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Sample records for factor ix inhibition

  1. Coagulation Factor IX for Hemophilia B Therapy


    OpenAIRE

    Orlova, N.; Kovnir, S.; Vorobiev, I.; Gabibov, A.

    2012-01-01

    Factor IX is a zymogen enzyme of the blood coagulation cascade. Inherited absence or deficit of the IX functional factor causes bleeding disorder hemophilia B, which requires constant protein replacement therapy. Reviewed herein are the current state in the manufacturing of FIX, improved variants of the recombinant protein for therapy, transgenic organisms for obtaining FIX, and the advances in the gene therapy of hemophilia B.

  2. Evaluation of factor IX deficiency by interdigitated electrode (IDE)

    Science.gov (United States)

    Gopinath, Subash C. B.; Hashim, Uda; Uda, M. N. A.

    2017-03-01

    Factor IX deficiency is the main cause of hemophilia A and B. This a severe excessive bleeding disorder that can even kill the patient if not treated with the right prescription of Factor IX hormone to stop the bleeding. The bleeding can be caused by an injury or even a sudden bleeding in some very rare cases. To find the Factor IX effectiveness and to understand the deficiency more carefully for the future of medicine, experiments are conducted to test the Factor IX using the Interdigitated Electrode (IDE) and gold Nanoparticle with the help of Nanoelectrical technology.

  3. Immunoaffinity purification of factor IX (Christmas factor) by using conformation-specific antibodies directed against the factor IX-metal complex.

    OpenAIRE

    1985-01-01

    Factor IX is a vitamin K-dependent blood clotting zymogen that is functionally defective or absent in patients with hemophilia B. A method of immunoaffinity chromatography has been developed for a one-step high yield purification of factor IX directly from plasma. The technique utilizes conformation-specific antibodies that bind solely to the metal-stabilized factor IX conformer, but not to the conformer of factor IX found in the absence of metal ions. Anti-factor IX-Ca(II) antibodies were im...

  4. Enhancement of pulmonary tumour seeding by human coagulation factors II, IX, X--an investigation into the possible mechanisms involved.

    OpenAIRE

    Purushotham, A D; McCulloch, P.; George, W. D.

    1991-01-01

    Warfarin inhibits metastasis in the animal model and injection of the Warfarin-dependent coagulation factor complex II, IX, X enhances pulmonary metastasis in the same model. We have studied two possible mechanisms responsible for the observed effect. Mtln3, rat mammary carcinoma cells, radiolabelled with 5-(125) Iodo-2'-deoxyuridine (IUDR) were injected intravenously in female Fisher 344 rats either alone or in combination with factor complex II, IX, X or bovine serum albumin. Following sacr...

  5. New polymorphic variants of human blood clotting factor IX

    Energy Technology Data Exchange (ETDEWEB)

    Surin, V.L.; Luk`yanenko, A.V.; Tagiev, A.F.; Smirnova, O.V. [Hematological Research Center, Moscow (Russian Federation); Plutalov, O.V.; Berlin, Yu.A. [Shemyakin Institute of Bioorganic Chemistry, Moscow (Russian Federation)

    1995-04-01

    The polymorphism of Alu-repeats, which are located in the introns of the human factor IX gene (copies 1-3), was studied. To identify polymorphic variants, direct sequencing of PCR products that contained appropriate repeats was used. In each case, 20 unrelated X chromosomes were studied. A polymorphic Dra I site was found near the 3{prime}-end of Alu copy 3 within the region of the polyA tract. A PCR-based testing system with internal control of restriction hydrolysis was suggested. Testing 81 unrelated X chromosomes revealed that the frequency of the polymorphic Dra I site is 0.23. Taq I polymorphism, which was revealed in Alu copy 4 of factor IX gene in our previous work, was found to be closely linked to Dra I polymorphism. Studies in linkage between different types of polymorphisms of the factor IX gene revealed the presence of a rare polymorphism in intron a that was located within the same minisatellite region as the known polymorphic insertion 50 bp/Dde I. However, the size of the insertion in our case was 26 bp. Only one polymorphic variant was found among over 150 unrelated X chromosomes derived from humans from Moscow and its vicinity. 10 refs., 4 figs., 1 tab.

  6. Recombinant Human Factor IX Produced from Transgenic Porcine Milk

    OpenAIRE

    Meng-Hwan Lee; Yin-Shen Lin; Ching-Fu Tu; Chon-Ho Yen

    2014-01-01

    Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX) produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitat...

  7. Inhibition of carbonic anhydrase isoforms I, II, IX and XII with secondary sulfonamides incorporating benzothiazole scaffolds.

    Science.gov (United States)

    Petrou, Anthi; Geronikaki, Athina; Terzi, Emine; Guler, Ozen Ozensoy; Tuccinardi, Tiziano; Supuran, Claudiu T

    2016-12-01

    Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the fundamental reaction of CO2 hydration in all living organisms, being actively involved in the regulation of a plethora of patho/physiological conditions. A series of benzothiazole-based sulfonamides were synthesized and tested as possible CA inhibitors. Their inhibitory activity was assessed against the cytosolic human isoforms hCA I and hCA II and the transmembrane hCA IX and hCA XII. Several of the investigated derivatives showed interesting inhibition activity and selectivities for inhibiting hCA IX and hCA XII over the off-target ones hCA I and hCA II. Furthermore, computational procedures were used to investigate the binding mode of this class of compounds, within the active site of hCA IX.

  8. Molecular interactions between coagulation factor IX and low density lipoprotein receptor-related protein

    NARCIS (Netherlands)

    Rohlena, Jakub

    2004-01-01

    Factor IX is an essential blood haemostatic protein, which is apparent from the observation that the absence of functional FIX is associated with the severe bleeding disorder haemophilia B. To achieve its full enzymatic activity, the serine protease precursor factor IX must first be activated into

  9. Isatin-pyrazole benzenesulfonamide hybrids potently inhibit tumor-associated carbonic anhydrase isoforms IX and XII.

    Science.gov (United States)

    Ibrahim, Hany S; Abou-Seri, Sahar M; Tanc, Muhammet; Elaasser, Mahmoud M; Abdel-Aziz, Hatem A; Supuran, Claudiu T

    2015-10-20

    New series of benzenesulfonamide derivatives incorporating pyrazole and isatin moieties were prepared using celecoxib as lead molecule. Biological evaluation of the target compounds was performed against the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more precisely against the human isoforms hCA I, II (cytosolic), IX and XII (transmembrane, tumor-associated enzymes). Most of the tested compounds efficiently inhibited hCA I, II and IX, with KIs of 2.5-102 nM, being more effective than the reference drug acetazolamide. Compounds 11e, 11f, 16e and 16f were found to inhibit hCA XII with Ki of 3.7, 6.5, 5.4 and 7.2 nM, respectively. Compounds 11e and 16e, with 5-NO2 substitution on the isatin ring, were found to be selective inhibitors of hCA IX and hCA XII. Docking studies revealed that the NO2 group of both compounds participate in interactions with Asp132 within the hCA IX active site, and with residues Lys67 and Asp130 in hCA XII, respectively.

  10. Coagulation Factor IX concentrate: method of preparation and assessment of potential in vivo thrombogenicity in animal models.

    Science.gov (United States)

    Menache, D; Behre, H E; Orthner, C L; Nunez, H; Anderson, H D; Triantaphyllopoulos, D C; Kosow, D P

    1984-12-01

    Thrombosis and/or disseminated intravascular coagulation (DIC) are complications specifically associated with the use of factor IX complex in some patients. Assuming that these complications might result from zymogen overload, we have produced, using diethylaminoethyl (DEAE)-Sephadex (Pharmacia, Piscataway, NJ) and sulfated dextran chromatography, a factor IX concentrate (coagulation factor IX) that is essentially free of prothrombin, factor VII, and factor X. Factor IX specific activity is at least 5 U/mg protein, a 250-fold purification compared to plasma. Amounts of factors II, VII, and X are less than 5 units each per 100 units of factor IX. The concentrate is essentially free of activated clotting factors and contains no added heparin. In the rabbit stasis model, a dose of 200 factor IX U/kg was less thrombogenic than 100 factor IX U/kg of the DEAE-Sephadex eluate from which the concentrate was derived. Infusion of 200 factor IX U/kg did not induce DIC in the nonstasis rabbit model, whereas 100 factor IX U/kg of the DEAE-Sephadex eluate resulted in DIC in this model. Several factor IX lots were found to have shortened nonactivated partial thromboplastin times (PTTs), but were nonthrombogenic in both animal models. These data indicate that coagulation factor IX concentrate is less thrombogenic than factor IX complex.

  11. Crystal Structure of the Fab Fragment of an Anti-factor IX Antibody 10C12

    Institute of Scientific and Technical Information of China (English)

    SHI Xiao-Li; ZENG Tu; HUANG Ming-Dong

    2008-01-01

    10C12 is an anticoagulant antibody identified from a phage display single-chain Fv human antibody library. It can be directed at the calcium-stabilized Gla domain of Factor-IX, an important coagulation factor in intrinsic pathway of blood coagulation cascade, and interfere with membrane anchoring of Factor IX, thus inhibiting blood coagulation function. 10C12 has been demonstrated as an effective anti-coagulant in attenuating thrombosis in several different animal models. Here, we report the crystal structure of the Fab fragment of 10C12. The crystal contains two Fab molecules in the asymmetric unit with identical conformation, forming a lattice with large cavities. In addition, comparison of this free Fab with the antigen-bound structure of 10C12 shows no change in CDR conformations and the relative disposition of the variable subunits of H and L chains, suggesting the rigid conformation of this 10C12 Fab and a lock-and-key mechanism of antibody-antigen recognition for 10C 12.

  12. Recombinant human factor IX produced from transgenic porcine milk.

    Science.gov (United States)

    Lee, Meng-Hwan; Lin, Yin-Shen; Tu, Ching-Fu; Yen, Chon-Ho

    2014-01-01

    Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX) produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitation step to remove casein. The purified protein had high specific activity and a low ratio of the active form (FIXa). The purified rhFIX had 11.9 γ-carboxyglutamic acid (Gla) residues/mol protein, which approached full occupancy of the 12 potential sites in the Gla domain. The rhFIX was shown to have a higher isoelectric point and lower sialic acid content than plasma-derived FIX (pdFIX). The rhFIX had the same N-glycosylation sites and phosphorylation sites as pdFIX, but had a higher specific activity. These results suggest that rhFIX produced from porcine milk is physiologically active and they support the use of transgenic animals as bioreactors for industrial scale production in milk.

  13. Recombinant Human Factor IX Produced from Transgenic Porcine Milk

    Directory of Open Access Journals (Sweden)

    Meng-Hwan Lee

    2014-01-01

    Full Text Available Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitation step to remove casein. The purified protein had high specific activity and a low ratio of the active form (FIXa. The purified rhFIX had 11.9 γ-carboxyglutamic acid (Gla residues/mol protein, which approached full occupancy of the 12 potential sites in the Gla domain. The rhFIX was shown to have a higher isoelectric point and lower sialic acid content than plasma-derived FIX (pdFIX. The rhFIX had the same N-glycosylation sites and phosphorylation sites as pdFIX, but had a higher specific activity. These results suggest that rhFIX produced from porcine milk is physiologically active and they support the use of transgenic animals as bioreactors for industrial scale production in milk.

  14. Enhancement of pulmonary tumour seeding by human coagulation factors II, IX, X--an investigation into the possible mechanisms involved.

    Science.gov (United States)

    Purushotham, A D; McCulloch, P; George, W D

    1991-09-01

    Warfarin inhibits metastasis in the animal model and injection of the Warfarin-dependent coagulation factor complex II, IX, X enhances pulmonary metastasis in the same model. We have studied two possible mechanisms responsible for the observed effect. Mtln3, rat mammary carcinoma cells, radiolabelled with 5-(125) Iodo-2'-deoxyuridine (IUDR) were injected intravenously in female Fisher 344 rats either alone or in combination with factor complex II, IX, X or bovine serum albumin. Following sacrifice at various intervals, measured lung radioactivity was significantly higher (20%) in animals administered cells with the factor complex than in the other two groups (P less than 0.001, ANOVA and Student's t-test). These results indicate increased entrapment of tumour cells in the pulmonary microcirculation. In a second experiment, rat factor complex II, IX, X was prepared, and Mtln3 cells were then injected in female Fisher 344 rats alone or in combination with either human factor complex or rat factor complex. Following sacrifice, the number of pulmonary nodules in animals receiving cells with rat factor complex was similar to that in animals receiving human factor complex, and significantly higher than that in the control (P less than 0.001, ANOVA and Mann-Whitney), indicating that the observed enhancement of pulmonary seeding is unrelated to the xenogeneic properties of the human factor complex.

