF in culture media and the cultured cells without adding LIF considered as control group. Both groups were evaluated and compared for proliferation rate using MTT assay, for CD90 marker by flow cytometric analysis and for the expression of Oct4, Nanog, PCNA and LIFr genes using real-time RT-PCR. Results: The proliferation rate of control and LIF-treated groups were 1.17±0.17 and 1.61±0.06 respectively and there was a significant increase in endometrial stromal cell proliferation following in vitro treatment by LIF compared to control group (p=0.049. The rate of CD90 positive cells was significantly increased in LIFtreated group (98.96±0.37% compared to control group (94.26±0.08% (p=0.0498. Also, the expression ratio of all studied genes was significantly increased in the LIFtreated group compared to control group (p=0.0479. Conclusion: The present study showed that LIF has a great impact on proliferation, survival, and maintenance of pluripotency of human endometrial stromal cells and it could be applicable in cell therapies.

  17. Correlation between HFE gene polymorphisms and increased risk of coronary artery disease among patients with type 2 diabetes in Iran.

    Saremi, Leila; Saremi, Marzieh; Lotfipanah, Shirin; İmani, Saber; Fu, Junjiang; Zhang, Tianyu

    2016-04-19

    Diabetes mellitus is a risk factor for cardiovascular diseases (CVDs), which are among the major causes of deaths in type 2 diabetes (T2D). The purpose of the present study was to determine the association of C282Y and H63D mutations in the HFE gene with increased risk of coronary artery disease (CAD) in T2D patients. Two hundred and ninety individuals were divided into two groups: a case group and a control group. Genomic DNA of peripheral venous blood cells was extracted and the HFE gene mutations were analyzed using the PCR-RFLP technique. Data analysis revealed a significant difference between the allele frequencies of H63D and C282Y mutations between the case group and the controls (P 0.05). Using a logistic regression model, BMI, FBS, HDL, and total cholesterol levels were significantly different with independent predictors of CVD (P < 0.05). Our results revealed a significant correlation between C282Y and H63D mutations and the development of CAD in T2D patients.

  18. Annuity payments can increase patient access to innovative cell and gene therapies under England's net budget impact test.

    Jørgensen, Jesper; Kefalas, Panos

    2017-01-01

    Background: Cell and gene therapies have the potential to provide therapeutic breakthroughs, but the high costs of researching, developing, manufacturing and delivering them translate into prices that may challenge healthcare budgets. Various measures exist that aim to address the affordability challenge, including reducing price, limiting patient numbers and/or linking remuneration to product performance. Objective: To explore how the net budget impact test recently introduced in England can affect patient access to high-value, one-off cell and gene therapies, and how managed entry agreements can improve access. Methods: We use a hypothetical example where a new high-value, one-off therapy launches in an indication where it displaces a relatively low cost chronic treatment. We calculate the number of patients that can be treated without exceeding the £20 million net budget impact threshold, and compare results for scenarios where a full upfront payment is used, and where annuity-based payments are used. Results : Charging a full upfront payment at the time of treatment can lead to suboptimal patient access. Conclusion: Annuity-based payments in combination with an outcomes-based remuneration scheme reduce consequences of decision uncertainty and can increase patient access, without exceeding the net budget impact test.

  19. Assets of the non-pathogenic microorganism Dictyostelium discoideum as a model for the study of eukaryotic extracellular vesicles [v1; ref status: indexed, http://f1000r.es/pa

    Irène Tatischeff

    2013-03-01

    Full Text Available Dictyostelium discoideum microvesicles have recently been presented as a valuable model for eukaryotic extracellular vesicles. Here, the advantages of D. discoideum for unraveling important biological functions of extracellular vesicles in general are detailed. D. discoideum, a non-pathogenic eukaryotic microorganism, belongs to a billion-year-old Amoeboza lineage, which diverged from the animal-fungal lineage after the plant animal-split. During growth and early starvation-induced development, it presents analogies with lymphocytes and macrophages with regard to motility and phagocytosis capability, respectively. Its 6-chromosome genome codes for about 12,500 genes, some showing analogies with human genes. The presence of extracellular vesicles during cell growth has been evidenced as a detoxification mechanism of various structurally unrelated drugs. Controls led to the discovery of constitutive extracellular vesicle secretion in this microorganism, which was an important point. It means that the secretion of extracellular vesicles occurs, in the absence of any drug, during both cell growth and early development. This constitutive secretion of D. discoideum cells is very likely to play a role in intercellular communication. The detoxifying secreted vesicles, which can transport drugs outside the cells, can also act as "Trojan horses", capable of transferring these drugs not only into naïve D. discoideum cells, but into human cells as well. Therefore, these extracellular vesicles were proposed as a new biological drug delivery tool. Moreover, Dictyostelium, chosen by the NIH (USA as a new model organism for biomedical research, has already been used for studying some human diseases. These cells, which are much easier to manipulate than human cells, can be easily designed in simple conditioned medium experiments. Owing to the increasing consensus that extracellular vesicles are probably important mediators of intercellular communication, D

  20. A hairy-leaf gene, BLANKET LEAF, of wild Oryza nivara increases photosynthetic water use efficiency in rice.

    Hamaoka, Norimitsu; Yasui, Hideshi; Yamagata, Yoshiyuki; Inoue, Yoko; Furuya, Naruto; Araki, Takuya; Ueno, Osamu; Yoshimura, Atsushi

    2017-12-01

    High water use efficiency is essential to water-saving cropping. Morphological traits that affect photosynthetic water use efficiency are not well known. We examined whether leaf hairiness improves photosynthetic water use efficiency in rice. A chromosome segment introgression line (IL-hairy) of wild Oryza nivara (Acc. IRGC105715) with the genetic background of Oryza sativa cultivar 'IR24' had high leaf pubescence (hair). The leaf hairs developed along small vascular bundles. Linkage analysis in BC 5 F 2 and F 3 populations showed that the trait was governed by a single gene, designated BLANKET LEAF (BKL), on chromosome 6. IL-hairy plants had a warmer leaf surface in sunlight, probably due to increased boundary layer resistance. They had a lower transpiration rate under moderate and high light intensities, resulting in higher photosynthetic water use efficiency. Introgression of BKL on chromosome 6 from O. nivara improved photosynthetic water use efficiency in the genetic background of IR24.

  1. Increased asthma and adipose tissue inflammatory gene expression with obesity and Inuit migration to a western country

    Backer, Vibeke; Baines, Katherine J; Powell, Heather

    2016-01-01

    inflammation can be modified by migration and diet. OBJECTIVE: To examine mast cell and inflammatory markers in adipose tissue and the association with asthma. METHODS: Two Inuit populations were recruited, one living in Greenland and another in Denmark. All underwent adipose subcutaneous biopsy, followed...... of mast cell markers in adipose tissue and asthma. Among Greenlandic Inuit, adipose tissue inflammation is also increased in those who migrate to Denmark, possibly as a result of dietary changes....... by clinical assessment of asthma, and measurement of AHR. Adipose tissue biopsies were homogenised, RNA extracted, and PCR was performed to determine the relative gene expression of mast cell (tryptase, chymase, CPA3) and inflammatory markers (IL-6, IL-1β, and CD163). RESULTS: Of the 1059 Greenlandic Inuit...

  2. Increasing Power by Sharing Information from Genetic Background and Treatment in Clustering of Gene Expression Time Series

    Sura Zaki Alrashid; Muhammad Arifur Rahman; Nabeel H Al-Aaraji; Neil D Lawrence; Paul R Heath

    2018-01-01

    Clustering of gene expression time series gives insight into which genes may be co-regulated, allowing us to discern the activity of pathways in a given microarray experiment. Of particular interest is how a given group of genes varies with different conditions or genetic background. This paper develops
a new clustering method that allows each cluster to be parameterised according to whether the behaviour of the genes across conditions is correlated or anti-correlated. By specifying correlati...

  3. Deletion of the hfsB gene increases ethanol production in Thermoanaerobacterium saccharolyticum and several other thermophilic anaerobic bacteria.

    Eminoğlu, Ayşenur; Murphy, Sean Jean-Loup; Maloney, Marybeth; Lanahan, Anthony; Giannone, Richard J; Hettich, Robert L; Tripathi, Shital A; Beldüz, Ali Osman; Lynd, Lee R; Olson, Daniel G

    2017-01-01

    With the discovery of interspecies hydrogen transfer in the late 1960s (Bryant et al. in Arch Microbiol 59:20-31, 1967), it was shown that reducing the partial pressure of hydrogen could cause mixed acid fermenting organisms to produce acetate at the expense of ethanol. Hydrogen and ethanol are both more reduced than glucose. Thus there is a tradeoff between production of these compounds imposed by electron balancing requirements; however, the mechanism is not fully known. Deletion of the hfsA or B subunits resulted in a roughly 1.8-fold increase in ethanol yield. The increase in ethanol production appears to be associated with an increase in alcohol dehydrogenase activity, which appears to be due, at least in part, to increased expression of the adhE gene, and may suggest a regulatory linkage between hfsB and adhE . We studied this system most intensively in the organism Thermoanaerobacterium saccharolyticum ; however, deletion of hfsB also increases ethanol production in other thermophilic bacteria suggesting that this could be used as a general technique for engineering thermophilic bacteria for improved ethanol production in organisms with hfs -type hydrogenases. Since its discovery by Shaw et al. (JAMA 191:6457-64, 2009), the hfs hydrogenase has been suspected to act as a regulator due to the presence of a PAS domain. We provide additional support for the presence of a regulatory phenomenon. In addition, we find a practical application for this scientific insight, namely increasing ethanol yield in strains that are of interest for ethanol production from cellulose or hemicellulose. In two of these organisms ( T. xylanolyticum and T. thermosaccharolyticum ), the ethanol yields are the highest reported to date.

  4. Deleterious genetic variants in ciliopathy genes increase risk of ritodrine-induced cardiac and pulmonary side effects.

    Seo, Heewon; Kwon, Eun Jin; You, Young-Ah; Park, Yoomi; Min, Byung Joo; Yoo, Kyunghun; Hwang, Han-Sung; Kim, Ju Han; Kim, Young Ju

    2018-01-24

    Ritodrine is a commonly used tocolytic to prevent preterm labour. However, it can cause unexpected serious adverse reactions, such as pulmonary oedema, pulmonary congestion, and tachycardia. It is unknown whether such adverse reactions are associated with pharmacogenomic variants in patients. Whole-exome sequencing of 13 subjects with serious ritodrine-induced cardiac and pulmonary side-effects was performed to identify causal genes and variants. The deleterious impact of nonsynonymous substitutions for all genes was computed and compared between cases (n = 13) and controls (n = 30). The significant genes were annotated with Gene Ontology (GO), and the associated disease terms were categorised into four functional classes for functional enrichment tests. To assess the impact of distributed rare variants in cases with side effects, we carried out rare variant association tests with a minor allele frequency ≤ 1% using the burden test, the sequence Kernel association test (SKAT), and optimised SKAT. We identified 28 genes that showed significantly lower gene-wise deleteriousness scores in cases than in controls. Three of the identified genes-CYP1A1, CYP8B1, and SERPINA7-are pharmacokinetic genes. The significantly identified genes were categorized into four functional classes: ion binding, ATP binding, Ca 2+ -related, and ciliopathies-related. These four classes were significantly enriched with ciliary genes according to SYSCILIA Gold Standard genes (P side effects may be associated with deleterious genetic variants in ciliary and pharmacokinetic genes.

  5. Long-term field application of sewage sludge increases the abundance of antibiotic resistance genes in soil.

    Chen, Qinglin; An, Xinli; Li, Hu; Su, Jianqiang; Ma, Yibing; Zhu, Yong-Guan

    2016-01-01

    Sewage sludge and manure are common soil amendments in crop production; however, their impact on the abundance and diversity of the antibiotic resistome in soil remains elusive. In this study, by using high-throughput sequencing and high-throughput quantitative PCR, the patterns of bacterial community and antibiotic resistance genes (ARGs) in a long-term field experiment were investigated to gain insights into these impacts. A total of 130 unique ARGs and 5 mobile genetic elements (MGEs) were detected and the long-term application of sewage sludge and chicken manure significantly increased the abundance and diversity of ARGs in the soil. Genes conferring resistance to beta-lactams, tetracyclines, and multiple drugs were dominant in the samples. Sewage sludge or chicken manure applications caused significant enrichment of 108 unique ARGs and MGEs with a maximum enrichment of up to 3845 folds for mexF. The enrichment of MGEs suggested that the application of sewage sludge or manure may accelerate the dissemination of ARGs in soil through horizontal gene transfer (HGT). Based on the co-occurrence pattern of ARGs subtypes revealed by network analysis, aacC, oprD and mphA-02, were proposed to be potential indicators for quantitative estimation of the co-occurring ARGs subtypes abundance by power functions. The application of sewage sludge and manure resulted in significant increase of bacterial diversity in soil, Proteobacteria, Acidobacteria, Actinobacteria and Chloroflexi were the dominant phyla (>10% in each sample). Five bacterial phyla (Chloroflexi, Planctomycetes, Firmicutes, Gemmatimonadetes and Bacteroidetes) were found to be significantly correlated with the ARGs in soil. Mantel test and variation partitioning analysis (VPA) suggested that bacterial community shifts, rather than MGEs, is the major driver shaping the antibiotic resistome. Additionally, the co-occurrence pattern between ARGs and microbial taxa revealed by network analysis indicated that four

  6. EAAC1 Gene Deletion Increases Neuronal Death and Blood Brain Barrier Disruption after Transient Cerebral Ischemia in Female Mice

    Bo Young Choi

    2014-10-01

    Full Text Available EAAC1 is important in modulating brain ischemic tolerance. Mice lacking EAAC1 exhibit increased susceptibility to neuronal oxidative stress in mice after transient cerebral ischemia. EAAC1 was first described as a glutamate transporter but later recognized to also function as a cysteine transporter in neurons. EAAC1-mediated transport of cysteine into neurons contributes to neuronal antioxidant function by providing cysteine substrates for glutathione synthesis. Here we evaluated the effects of EAAC1 gene deletion on hippocampal blood vessel disorganization after transient cerebral ischemia. EAAC1−/− female mice subjected to transient cerebral ischemia by common carotid artery occlusion for 30 min exhibited twice as much hippocampal neuronal death compared to wild-type female mice as well as increased reduction of neuronal glutathione, blood–brain barrier (BBB disruption and vessel disorganization. Pre-treatment of N-acetyl cysteine, a membrane-permeant cysteine prodrug, increased basal glutathione levels in the EAAC1−/− female mice and reduced ischemic neuronal death, BBB disruption and vessel disorganization. These findings suggest that cysteine uptake by EAAC1 is important for neuronal antioxidant function under ischemic conditions.