  15. Prevention and Reversal of Antibody Responses Against Factor IX in Gene Therapy for Hemophilia B

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    Sushrusha eNayak

    2011-12-01

    Full Text Available Intramuscular (IM administration of an adeno-associated viral (AAV vector represents a simple and safe method of gene transfer for treatment of the X-linked bleeding disorder hemophilia B (factor IX, F.IX, deficiency. However, the approach is hampered by an increased risk of immune responses against F.IX. Previously, we demonstrated that the drug cocktail of immune suppressants rapamycin, IL-10, and a specific peptide (encoding a dominant CD4+ T cell epitope caused an induction of regulatory T cells (Treg with a concomitant apoptosis of antigen-specific effector T cells (J. Thromb. Haemost. 7:1523, 2009. This protocol was effective in preventing inhibitory antibody formation against human F.IX (hF.IX in muscle gene transfer to C3H/HeJ hemophilia B mice (with targeted F9 gene deletion. Here, we show that this protocol can also be used to reverse inhibitor formation. IM injection of AAV1-hF.IX vector resulted in inhibitors of on average 8-10 BU within 1 month. Subsequent treatment with the tolerogenic cocktail accomplished a rapid reduction of hF.IX-specific antibodies to <2 BU, which lasted for >4.5 months. Systemic hF.IX expression increased from undetectable to >200 ng/ml, and coagulation times improved. In addition, we developed an alternative prophylactic protocol against inhibitor formation that did not require knowledge of T cell epitopes, consisting of daily oral administration of rapamycin for 1-month combined with frequent, low-dose intravenous injection of hF.IX protein. Experiments in T cell receptor transgenic mice showed that the route and dosing schedule of drug administration substantially affected Treg induction. When combined with intravenous antigen administration, oral delivery of rapamycin had to be performed daily in order to induce Treg, which were suppressive and phenotypically comparable to natural Treg.

  16. Inhibition of hepatitis C virus replication by chalepin and pseudane IX isolated from Ruta angustifolia leaves.

    Science.gov (United States)

    Wahyuni, Tutik Sri; Widyawaruyanti, Aty; Lusida, Maria Inge; Fuad, Achmad; Soetjipto; Fuchino, Hiroyuki; Kawahara, Nobuo; Hayashi, Yoshitake; Aoki, Chie; Hotta, Hak

    2014-12-01

    Hepatitis C virus (HCV) infection is highly prevalent among global populations, with an estimated number of infected patients being 170 million. Approximately 70-80% of patients acutely infected with HCV will progress to chronic liver disease, such as liver cirrhosis and hepatocellular carcinoma, which is a substantial cause of morbidity and mortality worldwide. New therapies for HCV infection have been developed, however, the therapeutic efficacies still need to be improved. Medicinal plants are promising sources for antivirals against HCV. A variety of plants have been tested and proven to be beneficial as antiviral drug candidates against HCV. In this study, we examined extracts, their subfractions and isolated compounds of Ruta angustifolia leaves for antiviral activities against HCV in cell culture. We isolated six compounds, chalepin, scopoletin, γ-fagarine, arborinine, kokusaginine and pseudane IX. Among them, chalepin and pseudane IX showed strong anti-HCV activities with 50% inhibitory concentration (IC₅₀) of 1.7 ± 0.5 and 1.4 ± 0.2 μg/ml, respectively, without apparent cytotoxicity. Their anti-HCV activities were stronger than that of ribavirin (2.8 ± 0.4 μg/ml), which has been widely used for the treatment of HCV infection. Mode-of-action analyses revealed that chalepin and pseudane IX inhibited HCV at the post-entry step and decreased the levels of HCV RNA replication and viral protein synthesis. We also observed that arborinine, kokusaginine and γ-fagarine possessed moderate levels of anti-HCV activities with IC₅₀ values being 6.4 ± 0.7, 6.4 ± 1.6 and 20.4 ± 0.4 μg/ml, respectively, whereas scopoletin did not exert significant anti-HCV activities at 30 μg/ml.

  17. An important role for the activation peptide domain in controlling factor IX levels in the blood of haemophilia B mice.

    Science.gov (United States)

    Begbie, Megan E; Mamdani, Asif; Gataiance, Sharon; Eltringham-Smith, Louise J; Bhakta, Varsha; Hortelano, Gonzalo; Sheffield, William P

    2005-12-01

    The factors responsible for the removal of injected factor IX (fIX) from the blood of individuals with haemophilia B are only partly understood, and may include binding to endothelial or subendothelial sites, passive extravasation related to size or charge, or interactions requiring fIX activation. To investigate these issues, we have produced and characterised recombinant fIX proteins with amino acid changes: delta155-177, an internal deletion which removes most of the activation peptide while retaining the activation cleavage sites; S365A, which inactivates the serine protease activity of fIXa; and K5A, previously shown to eliminate fIX binding of endothelial/subendothelial collagen IV. All proteins were expressed in stably transfected HEK 293 cells, purified by immunoaffinity chromatography, and compared to the wild type HEK 293-derived protein (fIX (WT)). Mutant fIX proteins K5A and delta155-177 exhibited 72 and 202% of the specific activity of fIX (WT), respectively; S365A was without activity. Following intravenous injection in haemophilia B (fIX knockout) mice, recoveries did not differ for fIX (WT) and delta155-177, but were higher for K5A and S365A. The terminal catabolic half-life of delta155-177, alone among the mutants, was increased, by 45% versus fIX (WT). Nine hours post-injection, the observed areas under the clearance curve (AUCs) of delta155-177 and K5, but not S365A, were elevated 2-fold. delta155-177 was equally effective as fIX (WT) in reducing blood loss following tail vein transection in haemophilia B mice. Our results suggest that deletion of the multiple sites of fIX post-translational modification found within the activation peptide eliminated important fIX clearance motifs.

  18. Regulation of human clotting factor IX cDNA expression in transgenic mice

    Institute of Scientific and Technical Information of China (English)

    胡以平; 邱信芳; 薛京伦; 刘祖洞

    1995-01-01

    To study the expression of human dotting factor IX cDNA in transgenic mice,Which is an es-sential work on gene therapy for hemophilia B,3 recombinant constructions containing different lengths ofhuman dotting factor IX cDNA have been introduced into the cultured cells.All of the recombinant constructionswere found to he expressed well in vitro.They were then microinjected into the male pronudei of the fertilizedmouse eggs respectively for generating trahsgenic mice.Unfortunately,none of them was expressed in any transgenicmice.These results show that the expression of the human clotting factor IX cDNA in the transgenic mice canbe determined by cis regulatory element(s).As compared With the results from other related works,it is sug-gested that the cis regulatory element(s)is resided in the 5’-end non-coding region.

  19. Analysis of the N-glycans of recombinant human Factor IX purified from transgenic pig milk

    OpenAIRE

    Gil, Geun-Cheol; Velander, William H.; Van Cott, Kevin E

    2008-01-01

    Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein. In this work, the N-glycan structures of recombinant human Factor IX (tg-FIX) produced in the transgenic pig mammary gland were determined. The majority of the N-glycans of transgenic pig-derived Factor IX (tg-FIX) are complex, bi-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan stru...

  20. Inhibition of carbonic anhydrase isoforms I, II, IX and XII with novel Schiff bases: identification of selective inhibitors for the tumor-associated isoforms over the cytosolic ones.

    Science.gov (United States)

    Sarikaya, Busra; Ceruso, Mariangela; Carta, Fabrizio; Supuran, Claudiu T

    2014-11-01

    A series of new Schiff bases was obtained from sulfanilamide, 3-fluorosulfanilamide or 4-(2-aminoethyl)-benzenesulfonamide and aromatic/heterocyclic aldehydes incorporating both hydrophobic and hydrophilic moieties. The obtained sulfonamides were investigated as inhibitors of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic CA I and II, as well as the transmembrane, tumor-associated CA IX and XII. Most derivatives were medium potency or weak hCA I/II inhibitors, but several of them showed nanomolar affinity for CA IX and/or XII, making them an interesting example of isoform-selective compounds. The nature of the aryl/hetaryl moiety present in the initial aldehyde was the main factor influencing potency and isoform selectivity. The best and most CA IX-selective compounds incorporated moieties such as 4-methylthiophenyl, 4-cyanophenyl-, 4-(2-pyridyl)-phenyl and the 4-aminoethylbenzenesulfonamide scaffold. The best hCA XII inhibitors, also showing selectivity for this isoform, incorporated 2-methoxy-4-nitrophenyl-, 2,3,5,6-tetrafluorophenyl and 4-(2-pyridyl)-phenyl functionalities and were also derivatives of 4-aminoethylbenzenesulfonamide. The sulfanilamide and 3-fluorosulfanilamide derived Schiff bases were less active compared to the corresponding 4-aminoethyl-benzenesulfonamide derivatives. As hCA IX/XII selective inhibition is attractive for obtaining antitumor agents/diagnostic tools with a new mechanism of action, compounds of the type described here may be considered interesting preclinical candidates.

  1. Isolation of a human anti-haemophilic factor IX cDNA clone using a unique 52-base synthetic oligonucleotide probe deduced from the amino acid sequence of bovine factor IX.

    Science.gov (United States)

    Jaye, M; de la Salle, H; Schamber, F; Balland, A; Kohli, V; Findeli, A; Tolstoshev, P; Lecocq, J P

    1983-04-25

    A unique 52mer oligonucleotide deduced from the amino acid sequence of bovine Factor IX was synthesized and used as a probe to screen a human liver cDNA bank. The Factor IX clone isolated shows 5 differences in nucleotide and deduced amino acid sequence as compared to a previously isolated clone. In addition, precisely one codon has been deleted.Images

  2. Purification, crystallization and preliminary crystallographic analysis of AHP IX-bp, a zinc ion and pH-dependent coagulation factor IX binding protein from Agkistrodon halys Pallas venom.

    Science.gov (United States)

    Zang, Jianye; Teng, Maikun; Niu, Liwen

    2003-04-01

    A new coagulation factor IX binding protein, AHP IX-bp, has been purified from Agkistrodon halys Pallas venom and estimated to be an AB heterodimer of about 25 kDa consisting of two chains (an A chain of 15.5 kDa and a B chain of 15 kDa) linked by one or more disulfide bonds. The N-terminal sequence of AHP IX-bp has been determined and aligned with C-type lectin-like proteins. The protein has a high sequence similarity to some snake-venom C-type lectin-like proteins. AHP IX-bp binds to coagulation factor IX but not to coagulation factor X. Moreover, AHP IX-bp shows binding to coagulation factor IX in both zinc ion-dependent and pH-dependent manners. The affinity between AHP IX-bp and coagulation factor IX is higher under neutral or weakly alkaline conditions than under weakly acidic conditions. Single crystals of AHP IX-bp grown at pH 6.5 and 7.5 diffract X-rays to 2.0 and 1.8 A resolution, respectively. Both crystals are isomorphous and belong to the space group P1; only one AB heterodimer is present in the unit cell.

  3. Characterization of three abnormal factor IX variants (Bm Lake Elsinore, Long Beach, and Los Angeles) of hemophilia-B. Evidence for defects affecting the latent catalytic site.

    OpenAIRE

    P. Usharani; Warn-Cramer, B J; Kasper, C K; BAJAJ, S. P.

    1985-01-01

    Abnormal factor IX variant proteins were isolated from the plasmas of three unrelated severe hemophilia-B families that had been previously shown to contain functionally impaired molecules immunologically similar to normal factor IX. The families studied were: (1) a patient with markedly prolonged ox brain prothrombin time, designated factor IX Bm Lake Elsinore (IXBmLE); (b) three patients (brothers) with moderately prolonged ox brain prothrombin time, designated factor IX Long Beach (IXLB); ...

  4. Gallium-Protoporphyrin IX Inhibits Pseudomonas aeruginosa Growth by Targeting Cytochromes.

    Science.gov (United States)

    Hijazi, Sarah; Visca, Paolo; Frangipani, Emanuela

    2017-01-01

    Pseudomonas aeruginosa is a challenging pathogen due to both innate and acquired resistance to antibiotics. It is capable of causing a variety of infections, including chronic lung infection in cystic fibrosis (CF) patients. Given the importance of iron in bacterial physiology and pathogenicity, iron-uptake and metabolism have become attractive targets for the development of new antibacterial compounds. P. aeruginosa can acquire iron from a variety of sources to fulfill its nutritional requirements both in the environment and in the infected host. The adaptation of P. aeruginosa to heme iron acquisition in the CF lung makes heme utilization pathways a promising target for the development of new anti-Pseudomonas drugs. Gallium [Ga(III)] is an iron mimetic metal which inhibits P. aeruginosa growth by interfering with iron-dependent metabolism. The Ga(III) complex of the heme precursor protoporphyrin IX (GaPPIX) showed enhanced antibacterial activity against several bacterial species, although no inhibitory effect has been reported on P. aeruginosa. Here, we demonstrate that GaPPIX is indeed capable of inhibiting the growth of clinical P. aeruginosa strains under iron-deplete conditions, as those encountered by bacteria during infection, and that GaPPIX inhibition is reversed by iron. Using P. aeruginosa PAO1 as model organism, we show that GaPPIX enters cells through both the heme-uptake systems has and phu, primarily via the PhuR receptor which plays a crucial role in P. aeruginosa adaptation to the CF lung. We also demonstrate that intracellular GaPPIX inhibits the aerobic growth of P. aeruginosa by targeting cytochromes, thus interfering with cellular respiration.

  5. Gallium-Protoporphyrin IX Inhibits Pseudomonas aeruginosa Growth by Targeting Cytochromes

    Science.gov (United States)

    Hijazi, Sarah; Visca, Paolo; Frangipani, Emanuela

    2017-01-01

    Pseudomonas aeruginosa is a challenging pathogen due to both innate and acquired resistance to antibiotics. It is capable of causing a variety of infections, including chronic lung infection in cystic fibrosis (CF) patients. Given the importance of iron in bacterial physiology and pathogenicity, iron-uptake and metabolism have become attractive targets for the development of new antibacterial compounds. P. aeruginosa can acquire iron from a variety of sources to fulfill its nutritional requirements both in the environment and in the infected host. The adaptation of P. aeruginosa to heme iron acquisition in the CF lung makes heme utilization pathways a promising target for the development of new anti-Pseudomonas drugs. Gallium [Ga(III)] is an iron mimetic metal which inhibits P. aeruginosa growth by interfering with iron-dependent metabolism. The Ga(III) complex of the heme precursor protoporphyrin IX (GaPPIX) showed enhanced antibacterial activity against several bacterial species, although no inhibitory effect has been reported on P. aeruginosa. Here, we demonstrate that GaPPIX is indeed capable of inhibiting the growth of clinical P. aeruginosa strains under iron-deplete conditions, as those encountered by bacteria during infection, and that GaPPIX inhibition is reversed by iron. Using P. aeruginosa PAO1 as model organism, we show that GaPPIX enters cells through both the heme-uptake systems has and phu, primarily via the PhuR receptor which plays a crucial role in P. aeruginosa adaptation to the CF lung. We also demonstrate that intracellular GaPPIX inhibits the aerobic growth of P. aeruginosa by targeting cytochromes, thus interfering with cellular respiration.

  6. Heme oxygenase and angiogenic activity of endothelial cells: stimulation by carbon monoxide and inhibition by tin protoporphyrin-IX.

    Science.gov (United States)

    Józkowicz, Alicja; Huk, Ihor; Nigisch, Anneliese; Weigel, Guenter; Dietrich, Wolf; Motterlini, Roberto; Dulak, Józef

    2003-04-01

    The activity of heme oxygenase enzymes (HOs) is responsible for the endogenous source of carbon monoxide (CO). Their activities can be inhibited by tin protoporphyrin-IX (SnPPIX). Recent data indicate the involvement of HOs in the regulation of angiogenesis. Here, we investigated the role of the HO pathway in the production and angiogenic activity of vascular endothelial growth factor (VEGF) in endothelial cells treated with SnPPIX, or cultured in the presence of a CO-releasing molecule (CO-RM). Addition of CO-RM or induction of HO-1 by hemin resulted in a threefold elevation in CO production in culture medium (up to 20.3 microg/L) and was associated with a 30% increase in VEGF synthesis. Much higher levels of CO (up to 60 microg/L) and a further increase in VEGF production (by 277%) were measured in cells treated with prostaglandin-J(2), a potent activator of HO-1. SnPPIX prevented the induction of CO generation and inhibited VEGF synthesis. Moreover, SnPPIX reduced the VEGF-elicited angiogenic activities of endothelial cells by decreasing their proliferation (by 26%), migration (by 46%), formation of tubes on Matrigel (by 48%), and outgrowth of capillaries from endothelial spheroids (by 30%). In contrast, overexpression of HO-1 or incubation of cells with CO-RM led to an increase in capillary sprouting. Thus, HO activity up-regulates VEGF production and augments the capability of endothelial cells to respond to exogenous stimulation.

  7. Expression of human factor IX in rat capillary endothelial cells: Toward somatic gene therapy for hemophilia B

    Energy Technology Data Exchange (ETDEWEB)

    Shounan Yao; Wilson, J.M.; Nabel, E.G.; Kurachi, Sumiko; Hachiya, H.L.; Kurachi, Kotoku (Univ. of Michigan, Ann Arbor (United States))

    1991-09-15

    In aiming to develop a gene therapy approach for hemophilia B, the authors expressed and characterized human factor IX in rat capillary endothelial cells (CECs). Moloney murine leukemia virus-derived retrovirus vectors that contain human factor IX cDNA linked to heterologous promoters and the neomycin-resistant gene were constructed and employed to prepare recombinant retroviruses. Rat CECs and NIH 3T3 cells infected with these viruses were selected with the neomycin analogue, G418 sulfate, and tested for expression of factor IX. A construct with the factor IX cDNA under direct control by long terminal repeat gave the highest level of expression as quantitated by immunoassays as well as clotting activity assays. A single RNA transcript of 4.4 kilobases predicted by the construct and a recombinant factor IX were found. The recombinant human factor IX produced showed full clotting activity, demonstrating that CECs have an efficient mechanism for posttranslational modifications, including {gamma}-carboxylation, essential for its biological activity. These results, in addition to other properties of the endothelium, including large number of cells, accessibility, and direct contact with the circulating blood, suggest that CECs can serve as an efficient drug delivery vehicle producing factor IX in a somatic gene therapy for hemophilia B.

  8. EFFECT OF ORAL CONTRACEPTIVES (LD AND CILEST ON CLOTTING FACTORS VIII AND IX

    Directory of Open Access Journals (Sweden)

    H.R. Sadeghipour Roudsari

    1997-10-01

    Full Text Available Based on epidemiologic data, women who take oral contraceptives seem to have an increased risk of developing thromboembollic disease. The thrombotic effects of oral contraceptive (OC are probably mediated, at least partly through their effects on the coagulation system. Plasma levels of several clotting factors have been shown to be elevated in OC users, and this increase is graduated according to the dose of estrogen. In this study, fifty healthy and non smoking women, aged 18-35 years, were randomly assigned to treatment with 2 different OCs: a monophasic pill containing 30 pg of ethinyl estradiol plus 150µg levonorgestrel (LD and a monophasic pill containing 35µg ethinylestradiol plus 250pg norgestimate (Cilest. Factor VIII plasma values were significantly decreased (P<0.05 only in women treated with the preparation LD, but the levels of factor VIII were not significantly different in the group treated with Cilest. Factor IX plasma values were significantly increased (P<0.05 only in women treated with the preparation Cilest, but the levels of factor Ix were not significantly different in the group treated with LD. In LD and cilest users factors VIII and IX were not significantly changed (P<0.05 in overweight and obese subjects in comparison to normal weight.

  9. In Vivo Gene Therapy of Hemophilia B: Sustained Partial Correction in Factor IX-Deficient Dogs

    Science.gov (United States)

    Kay, Mark A.; Rothenberg, Steven; Landen, Charles N.; Bellinger, Dwight A.; Leland, Frances; Toman, Carol; Finegold, Milton; Thompson, Arthur R.; Read, M. S.; Brinkhous, Kenneth M.; Woo, Savio L. C.

    1993-10-01

    The liver represents a model organ for gene therapy. A method has been developed for hepatic gene transfer in vivo by the direct infusion of recombinant retroviral vectors into the portal vasculature, which results in the persistent expression of exogenous genes. To determine if these technologies are applicable for the treatment of hemophilia B patients, preclinical efficacy studies were done in a hemophilia B dog model. When the canine factor IX complementary DNA was transduced directly into the hepatocytes of affected dogs in vivo, the animals constitutively expressed low levels of canine factor IX for more than 5 months. Persistent expression of the clotting. factor resulted in reductions of whole blood clotting and partial thromboplastin times of the treated animals. Thus, long-term treatment of hemophilia B patients may be feasible by direct hepatic gene therapy in vivo.

  10. Molecular Analysis of Factor VIII and Factor IX Genes in Hemophilia Patients: Identification of Novel Mutations and Molecular Dynamics Studies

    Science.gov (United States)

    Al-Allaf, Faisal A.; Taher, Mohiuddin M.; Abduljaleel, Zainularifeen; Bouazzaoui, Abdellatif; Athar, Mohammed; Bogari, Neda M.; Abalkhail, Halah A.; Owaidah, Tarek MA.

    2017-01-01

    Background Hemophilias A and B are X-linked bleeding disorders caused by mutations in the factor VIII and factor IX genes, respectively. Our objective was to identify the spectrum of mutations of the factor VIII and factor IX genes in Saudi Arabian population and determine the genotype and phenotype correlations by molecular dynamics (MD) simulation. Methods For genotyping, blood samples from Saudi Arabian patients were collected, and the genomic DNA was amplified, and then sequenced by Sanger method. For molecular simulations, we have used softwares such as CHARMM (Chemistry at Harvard Macromolecular Mechanics; http://www.charmm-gui.org) and GROMACS. In addition, the secondary structure was determined based on the solvent accessibility for the confirmation of the protein stability at the site of mutation. Results Six mutations (three novel and three known) were identified in factor VIII gene, and six mutations (one novel and five known) were identified in factor IX gene. The factor VIII novel mutations identified were c.99G>T, p. (W33C) in exon 1, c.2138 DelA, p. (N713Tfs*9) in eon14, also a novel mutation at splicing acceptor site of exon 23 c.6430 - 1G>A. In factor IX, we found a novel mutation c.855G>C, p. (E285D) in exon 8. These novel mutations were not reported in any factor VIII or factor IX databases previously. The deleterious effects of these novel mutations were confirmed by PolyPhen2 and SIFT programs. Conclusion The protein functional and structural studies and the models built in this work would be appropriate for predicting the effects of deleterious amino acid substitutions causing these genetic disorders. These findings are useful for genetic counseling in the case of consanguineous marriages which is more common in the Saudi Arabia. PMID:28270892

  11. Ares I-X Flight Test Development Challenges and Success Factors

    Science.gov (United States)

    Askins, Bruce; Davis, Steve; Olsen, Ronald; Taylor, James

    2010-01-01

    The NASA Constellation Program's Ares I-X rocket launched successfully on October 28, 2009 collecting valuable data and providing risk reduction for the Ares I project. The Ares I-X mission was formulated and implemented in less than four years commencing with the Exploration Systems Architecture Study in 2005. The test configuration was founded upon assets and processes from other rocket programs including Space Shuttle, Atlas, and Peacekeeper. For example, the test vehicle's propulsion element was a Shuttle Solid Rocket Motor. The Ares I-X rocket comprised a motor assembly, mass and outer mold line simulators of the Ares I Upper Stage, Orion Spacecraft and Launch Abort System, a roll control system, avionics, and other miscellaneous components. The vehicle was 327 feet tall and weighed approximately 1,800,000 pounds. During flight the rocket reached a maximum speed of Mach 4.8 and an altitude of 150,000 feet. The vehicle demonstrated staging at 130,000 feet, tested parachutes for recovery of the motor, and utilized approximately 900 sensors for data collection. Developing a new launch system and preparing for a safe flight presented many challenges. Specific challenges included designing a system to withstand the environments, manufacturing large structures, and re-qualifying heritage hardware. These and other challenges, if not mitigated, may have resulted in test cancellation. Ares I-X succeeded because the mission was founded on carefully derived objectives, led by decisive and flexible management, implemented by an exceptionally talented and dedicated workforce, and supported by a thorough independent review team. Other major success factors include the use of proven heritage hardware, a robust System Integration Laboratory, multi-NASA center and contractor team, concurrent operations, efficient vehicle assembly, effective risk management, and decentralized element development with a centralized control board. Ares I-X was a technically complex test that

  12. Ares I-X Flight Test Development Challenges and Success Factors

    Science.gov (United States)

    Askins, Bruce; Davis, Steve; Olsen, Ronald; Taylor, James

    2010-01-01

    The NASA Constellation Program's Ares I-X rocket launched successfully on October 28, 2009 collecting valuable data and providing risk reduction for the Ares I project. The Ares I-X mission was formulated and implemented in less than four years commencing with the Exploration Systems Architecture Study in 2005. The test configuration was founded upon assets and processes from other rocket programs including Space Shuttle, Atlas, and Peacekeeper. For example, the test vehicle's propulsion element was a Shuttle Solid Rocket Motor. The Ares I-X rocket comprised a motor assembly, mass and outer mold line simulators of the Ares I Upper Stage, Orion Spacecraft and Launch Abort System, a roll control system, avionics, and other miscellaneous components. The vehicle was 327 feet tall and weighed approximately 1,800,000 pounds. During flight the rocket reached a maximum speed of Mach 4.8 and an altitude of 150,000 feet. The vehicle demonstrated staging at 130,000 feet, tested parachutes for recovery of the motor, and utilized approximately 900 sensors for data collection. Developing a new launch system and preparing for a safe flight presented many challenges. Specific challenges included designing a system to withstand the environments, manufacturing large structures, and re-qualifying heritage hardware. These and other challenges, if not mitigated, may have resulted in test cancellation. Ares I-X succeeded because the mission was founded on carefully derived objectives, led by decisive and flexible management, implemented by an exceptionally talented and dedicated workforce, and supported by a thorough independent review team. Other major success factors include the use of proven heritage hardware, a robust System Integration Laboratory, multi-NASA center and contractor team, concurrent operations, efficient vehicle assembly, effective risk management, and decentralized element development with a centralized control board. Ares I-X was a technically complex test that

  13. Effects of genetic fusion of factor IX to albumin on in vivo clearance in mice and rabbits.

    Science.gov (United States)

    Sheffield, William P; Mamdani, Asif; Hortelano, Gonzalo; Gataiance, Sharon; Eltringham-Smith, Louise; Begbie, Megan E; Leyva, Rina A; Liaw, Peter S; Ofosu, Frederick A

    2004-08-01

    Individuals with haemophilia B require replacement therapy with recombinant or plasma-derived coagulation factor IX (fIX). More benefit per injected dose might be obtained if fIX clearance could be slowed. The contribution of overall size to fIX clearance was explored, using genetic fusion to albumin. Recombinant murine fIX (MIX), and three proteins with C-terminal epitope tags were expressed in HEK 293 cells: tagged MIX (MIXT), tagged mouse serum albumin (MSAT) and MFUST, in which MIX and MSAT were fused in a single polypeptide chain. Proteins MFUST and MIXT were two- to threefold less active in clotting assays than MIX. In mice, the area under the clearance curve (AUC) was reduced for MFUST compared with MSAT or plasma-derived MSA (pd-MSA); the terminal catabolic half-life (t(0.5)) did not differ amongst the three proteins. Two minutes after injection, >40% of the injected MFUST was found in the liver, compared with <10% of either MSAT or pd-MSA. In rabbits, the AUC for MFUST was reduced compared to MIXT, MSAT, or pd-MSA, while the t(0.5) of the fusion protein fell between that of MIXT and MSAT or pd-MSA. Similar results were obtained with non-radioactive fused or non-fused recombinant human fIX in fIX knockout mice. The clearance behaviour of the fusion protein thus more closely resembled that of fIX than that of albumin despite a modest increase in terminal half-life, suggesting that fIX-specific interactions that are important in determining clearance were maintained in spite of the increased size of the fusion protein.