  7. The Role of Extracellular Histones in Influenza Virus Pathogenesis.

    Ashar, Harshini K; Mueller, Nathan C; Rudd, Jennifer M; Snider, Timothy A; Achanta, Mallika; Prasanthi, Maram; Pulavendran, Sivasami; Thomas, Paul G; Ramachandran, Akhilesh; Malayer, Jerry R; Ritchey, Jerry W; Rajasekhar, Rachakatla; Chow, Vincent T K; Esmon, Charles T; Teluguakula, Narasaraju

    2018-01-01

    Although exaggerated host immune responses have been implicated in influenza-induced lung pathogenesis, the etiologic factors that contribute to these events are not completely understood. We previously demonstrated that neutrophil extracellular traps exacerbate pulmonary injury during influenza pneumonia. Histones are the major protein components of neutrophil extracellular traps and are known to have cytotoxic effects. Here, we examined the role of extracellular histones in lung pathogenesis during influenza. Mice infected with influenza virus displayed high accumulation of extracellular histones, with widespread pulmonary microvascular thrombosis. Occluded pulmonary blood vessels with vascular thrombi often exhibited endothelial necrosis surrounded by hemorrhagic effusions and pulmonary edema. Histones released during influenza induced cytotoxicity and showed strong binding to platelets within thrombi in infected mouse lungs. Nasal wash samples from influenza-infected patients also showed increased accumulation of extracellular histones, suggesting a possible clinical relevance of elevated histones in pulmonary injury. Although histones inhibited influenza growth in vitro, in vivo treatment with histones did not yield antiviral effects and instead exacerbated lung pathology. Blocking with antihistone antibodies caused a marked decrease in lung pathology in lethal influenza-challenged mice and improved protection when administered in combination with the antiviral agent oseltamivir. These findings support the pathogenic effects of extracellular histones in that pulmonary injury during influenza was exacerbated. Targeting histones provides a novel therapeutic approach to influenza pneumonia. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  8. Formation of neutrophil extracellular traps under low oxygen level

    Katja Branitzki-Heinemann

    2016-11-01

    Full Text Available Since their discovery, neutrophil extracellular traps (NETs have been characterized as a fundamental host innate immune defense mechanism. Conversely, excessive NET release may have a variety of detrimental consequences for the host. A fine balance between NET formation and elimination is necessary to sustain a protective effect during an infectious challenge. Our own recently published data revealed that stabilization of hypoxia inducible factor 1α (HIF-1α by the iron chelating HIF-1α-agonist desferoxamine or AKB-4924 enhanced the release of phagocyte extracellular traps. Since HIF-1α is a global regulator of the cellular response to low oxygen, we hypothesized that NET formation may be similarly increased under low oxygen conditions. Hypoxia occurs in tissues during infection or inflammation, mostly due to overconsumption of oxygen by pathogens and recruited immune cells. Therefore, experiments were performed to characterize the formation of NETs under hypoxic oxygen conditions compared to normoxia. Human blood-derived neutrophils were isolated and incubated under normoxic (21% oxygen level and compared to hypoxic (1% conditions. Dissolved oxygen levels were monitored in the primary cell culture using a Fibox4-PSt3 measurement system. The formation of NETs was quantified by fluorescence microscopy in response to the known NET-inducer phorbol 12-myristate 13-acetate (PMA or S. aureus wildtype and a nuclease-deficient mutant. In contrast to our hypothesis, spontaneous NET formation of neutrophils incubated under hypoxia was distinctly reduced compared to control neutrophils incubated under normoxia. Furthermore, neutrophils incubated under hypoxia showed significantly reduced formation of NETs in response to PMA. Gene expression analysis revealed that mRNA level of hif-1α as well as hif-1α target genes was not altered. However, in good correlation to the decreased NET formation under hypoxia, the cholesterol content of the neutrophils was

  9. Extracellular VirB5 enhances T-DNA transfer from Agrobacterium to the host plant.

    Benoît Lacroix

    Full Text Available VirB5 is a type 4 secretion system protein of Agrobacterium located on the surface of the bacterial cell. This localization pattern suggests a function for VirB5 which is beyond its known role in biogenesis and/or stabilization of the T-pilus and which may involve early interactions between Agrobacterium and the host cell. Here, we identify VirB5 as the first Agrobacterium virulence protein that can enhance infectivity extracellularly. Specifically, we show that elevating the amounts of the extracellular VirB5--by exogenous addition of the purified protein, its overexpression in the bacterium, or transgenic expression in and secretion out of the host cell--enhances the efficiency the Agrobacterium-mediated T-DNA transfer, as measured by transient expression of genes contained on the transferred T-DNA molecule. Importantly, the exogenous VirB5 enhanced transient T-DNA expression in sugar beet, a major crop recalcitrant to genetic manipulation. Increasing the pool of the extracellular VirB5 did not complement an Agrobacterium virB5 mutant, suggesting a dual function for VirB5: in the bacterium and at the bacterium-host cell interface. Consistent with this idea, VirB5 expressed in the host cell, but not secreted, had no effect on the transformation efficiency. That the increase in T-DNA expression promoted by the exogenous VirB5 was not due to its effects on bacterial growth, virulence gene induction, bacterial attachment to plant tissue, or host cell defense response suggests that VirB5 participates in the early steps of the T-DNA transfer to the plant cell.

  10. Extracellular VirB5 enhances T-DNA transfer from Agrobacterium to the host plant.

    Lacroix, Benoît; Citovsky, Vitaly

    2011-01-01

    VirB5 is a type 4 secretion system protein of Agrobacterium located on the surface of the bacterial cell. This localization pattern suggests a function for VirB5 which is beyond its known role in biogenesis and/or stabilization of the T-pilus and which may involve early interactions between Agrobacterium and the host cell. Here, we identify VirB5 as the first Agrobacterium virulence protein that can enhance infectivity extracellularly. Specifically, we show that elevating the amounts of the extracellular VirB5--by exogenous addition of the purified protein, its overexpression in the bacterium, or transgenic expression in and secretion out of the host cell--enhances the efficiency the Agrobacterium-mediated T-DNA transfer, as measured by transient expression of genes contained on the transferred T-DNA molecule. Importantly, the exogenous VirB5 enhanced transient T-DNA expression in sugar beet, a major crop recalcitrant to genetic manipulation. Increasing the pool of the extracellular VirB5 did not complement an Agrobacterium virB5 mutant, suggesting a dual function for VirB5: in the bacterium and at the bacterium-host cell interface. Consistent with this idea, VirB5 expressed in the host cell, but not secreted, had no effect on the transformation efficiency. That the increase in T-DNA expression promoted by the exogenous VirB5 was not due to its effects on bacterial growth, virulence gene induction, bacterial attachment to plant tissue, or host cell defense response suggests that VirB5 participates in the early steps of the T-DNA transfer to the plant cell.

  11. Phosphate starvation triggers production and secretion of an extracellular lipoprotein in Caulobacter crescentus.

    Sophie Le Blastier

    Full Text Available Life in oligotrophic environments necessitates quick adaptive responses to a sudden lack of nutrients. Secretion of specific degradative enzymes into the extracellular medium is a means to mobilize the required nutrient from nearby sources. The aquatic bacterium Caulobacter crescentus must often face changes in its environment such as phosphate limitation. Evidence reported in this paper indicates that under phosphate starvation, C. crescentus produces a membrane surface-anchored lipoprotein named ElpS subsequently released into the extracellular medium. A complete set of 12 genes encoding a type II secretion system (T2SS is located adjacent to the elpS locus in the C. crescentus genome. Deletion of this T2SS impairs release of ElpS in the environment, which surprisingly remains present at the cell surface, indicating that the T2SS is not involved in the translocation of ElpS to the outer membrane but rather in its release. Accordingly, treatment with protease inhibitors prevents release of ElpS in the extracellular medium suggesting that ElpS secretion relies on a T2SS-secreted protease. Finally, secretion of ElpS is associated with an increase in alkaline phosphatase activity in culture supernatants, suggesting a role of the secreted protein in inorganic phosphate mobilization. In conclusion, we have shown that upon phosphate starvation, C. crescentus produces an outer membrane bound lipoprotein, ElpS, which is further cleaved and released in the extracellular medium in a T2SS-dependent manner. Our data suggest that ElpS is associated with an alkaline phosphatase activity, thereby allowing the bacterium to gather inorganic phosphates from a poor environment.

  12. Extracellular vesicles: Exosomes, microvesicles, and friends

    Raposo, G.; Stoorvogel, W.|info:eu-repo/dai/nl/074352385

    2013-01-01

    Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for

  13. Regulation of pituitary hormones and cell proliferation by components of the extracellular matrix

    M. Paez-Pereda

    2005-10-01

    Full Text Available The extracellular matrix is a three-dimensional network of proteins, glycosaminoglycans and other macromolecules. It has a structural support function as well as a role in cell adhesion, migration, proliferation, differentiation, and survival. The extracellular matrix conveys signals through membrane receptors called integrins and plays an important role in pituitary physiology and tumorigenesis. There is a differential expression of extracellular matrix components and integrins during the pituitary development in the embryo and during tumorigenesis in the adult. Different extracellular matrix components regulate adrenocorticotropin at the level of the proopiomelanocortin gene transcription. The extracellular matrix also controls the proliferation of adrenocorticotropin-secreting tumor cells. On the other hand, laminin regulates the production of prolactin. Laminin has a dynamic pattern of expression during prolactinoma development with lower levels in the early pituitary hyperplasia and a strong reduction in fully grown prolactinomas. Therefore, the expression of extracellular matrix components plays a role in pituitary tumorigenesis. On the other hand, the remodeling of the extracellular matrix affects pituitary cell proliferation. Matrix metalloproteinase activity is very high in all types of human pituitary adenomas. Matrix metalloproteinase secreted by pituitary cells can release growth factors from the extracellular matrix that, in turn, control pituitary cell proliferation and hormone secretion. In summary, the differential expression of extracellular matrix components, integrins and matrix metalloproteinase contributes to the control of pituitary hormone production and cell proliferation during tumorigenesis.

  14. Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations

    Li Yongchao

    2012-01-01

    Full Text Available Abstract Background The model bacterium Clostridium cellulolyticum efficiently degrades crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H2 and CO2, the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels production. Therefore genetic engineering will likely be required to improve the ethanol yield. Plasmid transformation, random mutagenesis and heterologous expression systems have previously been developed for C. cellulolyticum, but targeted mutagenesis has not been reported for this organism, hindering genetic engineering. Results The first targeted gene inactivation system was developed for C. cellulolyticum, based on a mobile group II intron originating from the Lactococcus lactis L1.LtrB intron. This markerless mutagenesis system was used to disrupt both the paralogous L-lactate dehydrogenase (Ccel_2485; ldh and L-malate dehydrogenase (Ccel_0137; mdh genes, distinguishing the overlapping substrate specificities of these enzymes. Both mutations were then combined in a single strain, resulting in a substantial shift in fermentation toward ethanol production. This double mutant produced 8.5-times more ethanol than wild-type cells growing on crystalline cellulose. Ethanol constituted 93% of the major fermentation products, corresponding to a molar ratio of ethanol to organic acids of 15, versus 0.18 in wild-type cells. During growth on acid-pretreated switchgrass, the double mutant also produced four times as much ethanol as wild-type cells. Detailed metabolomic analyses identified increased flux through the oxidative branch of the mutant's tricarboxylic acid pathway. Conclusions The efficient intron-based gene inactivation system produced the first non-random, targeted mutations in C. cellulolyticum. As a key component of the genetic toolbox

  15. Genome-Wide Analysis of the AP2/ERF Gene Family in Physic Nut and Overexpression of the JcERF011 Gene in Rice Increased Its Sensitivity to Salinity Stress.

    Tang, Yuehui; Qin, Shanshan; Guo, Yali; Chen, Yanbo; Wu, Pingzhi; Chen, Yaping; Li, Meiru; Jiang, Huawu; Wu, Guojiang

    2016-01-01

    The AP2/ERF transcription factors play crucial roles in plant growth, development and responses to biotic and abiotic stresses. A total of 119 AP2/ERF genes (JcAP2/ERFs) have been identified in the physic nut genome; they include 16 AP2, 4 RAV, 1 Soloist, and 98 ERF genes. Phylogenetic analysis indicated that physic nut AP2 genes could be divided into 3 subgroups, while ERF genes could be classed into 11 groups or 43 subgroups. The AP2/ERF genes are non-randomly distributed across the 11 linkage groups of the physic nut genome and retain many duplicates which arose from ancient duplication events. The expression patterns of several JcAP2/ERF duplicates in the physic nut showed differences among four tissues (root, stem, leaf, and seed), and 38 JcAP2/ERF genes responded to at least one abiotic stressor (drought, salinity, phosphate starvation, and nitrogen starvation) in leaves and/or roots according to analysis of digital gene expression tag data. The expression of JcERF011 was downregulated by salinity stress in physic nut roots. Overexpression of the JcERF011 gene in rice plants increased its sensitivity to salinity stress. The increased expression levels of several salt tolerance-related genes were impaired in the JcERF011-overexpressing plants under salinity stress.

  16. Knockout of a P-glycoprotein gene increases susceptibility to abamectin and emamectin benzoate in Spodoptera exigua.

    Zuo, Y-Y; Huang, J-L; Wang, J; Feng, Y; Han, T-T; Wu, Y-D; Yang, Y-H

    2018-02-01

    P-glycoprotein [P-gp or the ATP-binding cassette transporter B1 (ABCB1)] is an important participant in multidrug resistance of cancer cells, yet the precise function of this arthropod transporter is unknown. The aim of this study was to determine the importance of P-gp for susceptibility to insecticides in the beet armyworm (Spodoptera exigua) using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene-editing technology. We cloned an open reading frame (ORF) encoding the S. exigua P-gp protein (SeP-gp) predicted to display structural characteristics common to P-gp and other insect ABCB1 transporters. A knockout line with a frame shift deletion of four nucleotides in the SeP-gp ORF was established using the CRISPR/Cas9 gene-editing system to test its potential role in determining susceptibility to chemical insecticides or insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). Results from comparative bioassays demonstrate that knockout of SeP-gp significantly increases susceptibility of S. exigua by around threefold to abamectin and emamectin benzoate (EB), but not to spinosad, chlorfenapyr, beta-cypermethrin, carbosulfan indoxacarb, chlorpyrifos, phoxim, diafenthiuron, chlorfluazuron, chlorantraniliprole or two Bt toxins (Cry1Ca and Cry1Fa). Our data support an important role for SeP-gp in susceptibility of S. exigua to abamectin and EB and imply that overexpression of SeP-gp may contribute to abamectin and EB resistance in S. exigua. © 2017 The Royal Entomological Society.

  17. Using targeted enrichment of nuclear genes to increase phylogenetic resolution in the neotropical rain forest genus Inga (Leguminosae: Mimosoideae

    James A Nicholls

    2015-09-01

    Full Text Available Evolutionary radiations are prominent and pervasive across many plant lineages in diverse geographical and ecological settings; in neotropical rainforests there is growing evidence suggesting that a significant fraction of species richness is the result of recent radiations. Understanding the evolutionary trajectories and mechanisms underlying these radiations demands much greater phylogenetic resolution than is currently available for these groups. The neotropical tree genus Inga (Leguminosae is a good example, with ~300 extant species and a crown age of 2-10 MY, yet over 6kb of plastid and nuclear DNA sequence data gives only poor phylogenetic resolution among species. Here we explore the use of larger-scale nuclear gene data obtained though targeted enrichment to increase phylogenetic resolution within Inga. Transcriptome data from three Inga species were used to select 264 nuclear loci for targeted enrichment and sequencing. Following quality control to remove probable paralogs from these sequence data, the final dataset comprised 259,313 bases from 194 loci for 24 accessions representing 22 Inga species and an outgroup (Zygia. Bayesian phylogenies reconstructed using either all loci concatenated or a subset of 60 loci in a gene-tree/species-tree approach yielded highly resolved phylogenies. We used coalescent approaches to show that the same targeted enrichment data also have significant power to discriminate among alternative within-species population histories in the widespread species I. umbellifera. In either application, targeted enrichment simplifies the informatics challenge of identifying orthologous loci associated with de novo genome sequencing. We conclude that targeted enrichment provides the large volumes of phylogenetically-informative sequence data required to resolve relationships within recent plant species radiations, both at the species level and for within-species phylogeographic studies.

  18. Aerobic training increases the expression of adiponectin receptor genes in the peripheral blood mononuclear cells of young men

    SH Lee

    2015-09-01

    Full Text Available Little is known about the effect of exercise training on the expression of adiponectin receptor genes in peripheral blood mononuclear cells (PBMCs. In this study, we investigated the effects of aerobic training on the expression of AdipoR1 and AidpoR2 mRNAs in PBMCs, whole body insulin sensitivity, and circulating adiponectins in men. Thirty young men were randomly assigned to either a control (n=15 or an exercise (n=15 group. Subjects assigned to the exercise group underwent a 12-week jogging and/or running programme on a motor-driven treadmill at an intensity of 60%-75% of the age-based maximum heart rate with duration of 40 minutes per session and a frequency of 5 days per week. Two-way mixed ANOVA with repeated measures was used to test any significant time-by-group interaction effects for the measured variables at p=0.05. We found significant time-by-group interaction effects for waist circumference (p=0.001, VO2 max (p<0.001, fasting insulin (p=0.016, homeostasis model assessment for insulin resistance (HOMA-IR (p=0.010, area under the curve (AUC for insulin response during the 75-g oral glucose tolerance test (p=0.002, high-molecular weight (HMW adiponectin (p=0.016, and the PBMC mRNA levels of AdipoR1 (p<0.001 and AdipoR2 (p=0.001. The exercise group had significantly increased mRNA levels of AdipoR1 and AdipoR2 in PBMCs, along with increased whole body insulin sensitivity and HMW adiponectin, decreased waist circumference, and increased VO2 max compared with the control group. In summary, the current findings suggest that exercise training modulates the expression of AdipoR1 and AdipoR2 mRNAs in PBMCs, implying that manipulation of the expression of these genes could be a potential surrogate for lifestyle intervention-mediated improvements of whole body insulin sensitivity and glucose homeostasis.

  19. Overexpressing Exogenous 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS Genes Increases Fecundity and Auxin Content of Transgenic Arabidopsis Plants

    Jia Fang

    2018-02-01

    Full Text Available Transgenic glyphosate-tolerant plants overproducing EPSPS (5-enolpyruvylshikimate-3-phosphate synthase may exhibit enhanced fitness in glyphosate-free environments. If so, introgression of transgenes overexpressing EPSPS into wild relative species may lead to increased competitiveness of crop-wild hybrids, resulting in unpredicted environmental impact. Assessing fitness effects of transgenes overexpressing EPSPS in a model plant species can help address this question, while elucidating how overproducing EPSPS affects the fitness-related traits of plants. We produced segregating T2 and T3Arabidopsis thaliana lineages with or without a transgene overexpressing EPSPS isolated from rice or Agrobacterium (CP4. For each of the three transgenes, we compared glyphosate tolerance, some fitness-related traits, and auxin (indole-3-acetic acid content in transgene-present, transgene-absent, empty vector (EV, and parental lineages in a common-garden experiment. We detected substantially increased glyphosate tolerance in T2 plants of transgene-present lineages that overproduced EPSPS. We also documented significant increases in fecundity, which was associated with increased auxin content in T3 transgene-present lineages containing rice EPSPS genes, compared with their segregating transgene-absent lineages, EV, and parental controls. Our results from Arabidopsis with nine transgenic events provide a strong support to the hypothesis that transgenic plants overproducing EPSPS can benefit from a fecundity advantage in glyphosate-free environments. Stimulated biosynthesis of auxin, an important plant growth hormone, by overproducing EPSPS may play a role in enhanced fecundity of the transgenic Arabidopsis plants. The obtained knowledge is useful for assessing environmental impact caused by introgression of transgenes overproducing EPSPS from any GE crop into populations of its wild relatives.

  20. Overexpressing Exogenous 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) Genes Increases Fecundity and Auxin Content of Transgenic Arabidopsis Plants.

    Fang, Jia; Nan, Peng; Gu, Zongying; Ge, Xiaochun; Feng, Yu-Qi; Lu, Bao-Rong

    2018-01-01

    Transgenic glyphosate-tolerant plants overproducing EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) may exhibit enhanced fitness in glyphosate-free environments. If so, introgression of transgenes overexpressing EPSPS into wild relative species may lead to increased competitiveness of crop-wild hybrids, resulting in unpredicted environmental impact. Assessing fitness effects of transgenes overexpressing EPSPS in a model plant species can help address this question, while elucidating how overproducing EPSPS affects the fitness-related traits of plants. We produced segregating T 2 and T 3 Arabidopsis thaliana lineages with or without a transgene overexpressing EPSPS isolated from rice or Agrobacterium ( CP4 ). For each of the three transgenes, we compared glyphosate tolerance, some fitness-related traits, and auxin (indole-3-acetic acid) content in transgene-present, transgene-absent, empty vector (EV), and parental lineages in a common-garden experiment. We detected substantially increased glyphosate tolerance in T 2 plants of transgene-present lineages that overproduced EPSPS. We also documented significant increases in fecundity, which was associated with increased auxin content in T 3 transgene-present lineages containing rice EPSPS genes, compared with their segregating transgene-absent lineages, EV, and parental controls. Our results from Arabidopsis with nine transgenic events provide a strong support to the hypothesis that transgenic plants overproducing EPSPS can benefit from a fecundity advantage in glyphosate-free environments. Stimulated biosynthesis of auxin, an important plant growth hormone, by overproducing EPSPS may play a role in enhanced fecundity of the transgenic Arabidopsis plants. The obtained knowledge is useful for assessing environmental impact caused by introgression of transgenes overproducing EPSPS from any GE crop into populations of its wild relatives.

  1. Variant at serotonin transporter gene predicts increased imitation in toddlers: relevance to the human capacity for cumulative culture.

    Schroeder, Kari Britt; Asherson, Philip; Blake, Peter R; Fenstermacher, Susan K; Saudino, Kimberly J

    2016-04-01

    Cumulative culture ostensibly arises from a set of sociocognitive processes which includes high-fidelity production imitation, prosociality and group identification. The latter processes are facilitated by unconscious imitation or social mimicry. The proximate mechanisms of individual variation in imitation may thus shed light on the evolutionary history of the human capacity for cumulative culture. In humans, a genetic component to variation in the propensity for imitation is likely. A functional length polymorphism in the serotonin transporter gene, the short allele at 5HTTLPR, is associated with heightened responsiveness to the social environment as well as anatomical and activational differences in the brain's imitation circuity. Here, we evaluate whether this polymorphism contributes to variation in production imitation and social mimicry. Toddlers with the short allele at 5HTTLPR exhibit increased social mimicry and increased fidelity of demonstrated novel object manipulations. Thus, the short allele is associated with two forms of imitation that may underlie the human capacity for cumulative culture. The short allele spread relatively recently, possibly due to selection, and its frequency varies dramatically on a global scale. Diverse observations can be unified via conceptualization of 5HTTLPR as influencing the propensity to experience others' emotions, actions and sensations, potentially through the mirror mechanism. © 2016 The Author(s).

  2. Extracellular secretion of recombinant proteins

    Linger, Jeffrey G.; Darzins, Aldis

    2014-07-22

    Nucleic acids encoding secretion signals, expression vectors containing the nucleic acids, and host cells containing the expression vectors are disclosed. Also disclosed are polypeptides that contain the secretion signals and methods of producing polypeptides, including methods of directing the extracellular secretion of the polypeptides. Exemplary embodiments include cellulase proteins fused to secretion signals, methods to produce and isolate these polypeptides, and methods to degrade lignocellulosic biomass.

  3. An Optimized Protocol to Increase Virus-Induced Gene Silencing Efficiency and Minimize Viral Symptoms in Petunia

    Broderick, Shaun R.; Jones, Michelle L.

    2013-01-01

    Virus-induced gene silencing (VIGS) is used to down-regulate endogenous plant genes. VIGS efficiency depends on viral proliferation and systemic movement throughout the plant. Although tobacco rattle virus (TRV)-based VIGS has been successfully used in petunia (Petunia × hybrida), the protocol has not been thoroughly optimized for efficient and uniform gene down-regulation in this species. Therefore, we evaluated six parameters that improved VIGS in petunia. Inoculation of mechanically wounde...

  4. Erwinia carotovora extracellular proteases : characterization and role in soft rot

    Kyöstiö, Sirkka R. M.

    1990-01-01

    Erwinia carotovora subsp. carotovora (Ecc) strain EC14, a Gram-negative bacterium, causes soft rot on several crops, including potato. Maceration of potato tuber tissue is caused by secreted pectolytic enzymes. Other cell-degrading enzymes may also have roles in pathogenesis, including cellulases, phospholipases, and protease(s). The objectives of this research were to (1) characterize Ecc extracellular protease (Prt) and (2) elucidate its role in potato soft rot. A gene enc...

  5. Compensatory increase in alpha 1-globin gene expression in individuals heterozygous for the alpha-thalassemia-2 deletion.

    Liebhaber, S A; Cash, F E; Main, D M

    1985-01-01

    alpha-Globin is encoded by the two adjacent genes, alpha 1 and alpha 2. Although it is clearly established that both alpha-globin genes are expressed, their relative contributions to alpha-globin messenger RNA (mRNA) and protein synthesis are not fully defined. Furthermore, changes that may occur in alpha-globin gene activity secondarily to the loss of function of one or more of these genes (alpha-thalassemia [Thal]) have not been directly investigated. This study further defines the expressi...

  6. The 341C/T polymorphism in the GSTP1 gene is associated with increased risk of oesophageal cancer

    Dandara Collet

    2010-06-01

    Full Text Available Abstract Background The Glutathione S-transferases (GSTs comprise a group of enzymes that are critical in the detoxification of carcinogens. In this study the effects of polymorphisms in these genes on the risk of developing oesophageal squamous cell carcinoma (OSCC were evaluated in a hospital-based case-control study in two South African population groups. Genetic polymorphisms in GSTs were investigated in 245 patients and 288 controls samples by PCR-RFLP analysis. Results The GSTP1 341T variant was associated with significantly increased risk of developing OSCC as observed from the odds ratios for the GSTP1 341C/T and GSTP1 341T/T genotypes (OR = 4.98; 95%CI 3.05-8.11 and OR = 10.9; 95%CI 2.43-49.1, respectively when compared to the homozygous GSTP1 341C/C genotype. The risk for OSCC in the combined GSTP1 341C/T and T/T genotypes was higher in tobacco smokers (OR = 7.51, 95% CI 3.82-14.7, alcohol consumers (OR = 15.3, 95% CI 1.81-12.9 and those using wood or charcoal for cooking and heating (OR = 12.1, 95% CI 3.26-49 when compared to those who did not smoke tobacco, or did not consume alcohol or user other forms of fuel for cooking and heating. Despite the close proximity of the two GSTP1 SNPs (313A>G and 341C>T, they were not in linkage disequilibrium in these two population groups (D':1.0, LOD: 0.52, r2: 0.225. The GSTP1 313A/G polymorphism on the other hand, did not display any association with OSSC. The homozygous GSTT1*0 genotype was associated with increased risk of OSCC (OR = 1.71, 95%CI 1.18-2.46 while the homozygous GSTM1*0 genotype was associated with significantly decreased risk of OSCC in the Mixed Ancestry subjects (OR= 0.39, 95%CI 0.25-0.62. Conclusions This study shows that the risk of developing OSCC in the South African population can be partly explained by genetic polymorphisms in GST coding genes and their interaction with environmental factors such as tobacco smoke and alcohol consumption.

  7. TRANSGENIC PLANTS OF RAPE (BRASSICA NAPUS L. WITH GENE OSMYB4 HAVE INCREASED RESISTANCE TO SALTS OF HEAVY METALS

    Raldugina G.N.

    2012-08-01

    Full Text Available This work aims to study the response of the transgenic spring rape plants (Brassica napus L. var. ‘Westar’ with the rice transfactor-encoding gene Osmyb4 to treatment with salts of heavy metals (HM CuSO4 or ZnSO4 and accumulation in the leaves of biomass, metals, photosynthetic pigments, lipid peroxidation, and antioxidant compounds: total phenols, anthocyanins, and antioxidant enzyme activity superoxide dismutase (SOD and guaiacol peroxidase (POX were determined. Vegetatively propagated transgenic plants and wild-type plants were grown on Hoagland-Snyder medium at 24°C, then at the 5-6th leaves-stage, CuSO4 (in concentration 25-150 mM or ZnSO4 (500 - 5000 mM were added to the growth medium, and plants were exposed to the salts for 15 days. Under the action of small concentrations of salts, the results obtained for the transgenic and untransformed plants did not differ, but at high concentrations strong differences between transgenic and untransformed plants were observed. In transgenic plants, accumulation of biomass was greater. Carotene and xanthophyll were destroyed in transgenic plants less than in the untransformed plants. They have accumulated in their leaves more metal, especially Zn, reaching almost to the accumulation of 7 mg per g of dry biomass, bringing these plants to the hyperaccumulation of Zn. In the tissues of transgenic plants exposed to high concentrations of salts, the content of total phenols, anthocyanins, and low molecular weight compounds, that are responsible for protection against ROS, increased significantly. All these results indicate a greater stability of the transgenic plants to the action of heavy metals, as evidenced also by less activity of lipid peroxidases in their tissue: at high salt concentrations, malondialdehyde (MDA accumulated significantly less in transgenic plants than in non-transformed plant tissues. The greater stability of transgenic plants to stressful effect of HM is also evidenced by the

  8. Recent and projected increases in atmospheric CO2 concentration can enhance gene flow between wild and genetically altered rice (Oryza sativa).

    Ziska, Lewis H; Gealy, David R; Tomecek, Martha B; Jackson, Aaron K; Black, Howard L

    2012-01-01

    Although recent and projected increases in atmospheric carbon dioxide can alter plant phenological development, these changes have not been quantified in terms of floral outcrossing rates or gene transfer. Could differential phenological development in response to rising CO(2) between genetically modified crops and wild, weedy relatives increase the spread of novel genes, potentially altering evolutionary fitness? Here we show that increasing CO(2) from an early 20(th) century concentration (300 µmol mol(-1)) to current (400 µmol mol(-1)) and projected, mid-21(st) century (600 µmol mol(-1)) values, enhanced the flow of genes from wild, weedy rice to the genetically altered, herbicide resistant, cultivated population, with outcrossing increasing from 0.22% to 0.71% from 300 to 600 µmol mol(-1). The increase in outcrossing and gene transfer was associated with differential increases in plant height, as well as greater tiller and panicle production in the wild, relative to the cultivated population. In addition, increasing CO(2) also resulted in a greater synchronicity in flowering times between the two populations. The observed changes reported here resulted in a subsequent increase in rice dedomestication and a greater number of weedy, herbicide-resistant hybrid progeny. Overall, these data suggest that differential phenological responses to rising atmospheric CO(2) could result in enhanced flow of novel genes and greater success of feral plant species in agroecosystems.

  9. Recent and projected increases in atmospheric CO2 concentration can enhance gene flow between wild and genetically altered rice (Oryza sativa.

    Lewis H Ziska

    Full Text Available Although recent and projected increases in atmospheric carbon dioxide can alter plant phenological development, these changes have not been quantified in terms of floral outcrossing rates or gene transfer. Could differential phenological development in response to rising CO(2 between genetically modified crops and wild, weedy relatives increase the spread of novel genes, potentially altering evolutionary fitness? Here we show that increasing CO(2 from an early 20(th century concentration (300 µmol mol(-1 to current (400 µmol mol(-1 and projected, mid-21(st century (600 µmol mol(-1 values, enhanced the flow of genes from wild, weedy rice to the genetically altered, herbicide resistant, cultivated population, with outcrossing increasing from 0.22% to 0.71% from 300 to 600 µmol mol(-1. The increase in outcrossing and gene transfer was associated with differential increases in plant height, as well as greater tiller and panicle production in the wild, relative to the cultivated population. In addition, increasing CO(2 also resulted in a greater synchronicity in flowering times between the two populations. The observed changes reported here resulted in a subsequent increase in rice dedomestication and a greater number of weedy, herbicide-resistant hybrid progeny. Overall, these data suggest that differential phenological responses to rising atmospheric CO(2 could result in enhanced flow of novel genes and greater success of feral plant species in agroecosystems.