  14. Antibody inhibiting enzymatic activity of tumour-associated carbonic anhydrase isoform IX

    NARCIS (Netherlands)

    Murri-Plesko, M.T.; Hulikova, A.; Oosterwijk, E.; Scott, A.M.; Zortea, A.; Harris, A.L.; Ritter, G.; Old, L.; Bauer, S.; Swietach, P.; Renner, C.

    2011-01-01

    Carbonic anhydrase IX (CAIX) is a hypoxia-induced, membrane-tethered enzyme that is highly expressed in many cancers. It catalyses the hydration of CO(2) to HCO(3)(-) and H(+), and the reverse dehydration reaction. Recent studies have shown an important role for CAIX in pH regulation and it has been

  15. Inhibition of hypoxia-inducible carbonic anhydrase-IX enhances hexokinase Ⅱ inhibitor-induced hepatocellular carcinoma cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    Su-jong YU; Hyo-suk LEE; Jung-hwan YOON; Jeong-hoon LEE; Sun-jung MYUNG; Eun-sun JANG; Min-sun KWAK; Eun-ju CHO; Ja-june JANG; Yoon-jun KIM

    2011-01-01

    Aim: The hypoxic condition within large or infiltrative hypovascular tumors produces intracellular acidification, which could activate many signaling pathways and augment cancer cell growth and invasion. Carbonic anhydrase-Ⅸ (CA-Ⅸ) is an enzyme lowering pH. This study is to examine whether hypoxia induces CA-Ⅸ in hepatocellular carcinoma (HCC) cells, and to evaluate its clinical implication in HCC patients.Methods: Human HCC cell lines (Huh-7 and HepG2 cells) were used, and cell growth was assessed using MTS assay. CA-IX expression and apoptotic/kinase signaling were evaluated using immunoblotting. The cells were transfected with CA-Ⅸ-specific siRNA, or treated with its inhibitor 4-(2-aminoethyl) benzenesulfonamide (CAI#1), and/or the hexokinase Ⅱ inhibitor, 3-bromopyruvate (3-BP). A clinic pathological analysis of 69 patients who underwent an HCC resection was performed using a tissue array.Results: Incubation of HCC cells under hypoxia (1% 02, 5% C02, 94% N2) for 36 h significantly increased CA-IX expression level. CAI#1(400 μmol/L) or CA-IX siRNA (100 μmol/L) did not influence HCC cell growth and induce apoptosis. However, CAI#1 or CA-IX siRNA at these concentrations enhanced the apoptosis induced by 3-BP (100 μmol/L). This enhancement was attributed to increased ER stress and JNK activation, as compared with 3-BP alone. Furthermore, a clinic pathological analysis of 69 HCC patients revealed that tumor CA-Ⅸ intensity was inversely related to E-cadherin intensity.Conclusion: Inhibition of hypoxia-induced CA-Ⅸ enhances hexokinase Ⅱ inhibitor-induced HCC apoptosis. Furthermore, CA-IX expres sion profiles may have prognostic implications in HCC patients. Thus, the inhibition of CA-Ⅸ, in combination with a hexokinase Ⅱ inhibitor, may be therapeutically useful in patients with HCCs that are aggressively growing in a hypoxic environment.

  16. The rates and patterns of deletions in the human factor IX gene

    Energy Technology Data Exchange (ETDEWEB)

    Ketterling, R.P.; Vielhaber, E.L.; Lind, T.J.; Thorland, E.C.; Sommer S.S. (Mayo Clinic/Foundation, Rochester, MN (United States))

    1994-02-01

    Deletions are commonly observed in genes with either segments of highly homologous sequences or excessive gene length. However, in the factor IX gene and in most genes, deletions (of [ge]21 bp) are uncommon. The authors have analyzed DNA from 290 families with hemophilia B (203 independent mutations) and have found 12 deletions >20 bp. Eleven of these are >2 kb (range >3-163 kb), and one is 1.1 kb. The junctions of the four deletions that are completely contained within the factor IX gene have been determined. A novel mutation occurred in patient HB128: the data suggest that a 26.8-kb deletion occurred between two segments of alternating purines and pyrimidines and that a 2.3-kb sense strand segment derived from the deleted region was inserted. For a sample of 203 independent mutations, the authors estimate the [open quotes]baseline[close quotes] rates of deletional mutation per base pair per generation as a function of size. The rate for large (>2 kb)I deletions is exceedingly low. For every mutational event in which a given base is at the junction of a large deletion, there are an estimated 58 microdeletions (<20 bp) and 985 single-base substitutions at that base. Analysis of the nine reported deletion junctions in the factor IX gene literature reveals that (i) five are associated with inversion, orphan sequences, or sense strand insertions; (ii) four are simple deletions that display an excess of short direct repeats at their junctions; (iii) there is no dramatic clustering of junctions within the gene; and (iv) with the exception of alternating purines and pyrimidines, deletion junctions are not preferentially associated with repetitive DNA. 58 refs., 5 figs., 5 tabs.

  17. Encapsulation of factor IX-engineered mesenchymal stem cells in fibrinogen-alginate microcapsules enhances their viability and transgene secretion.

    Science.gov (United States)

    Sayyar, Bahareh; Dodd, Megan; Wen, Jianping; Ma, Shirley; Marquez-Curtis, Leah; Janowska-Wieczorek, Anna; Hortelano, Gonzalo

    2012-01-01

    Cell microencapsulation holds significant promise as a strategy for cellular therapies; however, inadequate survival and functionality of the enclosed cells limit its application in hemophilia treatment. Here, we evaluated the use of alginate-based microcapsules to enhance the viability and transgene secretion of human cord blood-derived mesenchymal stem cells in three-dimensional cultures. Given the positive effects of extracellular matrix molecules on mesenchymal stem cell growth, we tested whether fibrinogen-supplemented alginate microcapsules can improve the efficiency of encapsulated factor IX-engineered mesenchymal stem cells as a treatment of hemophilia B. We found that fibrinogen-supplemented alginate microcapsules (a) significantly enhanced the viability and proliferation of factor IX-engineered mesenchymal stem cells and (b) increased factor IX secretion by mesenchymal stem cells compared to mesenchymal stem cells in nonsupplemented microcapsules. Moreover, we observed the osteogenic, but not chondrogenic or adipogenic, differentiation capability of factor IX-engineered cord blood mesenchymal stem cells and their efficient factor IX secretion while encapsulated in fibrinogen-supplemented alginate microcapsules. Thus, the use of engineered mesenchymal stem cells encapsulated in fibrinogen-modified microcapsules may have potential application in the treatment of hemophilia or other protein deficiency diseases.

  18. Systemic delivery of factor IX messenger RNA for protein replacement therapy

    Science.gov (United States)

    Ramaswamy, Suvasini; Tonnu, Nina; Tachikawa, Kiyoshi; Limphong, Pattraranee; Vega, Jerel B.; Karmali, Priya P.; Chivukula, Pad; Verma, Inder M.

    2017-01-01

    Safe and efficient delivery of messenger RNAs for protein replacement therapies offers great promise but remains challenging. In this report, we demonstrate systemic, in vivo, nonviral mRNA delivery through lipid nanoparticles (LNPs) to treat a Factor IX (FIX)-deficient mouse model of hemophilia B. Delivery of human FIX (hFIX) mRNA encapsulated in our LUNAR LNPs results in a rapid pulse of FIX protein (within 4–6 h) that remains stable for up to 4–6 d and is therapeutically effective, like the recombinant human factor IX protein (rhFIX) that is the current standard of care. Extensive cytokine and liver enzyme profiling showed that repeated administration of the mRNA–LUNAR complex does not cause any adverse innate or adaptive immune responses in immune-competent, hemophilic mice. The levels of hFIX protein that were produced also remained consistent during repeated administrations. These results suggest that delivery of long mRNAs is a viable therapeutic alternative for many clotting disorders and for other hepatic diseases where recombinant proteins may be unaffordable or unsuitable. PMID:28202722

  19. Analysis of the N-glycans of recombinant human Factor IX purified from transgenic pig milk.

    Science.gov (United States)

    Gil, Geun-Cheol; Velander, William H; Van Cott, Kevin E

    2008-07-01

    Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein. In this work, the N-glycan structures of recombinant human Factor IX (tg-FIX) produced in the transgenic pig mammary gland were determined. The majority of the N-glycans of transgenic pig-derived Factor IX (tg-FIX) are complex, bi-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan structures of tg-FIX produced in the porcine mammary epithelial cells differed with respect to N-glycans from glycoproteins produced in other porcine tissues. tg-FIX contains no detectable Neu5Gc, the sialic acid commonly found in porcine glycoproteins produced in other tissues. Additionally, we were unable to detect glycans in tg-FIX that have a terminal Galalpha(1,3)Gal disaccharide sequence, which is strongly antigenic in humans. The N-glycan structures of tg-FIX are also compared to the published N-glycan structures of recombinant human glycoproteins produced in other transgenic animal species. While tg-FIX contains only complex structures, antithrombin III (goat), C1 inhibitor (rabbit), and lactoferrin (cow) have both high mannose and complex structures. Collectively, these data represent a beginning point for the future investigation of species-specific and tissue/cell-specific differences in N-glycan structures among animals used for transgenic animal bioreactors.

  20. Physiological levels of blood coagulation factors IX and X control coagulation kinetics in an in vitro model of circulating tissue factor

    Science.gov (United States)

    Tormoen, Garth W.; Khader, Ayesha; Gruber, András; McCarty, Owen J. T.

    2013-06-01

    Thrombosis significantly contributes to cancer morbidity and mortality. The mechanism behind thrombosis in cancer may be circulating tissue factor (TF), as levels of circulating TF are associated with thrombosis. However, circulating TF antigen level alone has failed to predict thrombosis in patients with cancer. We hypothesize that coagulation factor levels regulate the kinetics of circulating TF-induced thrombosis. Coagulation kinetics were measured as a function of individual coagulation factor levels and TF particle concentration. Clotting times increased when pooled plasma was mixed at or above a ratio of 4:6 with PBS. Clotting times increased when pooled plasma was mixed at or above a ratio of 8:2 with factor VII-depleted plasma, 7:3 with factor IX- or factor X-depleted plasmas, or 2:8 with factor II-, V- or VIII-depleted plasmas. Addition of coagulation factors VII, X, IX, V and II to depleted plasmas shortened clotting and enzyme initiation times, and increased enzyme generation rates in a concentration-dependent manner. Only additions of factors IX and X from low-normal to high-normal levels shortened clotting times and increased enzyme generation rates. Our results demonstrate that coagulation kinetics for TF particles are controlled by factor IX and X levels within the normal physiological range. We hypothesize that individual patient factor IX and X levels may be prognostic for susceptibility to circulating TF-induced thrombosis.

  1. Survey of the anti-factor IX immunoglobulin profiles in patients with hemophilia B using a fluorescence-based immunoassay.

    Science.gov (United States)

    Boylan, B; Rice, A S; Neff, A T; Manco-Johnson, M J; Kempton, C L; Miller, C H

    2016-10-01

    Essentials Studies characterizing neutralizing antibodies (inhibitors) in hemophilia B (HB) are lacking. The current study describes anti-factor (F) IX antibody profiles in 37 patients who have HB. Anti-FIX IgG4 levels exhibited a strong positive correlation with Nijmegen-Bethesda results. These data will help to more clearly define, predict, and treat alloantibody formation in HB.

  2. Epithelial mesenchymal transition and pancreatic tumor initiating CD44+/EpCAM+ cells are inhibited by γ-secretase inhibitor IX.