  10. Increased yield of PCR products by addition of T4 gene 32 protein to the SMART PCR cDNA synthesis system.

    Villalva, C; Touriol, C; Seurat, P; Trempat, P; Delsol, G; Brousset, P

    2001-07-01

    Under certain conditions, T4 gene 32 protein is known to increase the efficiency of different enzymes, such as Taq DNA polymerase, reverse transcriptase, and telomerase. In this study, we compared the efficiency of the SMART PCR cDNA synthesis kit with and without the T4 gene 32 protein. The use of this cDNA synthesis procedure, in combination with T4 gene 32 protein, increases the yield of RT-PCR products from approximately 90% to 150%. This effect is even observed for long mRNA templates and low concentrations of total RNA (25 ng). Therefore, we suggest the addition of T4 gene 32 protein in the RT-PCR mixture to increase the efficiency of cDNA synthesis, particularly in cases when low amounts of tissue are used.

  11. Nanostructured gold microelectrodes for extracellular recording

    Brueggemann, Dorothea; Wolfrum, Bernhard; Maybeck, Vanessa; Offenhaeusser, Andreas [CNI Center of Nanoelectronic Systems for Information Technology and Institute of Bio- and Nanosystems 2, Forschungszentrum Juelich (Germany)

    2010-07-01

    Electrophysiological activity of electrogenic cells is currently recorded with planar bioelectronic interfaces such as microelectrode arrays (MEAs). In this work, a novel concept of biocompatible nanostructured gold MEAs for extracellular signal recording is presented. MEAs were fabricated using clean room technologies, e.g. photolithography and metallization. Subsequently, they were modified with gold nanopillars of approximately 300 to 400 nm in height and 60 nm width. The nanostructuring process was carried out with a template-assisted approach using nanoporous aluminium oxide. Impedance spectroscopy of the resulting nanostructures showed higher capacitances compared to planar gold. This confirmed the expected increase of the surface area via nanostructuring. We used the nanostructured microelectrodes to record extracellular potentials from heart muscle cells (HL1), which were plated onto the chips. Good coupling between the HL1 cells and the nanostructured electrodes was observed. The resulting signal-to-noise ratio of nanopillar-MEAs was increased by a factor of 2 compared to planar MEAs. In future applications this nanopillar concept can be adopted for distinct interface materials and coupling to cellular and molecular sensing components.

  12. Extracellular histones induce erythrocyte fragility and anemia.

    Kordbacheh, Farzaneh; O'Meara, Connor H; Coupland, Lucy A; Lelliott, Patrick M; Parish, Christopher R

    2017-12-28

    Extracellular histones have been shown to play an important pathogenic role in many diseases, primarily through their cytotoxicity toward nucleated cells and their ability to promote platelet activation with resultant thrombosis and thrombocytopenia. In contrast, little is known about the effect of extracellular histones on erythrocyte function. We demonstrate in this study that histones promote erythrocyte aggregation, sedimentation, and using a novel in vitro shear stress model, we show that histones induce erythrocyte fragility and lysis in a concentration-dependent manner. Furthermore, histones impair erythrocyte deformability based on reduced passage of erythrocytes through an artificial spleen. These in vitro results were mirrored in vivo with the injection of histones inducing anemia within minutes of administration, with a concomitant increase in splenic hemoglobin content. Thrombocytopenia and leukopenia were also observed. These findings suggest that histones binding to erythrocytes may contribute to the elevated erythrocyte sedimentation rates observed in inflammatory conditions. Furthermore, histone-induced increases in red blood cell lysis and splenic clearance may be a significant factor in the unexplained anemias seen in critically ill patients. © 2017 by The American Society of Hematology.

  13. Increasing Power by Sharing Information from Genetic Background and Treatment in Clustering of Gene Expression Time Series

    Sura Zaki Alrashid

    2018-02-01

    Full Text Available Clustering of gene expression time series gives insight into which genes may be co-regulated, allowing us to discern the activity of pathways in a given microarray experiment. Of particular interest is how a given group of genes varies with different conditions or genetic background. This paper develops
a new clustering method that allows each cluster to be parameterised according to whether the behaviour of the genes across conditions is correlated or anti-correlated. By specifying correlation between such genes,more information is gain within the cluster about how the genes interrelate. Amyotrophic lateral sclerosis (ALS is an irreversible neurodegenerative disorder that kills the motor neurons and results in death within 2 to 3 years from the symptom onset. Speed of progression for different patients are heterogeneous with significant variability. The SOD1G93A transgenic mice from different backgrounds (129Sv and C57 showed consistent phenotypic differences for disease progression. A hierarchy of Gaussian isused processes to model condition-specific and gene-specific temporal co-variances. This study demonstrated about finding some significant gene expression profiles and clusters of associated or co-regulated gene expressions together from four groups of data (SOD1G93A and Ntg from 129Sv and C57 backgrounds. Our study shows the effectiveness of sharing information between replicates and different model conditions when modelling gene expression time series. Further gene enrichment score analysis and ontology pathway analysis of some specified clusters for a particular group may lead toward identifying features underlying the differential speed of disease progression.

  14. Epidermal growth factor receptor activation in glioblastoma through novel missense mutations in the extracellular domain.

    Jeffrey C Lee

    2006-12-01

    Full Text Available Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy.Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132 of glioblastomas and 12.5% (1/8 of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors.Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma.

  15. A combination of biomolecules enhances expression of E-cadherin and peroxisome proliferator-activated receptor gene leading to increased cell proliferation in primary human meniscal cells: an in vitro study.

    Pillai, Mamatha M; Elakkiya, V; Gopinathan, J; Sabarinath, C; Shanthakumari, S; Sahanand, K Santosh; Dinakar Rai, B K; Bhattacharyya, Amitava; Selvakumar, R

    2016-10-01

    The present study investigates the impact of biomolecules (biotin, glucose, chondroitin sulphate, proline) as supplement, (individual and in combination) on primary human meniscus cell proliferation. Primary human meniscus cells isolated from patients undergoing meniscectomy were maintained in Dulbecco's Modified Eagle's Medium (DMEM). The isolated cells were treated with above mentioned biomolecules as individual (0-100 µg/ml) and in combinations, as a supplement to DMEM. Based on the individual biomolecule study, a unique combination of biomolecules (UCM) was finalized using one way ANOVA analysis. With the addition of UCM as supplement to DMEM, meniscal cells reached 100 % confluency within 4 days in 60 mm culture plate; whereas the cells in medium devoid of UCM, required 36 days for reaching confluency. The impact of UCM on cell viability, doubling time, histology, gene expression, biomarkers expression, extra cellular matrix synthesis, meniscus cell proliferation with respect to passages and donor's age were investigated. The gene expression studies for E-cadherin and peroxisome proliferator-activated receptor (PPAR∆) using RT-qPCR and immunohistochemical analysis for Ki67, CD34 and Vimentin confirmed that UCM has significant impact on cell proliferation. The extracellular collagen and glycosaminoglycan secretion in cells supplemented with UCM were found to increase by 31 and 37 fold respectively, when compared to control on the 4th day. The cell doubling time was reduced significantly when supplemented with UCM. The addition of UCM showed positive influence on different passages and age groups. Hence, this optimized UCM can be used as an effective supplement for meniscal tissue engineering.

  16. Colonic vascular conductance increased by Daikenchuto via calcitonin gene-related peptide and receptor-activity modifying protein 1.

    Kono, Toru; Koseki, Takashi; Chiba, Shinichi; Ebisawa, Yoshiaki; Chisato, Naoyuki; Iwamoto, Jun; Kasai, Shinichi

    2008-11-01

    Daikencyuto (DKT) is a traditional Japanese medicine (Kampo) and is a mixture of extract powders from dried Japanese pepper, processed ginger, ginseng radix, and maltose powder and has been used as the treatment of paralytic ileus. DKT may increase gastrointestinal motility by an up-regulation of the calcitonin gene-related peptide (CGRP). CGRP is also the most powerful vasoactive substance. In the present study, we investigated whether DKT has any effect on the colonic blood flow in rats. Experiments were performed on fasted anesthetized and artificially ventilated Wistar rats. Systemic mean arterial blood pressure and heart rate were recorded. Red blood cell flux in colonic blood flow was measured using noncontact laser tissue blood flowmetry, and colonic vascular conductance (CVC) was calculated as the ratio of flux to mean arterial blood pressure. We examined four key physiological mechanisms underlying the response using blocker drugs: CGRP1 receptor blocker (CGRP(8-37)), nitric oxide synthase inhibitor, vasoactive intestinal polypeptide (VIP) receptor blocker ([4-Cl-DPhe6, Leu17]-VIP), and substance P receptor blocker (spantide). Reverse transcription-polymerase chain reaction was used for the detection of mRNA of calcitonin receptor-like receptor, receptor-activity modifying protein 1, the component of CGRP 1 receptor and CGRP. After laparotomy, a cannula was inserted into the proximal colon to administer the DKT and to measure CVC at the distal colon. Intracolonal administration of DKT (10, 100, and 300 mg/kg) increased CVC (basal CVC, 0.10 mL/mmHg) from the first 15-min observation period (0.14, 0.17, and 0.17 mL/mmHg, respectively) and with peak response at either 45 min (0.17 mL/mmHg by 10 mg/kg), or 75 and 60 min (0.23 and 0.21 mL/mmHg by 100 and 300 mg/kg, respectively). CGRP(8-37) completely abolished the DKT-induced hyperemia, whereas nitric oxide synthase inhibitor partially attenuated the DKT-induced hyperemia. [4-Cl-DPhe6, Leu17]-VIP and spantide

  17. Experimental diabetes increases insulin-like growth factor I and II receptor concentration and gene expression in kidney

    Werner, H.; Shen-Orr, Z.; Stannard, B.; Burguera, B.; Roberts, C.T. Jr.; LeRoith, D.

    1990-01-01

    Insulinlike growth factor I (IGF-I) is a mitogenic hormone with important regulatory roles in growth and development. One of the target organs for IGF-I action is the kidney, which synthesizes abundant IGF-I receptors and IGF-I itself. To study the involvement of IGF-I and the IGF-I receptor in the development of nephropathy, one of the major complications of diabetes mellitus, we measured the expression of these genes in the kidney and in other tissues of the streptozocin-induced diabetic rat. The binding of 125I-labeled IGF-I to crude membranes was measured in the same tissues. We observed a 2.5-fold increase in the steady-state level of IGF-I-receptor mRNA in the diabetic kidney, which was accompanied by a 2.3-fold increase in IGF-I binding. In addition to this increase in IGF-I binding to the IGF-I receptor, there was also binding to a lower-molecular-weight material that may represent an IGF-binding protein. No change was detected in the level of IGF-I-peptide mRNA. Similarly, IGF-II-receptor mRNA levels and IGF-II binding were significantly increased in the diabetic kidney. IGF-I- and IGF-II-receptor mRNA levels and IGF-I and IGF-II binding returned to control values after insulin treatment. Because the IGF-I receptor is able to transduce mitogenic signals on activation of its tyrosine kinase domain, we hypothesize that, among other factors, high levels of receptor in the diabetic kidney may also be involved in the development of diabetic nephropathy. Increased IGF-II-receptor expression in the diabetic kidney may be important for the intracellular transport and packaging of lysosomal enzymes, although a role for this receptor in signal transduction cannot be excluded. Finally, the possible role of IGF-binding proteins requires further study

  18. Carboxylesterase 1A2 encoding gene with increased transcription and potential rapid drug metabolism in Asian populations

    Rasmussen, Henrik Berg; Madsen, Majbritt Busk; Lyauk, Yassine Kamal

    2017-01-01

    The carboxylesterase 1 gene (CES1) encodes a hydrolase implicated in the metabolism of commonly used drugs. CES1A2, a hybrid of CES1 and a CES1-like pseudogene, has a promoter that is weak in most individuals. However, some individuals harbor a promoter haplotype of this gene with two overlapping...

  19. Recurrent RECQL4 Imbalance and Increased Gene Expression Levels Are Associated with Structural Chromosomal Instability in Sporadic Osteosarcoma

    Georges Maire

    2009-03-01

    Full Text Available Osteosarcoma (OS is an aggressive bone tumor with complex abnormal karyotypes and a highly unstable genome, exhibiting both numerical- and structural-chromosomal instability (N- and S-CIN. Chromosomal rearrangements and genomic imbalances affecting 8q24 are frequent in OS. RECQL4 gene maps to this cytoband and encodes a putative helicase involved in the fidelity of DNA replication and repair. This protective genomic function of the protein is relevant because often patients with Rothmund-Thomson syndrome have constitutional mutations of RECQL4 and carry a very high risk of developing OS. To determine the relative level of expression of RECQL4 in OS, 18 sporadic tumors were studied by reverse transcription–polymerase chain reaction. All tumors overexpressed RECQL4 in comparison to control osteoblasts, and fluorescence in situ hybridization analysis of tumor DNA showed that expression levels were strongly copy number–dependent. Relative N- and S-CIN levels were determined by classifying copy number transitions within array comparative genomic hybridization profiles and by enumerating the frequency of break-apart fluorescence in situ hybridization within 8q24 using region-specific and control probes. Although there was no evidence that disruption of 8q24 in OS led to an elevated expression of RECQL4, there was a marked association between increased overall levels of S-CIN, determined by copy number transition frequency and higher levels of RECQL4.

  20. Lack of increased immediate early gene expression in rats reinstating cocaine-seeking behavior to discrete sensory cues.

    Matthew D Riedy

    Full Text Available Drug-seeking behavior elicited by drug-associated cues contributes to relapse in addiction; however, whether relapse elicited by drug-associated conditioned reinforcers (CR versus discriminative stimuli (DS involves distinct or overlapping neuronal populations is unknown. To address this question, we developed a novel cocaine self-administration and cue-induced reinstatement paradigm that exposed the same rats to distinct cocaine-associated CR and DS. Rats were trained to self-administer cocaine in separate sessions. In one, a DS signaled cocaine availability; in the other, cocaine delivery was paired with a different CR. After extinction training and reinstatement testing, where both cues were presented in separate sessions, rats were sacrificed and processed for cellular analysis of temporal activity by fluorescent in situ hybridization (CatFISH for activity regulated cytoskeleton-associated protein (Arc mRNA and for radioactive in situ hybridization for Arc and zif268 mRNAs. CatFISH did not reveal significant changes in Arc mRNA expression. Similar results were obtained with radioactive in situ hybridization. We have shown that while rats reinstate drug seeking in response to temporally discrete presentations of distinct drug-associated cues, such reinstatement is not associated with increased transcriptional activation of Arc or zif268 mRNAs, suggesting that expression of these genes may not be necessary for cue-induced reinstatement of drug-seeking behavior.

  1. Extracellular small RNAs: what, where, why?

    Hoy, Anna M.; Buck, Amy H.

    2012-01-01

    miRNAs (microRNAs) are a class of small RNA that regulate gene expression by binding to mRNAs and modulating the precise amount of proteins that get expressed in a cell at a given time. This form of gene regulation plays an important role in developmental systems and is critical for the proper function of numerous biological pathways. Although miRNAs exert their functions inside the cell, these and other classes of RNA are found in body fluids in a cell-free form that is resistant to degradation by RNases. A broad range of cell types have also been shown to secrete miRNAs in association with components of the RISC (RNA-induced silencing complex) and/or encapsulation within vesicles, which can be taken up by other cells. In the present paper, we provide an overview of the properties of extracellular miRNAs in relation to their capacity as biomarkers, stability against degradation and mediators of cell–cell communication. PMID:22817753

  2. Overexpression of the Squalene Epoxidase Gene Alone and in Combination with the 3-Hydroxy-3-methylglutaryl Coenzyme A Gene Increases Ganoderic Acid Production in Ganoderma lingzhi.