    Directory of Open Access Journals (Sweden)

    Vindhya Palagani

    Full Text Available Pancreatic ductal adenocarcinoma (PDAC is an aggressive disease with a high rate of metastasis. Recent studies have indicated that the Notch signalling pathway is important in PDAC initiation and maintenance, although the specific cell biological roles of the pathway remain to be established. Here we sought to examine this question in established pancreatic cancer cell lines using the γ-secretase inhibitor IX (GSI IX to inactivate Notch. Based on the known roles of Notch in development and stem cell biology, we focused on effects on epithelial mesenchymal transition (EMT and on pancreatic tumor initiating CD44+/EpCAM+ cells. We analyzed the effect of the GSI IX on growth and epithelial plasticity of human pancreatic cancer cell lines, and on the tumorigenicity of pancreatic tumor initiating CD44+/EpCAM+ cells. Notably, apoptosis was induced after GSI IX treatment and EMT markers were selectively targeted. Furthermore, under GSI IX treatment, decline in the growth of pancreatic tumor initiating CD44+/EpCAM+ cells was observed in vitro and in a xenograft mouse model. This study demonstrates a central role of Notch signalling pathway in pancreatic cancer pathogenesis and identifies an effective approach to inhibit selectively EMT and suppress tumorigenesis by eliminating pancreatic tumor initiating CD44+/EpCAM+ cells.

  3. Optimization of The Electroporation Conditions for Transfection of Human Factor IX into The Goat Fetal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Amir Amiri Yekta

    2013-01-01

    Full Text Available Objective: Electroporation is the most common method used for the transfection of DNA. Although this method has been attributed for various cell using different buffer compositions, the effects of DNA concentration on the transfection efficiency has not yet been studied. Here the correlation between the efficiency of electroporation reaction and increments of DNA concentration has been investigated. Following this investigation, a study was set out to produce a transgenic goat which is capable of secreting recombinant human coagulation factor IX in its milk.Materials and Methods: In this experimental study, a linearized recombinant vector (pBC1 entailing human coagulation factor IX (5.7 kb cDNA was introduced into goat fetal fibroblast cells (three sub passages via electroporation in four separate experiments (while other variables were kept constant, beginning with 10 μg DNA per pulse in the first experiment and increments of 10 μg/pulse for the next three reactions. Thereafter, polymerase chain reaction (PCR-positive cell culture plates were diluted by several factors for further analysis of the transfected wells. Subsequently, positive cells were isolated for fluorescence in situ hybridization. Logistic regression model was used for data analyzing. Significance was defined as p< 0.05.Results: The results showed no significant difference among three first concentrations except for group 1 (10 μg/pulse and group 3 (30 μg/pulse, but there was a significant difference between these groups and the fourth group (p<0.05, as this group (40 μg/pulse statistically showed the highest rate of transfection. As the fluorescence in situ hybridization (FISH results indicated the transgene was integrated in a single position in PCR positive cells.Conclusion: Increasing amount of using the vector 40μg/pulse efficiently increased the number of transfected cells. Besides of voltage and buffer strength which had been previously shown to play a critical role

  4. Factor IX[sub Madrid 2]: A deletion/insertion in Facotr IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site

    Energy Technology Data Exchange (ETDEWEB)

    Solera, J. (Unidades de Genetica Molecular, Madrid (Spain)); Magallon, M.; Martin-Villar, J. (Hemofilia Hospital, Madrid (Spain)); Coloma, A. (Departamento deBioquimica de la Facultad de Medicina de la Universidad Autonoma, Madrid (Spain))

    1992-02-01

    DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5[prime] end of intron d and the two last coding nucleotides located at the 3[prime] end of exon IV in the normal factor IX gene; this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends of the deleted DNA fragment.

  5. Evaluation of engineered AAV capsids for hepatic factor IX gene transfer in murine and canine models.

    Science.gov (United States)

    Markusic, David M; Nichols, Timothy C; Merricks, Elizabeth P; Palaschak, Brett; Zolotukhin, Irene; Marsic, Damien; Zolotukhin, Sergei; Srivastava, Arun; Herzog, Roland W

    2017-05-01

    Adeno-associated virus (AAV) gene therapy vectors have shown the best outcomes in human clinical studies for the treatment of genetic diseases such as hemophilia. However, these pivotal investigations have also identified several challenges. For example, high vector doses are often used for hepatic gene transfer, and cytotoxic T lymphocyte responses against viral capsid may occur. Therefore, achieving therapy at reduced vector doses and other strategies to reduce capsid antigen presentation are desirable. We tested several engineered AAV capsids for factor IX (FIX) expression for the treatment of hemophilia B by hepatic gene transfer. These capsids lack potential phosphorylation or ubiquitination sites, or had been generated through molecular evolution. AAV2 capsids lacking either a single lysine residue or 3 tyrosine residues directed substantially higher coagulation FIX expression in mice compared to wild-type sequence or other mutations. In hemophilia B dogs, however, expression from the tyrosine-mutant vector was merely comparable to historical data on AAV2. Evolved AAV2-LiC capsid was highly efficient in hemophilia B mice but lacked efficacy in a hemophilia B dog. Several alternative strategies for capsid modification improve the in vivo performance of AAV vectors in hepatic gene transfer for correction of hemophilia. However, capsid optimization solely in mouse liver may not predict efficacy in other species and thus is of limited translational utility.

  6. Expression of human coagulation Factor IX in transgenic tomato (Lycopersicon esculentum).

    Science.gov (United States)

    Zhang, Hui; Zhao, Lingxia; Chen, Yuhui; Cui, Lijie; Ren, Weiwei; Tang, Kexuan

    2007-10-01

    In the present study, a plant binary expression vector PG-pRD12-hFIX (where PG is polygalacturonase) harbouring the hFIX (human coagulation Factor IX) gene was constructed and introduced into tomato (Lycopersicon esculentum) via Agrobacterium tumefaciens-mediated transformation. After kanamycin selection, 32 putative independent transgenic tomato plants were regenerated. PCR and Southern-blot analyses confirmed the transgenic status of some plants. RT (reverse transcription)-PCR analysis for the expression of the introduced gene (hFIX) demonstrated that the hFIX gene was expressed specifically in fruits of the tomato. Western-blot analysis confirmed the presence of a 56 kDa band specific to hFIX in the transformed tomatoes. ELISA results showed that the expression of hFIX protein reached a maximum of 15.84 ng/g fresh weight in mature fruit. A blood-clotting assay demonstrated the clotting activity of the expressed hFIX protein in transgenic tomato fruits. This is the first report on the expression of hFIX in plants, and our research provides potentially valuable knowledge for further development of the plant-derived therapeutic proteins.

  7. In vitro differentiation of mouse ES cells into hepatocytes with coagulation factors VIII and IX expression profiles

    Institute of Scientific and Technical Information of China (English)

    MENG; Ying; HUANG; Shaoliang; MIN; Jun; GUO; Zhongmin

    2006-01-01

    Coagulation factors II, V, VII, VIII, IX and X are produced by hepatocytes. So factors VIII and IX deficiencies, which result in hemophilia A and B, have the potential to respond to cellular re- placement therapy. Embryonic stem (ES) cells provide a unique source for therapeutic applications. Here, E14 mouse ES cells have been induced into hepatocytes in vitro. Morphology revealed that ES-derived hepatic-like cells were round or polyhedral shaped with distinct boundary of individual cells, and some arranged in trabeculae. These cells expressed endodermal- or liver-specific mRNA --transthyretin (TTR), α1-anti-trypsin (AAT), α-fetoprotein (AFP), albumin (ALB), glucose-6- phoshpatase (G6P) and tyrosine aminotransferase (TAT). Approximately (85.1(0.5)% of the ES-de- rived cells was stained positive green with ICG uptake. These cells were also stained magenta as a result of PAS reaction. In this paper, expression of coagulation factors VIII and IX mRNA in the ES-derived cells is documented. Therefore, ES cells might be developed as substitute donor cells for the therapy of coagulation factor deficiencies.

  8. Once-weekly prophylactic dosing of recombinant factor IX improves adherence in hemophilia B

    Science.gov (United States)

    Djambas Khayat, Claudia

    2016-01-01

    Regular prophylactic treatment in severe hemophilia should be considered an optimal treatment. There is no general agreement on the optimal prophylaxis regimen, and adherence to prophylaxis is a main challenge due to medical, psychosocial, and cost controversies. Improved approaches in prophylaxis regimen of hemophilia B are needed to make patients’ lives easier. There is some evidence to support the efficacy of once-weekly prophylaxis. Longer sampling schedules are required for the determination of pharmacokinetic (PK) properties of factor IX (FIX). The half-life of FIX seems to be longer than previously described and is expected to be 34 hours. The clinical significance of maintaining a 1% trough level is widely debated in hemophilia B. The overall relationship between factor concentrate levels and incidence of joint bleeding was found to be very weak. Data also indicate that the distribution of FIX into an extravascular FIX compartment may contribute to hemostasis independently of circulating plasma FIX levels. Clinical assessment of the frequency and severity of bleeds remain an important measure of the efficacy of treatment. Role of PK-guided therapy remains to be established. Two prospective randomized studies had evaluated the efficacy and safety of 100 IU/kg once-weekly prophylaxis with nonacog alfa, and this prophylaxis regimen was found to be associated with lower annual bleeding rate compared with on-demand treatment in adolescents and adults with moderately severe-to-severe hemophilia B. Secondary prophylaxis therapy with 100 IU/kg nonacog alfa once weekly reduced annual bleeding rate by 89.4% relative to on-demand treatment. Residual FIX may be supportive of effectiveness. Once-weekly prophylaxis was well tolerated in the two studies, with a safety profile similar to that reported during the on-demand treatment period. To individually tailor treatment to clinical response and to minimize costs of factor concentrate, it would be of interest to

  9. Anion-exchange purification of recombinant factor IX from cell culture supernatant using different chromatography supports.

    Science.gov (United States)

    Ribeiro, Daniel A; Passos, Douglas F; Ferraz, Helen C; Castilho, Leda R

    2013-11-01

    Both recombinant and plasma-derived factor IX concentrates are used in replacement therapies for the treatment of haemophilia B. In the present work, the capture step for a recombinant FIX (rFIX) purification process was investigated. Different strong anion-exchange chromatography media (the resins Q Sepharose(®) FF and Fractogel(®) TMAE, the monolith CIM(®) QA and the membrane adsorber Sartobind(®) Q) were tested for their rFIX binding capacity under dynamic conditions. In these experiments, crude supernatant from CHO cells was used, thus in the presence of supernatant contaminants and mimicking process conditions. The highest dynamic binding capacity was obtained for the monolith, which was then further investigated. To study pseudoaffinity elution of functional rFIX with Ca(2+) ions, a design of experiments to evaluate the effects of pH, NaCl and CaCl2 on yield and purification factor was carried out. The effect of pH was not statistically significant, and a combination of no NaCl and 45mM CaCl2 yielded a good purification factor combined with a high yield of active rFIX. Under these conditions, activity yield of rFIX was higher than the mass yield, confirming selective elution of functional, γ-carboxylated rFIX. Scaling-up of this process 8 fold resulted in very similar process performance. Monitoring of the undesired activated FIX (FIXa) revealed that the FIXa/FIX ratio (1.94%) was higher in the eluate than in the loaded sample, but was still within an acceptable range. HCP and DNA clearances were high (1256 and 7182 fold, respectively), indicating that the proposed process is adequate for the intended rFIX capture step. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Construction and high expression of retroviral vector with human clotting factor IX cDNA in vitro

    Institute of Scientific and Technical Information of China (English)

    卢大儒; 邱信芳; 郑冰; 邱晓赟; 薛京伦

    1995-01-01

    The construction of the high liter and highly expressed safety retroviral vector carrying human clotting factor IX cDNA is reported. Retroviral vectors LNCTX, LIXSN and LCTXSN, driven by hCMV, LTR and hCMV combined with LTR promoter respectively, were constructed, based on the retroviral vector LNL6, and transferred into packaging cell line PA317 with electroporalion. Human dolling factor IX was delected in the cultured cells transduced with LNCIX and LIXSN but not in the cells transduced with LCIXSN. The viral titer of PA317/LNC1X was 800000 CFU per mL. With ELISA detection, it was found that the cells transduced with this vector can express human clotting factor IX at the level of 3.3μg per 106 cells in 24 h in human fibrosarcoma cells HT-1080 and 2.5μg per 106 cells in 24 h in hemophilia B patients’ skin fibroblast HSF cells, and more than 80% of them were biologically active. The viral liter and expression of human FIX were increased, and the construction of retroviral vector backbone was improved

  11. A study of gene transfer and expression of human clotting factor IX in Hemophilia B mice mediated by mini-adenoviral vector

    Institute of Scientific and Technical Information of China (English)

    高啸波; 叶晨波; 侍鼎; 陈立; 邱信芳; 薛京伦

    2003-01-01

    Vector Gti'IX containing human clotting factor IX cDNA with intron 1 (hFIX mini-gene or Fi'IX) driven by CMV promoter was constructed based on the mini-adenoviral vector GT2073 (mini-Ad vector) with all viral protein coding sequences deleted. Mini-Ad packaging cell 293Cre4 was first transduced with Gti'IX, and then was transfected with helper-adenovirus AdLC8, thus mini-Ad virions AdGTi'IX were obtained. At the same time, previous normal adenoviral vector pAdSPi'IX containing viral genome and hFIX mini-gene was constructed, and then previous adenovirus (pre-Ad) AdSPi'IX was obtained as control. The ratio of helper-adenovirus among purified virons AdGTi'IX was less than 0.8%. 3T3 cells were transfected with AdGTi'IX and AdSPi'IX at a MOI of 50 per cell and ELISA result showed that transient expression level in vitro was 1.4±0.2 μg /106@24 h and 1.6±0.3 μg/106@24 h respectively. Each hemophilia B (FIX knock-out) mouse received celiac injection of 1×1010pfu AdGTi'IX or AdSPi'IX. The highest expression level of hFIX in mouse plasma was 590 ng/mL and 690 ng/mL respectively, and the expression time lasted for 16 weeks and 9 weeks respectively. The bleeding time reduced from over 30 min to 7.5 min, and 5-min blood lost reduced from 430 μL to 60 μL. The results of anti-Ad IgG assays indicated that immune response triggered by AdGTi'IX was obviously weaker than that triggered by AdSPi'IX. These results indicated that, compared with previous adenovirus (pre-Ad), the mini-Ad vector system prolonged the expression time of hFIX and reduced immune response, thus offering a promising result for further pre-clinical study.