    Zhang, De-Huai; Jiang, Lu-Xi; Li, Na; Yu, Xuya; Zhao, Peng; Li, Tao; Xu, Jun-Wei

    2017-06-14

    The squalene epoxidase (SE) gene from the biosynthetic pathway of ganoderic acid (GA) was cloned and overexpressed in Ganoderma lingzhi. The strain that overexpressed the SE produced approximately 2 times more GA molecules than the wild-type (WT) strain. Moreover, SE overexpression upregulated lanosterol synthase gene expression in the biosynthetic pathway. These results indicated that SE stimulates GA accumulation. Then, the SE and 3-hydroxy-3-methylglutaryl coenzyme A (HMGR) genes were simultaneously overexpressed in G. lingzhi. Compared with the individual overexpression of SE or HMGR, the combined overexpression of the two genes further enhanced individual GA production. The overexpressing strain produced maximum GA-T, GA-S, GA-Mk, and GA-Me contents of 90.4 ± 7.5, 35.9 ± 5.4, 6.2 ± 0.5, and 61.8 ± 5.8 μg/100 mg dry weight, respectively. These values were 5.9, 4.5, 2.4, and 5.8 times higher than those produced by the WT strain. This is the first example of the successful manipulation of multiple biosynthetic genes to improve GA content in G. lingzhi.

  3. Reducing cytoplasmic polyamine oxidase activity in Arabidopsis increases salt and drought tolerance by reducing reactive oxygen species production and increasing defense gene expression

    G.H.M. eSagor

    2016-02-01

    Full Text Available The link between polyamine oxidases (PAOs, which function in polyamine catabolism, and stress responses remains elusive. Here, we address this issue using Arabidopsis pao mutants in which the expression of the five PAO genes is knocked-out or knocked-down. As the five single pao mutants and wild type (WT showed similar response to salt stress, we tried to generate the mutants that have either the cytoplasmic PAO pathway (pao1 pao5 or the peroxisomal PAO pathway (pao2 pao3 pao4 silenced. However, the latter triple mutant was not obtained. Thus, in this study, we used two double mutants, pao1 pao5 and pao2 pao4. Of interest, pao1 pao5 mutant was NaCl- and drought-tolerant, whereas pao2 pao4 showed similar sensitivity to those stresses as WT. To reveal the underlying mechanism of salt tolerance, further analyses were performed. Na uptake of the mutant (pao1 pao5 decreased to 75% of WT. PAO activity of the mutant was reduced to 62% of WT. The content of reactive oxygen species (ROS such as hydrogen peroxide, a reaction product of PAO action, and superoxide anion in the mutant became 81% and 72% of the levels in WT upon salt treatment. The mutant contained 2.8-fold higher thermospermine compared to WT. Moreover, the mutant induced the genes of salt overly sensitive-, abscisic acid (ABA-dependent- and ABA-independent- pathways more strongly than WT upon salt treatment. The results suggest that the Arabidopsis plant silencing cytoplasmic PAOs shows salinity tolerance by reducing ROS production and strongly inducing subsets of stress-responsive genes under stress conditions.

  4. Ascorbic acid deficiency increases endotoxin influx to portal blood and liver inflammatory gene expressions in ODS rats.

    Tokuda, Yuki; Miura, Natsuko; Kobayashi, Misato; Hoshinaga, Yukiko; Murai, Atsushi; Aoyama, Hiroaki; Ito, Hiroyuki; Morita, Tatsuya; Horio, Fumihiko

    2015-02-01

    The aim of this study was to determine whether ascorbic acid (AsA) deficiency-induced endotoxin influx into portal blood from the gastrointestinal tract contributes to the inflammatory changes in the liver. The mechanisms by which AsA deficiency provokes inflammatory changes in the liver were investigated in Osteogenic Disorder Shionogi (ODS) rats (which are unable to synthesize AsA). Male ODS rats (6-wk-old) were fed a diet containing sufficient (300 mg/kg) AsA (control group) or a diet without AsA (AsA-deficient group) for 14 or 18 d. On day 14, the hepatic mRNA levels of acute-phase proteins and inflammation-related genes were significantly higher in the AsA-deficient group than the control group, and these elevations by AsA deficiency were exacerbated on day 18. The serum concentrations of interleukin (IL)-1β and IL-6, which induce acute-phase proteins in the liver, were also significantly elevated on day 14 in the AsA-deficient group compared with the respective values in the control group. IL-1β mRNA levels in the liver, spleen, and lung were increased by AsA deficiency. Moreover, on both days 14 and 18, the portal blood endotoxin concentration was significantly higher in the AsA-deficient group than in the control group, and a significant correlation between serum IL-1β concentrations and portal endotoxin concentrations was found in AsA-deficient rats. In the histologic analysis of the ileum tissues, the number of goblet cells per villi was increased by AsA deficiency. These results suggest that AsA deficiency-induced endotoxin influx into portal blood from the gastrointestinal tract contributes to the inflammatory changes in the liver. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. ABNORMAL TYPE-III COLLAGEN PRODUCED BY AN EXON-17-SKIPPING MUTATION OF THE COL3A1 GENE IN EHLERS-DANLOS SYNDROME TYPE-IV IS NOT INCORPORATED INTO THE EXTRACELLULAR-MATRIX

    CHIODO, AA; SILLENCE, DO; COLE, WG; BATEMAN, JF

    1995-01-01

    A novel heterozygous mutation of the COL3Al gene that encodes the alpha 1(III) chains of type III collagen was identified in a family with the: acrogeric form of Ehlers-Danlos syndrome type IV (EDS-IV). Cultured dermal fibroblasts produced normal and shortened alpha 1(III) chains. The triple helix

  6. Skeletal muscle expresses the extracellular cyclic AMP–adenosine pathway

    Chiavegatti, T; Costa, V L; Araújo, M S; Godinho, R O

    2007-01-01

    Background and purpose: cAMP is a key intracellular signalling molecule that regulates multiple processes of the vertebrate skeletal muscle. We have shown that cAMP can be actively pumped out from the skeletal muscle cell. Since in other tissues, cAMP efflux had been associated with extracellular generation of adenosine, in the present study we have assessed the fate of interstitial cAMP and the existence of an extracellular cAMP-adenosine signalling pathway in skeletal muscle. Experimental approach: cAMP efflux and/or its extracellular degradation were analysed by incubating rat cultured skeletal muscle with exogenous cAMP, forskolin or isoprenaline. cAMP and its metabolites were quantified by radioassay or HPLC, respectively. Key results: Incubation of cells with exogenous cAMP was followed by interstitial accumulation of 5′-AMP and adenosine, a phenomenon inhibited by selective inhibitors of ecto-phosphodiesterase (DPSPX) and ecto-nucleotidase (AMPCP). Activation of adenylyl cyclase (AC) in cultured cells with forskolin or isoprenaline increased cAMP efflux and extracellular generation of 5′-AMP and adenosine. Extracellular cAMP-adenosine pathway was also observed after direct and receptor-dependent stimulation of AC in rat extensor muscle ex vivo. These events were attenuated by probenecid, an inhibitor of ATP binding cassette family transporters. Conclusions and implications: Our results show the existence of an extracellular biochemical cascade that converts cAMP into adenosine. The functional relevance of this extracellular signalling system may involve a feedback modulation of cellular response initiated by several G protein-coupled receptor ligands, amplifying cAMP influence to a paracrine mode, through its metabolite, adenosine. PMID:18157164

  7. Alterations of tumor suppressor genes (Rb, p16, p27 and p53) and an increased FDG uptake in lung cancer

    Sasaki, Masayuki; Sugio, Kenji; Kuwabara, Yasuo

    2003-01-01

    The FDG uptake in lung cancer is considered to reflect the degree of malignancy, while alterations of some tumor suppressor genes are considered to be related to the malignant biological behavior of tumors. The aim of this study is to examine the relationship between FDG-PET and alterations in the tumor suppression genes of lung cancer. We examined 28 patients with primary lung cancer who underwent FDG-PET before surgery consisting of 17 patients with adenocarcinoma, 10 with squamous cell carcinoma and 1 with large cell carcinoma. The FDG-PET findings were evaluated based on the standardized uptake value (SUV). Alterations in the tumor suppressor genes, Rb, p16, p27 and p53, were evaluated immunohistochemically. The FDG uptake in lung cancer with alteration in each tumor suppressor gene tended to be higher than in those genes without alterations, although the differences were not significant. In 15 tumors with alterations in either tumor suppressor genes, the FDG uptake was 6.83±3.21. On the other hand, the mean FDG uptake was 1.95 in 2 tumors without alterations in any genes. The difference in the FDG uptake between the 2 groups was statistically significant (p<0.001). In conclusion, the presence of abnormalities in the tumor suppressor genes, which results in an accelerated cell proliferation, is thus considered to increase the FDG uptake in lung cancer. (author)

  8. Vesiclepedia: a compendium for extracellular vesicles with continuous community annotation.

    Hina Kalra

    Full Text Available Extracellular vesicles (EVs are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.

  9. Increased methylation and decreased expression of homeobox genes TLX1, HOXA10 and DLX5 in human placenta are associated with trophoblast differentiation.

    Novakovic, Boris; Fournier, Thierry; Harris, Lynda K; James, Joanna; Roberts, Claire T; Yong, Hannah E J; Kalionis, Bill; Evain-Brion, Danièle; Ebeling, Peter R; Wallace, Euan M; Saffery, Richard; Murthi, Padma

    2017-07-03

    Homeobox genes regulate embryonic and placental development, and are widely expressed in the human placenta, but their regulatory control by DNA methylation is unclear. DNA methylation analysis was performed on human placentae from first, second and third trimesters to determine methylation patterns of homeobox gene promoters across gestation. Most homeobox genes were hypo-methylated throughout gestation, suggesting that DNA methylation is not the primary mechanism involved in regulating HOX genes expression in the placenta. Nevertheless, several genes showed variable methylation patterns across gestation, with a general trend towards an increase in methylation over gestation. Three genes (TLX1, HOXA10 and DLX5) showed inverse gains of methylation with decreasing mRNA expression throughout pregnancy, supporting a role for DNA methylation in their regulation. Proteins encoded by these genes were primarily localised to the syncytiotrophoblast layer, and showed decreased expression later in gestation. siRNA mediated downregulation of DLX5, TLX1 and HOXA10 in primary term villous cytotrophoblast resulted in decreased proliferation and increased expression of differentiation markers, including ERVW-1. Our data suggest that loss of DLX5, TLX1 and HOXA10 expression in late gestation is required for proper placental differentiation and function.

  10. Over-expression of a tobacco nitrate reductase gene in wheat (Triticum aestivum L. increases seed protein content and weight without augmenting nitrogen supplying.

    Xiao-Qiang Zhao

    Full Text Available Heavy nitrogen (N application to gain higher yield of wheat (Triticum aestivum L. resulted in increased production cost and environment pollution. How to diminish the N supply without losing yield and/or quality remains a challenge. To meet the challenge, we integrated and expressed a tobacco nitrate reductase gene (NR in transgenic wheat. The 35S-NR gene was transferred into two winter cultivars, "Nongda146" and "Jimai6358", by Agrobacterium-mediation. Over-expression of the transgene remarkably enhanced T1 foliar NR activity and significantly augmented T2 seed protein content and 1000-grain weight in 63.8% and 68.1% of T1 offspring (total 67 individuals analyzed, respectively. Our results suggest that constitutive expression of foreign nitrate reductase gene(s in wheat might improve nitrogen use efficiency and thus make it possible to increase seed protein content and weight without augmenting N supplying.

  11. Over-expression of a tobacco nitrate reductase gene in wheat (Triticum aestivum L.) increases seed protein content and weight without augmenting nitrogen supplying.

    Zhao, Xiao-Qiang; Nie, Xuan-Li; Xiao, Xing-Guo

    2013-01-01

    Heavy nitrogen (N) application to gain higher yield of wheat (Triticum aestivum L.) resulted in increased production cost and environment pollution. How to diminish the N supply without losing yield and/or quality remains a challenge. To meet the challenge, we integrated and expressed a tobacco nitrate reductase gene (NR) in transgenic wheat. The 35S-NR gene was transferred into two winter cultivars, "Nongda146" and "Jimai6358", by Agrobacterium-mediation. Over-expression of the transgene remarkably enhanced T1 foliar NR activity and significantly augmented T2 seed protein content and 1000-grain weight in 63.8% and 68.1% of T1 offspring (total 67 individuals analyzed), respectively. Our results suggest that constitutive expression of foreign nitrate reductase gene(s) in wheat might improve nitrogen use efficiency and thus make it possible to increase seed protein content and weight without augmenting N supplying.

  12. Isolation and genetic analysis of Aspergillus niger mutants with reduced extracellular glucoamylase

    Valent, G.U.; Calil, M.R.; Bonatelli Junior, R.

    1992-01-01

    Mutants with impaired production of extracellular glucoamylase were isolated at a high frequency (2% of survivors) from an Aspergillus niger strain treated with UV light. These were designated as low glucoamylase producers (lgp, up to 30% of the parental yield) and medium producers (mgp, a 35 to 50% decrease in enzyme level). All the mutants were shown to be recessive; one strain segregated two unlinked genes. Complementation tests, and segregation from heterozygous diploid, suggested at least three to four unlinked genes, each able to impair glucoamylase production. There is evidence of a single structural gene for glucoamylase in A. niger. Therefore, as production of extracellular enzymes is normally the final result of several steps at intracellular and membrane levels, including regulation of enzyme synthesis, we suggest intergenic interaction that controls extracellular enzyme accumulation and that mutation in any of these genes would result in impaired production. (author)

  13. Crystallization and preliminary crystallographic analysis of Gibberella zeae extracellular lipase

    Sun, Yuna; Li, Ming; Zhang, Yan; Liu, Lifang; Liu, Ye; Liu, Zheng; Li, Xumei; Lou, Zhiyong

    2008-01-01

    G. zeae extracellular lipase has been overexpressed, purified and crystallized. Diffraction data were collected to 2.8 Å resolution. Fusarium head blight, one of the most destructive crop diseases, is mainly caused by Fusarium graminearum (known in its sexual stage as Gibberella zeae). F. graminearum secretes various extracellular enzymes that have been hypothesized to be involved in host infection. One of the extracellular enzymes secreted by this organism is the G. zeae extracellular lipase (GZEL), which is encoded by the FGL1 gene. In order to solve the crystal structure of GZEL and to gain a better understanding of the biological functions of the protein and of possible inhibitory mechanisms of lipase inhibitors, recombinant GZEL was crystallized at 291 K using PEG 3350 as a precipitant. A data set was collected to 2.8 Å resolution from a single flash-cooled crystal (100 K). The crystal belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 78.4, b = 91.0, c = 195.8 Å, α = β = γ = 90°. The presence of four molecules was assumed per asymmetric unit, which gave a Matthews coefficient of 2.6 Å 3 Da −1

  14. Extracellular Membrane Vesicles and Phytopathogenicity of Acholeplasma laidlawii PG8

    Vladislav M. Chernov

    2012-01-01

    Full Text Available For the first time, the phytopathogenicity of extracellular vesicles of Acholeplasma laidlawii PG8 (a ubiquitous mycoplasma that is one of the five common species of cell culture contaminants and is a causative agent for phytomycoplasmoses in Oryza sativa L. plants was studied. Data on the ability of extracellular vesicles of Acholeplasma laidlawii PG8 to penetrate from the nutrient medium into overground parts of Oryza sativa L. through the root system and to cause alterations in ultrastructural organization of the plants were presented. As a result of the analysis of ultrathin leaf sections of plants grown in medium with A. laidlawii PG8 vesicles, we detected significant changes in tissue ultrastructure characteristic to oxidative stress in plants as well as their cultivation along with bacterial cells. The presence of nucleotide sequences of some mycoplasma genes within extracellular vesicles of Acholeplasma laidlawii PG8 allowed a possibility to use PCR (with the following sequencing to perform differential detection of cells and bacterial vesicles in samples under study. The obtained data may suggest the ability of extracellular vesicles of the mycoplasma to display in plants the features of infection from the viewpoint of virulence criteria—invasivity, infectivity—and toxigenicity—and to favor to bacterial phytopathogenicity.