  12. A simple technique to reduce epistaxis and nasopharyngeal trauma during nasotracheal intubation in a child with factor IX deficiency having dental restoration.

    Science.gov (United States)

    Delgado, Anita V; Sanders, John C

    2004-10-01

    Epistaxis and airway trauma are often associated with nasotracheal intubation. We describe a patient with Factor IX deficiency who required nasotracheal intubation. An inexpensive, nonproprietary, rapid technique was used to reduce the trauma of intubation.

  13. Sustained expression of coagulation factor IX by modified cord blood-derived mesenchymal stromal cells.

    Science.gov (United States)

    Dodd, Megan; Marquez-Curtis, Leah; Janowska-Wieczorek, Anna; Hortelano, Gonzalo

    2014-01-01

    Hemophilia B patients are subject to frequent and spontaneous bleeding caused by a deficiency of clotting factor IX (FIX). Mesenchymal stromal cells (MSCs) have been used in cellular therapies as a result of their immunomodulatory properties, the ability to home to sites of injury and their amenability to various ex vivo modifications, including lentiviral-mediated gene transfer. MSCs were isolated from human umbilical cord blood and differentiated into adipogenic, chondrogenic and osteogenic lineages. A lentiviral DNA vector containing the human FIX gene was generated using traditional restriction enzyme digest and ligation techniques to generate viable replication-incompetent lentiviral particles that were used to transduce MSCs. Quantitative measurement of FIX expression was conducted using an enzyme-linked immunosorbent assay. The over-expression of FIX was sustained in vitro at levels > 4 µg/10(6) cells/24 h and FIX coagulant activity was > 2.5 mIU/10(6) cells/24 h for the 6-week duration of study. Lentiviral modification of cells with a multiplicity of infection of 10 did not adversely affect the potential of cord blood (CB) MSCs to differentiate to adipocytes, chondrocytes and osteoblastic cells, and the expression of functional FIX was sustained after differentiation and was similar to that in nondifferentiated cells. Modification of human CB MSCs with a lentiviral vector resulted in sustained high FIX expression in vitro after differentiation to adipogenic, chondrogenic and osteoblastic cells. These modified MSCs could have applications in cellular therapies for hemophilia B. Copyright © 2014 John Wiley & Sons, Ltd.

  14. Expression of the human coagulation factor IX in the bone marrow mesenchymal stem cells

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    Azadehsadat Azadbakhsh

    2014-05-01

    Full Text Available Background: Mesenchymal stem cells (MSCs are appropriate target for gene and cell-based therapy of hemophilia B patients. MSCs possess several unique properties such as capability of differentiating into multiple lineages and lower immunogenecity in transplant procedure that make them attractive candidates for cell and gene therapy. One of the challenges in the gene therapy is the low expression level of transgene. To improve expression, strong regulatory elements in the context of vectors could contribute to improve efficacy of gene therapy strategies. In this study four human factor IX (hFIX-expressing plasmids equipped with various combination of human -globin (hBG introns and Kozak sequence were transfected into the MSCs and expression of the hFIX was evaluated in vitro. Material and Methods: MSCs were obtained from tibias and the femora of rats and phenotypic characterization of the MSCs was determined by flow cytometry. Four hFIX-expressing plasmids were introduced into the culture-expanded MSCs using transfection agent. 48 hours after transfection, ability of the MSCs for expression of the hFIX and efficacies of the plasmids were evaluated by performing sandwich ELISA on cultured media as well as semi-quantitative RT-PCR. All analyses were performed with One-way ANOVA using SPSS software. Results:The highest expression level of the hFIX was obtained from intron-less and hBG intron-I containing construct. The highest biological activity was obtained from hBG intron-I,II containing construct. Conclusion:Successful expression of the hFIX was obtained from recombinant MSCs. MSCs were able to splice heterologous hBG intron-I from the hFIX-cDNA. Application of thehBG introns reduced the hFIX expression levels, probably due to improper splicing of the hBG introns.

  15. Ultrasound-targeted hepatic delivery of factor IX in hemophiliac mice.

    Science.gov (United States)

    Anderson, C D; Moisyadi, S; Avelar, A; Walton, C B; Shohet, R V

    2016-06-01

    Ultrasound-targeted microbubble destruction (UTMD) was used to direct the delivery of plasmid and transposase-based vectors encoding human factor IX (hFIX) to the livers of hemophilia B (FIX-/-) mice. The DNA vectors were incorporated into cationic lipid microbubbles, injected intravenously, and transfected into hepatocytes by acoustic cavitation of the bubbles as they transited the liver. Ultrasound parameters were identified that produced transfection of hepatocytes in vivo without substantial damage or bleeding in the livers of the FIX-deficient mice. These mice were treated with a conventional expression plasmid, or one containing a piggyBac transposon construct, and hFIX levels in the plasma and liver were evaluated at multiple time points after UTMD. We detected hFIX in the plasma by western blotting from mice treated with either plasmid during the 12 days after UTMD, and in the hepatocytes of treated livers by immunofluorescence. Reductions in clotting time and improvements in the percentage of FIX activity were observed for both plasmids, conventional (4.15±1.98%), and transposon based (2.70±.75%), 4 to 5 days after UTMD compared with untreated FIX (-/-) control mice (0.92±0.78%) (P=0.001 and P=0.012, respectively). Reduced clotting times persisted for both plasmids 12 days after treatment (reflecting percentage FIX activity of 3.12±1.56%, P=0.02 and 3.08±0.10%, P=0.001, respectively). Clotting times from an additional set of mice treated with pmGENIE3-hFIX were evaluated for long-term effects and demonstrated a persistent reduction in average clotting time 160 days after a single treatment. These data suggest that UTMD could be a minimally invasive, nonviral approach to enhance hepatic FIX expression in patients with hemophilia.

  16. Efficacy of a high-purity factor IX concentrate in hemophilia B patients undergoing surgery Eficácia do concentrado de alta pureza do fator IX em pacientes cirúrgicos portadores de hemofilia B

    Directory of Open Access Journals (Sweden)

    Vesa Rasi

    2002-04-01

    Full Text Available A plasma derived, high purity, solvent-detergent treated and subsequently nanofiltered factor IX concentrate (BEMOFIL was evaluated in 19 hemophilia B patients, including four with severe, thirteen with mild or moderate type of disease and two hemophilia B carriers undergoing 31 surgical procedures. The mean in vivo recovery was 52 %, range 36 - 76 %. The mean preoperative plasma factor IX activity after the initial loading dose was 0.86 IU mL-1, range 0.59 - 1.32 IU mL-1. In eight major orthopedic procedures, the mean usage of factor IX was 44600 IU or 574 IU kg-1 during the hospital stay, mean 11.6 days. Thromboprophylaxis was not used. The hemostatic efficacy was evaluated good in all cases and there were no thromboembolic complications. In conclusion, BEMOFIL used as bolus dosing was found to be safe and effective in achieving hemostasis in subjects with hereditary F IX deficiency undergoing surgery.O concentrado de fator IX ( Bemofil , um derivado plasmático de alta pureza tratado com solventes- detergente e nano-filtrado , foi avaliado em 19 pacientes portadores de Hemofilia B .Quatro pacientes apresentavam a forma grave da moléstia, 13 a forma leve e moderada e dois portadores em um total de 31 atos cirúrgicos.A recuperação média "in vivo" foi de 52% (36-76%. A atividade plasmática média pré-operatória do fator IX após a dose inicial foi de 0,86 UI ml -1 , média de 0,59 - 1,32 UI ml-1. Em oito procedimentos ortopédicos extensos , a média de utilização do fator IX foi de 44.600 UI ou 574 UI kg -1 durante a hospitalização que teve a média de 11,6 dias. A tromboprofilaxia não foi utilizada. A eficácia hemostática avaliada em todos os casos foi boa ,e não ocorreu nenhum tipo de complicação tromboembólica. Concluímos que o Bemofil em bolus foi considerado seguro e eficaz para a hemostasia em pacientes portadores de hemofilia B que necessitam de um procedimento cirúrgico.

  17. Recombinant factor IX (BAX326) in previously treated paediatric patients with haemophilia B: a prospective clinical trial.

    Science.gov (United States)

    Urasinski, T; Stasyshyn, O; Andreeva, T; Rusen, L; Perina, F G; Oh, M S; Chapman, M; Pavlova, B G; Valenta-Singer, B; Abbuehl, B E

    2015-03-01

    A newly developed recombinant factor IX (BAX326(1) ) was investigated for prophylactic use in paediatric patients aged 96% of bleeds (100% of minor, 88.9% of moderate and 100% of major bleeds); the majority (88.5%) resolved after 1-2 infusions. Longer T1/2 and lower IR were observed in younger children (<6 years) compared to those aged 6 to 12 years. BAX326 administered as prophylactic treatment as well as for controlling bleeds is efficacious and safe in paediatric patients aged <12 years with haemophilia B.

  18. Characterization of three abnormal factor IX variants (Bm Lake Elsinore, Long Beach, and Los Angeles) of hemophilia-B. Evidence for defects affecting the latent catalytic site.

    Science.gov (United States)

    Usharani, P; Warn-Cramer, B J; Kasper, C K; Bajaj, S P

    1985-01-01

    Abnormal factor IX variant proteins were isolated from the plasmas of three unrelated severe hemophilia-B families that had been previously shown to contain functionally impaired molecules immunologically similar to normal factor IX. The families studied were: (1) a patient with markedly prolonged ox brain prothrombin time, designated factor IX Bm Lake Elsinore (IXBmLE); (b) three patients (brothers) with moderately prolonged ox brain prothrombin time, designated factor IX Long Beach (IXLB); and (c) a patient with normal ox brain prothrombin time designated factor IX Los Angeles (IXLA). Each variant molecule comigrates with normal factor IX (IXN) both in the sodium dodecyl sulfate and in the nondenaturing alkaline gel electrophoresis. All three variant proteins are indistinguishable from IXN in their amino acid compositions, isoelectric points, carbohydrate distributions and number of gamma-carboxyglutamic acid residues. Each variant protein undergoes a similar pattern of cleavage by factor XIa/Ca2+ and by factor VIIa/Ca2+/tissue factor, and is activated at a rate similar to that observed for IXN. All of the three variant proteins also react with an anti-IXN monoclonal antibody that interferes with the binding of activated IXN(IXaN) to thrombin-treated factor VIIIC. However, in contrast to IXaN, the cleaved IXBmLE has negligible activity (approximately 0.2%), and cleaved forms of IXLA and IXLB have significantly reduced activity (approximately 5-6%) in binding to antithrombin-III/heparin, and in activating factor VII (plus Ca2+ and phospholipid) or factor X (plus Ca2+ and phospholipid) +/- factor VIII. These data, taken together, strongly indicate that the defect in these three variant proteins resides near or within the latent catalytic site. This results in virtually a complete loss of catalytic activity of the cleaved IXBmLE molecule and approximately 95% loss of catalytic activity of the cleaved IXLA and IXLB molecules.

  19. PpIX induces mitochondria-related apoptosis in murine leukemia L1210 cells.

    Science.gov (United States)

    Su, Xiaomin; Chen, Yan; Wang, Xiaobing; Wang, Yuan; Wang, Pan; Li, Long; Liu, Quanhong

    2014-07-01

    Protoporphyrin IX (PpIX), a well-known sensitizer that can enhance laser light or ultrasound induced cytotoxicity in photodynamic and sonodynamic therapy. However, PpIX alone could effectively cause anti-tumor effect and the underlying mechanisms are rarely been reported. Therefore, this study was to investigate the possible mechanism by which PpIX revealed anti-proliferative effect on murine leukemia L1210 cells. The accumulation of PpIX in L1210 cells and normal peripheral blood mononuclear cells (PBMCs) was evaluated with flow cytometry. The subcellular localization of PpIX and apoptosis-inducing factor (AIF) translocation were determined by confocal microscope. The cell viability was examined by MTT assay. Annexin V-PE/7-AAD and DAPI staining were used to detect apoptotic cells. The mitochondrial membrane potential (MMP) changes were tested by rhodamine123 staining. DNA damage was measured by comet assay. PpIX preferentially accumulated in L1210 cells compared to PBMCs and PpIX mainly located in the mitochondria of L1210 cells. PpIX at a concentration of 1 µg/ml or above exerted significant anti-tumor effect and the cell viability loss presented PpIX dose-dependent manner. Typical apoptotic features such as chromatin condensation were observed by DAPI staining. Annexin V-PE/7-AAD analysis showed 5 µg/ml PpIX could induce about 24% cell apoptosis, which was inhibited by cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore. In addition, the PpIX caused MMP loss, AIF translocation to nucleus and serious DNA damage were also suppressed by CsA. The results indicate mitochondria-dependent apoptosis were involved in PpIX caused cell damage on L1210 cells.