  15. Sodium bicarbonate cotransporter NBCe2 gene variants increase sodium and bicarbonate transport in human renal proximal tubule cells.

    Gildea, John J; Xu, Peng; Kemp, Brandon A; Carlson, Julia M; Tran, Hanh T; Bigler Wang, Dora; Langouët-Astrié, Christophe J; McGrath, Helen E; Carey, Robert M; Jose, Pedro A; Felder, Robin A

    2018-01-01

    Salt sensitivity of blood pressure affects >30% of the hypertensive and >15% of the normotensive population. Variants of the electrogenic sodium bicarbonate cotransporter NBCe2 gene, SLC4A5, are associated with increased blood pressure in several ethnic groups. SLC4A5 variants are also highly associated with salt sensitivity, independent of hypertension. However, little is known about how NBCe2 contributes to salt sensitivity, although NBCe2 regulates renal tubular sodium bicarbonate transport. We hypothesized that SLC4A5 rs10177833 and rs7571842 increase NBCe2 expression and human renal proximal tubule cell (hRPTC) sodium transport and may be a cause of salt sensitivity of blood pressure. To characterize the hRPTC ion transport of wild-type (WT) and homozygous variants (HV) of SLC4A5. The expressions of NBCe2 mRNA and protein were not different between hRPTCs carrying WT or HV SLC4A5 before or after dopaminergic or angiotensin (II and III) stimulation. However, luminal to basolateral sodium transport, NHE3 protein, and Cl-/HCO3- exchanger activity in hRPTCs were higher in HV than WT SLC4A5. Increasing intracellular sodium enhanced the apical location of NBCe2 in HV hRPTCs (4.24±0.35% to 11.06±1.72% (P<0.05, N = 3, 2-way ANOVA, Holm-Sidak test)) as determined by Total Internal Reflection Fluorescence Microscopy (TIRFM). In hRPTCs isolated from kidney tissue, increasing intracellular sodium enhanced bicarbonate-dependent pH recovery rate and increased NBCe2 mRNA and protein expressions to a greater extent in HV than WT SLC4A5 (+38.00±6.23% vs HV normal salt (P<0.01, N = 4, 2-way ANOVA, Holm-Sidak test)). In hRPTCs isolated from freshly voided urine, bicarbonate-dependent pH recovery was also faster in those from salt-sensitive and carriers of HV SLC4A5 than from salt-resistant and carriers of WT SLC4A5. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was normalized by SLC4A5- but not SLC4A4-shRNA. The binding of purified hepatocyte

  16. Extracellular Vesicles in Hematological Disorders

    Anat Aharon

    2014-10-01

    Full Text Available Extracellular vesicles (EVs, comprised of exosomes, microparticles, apoptotic bodies, and other microvesicles, are shed from a variety of cells upon cell activation or apoptosis. EVs promote clot formation, mediate pro-inflammatory processes, transfer proteins and miRNA to cells, and induce cell signaling that regulates cell differentiation, proliferation, migration, invasion, and apoptosis. This paper will review the contribution of EVs in hematological disorders, including hemoglobinopathies (sickle cell disease, thalassemia, paroxysmal nocturnal hemoglobinuria, and hematological malignancies (lymphomas, myelomas, and acute and chronic leukemias.

  17. Blood extracellular DNA after irradiation

    Vladimirov, V.G.; Tishchenko, L.I.; Surkova, E.A.; Vasil'eva, I.N.

    1993-01-01

    It has been shown that blood extracellular DNA of irradiated rats largely consists of the low-molecular DNA and its oligomers. Molecular masses of oligomers are multiple to molecular mass of monomer fragment with nucleosome size. The low-molecular DNA has linear form. The average content of GC-pairs in low-molecular DNA is higher than in total rat's DNA (48.5% against 41.5%). The low-molecular DNA is a part of complex containing RNA, acidic proteins and lipids. It is assumed that the formation of low-molecular DNA is a result of Ca/Mg - dependent nuclear endonuclease action

  18. Curd development associated gene (CDAG1) in cauliflower (Brassica oleracea L. var. botrytis) could result in enlarged organ size and increased biomass.

    Li, Hui; Liu, Qian; Zhang, Qingli; Qin, Erjun; Jin, Chuan; Wang, Yu; Wu, Mei; Shen, Guangshuang; Chen, Chengbin; Song, Wenqin; Wang, Chunguo

    2017-01-01

    The curd is a specialized organ and the most important product organ of cauliflower (Brassica oleracea L. var. botrytis). However, the mechanism underlying the regulation of curd formation and development remains largely unknown. In the present study, a novel homologous gene containing the Organ Size Related (OSR) domain, namely, CDAG1 (Curd Development Associated Gene 1) was identified in cauliflower. Quantitative analysis indicated that CDAG1 showed significantly higher transcript levels in young tissues. Functional analysis demonstrated that the ectopic overexpression of CDAG1 in Arabidopsis and cauliflower could significantly promote organ growth and result in larger organ size and increased biomass. Organ enlargement was predominantly due to increased cell number. In addition, 228 genes involved in the CDAG1-mediated regulatory network were discovered by transcriptome analysis. Among these genes, CDAG1 was confirmed to inhibit the transcriptional expression of the endogenous OSR genes, ARGOS and ARL, while a series of ethylene-responsive transcription factors (ERFs) were found to increased expression in 35S:CDAG1 transgenic Arabidopsis plants. This implies that CDAG1 may function in the ethylene-mediated signal pathway. These findings provide new insight into the function of OSR genes, and suggest potential applications of CDAG1 in breeding high-yielding crops. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  19. Maternal obesity programs increased leptin gene expression in rat male offspring via epigenetic modifications in a depot-specific manner

    Simon Lecoutre

    2017-08-01

    Conclusions: Consistent with the DOHaD hypothesis, persistent epigenetic remodeling occurs at regulatory regions especially within intergenic sequences, linked to higher leptin gene expression in adult HF offspring in a depot-specific manner.

  20. Genes encoding novel lipid transporters and their use to increase oil production in vegetative tissues of plants

    Xu, Changcheng; Fan, Jilian; Yan, Chengshi; Shanklin, John

    2017-12-26

    The present invention discloses a novel gene encoding a transporter protein trigalactosyldiacylglycerol-5 (TGD5), mutations thereof and their use to enhance TAG production and retention in plant vegetative tissue.

  1. EVpedia: an integrated database of high-throughput data for systemic analyses of extracellular vesicles

    Dae-Kyum Kim

    2013-03-01

    Full Text Available Secretion of extracellular vesicles is a general cellular activity that spans the range from simple unicellular organisms (e.g. archaea; Gram-positive and Gram-negative bacteria to complex multicellular ones, suggesting that this extracellular vesicle-mediated communication is evolutionarily conserved. Extracellular vesicles are spherical bilayered proteolipids with a mean diameter of 20–1,000 nm, which are known to contain various bioactive molecules including proteins, lipids, and nucleic acids. Here, we present EVpedia, which is an integrated database of high-throughput datasets from prokaryotic and eukaryotic extracellular vesicles. EVpedia provides high-throughput datasets of vesicular components (proteins, mRNAs, miRNAs, and lipids present on prokaryotic, non-mammalian eukaryotic, and mammalian extracellular vesicles. In addition, EVpedia also provides an array of tools, such as the search and browse of vesicular components, Gene Ontology enrichment analysis, network analysis of vesicular proteins and mRNAs, and a comparison of vesicular datasets by ortholog identification. Moreover, publications on extracellular vesicle studies are listed in the database. This free web-based database of EVpedia (http://evpedia.info might serve as a fundamental repository to stimulate the advancement of extracellular vesicle studies and to elucidate the novel functions of these complex extracellular organelles.

  2. Gender and plasma iron biomarkers, but not HFE gene mutations, increase the risk of colorectal cancer and polyps.

    Castiella, Agustin; Múgica, Fernando; Zapata, Eva; Zubiaurre, Leire; Iribarren, Arantxa; de Juan, M Dolores; Alzate, Luis; Gil, Ines; Urdapilleta, Gregorio; Otazua, Pedro; Emparanza, José Ignacio

    2015-09-01

    A cohort study of patients included in the Basque Country colorectal cancer (CRC) screening programme was carried out to assess the risk of adenomatous polyps and CRC (P-CRC) associated with HFE gene mutations, with gender and with iron biomarkers (serum ferritin (SF), iron (Fe) and transferrin saturation index (TSI)). Among 432 included patients (mean age 59.8 years), 263 were men (60.9 %) and 169 women (39.1 %). P-CRC were identified in 221 patients (51.2 %) and no polyps (NP) in 211 patients (48.8 %). HFE mutations were identified in 43.8 % of the patients. C282Y/wt genotypic frequency was 6.8 % in the P-CRC group and 1.4 % in the NP group (p < 0.05). The allelic frequency was 3.8 versus 1.2 % (p < 0.05). For laboratory, all three iron biomarkers showed a statistically significant difference: mean Fe, 91.29 ± 34 for P-CRC and 80.81 ± 30.59 for NP group. Mean TSI for P-CRC was 24.95 ± 8.90 and 22.74 ± 8.79 for NP group. Mean SF 308.09 ± 536.32 for P-CRC and 177.55 ± 159.95 for NP group. In a multivariate logistic regression analysis, only male gender (odds ratio (OR) = 2.04, 1.29-3.22), SF (OR = 1.001, 1.0004-1.003) and Fe (OR = 1.01, 1.004-1.02) were related with the presence of CRC and adenoma. Men gender and raised serum iron biomarkers increase the risk of P-CRC.

  3. In vitro Determination of Extracellular Proteins from Xylella fastidiosa.

    Mendes, Juliano S; Santiago, André S; Toledo, Marcelo A S; Horta, Maria A C; de Souza, Alessandra A; Tasic, Ljubica; de Souza, Anete P

    2016-01-01

    The phytopathogen Xylella fastidiosa causes economic losses in important agricultural crops. Xylem vessel occlusion caused by biofilm formation is the major mechanism underlying the pathogenicity of distinct strains of X. fastidiosa . Here, we provide a detailed in vitro characterization of the extracellular proteins of X. fastidiosa . Based on the results, we performed a comparison with a strain J1a12, which cannot induce citrus variegated chlorosis symptoms when inoculated into citrus plants. We then extend this approach to analyze the extracellular proteins of X. fastidiosa in media supplemented with calcium. We verified increases in extracellular proteins concomitant with the days of growth and, consequently, biofilm development (3-30 days). Outer membrane vesicles carrying toxins were identified beginning at 10 days of growth in the 9a5c strain. In addition, a decrease in extracellular proteins in media supplemented with calcium was observed in both strains. Using mass spectrometry, 71 different proteins were identified during 30 days of X. fastidiosa biofilm development, including proteases, quorum-sensing proteins, biofilm formation proteins, hypothetical proteins, phage-related proteins, chaperones, toxins, antitoxins, and extracellular vesicle membrane components.

  4. Extracellular nucleotide signaling in plants

    Stacey, Gary [Univ. of Missouri, Columbia, MO (United States)

    2016-09-08

    Over the life of this funded project, our research group identified and characterized two key receptor proteins in plants; one mediating the innate immunity response to chitin and the other elucidating the key receptor for extracellular ATP. In the case of chitin recognition, we recently described the quaternary structure of this receptor, shedding light on how the receptor functions. Perhaps more importantly, we demonstrated that all plants have the ability to recognize both chitin oligomers and lipochitooligosacchardes, fundamentally changing how the community views the evolution of these systems and strategies that might be used, for example, to extend symbiotic nitrogen fixation to non-legumes. Our discovery of DORN1 opens a new chapter in plant physiology documenting conclusively that eATP is an important extracellular signal in plants, as it is in animals. At this point, we cannot predict just how far reaching this discovery may prove to be but we are convinced that eATP signaling is fundamental to plant growth and development and, hence, we believe that the future will be very exciting for the study of DORN1 and its overall function in plants.

  5. Baseline extracellular potassium level as an indicator of the rate of increase of the same on further storage in CPDA-1 whole blood units: a potential approach to complement FIFO system for prioritisation of blood bags for release from blood-banks.

    Baliarsingh, S; Jaiswal, M

    2014-02-01

    Potassium levels in stored blood bags increases as they age. Hyperkalemia in transfused blood has undesirable cardiac effects. Within a 19-month period, baseline and weekly samples from 15 CPDA-1 whole blood bags were collected till 28 days of storage and analysed for potassium, sodium, uric acid, albumin and whole blood haemoglobin. One unit increase in baseline (0 day) potassium in extracellular fluid of blood units was associated with the following increases in potassium levels on later days of storage: around two unit increase at 1 week (r2 = 0·50, P values. For CPDA-1 blood bags (i) low baseline potassium blood bags might be preferred for transfusion in cases demanding a low potassium load and (ii) coordinating the ‘first-in-first-out’ (FIFO) policy with ‘early release of blood-bags with high initial potassium’ might be helpful in improving the release of suitable blood units from blood-banks.

  6. ISEV position paper: extracellular vesicle RNA analysis and bioinformatics

    Andrew F. Hill

    2013-12-01

    Full Text Available Extracellular vesicles (EVs are the collective term for the various vesicles that are released by cells into the extracellular space. Such vesicles include exosomes and microvesicles, which vary by their size and/or protein and genetic cargo. With the discovery that EVs contain genetic material in the form of RNA (evRNA has come the increased interest in these vesicles for their potential use as sources of disease biomarkers and potential therapeutic agents. Rapid developments in the availability of deep sequencing technologies have enabled the study of EV-related RNA in detail. In October 2012, the International Society for Extracellular Vesicles (ISEV held a workshop on “evRNA analysis and bioinformatics.” Here, we report the conclusions of one of the roundtable discussions where we discussed evRNA analysis technologies and provide some guidelines to researchers in the field to consider when performing such analysis.

  7. Decreased extracellular adenosine levels lead to loss of hypoxia-induced neuroprotection after repeated episodes of exposure to hypoxia.