  20. Sustained and therapeutic delivery of factor IX in nude haemophilia B mice by encapsulated C2C12 myoblasts: concurrent tumourigenesis.

    Science.gov (United States)

    Hortelano, G; Wang, L; Xu, N; Ofosu, F A

    2001-03-01

    This study reports the generation of an immunodeficient murine model for haemophilia B, obtained by breeding factor IX-deficient mice with an immunodeficient mouse strain, and use of this mouse model to evaluate the long-term efficacy and safety of a gene therapy strategy for treating haemophilia B. Nude haemophilic mice were implanted with biocompatible microcapsules enclosing recombinant myoblasts secreting human factor IX. The activated partial thromboplastin time (APTT) of plasma of mice thus treated was invariably shortened 3 weeks after microcapsule implantation, and remained shortened for at least 77 days. Shortening of the APTT of the haemophilia mice coincided with the appearance of human factor IX in mice plasmas (up to 600 ng mL(-1) on day 77), and normalization of the tail-bleeding time. Thus, the microencapsulated myoblasts reversed the clinical phenotype of haemophilia B. In contrast, plasmas of immunocompetent haemophilic mice similarly implanted with microcapsules only showed a transient shortening of APTT, and coincident transient delivery of human factor IX antigen. Rapid disappearance of human factor IX from plasmas of immunocompetent mice also coincided with production of antibodies to the human transgene. Significantly, 86% of the nude haemophilia mice developed tumours of myoblast origin. Thus, while this study revealed the feasibility of this gene therapy approach to treat severe haemophilia B, it also highlights the importance of using safer cell lines to prevent tumour development.

  1. Expression of human factor IX in rabbit hepatocytes by retrovirus-mediated gene transfer: Potential for gene therapy of hemophilia B

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, A.R. (Univ. of Washington, Seattle (USA) Puget Sound Blood Center, Seattle, WA (USA)); Darlington, G. (Baylor College of Medicine, Houston, TX (USA)); Armentano, D.; Woo, S.L.C.

    1990-08-01

    Hemophilia B (Christmas disease) is a chromosome X-linked blood clotting disorder which results when factor IX is deficient or functionally defective. The enzyme is synthesized in the liver, and the existence of animal models for this genetic disease will permit the development of somatic gene therapy protocols aimed at transfer of the functional gene into the liver. The authors report the construction of an N2-based recombinant retroviral vector, NCMVFIX, for efficient transfer and expression of human factor IX cDNA in primary rabbit hepatocytes. In this construct the human cytomegalovirus immediate early promoter directs the expression of factor IX. Hepatocytes were isolated from 3-week-old New Zealand White rabbits, infected with the recombinant virus, and analyzed for secretion of active factor IX. The infected rabbit hepatocytes produced human factor IX that is indistinguishable from enzyme derived from normal human plasma. The recombinant protein is sufficiently {gamma}-carboxylated and is functionally active in clotting assays. These results establish the feasibility of using infected hepatocytes for the expression of this protein and are a step toward the goal of correcting hemophilia B by hepatic gene transfer.

  2. A pilot study on potential plasma hypoxia markers in the radiotherapy of non-small cell lung cancer. Osteopontin, carbonic anhydrase IX and vascular endothelial growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Ostheimer, C.; Bache, M.; Guettler, A.; Vordermark, D. [Martin-Luther-University Halle-Wittenberg, Department of Radiation Oncology, Halle (Saale) (Germany); Kotzsch, M. [Technical University Dresden, Department of Pathology, Dresden (Germany)

    2014-03-15

    Hypoxic radioresistance plays a critical role in the radiotherapy of cancer and adversely impacts prognosis and treatment response. This prospective study investigated the interrelationship and the prognostic significance of several hypoxia-related proteins in non-small cell lung cancer (NSCLC) patients treated by radiotherapy ± chemotherapy. Pretreatment osteopontin (OPN), vascular endothelial growth factor (VEGF) and carbonic anhydrase IX (CA IX) plasma levels were determined by ELISA in 55 NSCLC (M0) patients receiving 66 Gy curative-intent radiotherapy or chemoradiation. Marker correlation, association with clinicopathological parameters and the prognostic value of a biomarker combination was evaluated. All biomarkers were linearly correlated and linked to different clinical parameters including lung function, weight loss (OPN), gross tumor volume (VEGF) and T stage (CA IX). High OPN (p = 0.03), VEGF (p = 0.02) and CA IX (p = 0.04) values were significantly associated with poor survival. Double marker combination additively increased the risk of death by a factor of 2 and high plasma levels of the triple combination OPN/VEGF/CA IX yielded a 5.9-fold risk of death (p = 0.009). The combined assessment of OPN/VEGF/CA IX correlated independently with prognosis (p = 0.03) in a multivariate Cox regression model including N stage, T stage and GTV. This pilot study suggests that a co-detection augments the prognostic value of single markers and that the integration of OPN, VEGF and CA IX into a hypoxic biomarker profile for the identification of patients with largely hypoxic and radioresistant tumors should be further evaluated. (orig.) [German] Hypoxische Radioresistenz spielt eine kritische Rolle in der Radiotherapie maligner Tumoren und beeinflusst Prognose und Therapieansprechen negativ. Diese prospektive Studie untersuchte den Zusammenhang und die prognostische Bedeutung einiger hypoxieassoziierter Proteine bei Patienten mit nicht-kleinzelligem Bronchialkarzinom

  3. Carbonic anhydrase inhibitors: synthesis and inhibition of the human carbonic anhydrase isoforms I, II, IX and XII with benzene sulfonamides incorporating 4- and 3-nitrophthalimide moieties.

    Science.gov (United States)

    Sethi, Kalyan K; Verma, Saurabh M; Tanç, Muhammet; Purper, Gaultier; Calafato, Gaetan; Carta, Fabrizio; Supuran, Claudiu T

    2014-03-01

    A series of 4 and 5 nitro-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1-8) was synthesized by reaction of benzenesulfonamide derivatives with 4 and 3-nitrophthalic anhydrides. These new sulfonamides were investigated as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more specifically against the human (h) cytosolic isoforms hCA I and II and the transmembrane, tumor-associated hCA IX and XII. Most of the novel compounds were medium potency-weak hCA I inhibitors (Kis in the range of 295-10,000 nM), but were more effective hCA II inhibitors (Kis of 1.7-887 nM). The tumor-associated hCA IX was also inhibited, with Kis in the micromolar range, whereas against hCA XII the inhibition constants were in the range of 90-3,746 nM. The structure-activity relationship (SAR) with this series of sulfonamides is straightforward, with the main features leading to good activity for each isoforms being established. The high sequence hCA alignment homology and molecular docking studies was performed in order to rationalize the activities reported and binding mode to different hCA as inhibitors. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. A novel endogenous antimalarial: Fe(II)-protoporphyrin IX alpha (heme) inhibits hematin polymerization to beta-hematin (malaria pigment) and kills malaria parasites.

    Science.gov (United States)

    Monti, D; Vodopivec, B; Basilico, N; Olliaro, P; Taramelli, D

    1999-07-13

    The polymerization of hemoglobin-derived ferric-protoporphyrin IX [Fe(III)PPIX] to inert hemozoin (malaria pigment) is a crucial and unique process for intraerythrocytic plasmodia to prevent heme toxicity and thus a good target for new antimalarials. Quinoline drugs, i.e., chloroquine, and non-iron porphyrins have been shown to block polymerization by forming electronic pi-pi interactions with heme monomers. Here, we report the identification of ferrous-protoporphyrin IX [Fe(II)PPIX] as a novel endogenous anti-malarial. Fe(II)PPIX molecules, released from the proteolysis of hemoglobin, are first oxidized and then polymerized to hemozoin. We obtained Fe(II)PPIX on preparative scale by electrochemical reduction of Fe(III)PPIX, and the reaction was monitored by cyclic voltammetry. Polymerization assays at acidic pH were conducted with the resulting Fe(II)PPIX using a spectrophotometric microassay of heme polymerization adapted to anaerobic conditions and the products characterized by infrared spectroscopy. Fe(II)PPIX (a) did not polymerize and (b) produced a dose-dependent inhibition of Fe(III)PPIX polymerization (IC(50) = 0.4 molar equiv). Moreover, Fe(II)PPIX produced by chemical reduction with thiol-containing compounds gave similar results: a dose-dependent inhibition of heme polymerization was observed using either L-cysteine, N-acetylcysteine, or DL-homocysteine, but not with L-cystine. Cyclic voltammetry confirmed that the inhibition of heme polymerization was due to the Fe(II)PPIX molecules generated by the thiol-mediated reduction of Fe(III)PPIX. These results point to Fe(II)PPIX as a potential endogenous antimalarial and to Fe(III)PPIX reduction as a potential new pharmacological target.

  5. Carbonic anhydrase inhibitors. Inhibition of the human cytosolic isoforms I and II and transmembrane, tumor-associated isoforms IX and XII with boronic acids.

    Science.gov (United States)

    Winum, Jean-Yves; Innocenti, Alessio; Scozzafava, Andrea; Montero, Jean-Louis; Supuran, Claudiu T

    2009-05-15

    A series of aromatic, arylalkenyl- and arylalkyl boronic acids were assayed as inhibitors of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic human (h) hCA I and II, and the transmembrane, tumor-associated hCA IX and XII. The best hCA I and II inhibitor was biphenyl boronic acid with, a K(I) of 3.7-4.5 microM, whereas the remaining derivatives showed inhibition constants in the range of 6.0-1560 microM for hCA I and of 6.0-1050 microM for hCA II, respectively. hCA IX and XII were effectively inhibited by most of the aromatic boronic acids (K(I)s of 7.6-12.3 microM) whereas the arylalkenyl and aryl-alkyl derivatives generally showed weaker inhibitory properties (K(I)s of 34-531 microM). The nature of the moiety substituting the boronic acid group strongly influenced the CA inhibitory activity, with inhibitors possessing low micromolar to millimolar activity being detected in this small series of investigated compounds. This study proves that the B(OH)(2) moiety represents a new zinc-binding group for the generation of effective CA inhibitors targeting isoforms with medicinal chemistry applications. The boronic acids probably bind to the Zn(II) ion within the CA active site leading to a tetrahedral geometry of the metal ion and of the B(III) derivative.

  6. Abnormal joint and bone wound healing in hemophilia mice is improved by extending factor IX activity after hemarthrosis.

    Science.gov (United States)

    Sun, Junjiang; Hua, Baolai; Livingston, Eric W; Taves, Sarah; Johansen, Peter B; Hoffman, Maureane; Ezban, Mirella; Monroe, Dougald M; Bateman, Ted A; Monahan, Paul E

    2016-12-30

    Wound healing requires interactions between coagulation, inflammation, angiogenesis, cellular migration, and proliferation. Healing in dermal wounds of hemophilia B mice is delayed when compared to hemostatically normal wild type (WT) mice, with abnormal persistence of iron deposition, inflammation, and neovascularity. We observed healing following induced joint hemorrhage in WT and factor IX (FIX) knockout (FIX(-/-)) mice, examining also parameters previously studied in an excisional skin wound model. Hemostatically normal mice tolerated this joint bleeding challenge, cleared blood from the joint, and healed with minimal pathology, even if additional autologous blood was injected intra-articularly at the time of wounding. Following hemarthrosis, joint wound healing in hemophilia B mice was impaired and demonstrated similar abnormal histologic features as previously described in hemophilic dermal wounds. Therefore, studies of pathophysiology and therapy of hemophilic joint bleeding performed in hemostatically normal animals are not likely to accurately reflect the healing defect of hemophilia. We additionally explored the hypothesis that the use of a FIX replacement protein with extended circulating FIX activity could improve synovial and osteochondral wound healing in hemophilic mice, when compared to treatment with unmodified recombinant FIX (rFIX) in the established joint bleeding model. Significantly improved synovial wound healing and preservation of normal osteochondral architecture are achieved by extending FIX activity after hemarthrosis using glycoPEGylated FIX when compared to an equivalent dose of rFIX. These results suggest that treating joint bleeding only until hemostasis is achieved may not result in optimal joint healing, which is improved by extending factor activity.

  7. Modulating immunogenicity of factor IX by fusion to an immunoglobulin Fc domain: a study using a hemophilia B mouse model.