    Mei Cui

    Full Text Available Achieving a prolonged neuroprotective state following transient ischemic attacks (TIAs is likely to effectively reduce the brain damage and neurological dysfunction associated with recurrent stroke. HPC is a phenomenon in which advanced exposure to mild hypoxia reduces the stroke volume produced by a subsequent TIA. However, this neuroprotection is not long-lasting, with the effects reaching a peak after 3 days. Therefore, in this study, we investigated the use of multiple episodes of hypoxic exposure at different time intervals to induce longer-term protection in a mouse stroke model. C57BL/6 mice were subjected to different hypoxic preconditioning protocols: a single episode of HPC or five identical episodes at intervals of 3 days (E3d HPC or 6 days (E6d HPC. Three days after the last hypoxic exposure, temporary middle cerebral artery occlusion (MCAO was induced. The effects of these HPC protocols on hypoxia-inducible factor (HIF regulated gene mRNA expression were measured by quantitative PCR. Changes in extracellular adenosine concentrations, known to exert neuroprotective effects, were also measured using in vivo microdialysis and high pressure liquid chromatography (HPLC. Neuroprotection was provided by E6d HPC but not E3d HPC. HIF-regulated target gene expression increased significantly following all HPC protocols. However, E3d HPC significantly decreased extracellular adenosine and reduced cerebral blood flow in the ischemic region with upregulated expression of the adenosine transporter, equilibrative nucleoside transporter 1 (ENT1. An ENT1 inhibitor, propentofylline increased the cerebral blood flow and re-established neuroprotection in E3d HPC. Adenosine receptor specific antagonists showed that adenosine mainly through A1 receptor mediates HPC induced neuroprotection. Our data indicate that cooperation of HIF-regulated genes and extracellular adenosine is necessary for HPC-induced neuroprotection.

  8. The cell wall stress response in Aspergillus niger involves increased expression of the glutamine: Fructose-6-phosphate amidotransferase-encoding gene (gfaA) and increased deposition of chitin in the cell wall

    Ram, A.F.J.; Arentshorst, M.; Damveld, R.A.; Kuyk, P.A. van; Klis, F.M.; Hondel, C.A.M.J.J. van den

    2004-01-01

    Perturbation of cell wall synthesis in Saccharomyces cerevisiae, either by mutations in cell wall synthesis-related genes or by adding compounds that interfere with normal cell wall assembly, triggers a compensatory response to ensure cell wall integrity. This response includes an increase in chitin

  9. Usage of the Heterologous Expression of the Antimicrobial Gene afp From Aspergillus giganteus for Increasing Fungal Resistance in Olive

    Narvaez, Isabel; Khayreddine, Titouh; Pliego, Clara; Cerezo, Sergio; Jiménez-Díaz, Rafael M.; Trapero-Casas, José L.; López-Herrera, Carlos; Arjona-Girona, Isabel; Martín, Carmen; Mercado, José A.; Pliego-Alfaro, Fernando

    2018-01-01

    The antifungal protein (AFP) produced by Aspergillus giganteus, encoded by the afp gene, has been used to confer resistance against a broad range of fungal pathogens in several crops. In this research, transgenic olive plants expressing the afp gene under the control of the constitutive promoter CaMV35S were generated and their disease response against two root infecting fungal pathogens, Verticillium dahliae and Rosellinia necatrix, was evaluated. Embryogenic cultures derived from a mature zygotic embryo of cv. ‘Picual’ were used for A. tumefaciens transformation. Five independent transgenic lines were obtained, showing a variable level of afp expression in leaves and roots. None of these transgenic lines showed enhanced resistance to Verticillium wilt. However, some of the lines displayed a degree of incomplete resistance to white root rot caused by R. necatrix compared with disease reaction of non-transformed plants or transgenic plants expressing only the GUS gene. The level of resistance to this pathogen correlated with that of the afp expression in root and leaves. Our results indicate that the afp gene can be useful for enhanced partial resistance to R. necatrix in olive, but this gene does not protect against V. dahliae. PMID:29875785

  10. Adapted Boolean network models for extracellular matrix formation

    Wollbold Johannes

    2009-07-01

    Full Text Available Abstract Background Due to the rapid data accumulation on pathogenesis and progression of chronic inflammation, there is an increasing demand for approaches to analyse the underlying regulatory networks. For example, rheumatoid arthritis (RA is a chronic inflammatory disease, characterised by joint destruction and perpetuated by activated synovial fibroblasts (SFB. These abnormally express and/or secrete pro-inflammatory cytokines, collagens causing joint fibrosis, or tissue-degrading enzymes resulting in destruction of the extra-cellular matrix (ECM. We applied three methods to analyse ECM regulation: data discretisation to filter out noise and to reduce complexity, Boolean network construction to implement logic relationships, and formal concept analysis (FCA for the formation of minimal, but complete rule sets from the data. Results First, we extracted literature information to develop an interaction network containing 18 genes representing ECM formation and destruction. Subsequently, we constructed an asynchronous Boolean network with biologically plausible time intervals for mRNA and protein production, secretion, and inactivation. Experimental gene expression data was obtained from SFB stimulated by TGFβ1 or by TNFα and discretised thereafter. The Boolean functions of the initial network were improved iteratively by the comparison of the simulation runs to the experimental data and by exploitation of expert knowledge. This resulted in adapted networks for both cytokine stimulation conditions. The simulations were further analysed by the attribute exploration algorithm of FCA, integrating the observed time series in a fine-tuned and automated manner. The resulting temporal rules yielded new contributions to controversially discussed aspects of fibroblast biology (e.g., considerable expression of TNF and MMP9 by fibroblasts stimulation and corroborated previously known facts (e.g., co-expression of collagens and MMPs after TNF

  11. Adaptive evolution of the matrix extracellular phosphoglycoprotein in mammals

    Machado João

    2011-11-01

    Full Text Available Abstract Background Matrix extracellular phosphoglycoprotein (MEPE belongs to a family of small integrin-binding ligand N-linked glycoproteins (SIBLINGs that play a key role in skeleton development, particularly in mineralization, phosphate regulation and osteogenesis. MEPE associated disorders cause various physiological effects, such as loss of bone mass, tumors and disruption of renal function (hypophosphatemia. The study of this developmental gene from an evolutionary perspective could provide valuable insights on the adaptive diversification of morphological phenotypes in vertebrates. Results Here we studied the adaptive evolution of the MEPE gene in 26 Eutherian mammals and three birds. The comparative genomic analyses revealed a high degree of evolutionary conservation of some coding and non-coding regions of the MEPE gene across mammals indicating a possible regulatory or functional role likely related with mineralization and/or phosphate regulation. However, the majority of the coding region had a fast evolutionary rate, particularly within the largest exon (1467 bp. Rodentia and Scandentia had distinct substitution rates with an increased accumulation of both synonymous and non-synonymous mutations compared with other mammalian lineages. Characteristics of the gene (e.g. biochemical, evolutionary rate, and intronic conservation differed greatly among lineages of the eight mammalian orders. We identified 20 sites with significant positive selection signatures (codon and protein level outside the main regulatory motifs (dentonin and ASARM suggestive of an adaptive role. Conversely, we find three sites under selection in the signal peptide and one in the ASARM motif that were supported by at least one selection model. The MEPE protein tends to accumulate amino acids promoting disorder and potential phosphorylation targets. Conclusion MEPE shows a high number of selection signatures, revealing the crucial role of positive selection in the

  12. Glycosylation of extracellular vesicles : current knowledge, tools and clinical perspectives

    Williams, Charles; Royo, Felix; Aizpurua-Olaizola, Oier; Pazos, Raquel; Boons, Geert-Jan; Reichardt, Niels-Christian; Falcon-Perez, Juan M

    2018-01-01

    It is now acknowledged that extracellular vesicles (EVs) are important effectors in a vast number of biological processes through intercellular transfer of biomolecules. Increasing research efforts in the EV field have yielded an appreciation for the potential role of glycans in EV function. Indeed,

  13. Optimization of culture media for extracellular expression of ...

    Purpose: To investigate the enhancement of streptokinase extracellular expression in Escherichia coli by adjusting culture media. Methods: Screening of 10 chemical factors (EDTA, peptone, glycine, triton X-100, glycerol, K2HPO4,. KH2PO4, Ca2+ (calcium chloride), yeast and NaCl) in order to increase the secretion of ...

  14. Neutrophil extracellular traps promote deep vein thrombosis in mice

    Brill, A.; Fuchs, T.A.; Savchenko, A.S.; Thomas, G.M.; Martinod, K.; De Meyer, S.F.; Bhandari, A.A.; Wagner, D.D.

    2011-01-01

    Summary Background Upon activation, neutrophils can release nuclear material known as neutrophil extracellular traps (NETs), which were initially described as a part of antimicrobial defense. Extracellular chromatin was recently reported to be pro-thrombotic in vitro and to accumulate in plasma and thrombi of baboons with experimental deep vein thrombosis (DVT). Objective To explore the source and role of extracellular chromatin in DVT. Methods We used an established murine model of DVT induced by flow restriction (stenosis) in the inferior vena cava (IVC). Results We demonstrate that the levels of extracellular DNA increase in plasma after 6 h IVC stenosis, compared to sham-operated mice. Immunohistochemical staining revealed the presence of Gr-1-positive neutrophils in both red (RBC-rich) and white (platelet-rich) parts of thrombi. Citrullinated histone H3 (CitH3), an element of NETs’ structure, was present only in the red part of thrombi and was frequently associated with the Gr-1 antigen. Immunofluorescent staining of thrombi showed proximity of extracellular CitH3 and von Willebrand factor (VWF), a platelet adhesion molecule crucial for thrombus development in this model. Infusion of Deoxyribonuclease 1 (DNase 1) protected mice from DVT after 6 h and also 48 h IVC stenosis. Infusion of an unfractionated mixture of calf thymus histones increased plasma VWF and promoted DVT early after stenosis application. Conclusions Extracellular chromatin, likely originating from neutrophils, is a structural part of a venous thrombus and both the DNA scaffold and histones appear to contribute to the pathogenesis of DVT in mice. NETs may provide new targets for DVT drug development. PMID:22044575

  15. Further increased production of free fatty acids by overexpressing a predicted transketolase gene of the pentose phosphate pathway in Aspergillus oryzae faaA disruptant.

    Tamano, Koichi; Miura, Ai

    2016-09-01

    Free fatty acids are useful as source materials for the production of biodiesel fuel and various chemicals such as pharmaceuticals and dietary supplements. Previously, we attained a 9.2-fold increase in free fatty acid productivity by disrupting a predicted acyl-CoA synthetase gene (faaA, AO090011000642) in Aspergillus oryzae. In this study, we achieved further increase in the productivity by overexpressing a predicted transketolase gene of the pentose phosphate pathway in the faaA disruptant. The A. oryzae genome is predicted to have three transketolase genes and overexpression of AO090023000345, one of the three genes, resulted in phenotypic change and further increase (corresponding to an increased production of 0.38 mmol/g dry cell weight) in free fatty acids at 1.4-fold compared to the faaA disruptant. Additionally, the biomass of hyphae increased at 1.2-fold by the overexpression. As a result, free fatty acid production yield per liter of liquid culture increased at 1.7-fold by the overexpression.

  16. Increasing of miR-148a 0061nd Decreasing of miR-146a Gene Expression in the Stomach with Ageing in Men

    Shirin Abdolvand

    2017-06-01

    Full Text Available Abstract Background: The incidence of gastric cancer is different in two sexes with ratio 2 to 1 that it is more common in men. The most important biologically reason is sexual hormones between two sexes that lead to sexual dimorphism and in turn can cause a sex bias in incidence of disease between two sexes. Recently, studies have shown that microRNA is involved in sexual dimorphism in gene expression. Given the sexual dimorphism in the incidence of gastric cancer and sex hormones response elements in the regulatory regions of miR-146a and miR-148a genes, in this study, the expression of these two genes in the stomach of healthy men and women at different age groups were compared. Materials and Methods: Using endoscopy, gastric antrum tissues of 35 healthy women and 35 healthy men were collected. After RNA extraction and synthesis of cDNA, the expression of miR-146a and miR-148a genes were compared between sexes by Real time RT-PCR and data were analyzed using independent sample t and ANOVA tests. Results: There was no difference between men and women in genes expression of miR-146a and miR-148a. However, expression of miR-146a gene was significantly more in men under 45 years than men over 45 years (p= 0.017, df= 14, t= 1.47. Also, expression of miR-148a gene was significantly more in men over 45 years than men under 45 years (p=0.001, df= 12, t= 1.28. But the expression of both genes had no significant difference between women under 45 years and women over 45 years. Conclusion: Expression of miR-146a and miR-148a genes in the stomach is increased and decreased with aging in men, respectively.

  17. An accessory to the 'Trinity': SR-As are essential pathogen sensors of extracellular dsRNA, mediating entry and leading to subsequent type I IFN responses.

    Stephanie J DeWitte-Orr

    2010-03-01

    Full Text Available Extracellular RNA is becoming increasingly recognized as a signaling molecule. Virally derived double stranded (dsRNA released into the extracellular space during virus induced cell lysis acts as a powerful inducer of classical type I interferon (IFN responses; however, the receptor that mediates this response has not been identified. Class A scavenger receptors (SR-As are likely candidates due to their cell surface expression and ability to bind nucleic acids. In this study, we investigated a possible role for SR-As in mediating type I IFN responses induced by extracellular dsRNA in fibroblasts, a predominant producer of IFNbeta. Fibroblasts were found to express functional SR-As, even SR-A species thought to be macrophage specific. SR-A specific competitive ligands significantly blocked extracellular dsRNA binding, entry and subsequent interferon stimulated gene (ISG induction. Candidate SR-As were systematically investigated using RNAi and the most dramatic inhibition in responses was observed when all candidate SR-As were knocked down in unison. Partial inhibition of dsRNA induced antiviral responses was observed in vivo in SR-AI/II(-/- mice compared with WT controls. The role of SR-As in mediating extracellular dsRNA entry and subsequent induced antiviral responses was observed in both murine and human fibroblasts. SR-As appear to function as 'carriers', facilitating dsRNA entry and delivery to the established dsRNA sensing receptors, specifically TLR3, RIGI and MDA-5. Identifying SR-As as gatekeepers of the cell, mediating innate antiviral responses, represents a novel function for this receptor family and provides insight into how cells recognize danger signals associated with lytic virus infections. Furthermore, the implications of a cell surface receptor capable of recognizing extracellular RNA may exceed beyond viral immunity to mediating other important innate immune functions.

  18. Analysis of extracellular RNA by digital PCR

    Kenji eTakahashi

    2014-06-01

    Full Text Available The transfer of extracellular RNA is emerging as an important mechanism for intracellular communication. The ability for the transfer of functionally active RNA molecules from one cell to another within vesicles such as exosomes enables a cell to modulate cellular signaling and biological processes within recipient cells. The study of extracellular RNA requires sensitive methods for the detection of these molecules. In this methods article, we will describe protocols for the detection of such extracellular RNA using sensitive detection technologies such as digital PCR. These protocols should be valuable to researchers interested in the role and contribution of extracellular RNA to tumor cell biology.

  19. Extracellular Molecules Involved in Cancer Cell Invasion

    Stivarou, Theodora; Patsavoudi, Evangelia

    2015-01-01

    Nowadays it is perfectly clear that understanding and eradicating cancer cell invasion and metastasis represent the crucial, definitive points in cancer therapeutics. During the last two decades there has been a great interest in the understanding of the extracellular molecular mechanisms involved in cancer cell invasion. In this review, we highlight the findings concerning these processes, focusing in particular on extracellular molecules, including extracellular matrix proteins and their receptors, growth factors and their receptors, matrix metalloproteinases and extracellular chaperones. We report the molecular mechanisms underlying the important contribution of this pool of molecules to the complex, multi-step phenomenon of cancer cell invasion

  20. Extracellular Molecules Involved in Cancer Cell Invasion

    Theodora Stivarou

    2015-01-01

    Full Text Available Nowadays it is perfectly clear that understanding and eradicating cancer cell invasion and metastasis represent the crucial, definitive points in cancer therapeutics. During the last two decades there has been a great interest in the understanding of the extracellular molecular mechanisms involved in cancer cell invasion. In this review, we highlight the findings concerning these processes, focusing in particular on extracellular molecules, including extracellular matrix proteins and their receptors, growth factors and their receptors, matrix metalloproteinases and extracellular chaperones. We report the molecular mechanisms underlying the important contribution of this pool of molecules to the complex, multi-step phenomenon of cancer cell invasion.

  1. Specialisation of extracellular matrix for function in tendons and ligaments

    Birch, Helen L.; Thorpe, Chavaunne T.; Rumian, Adam P.

    2013-01-01

    Summary Tendons and ligaments are similar structures in terms of their composition, organisation and mechanical properties. The distinction between them stems from their anatomical location; tendons form a link between muscle and bone while ligaments link bones to bones. A range of overlapping functions can be assigned to tendon and ligaments and each structure has specific mechanical properties which appear to be suited for particular in vivo function. The extracellular matrix in tendon and ligament varies in accordance with function, providing appropriate mechanical properties. The most useful framework in which to consider extracellular matrix differences therefore is that of function rather than anatomical location. In this review we discuss what is known about the relationship between functional requirements, structural properties from molecular to gross level, cellular gene expression and matrix turnover. The relevance of this information is considered by reviewing clinical aspects of tendon and ligament repair and reconstructive procedures. PMID:23885341

  2. A perspective on extracellular vesicles proteomics

    Rosa-Fernandes, Livia; Rocha, Victória Bombarda; Carregari, Victor Corasolla; Urbani, Andrea; Palmisano, Giuseppe

    2017-11-01

    Increasing attention has been given to secreted extracellular vesicles (EVs) in the past decades, especially in the portrayal of their molecular cargo and role as messengers in both homeostasis and pathophysiological conditions. This review presents the state-of-the-art proteomic technologies to identify and quantify EVs proteins along with their PTMs, interacting partners and structural details. The rapid growth of mass spectrometry-based analytical strategies for protein sequencing, PTMs and structural characterization has improved the level of molecular details that can be achieve from limited amount of EVs isolated from different biological sources. Here we will provide a perspective view on the achievements and challenges on EVs proteome characterization using mass spectrometry. A detailed bioinformatics approach will help us to picture the molecular fingerprint of EVs and understand better their pathophysiological function.

  3. Prevalence of antiseptic resistance genes increases in staphylococcal isolates from orthokeratology lens wearers over initial six-month period of use.

    Guang-Sen, Shi; Boost, Maureen; Cho, Pauline

    2016-06-01

    The purpose of this study was to investigate the prevalence of antiseptic-resistance (QAC) genes in staphylococci colonizing periorbital tissues and accessories of orthokeratology (ortho-k) lens wearers over a 6-month period and determine the effect of their presence on minimum inhibitory (MIC) and bactericidal concentrations (MBC) of disinfectants and log reduction of multipurpose contact lens solutions (MPS). Staphylococci were isolated from periorbital tissues and accessories of patients before commencing ortho-k therapy and at two subsequent visits. Presence of QAC genes in 116 S. aureus and 67 CNS isolates was determined by PCR and association with period of ortho-k use determined. MICs and MBCs of staphylococci gene-positive were compared with gene-negative strains and the effectiveness of four MPS for rigid contact lenses investigated. S. aureus carriage rates in the conjunctiva decreased significantly from 41.2 % (baseline) to 11.8 % (3-months), and 13.3 % (6-months) (p trend 0.03), while CNS increased from 58.8 %(baseline) to 94.1 % (3-months), and 93.3 % (6-months) (p trend 0.02). Prevalence of qacA/B increased considerably over time (S. aureus: 4.4 % to 15.4 %, CNS: 6.7 % to 25 %), but frequency of smr was relatively stable. Only five CNS isolates harboured qacH. MICs and MBCs of gene-positive isolates were significantly increased and three MPS did not achieve a 3-log reduction of many QAC-positive strains. Ortho-k lens wear contributed to changes of staphylococcal carriage rates in the conjunctival sac. Use of MPS containing quaternary ammonium compounds may select for the carriage of organisms harbouring QAC genes, as the low concentration of disinfecting agents is not adequate for killing gene-positive strains.

  4. An extracellular subtilase switch for immune priming in Arabidopsis.

    Vicente Ramírez

    Full Text Available In higher eukaryotes, induced resistance associates with acquisition of a priming state of the cells for a more effective activation of innate immunity; however, the nature of the components for mounting this type of immunological memory is not well known. We identified an extracellular subtilase from Arabidopsis, SBT3.3, the overexpression of which enhances innate immune responses while the loss of function compromises them. SBT3.3 expression initiates a durable autoinduction mechanism that promotes chromatin remodeling and activates a salicylic acid(SA-dependent mechanism of priming of defense genes for amplified response. Moreover, SBT3.3 expression-sensitized plants for enhanced expression of the OXI1 kinase gene and activation of MAP kinases following pathogen attack, providing additional clues for the regulation of immune priming by SBT3.3. Conversely, in sbt3.3 mutant plants pathogen-mediated induction of SA-related defense gene expression is drastically reduced and activation of MAP kinases inhibited. Moreover, chromatin remodeling of defense-related genes normally associated with activation of an immune priming response appear inhibited in sbt3.3 plants, further indicating the importance of the extracellular SBT3.3 subtilase in the establishment of immune priming. Our results also point to an epigenetic control in the regulation of plant immunity, since SBT3.3 is up-regulated and priming activated when epigenetic control is impeded. SBT3.3 represents a new regulator of primed immunity.

  5. Enhanced pest resistance and increased phenolic production in maize callus transgenically expressing a maize chalcone isomerase -3 like gene

    Significant losses in maize production are due to damage by insects and ear rot fungi. A gene designated as chalcone-isomerase-like, located in a quantitative trait locus for resistance to Fusarium ear rot fungi, was cloned from a Fusarium ear rot resistant inbred and transgenically expressed in mai...

  6. Silencing abnormal wing disc gene of the Asian citrus psyllid, Diaphorina citri disrupts adult wing development and increases nymph mortality.

    Ibrahim El-Shesheny

    Full Text Available Huanglongbing (HLB causes considerable economic losses to citrus industries worldwide. Its management depends on controlling of the Asian citrus Psyllid (ACP, the vector of the bacterium, Candidatus Liberibacter asiaticus (CLas, the causal agent of HLB. Silencing genes by RNA interference (RNAi is a promising tool to explore gene functions as well as control pests. In the current study, abnormal wing disc (awd gene associated with wing development in insects is used to interfere with the flight of psyllids. Our study showed that transcription of awd is development-dependent and the highest level was found in the last instar (5(th of the nymphal stage. Micro-application (topical application of dsRNA to 5(th instar of nymphs caused significant nymphal mortality and adult wing-malformation. These adverse effects in ACP were positively correlated with the amounts of dsRNA used. A qRT-PCR analysis confirmed the dsRNA-mediated transcriptional down-regulation of the awd gene. Significant down-regulation was required to induce a wing-malformed phenotype. No effect was found when dsRNA-gfp was used, indicating the specific effect of dsRNA-awd. Our findings suggest a role for awd in ACP wing development and metamorphosis. awd could serve as a potential target for insect management either via direct application of dsRNA or by producing transgenic plants expressing dsRNA-awd. These strategies will help to mitigate HLB by controlling ACP.

  7. Competition between the sperm of a single male can increase the evolutionary rate of haploid expressed genes.

    Ezawa, Kiyoshi; Innan, Hideki

    2013-07-01

    The population genetic behavior of mutations in sperm genes is theoretically investigated. We modeled the processes at two levels. One is the standard population genetic process, in which the population allele frequencies change generation by generation, depending on the difference in selective advantages. The other is the sperm competition during each genetic transmission from one generation to the next generation. For the sperm competition process, we formulate the situation where a huge number of sperm with alleles A and B, produced by a single heterozygous male, compete to fertilize a single egg. This "minimal model" demonstrates that a very slight difference in sperm performance amounts to quite a large difference between the alleles' winning probabilities. By incorporating this effect of paternity-sharing sperm competition into the standard population genetic process, we show that fierce sperm competition can enhance the fixation probability of a mutation with a very small phenotypic effect at the single-sperm level, suggesting a contribution of sperm competition to rapid amino acid substitutions in haploid-expressed sperm genes. Considering recent genome-wide demonstrations that a substantial fraction of the mammalian sperm genes are haploid expressed, our model could provide a potential explanation of rapid evolution of sperm genes with a wide variety of functions (as long as they are expressed in the haploid phase). Another advantage of our model is that it is applicable to a wide range of species, irrespective of whether the species is externally fertilizing, polygamous, or monogamous. The theoretical result was applied to mammalian data to estimate the selection intensity on nonsynonymous mutations in sperm genes.

  8. The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles

    L?sser, Cecilia; Th?ry, Clotilde; Buz?s, Edit I.; Mathivanan, Suresh; Zhao, Weian; Gho, Yong Song; L?tvall, Jan

    2016-01-01

    The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, “Basics of Extracellular Vesicles,” uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field co...

  9. Brain Extracellular Space: The Final Frontier of Neuroscience.

    Nicholson, Charles; Hrabětová, Sabina

    2017-11-21

    Brain extracellular space is the narrow microenvironment that surrounds every cell of the central nervous system. It contains a solution that closely resembles cerebrospinal fluid with the addition of extracellular matrix molecules. The space provides a reservoir for ions essential to the electrical activity of neurons and forms an intercellular chemical communication channel. Attempts to reveal the size and structure of the extracellular space using electron microscopy have had limited success; however, a biophysical approach based on diffusion of selected probe molecules has proved useful. A point-source paradigm, realized in the real-time iontophoresis method using tetramethylammonium, as well as earlier radiotracer methods, have shown that the extracellular space occupies ∼20% of brain tissue and small molecules have an effective diffusion coefficient that is two-fifths that in a free solution. Monte Carlo modeling indicates that geometrical constraints, including dead-space microdomains, contribute to the hindrance to diffusion. Imaging the spread of macromolecules shows them increasingly hindered as a function of size and suggests that the gaps between cells are predominantly ∼40 nm with wider local expansions that may represent dead-spaces. Diffusion measurements also characterize interactions of ions and proteins with the chondroitin and heparan sulfate components of the extracellular matrix; however, the many roles of the matrix are only starting to become apparent. The existence and magnitude of bulk flow and the so-called glymphatic system are topics of current interest and controversy. The extracellular space is an exciting area for research that will be propelled by emerging technologies. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. [Expression of plant antimicrobial peptide pro-SmAMP2 gene increases resistance of transgenic potato plants to Alternaria and Fusarium pathogens].

    Vetchinkina, E M; Komakhina, V V; Vysotskii, D A; Zaitsev, D V; Smirnov, A N; Babakov, A V; Komakhin, R A

    2016-09-01

    The chickweed (Stellaria media L.) pro-SmAMP2 gene encodes the hevein-like peptides that have in vitro antimicrobial activity against certain harmful microorganisms. These peptides play an important role in protecting the chickweed plants from infection, and the pro-SmAMP2 gene was previously used to protect transgenic tobacco and Arabidopsis plants from phytopathogens. In this study, the pro-SmAMP2 gene under control of viral CaMV35S promoter or under control of its own pro-SmAMP2 promoter was transformed into cultivated potato plants of two cultivars, differing in the resistance to Alternaria: Yubiley Zhukova (resistant) and Skoroplodny (susceptible). With the help of quantitative real-time PCR, it was demonstrated that transgenic potato plants expressed the pro-SmAMP2 gene under control of both promoters at the level comparable to or exceeding the level of the potato actin gene. Assessment of the immune status of the transformants demonstrated that expression of antimicrobial peptide pro-SmAMP2 gene was able to increase the resistance to a complex of Alternaria sp. and Fusarium sp. phytopathogens only in potato plants of the Yubiley Zhukova cultivar. The possible role of the pro-SmAMP2 products in protecting potatoes from Alternaria sp. and Fusarium sp. is discussed.

  11. Extracellular hyperosmolality and body temperature during physical exercise in dogs

    Kozlowski, S.; Greenleaf, J. E.; Turlejska, E.; Nazar, K.

    1980-01-01

    The purpose of this study was to test the hypothesis that thermoregulation during exercise can be affected by extracellular fluid hyperosmolality without changing the plasma Na(+) concentration. The effects of preexercise venous infusions of hypertonic mannitol and NaCl solutions on rectal temperature responses were compared in dogs running at moderate intensity for 60 min on a treadmill. Plasma Na(+) concentration was increased by 12 meq after NaCl infusion, and decreased by 9 meq after mannitol infusion. Both infusions increased plasma by 15 mosmol/kg. After both infusions, rectal temperature was essentially constant during 60 min rest. However, compared with the noninfusion exercise increase in osmolality of 1.3 C, rectal temperature increased by 1.9 C after both postinfusion exercise experiments. It was concluded that inducing extracellular hyperosmolality, without elevating plasma, can induce excessive increases in rectal temperature during exericse but not at rest.

  12. Silencing of a putative immunophilin gene in the cattle tick Rhipicephalus (Boophilus microplus increases the infection rate of Babesia bovis in larval progeny

    Knowles Donald P

    2009-11-01

    Full Text Available Abstract Background The cattle tick Rhipicephalus (Boophilus microplus is involved in the transmission of the protozoan Babesia bovis, the etiological agent of bovine babesiosis. Interactions between ticks and protozoa are poorly understood and the investigation of tick genes that affect tick fitness and protozoan infection can set the stage for dissecting the molecular interactions between the two species. Results In this study, RNA interference was used to silence R. microplus genes that had been previously shown to be up-regulated in response to B. bovis infection. The silencing of a putative immunophilin gene (Imnp in female ticks fed on a calf acutely infected with B. bovis decreased the hatching rate and survival of larval progeny. Interestingly, Imnp was up-regulated significantly in ovaries of R. microplus in response to B. bovis infection and its silencing in female ticks significantly increased the infection rate of the protozoan in larval progeny. The results also showed that the silencing of a putative Kunitz-type serine protease inhibitor (Spi gene and a putative lipocalin (Lpc gene decreased the fitness of R. microplus females, but had no significant effect on the infection rate of B. bovis in larval progeny. Conclusion The silencing of the Imnp, Spi or Lpc genes decreased the fitness of R. microplus females fed on a calf during acute B. bovis infection. The Imnp gene data suggest that this putative immunophilin gene is involved in the defense system of R. microplus against B. bovis and may play a role in controlling the protozoan infection in tick ovaries and larval progeny.

  13. An immunofluorescence assay for extracellular matrix components highlights the role of epithelial cells in producing a stable, fibrillar extracellular matrix

    Omar S. Qureshi

    2017-10-01

    Full Text Available Activated fibroblasts are considered major drivers of fibrotic disease progression through the production of excessive extracellular matrix (ECM in response to signals from damaged epithelial and inflammatory cells. Nevertheless, epithelial cells are capable of expressing components of the ECM, cross-linking enzymes that increase its stability and are sensitive to factors involved in the early stages of fibrosis. We therefore wanted to test the hypothesis that epithelial cells can deposit ECM in response to stimulation in a comparable manner to fibroblasts. We performed immunofluorescence analysis of components of stable, mature extracellular matrix produced by primary human renal proximal tubular epithelial cells and renal fibroblasts in response to cytokine stimulation. Whilst fibroblasts produced a higher basal level of extracellular matrix components, epithelial cells were able to deposit significant levels of fibronectin, collagen I, III and IV in response to cytokine stimulation. In response to hypoxia, epithelial cells showed an increase in collagen IV deposition but not in response to the acute stress stimuli aristolochic acid or hydrogen peroxide. When epithelial cells were in co-culture with fibroblasts we observed significant increases in the level of matrix deposition which could be reduced by transforming growth factor beta (TGF-β blockade. Our results highlight the role of epithelial cells acting as efficient producers of stable extracellular matrix which could contribute to renal tubule thickening in fibrosis.