    Science.gov (United States)

    Levin, D; Lagassé, H A D; Burch, E; Strome, S; Tan, S; Jiang, H; Sauna, Z E; Golding, B

    2017-04-01

    Essentials Fc-fusion increases a therapeutic's half-life, but FcγR interactions may impact immunogenicity. Species-specific Fc-FcγR interactions allow for mechanistic in vivo studies using mouse models. Fc fusion modulates the immune response to factor IX in hemophilia B mice by eliciting Th1 bias. This model could inform future studies of IgE-associated anaphylaxis in hemophilia B patients. Background Fc fusion is a platform technology used to increase the circulating half-life of protein and peptide therapeutics. However, there are potential immunological consequences with this approach, such as changes in the molecule's immunogenicity as well as possible interactions with a repertoire of Fc receptors (FcR) that can modulate immune responses. Objectives/Methods Using a mouse hemophilia B (HB) model, we compared the immune responses to infusions of recombinant human factor IX (hFIX) and hFIX fused to mouse IgG2a-Fc (hFIX-mFc). The mFc was employed to allow species-specific Fc-FcγR interactions. Results Although treatment with hFIX-mFc altered the early development of anti-FIX IgG, no significant differences in anti-FIX antibody titers were observed at the end of the treatment regimen (5 weeks) or upon anamnestic response (5 months). However, treatment with hFIX-mFc elicited higher FIX-neutralizing antibody levels and resulted in reduced IgE titers compared with the hFIX-treated group. Additionally, differences in plasma cytokine levels and in vitro CD4(+) T-cell responses suggest that whereas hFIX treatment triggered a Th2-biased immune response, hFIX-mFc treatment induced Th1-biased CD4(+) T cells. We also show that hFIX-mFc bound to soluble FcγRs and engaged with FcγRs on different cell types, which may impact antigen presentation. Conclusions These studies provide a model system to study how Fc-fusion proteins may affect immune mechanisms. We used this model to demonstrate a plausible mechanism by which Fc fusion may modulate the IgE response to hFIX. This

  8. DNA Damage and Cell Cycle Arrest Induced by Protoporphyrin IX in Sarcoma 180 Cells

    Directory of Open Access Journals (Sweden)

    Qing Li

    2013-09-01

    Full Text Available Background: Porphyrin derivatives have been widely used in photodynamic therapy as effective sensitizers. Protoporphyrin IX (PpIX, a well-known hematoporphyrin derivative component, shows great potential to enhance light induced tumor cell damage. However, PpIX alone could also exert anti-tumor effects. The mechanisms underlying those direct effects are incompletely understood. This study thus investigated the putative mechanisms underlying the anti-tumor effects of PpIX on sarcoma 180 (S180 cells. Methods: S180 cells were treated with different concentrations of PpIX. Following the treatment, cell viability was evaluated by the 3-(4, 5- dimethylthiazol-2-yl-2, 5-diphenyltetrazoliumbromide (MTT assay; Disruption of mitochondrial membrane potential was measured by flow cytometry; The trans-location of apoptosis inducer factor (AIF from mitochondria to nucleus was visualized by confocal laser scanning microscopy; DNA damage was detected by single cell gel electrophoresis; Cell cycle distribution was analyzed by DNA content with flow cytometry; Cell cycle associated proteins were detected by western blotting. Results: PpIX (≥ 1 µg/ml significantly inhibited proliferation and reduced viability of S180 cells in a dose-dependent manner. PpIX rapidly and significantly triggered mitochondrial membrane depolarization, AIF (apoptosis inducer factor translocation from mitochondria to nucleus and DNA damage, effects partially relieved by the specific inhibitor of MPTP (mitochondrial permeability transition pore. Furthermore, S phase arrest and upregulation of the related proteins of P53 and P21 were observed following 12 and 24 h PpIX exposure. Conclusion: PpIX could inhibit tumor cell proliferation by induction of DNA damage and cell cycle arrest in the S phase.

  9. Fibronectin-Alginate microcapsules improve cell viability and protein secretion of encapsulated Factor IX-engineered human mesenchymal stromal cells.

    Science.gov (United States)

    Sayyar, Bahareh; Dodd, Megan; Marquez-Curtis, Leah; Janowska-Wieczorek, Anna; Hortelano, Gonzalo

    2015-01-01

    Continuous delivery of proteins by engineered cells encapsu-lated in biocompatible polymeric microcapsules is of considerable therapeutic potential. However, this technology has not lived up to expectations due to inadequate cell--matrix interactions and subsequent cell death. In this study we hypoth-esize that the presence of fibronectin in an alginate matrix may enhance the viability and functionality of encapsulated human cord blood-derived mesenchymal stromal cells (MSCs) expressing the human Factor IX (FIX) gene. MSCs were encapsulated in alginate-PLL microcapsules containing 10, 100, or 500 μg/ml fibronectin to ameliorate cell survival. MSCs in microcapsules with 100 and 500 μg/ml fibronectin demonstrated improved cell viability and proliferation and higher FIX secretion compared to MSCs in non-supplemented microcapsules. In contrast, 10 μg/ml fibronectin did not significantly affect the viability and protein secretion from the encapsulated cells. Differentiation studies demonstrated osteogenic (but not chondrogenic or adipogenic) differentiation capability and efficient FIX secretion of the enclosed MSCs in the fibronectin-alginate suspension culture. Thus, the use of recombinant MSCs encapsulated in fibronectin-alginate microcapsules in basal or osteogenic cultures may be of practical use in the treatment of hemophilia B.

  10. Targeting of the human coagulation factor IX gene at rDNA locus of human embryonic stem cells.

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    Xionghao Liu

    Full Text Available BACKGROUND: Genetic modification is a prerequisite to realizing the full potential of human embryonic stem cells (hESCs in human genetic research and regenerative medicine. Unfortunately, the random integration methods that have been the primary techniques used keep creating problems, and the primary alternative method, gene targeting, has been effective in manipulating mouse embryonic stem cells (mESCs but poorly in hESCs. METHODOLOGY/PRINCIPAL FINDINGS: Human ribosomal DNA (rDNA repeats are clustered on the short arm of acrocentric chromosomes. They consist of approximately 400 copies of the 45S pre-RNA (rRNA gene per haploid. In the present study, we targeted a physiological gene, human coagulation factor IX, into the rDNA locus of hESCs via homologous recombination. The relative gene targeting efficiency (>50% and homologous recombination frequency (>10(-5 were more than 10-fold higher than those of loci targeted in previous reports. Meanwhile, the targeted clones retained both a normal karyotype and the main characteristics of ES cells. The transgene was found to be stably and ectopically expressed in targeted hESCs. CONCLUSION/SIGNIFICANCE: This is the first targeting of a human physiological gene at a defined locus on the hESC genome. Our findings indicate that the rDNA locus may serve as an ideal harbor for transgenes in hESCs.

  11. Variations among Japanese of the factor IX gene (F9) detected by PCR-denaturing gradient gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Satoh, Chiyoko; Takahashi, Norio; Asakawa, Junichi; Hiyama, Keiko; Kodaira, Meiko (Radiation Effects Research Foundation, Hiroshima (Japan))

    1993-01-01

    In the course of feasibility studies to examine the efficiencies and practicalities of various techniques for screening for genetic variations, the human coagulation factor IX (F9) genes of 63 Japanese families were examined by PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Four target sequences with lengths of 983-2,891 bp from the F9 genes of 126 unrelated individuals from Hiroshima and their 100 children were amplified by PCR, digested with restriction enzymes to approximately 500-bp fragments, and examined by DGGE - a total of 6,724 bp being examined per individual. GC-rich sequences (GC-clamps) of 40 bp were attached to both ends of the target sequences, as far as was feasible. Eleven types of new nucleotide substitutions were detected in the population, none of which produced RFLPs or caused hemophilia B. By examining two target sequences in a single lane, approximately 8,000 bp in a diploid individual could be examined. This approach is very effective for the detection of variations in DNA and is applicable to large-scale population studies. 46 refs., 3 figs., 1 tab.

  12. Identification and Genetic Analysis of a Factor IX Gene Intron 3 Mutation in a Hemophilia B Pedigree in China

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    Dong Hua Cao

    2014-09-01

    Full Text Available OBJECTIVE: Hemophilia B is caused by coagulation defects in the factor IX gene located in Xq27.1 on the X chromosome. A wide range of mutations, showing extensive molecular heterogeneity, have been described in hemophilia B patients. Our study was aimed at genetic analysis and prenatal diagnosis of hemophilia B in order to further elucidate the pathogenesis of the hemophilia B pedigree in China. METHODS: Polymerase chain reaction amplification and direct sequencing of all the coding regions was conducted in hemophilia B patients and carriers. Prenatal diagnosis of the proband was conducted at 20 weeks. RESULTS: We identified the novel point mutation 10.389 A>G, located upstream of the intron 3 acceptor site in hemophilia B patients. The fetus of the proband’s cousin was identified as a carrier. CONCLUSION: Our identification of a novel mutation in the F9 gene associated with hemophilia B provides novel insight into the pathogenesis of this genetically inherited disorder and also represents the basis of prenatal diagnosis.

  13. [Construction of nonsense-mutated eukaryotic expression vector of factor IX gene and its expression in COS-7 cells].

    Science.gov (United States)

    Nie, Xin; Yang, Lin-Hua; Chai, Bao-Feng; Shen, Quan; Zhang, Yuan; Zhang, Yao-Fang; Chen, Jian-Fang

    2010-06-01

    The purpose of this study was to construct 4 types of nonsense-mutated eukaryotic expression plasmids of fIX gene, using pcDNA3.1 plasmid containing fIX cDNA as template, and to identify, then to perform their expression in COS-7 cells. These stop mutants constructed by site-directed mutagenesis based on PCR, and further confirmed by DNA sequencing. COS-7 cells were transfected with either the wild-type or mutated fIX expression constructs, then the relative expression levels of fIX mRNA were detected by real time fluorescent quantitative PCR. The result showed that except the designed sites, there were no other nucleotide mutation in the sequences of four nonsense mutants. The results of real time PCR proved that the nonsense-mutated vectors can be effectively expressed in COS-7 cells. It is concluded that the nonsense-mutated eukaryotic expression vectors of fIX gene have been successfully constructed and can express in COS-7 cells, which provides the material basis for further researches on mechanism and treatment of FIX deficiency and the function defects caused by nonsense mutation.

  14. Validation of the manufacturing process used to produce long-acting recombinant factor IX Fc fusion protein.

    Science.gov (United States)

    McCue, J; Osborne, D; Dumont, J; Peters, R; Mei, B; Pierce, G F; Kobayashi, K; Euwart, D

    2014-07-01

    Recombinant factor IX Fc (rFIXFc) fusion protein is the first of a new class of bioengineered long-acting factors approved for the treatment and prevention of bleeding episodes in haemophilia B. The aim of this work was to describe the manufacturing process for rFIXFc, to assess product quality and to evaluate the capacity of the process to remove impurities and viruses. This manufacturing process utilized a transferable and scalable platform approach established for therapeutic antibody manufacturing and adapted for production of the rFIXFc molecule. rFIXFc was produced using a process free of human- and animal-derived raw materials and a host cell line derived from human embryonic kidney (HEK) 293H cells. The process employed multi-step purification and viral clearance processing, including use of a protein A affinity capture chromatography step, which binds to the Fc portion of the rFIXFc molecule with high affinity and specificity, and a 15 nm pore size virus removal nanofilter. Process validation studies were performed to evaluate identity, purity, activity and safety. The manufacturing process produced rFIXFc with consistent product quality and high purity. Impurity clearance validation studies demonstrated robust and reproducible removal of process-related impurities and adventitious viruses. The rFIXFc manufacturing process produces a highly pure product, free of non-human glycan structures. Validation studies demonstrate that this product is produced with consistent quality and purity. In addition, the scalability and transferability of this process are key attributes to ensure consistent and continuous supply of rFIXFc.

  15. Sustained and therapeutic levels of human factor IX in hemophilia B mice implanted with microcapsules: key role of encapsulated cells.

    Science.gov (United States)

    Wen, Jianping; Vargas, Andrew Gómez; Ofosu, Frederick A; Hortelano, Gonzalo

    2006-03-01

    A gene therapy delivery system based on microcapsules enclosing recombinant cells engineered to secrete a therapeutic protein was explored in this study. In order to prevent immune rejection of the delivered cells, they were enclosed in non-antigenic biocompatible alginate microcapsules prior to being implanted intraperitoneally into mice. We have shown that encapsulated C2C12 myoblasts can temporarily deliver therapeutic levels of factor IX (FIX) in mice, but the C2C12 myoblasts elicited an immune response to FIX. In this study we report the use of mouse fetal G8 myoblasts secreting hFIX in hemophilia mice. Mouse G8 myoblasts were transduced with MFG-FIX vector. A pool of recombinant G8 myoblasts secreting approximately 1500 ng hFIX/10(6) cells/24 h in vitro were enclosed in biocompatible alginate microcapsules and implanted intraperitoneally into immunocompetent C57BL/6 and hemophilic mice. Circulating levels of hFIX in treated mice reached approximately 400 ng/ml for at least 120 days (end of experiment). Interestingly, mice treated with encapsulated G8 myoblasts did not develop anti-hFIX antibodies. Activated partial thromboplastin time (APTT) of plasmas obtained from treated hemophilic mice was reduced from 107 to 82 sec on day 60 post-treatment, and whole blood clotting time (WBCT) was also corrected from 7-9 min before treatment to 3-5 min following microcapsule implantation. Further, mice were protected against bleeding following major trauma. Thus, the FIX delivery in vivo was biologically active. Our findings suggest that the type of cells encapsulated play a key role in the generation of immune responses against the transgene. Further, a judicious selection of encapsulated cells is critical for achieving sustained gene expression. Our findings support the feasibility of encapsulated G8 myoblasts as a gene therapy approach for hemophilia B.

  16. Preliminary study on non-viral transfection of F9 (factor IX gene by nucleofection in human adipose-derived mesenchymal stem cells

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    Susana Olmedillas López

    2016-04-01

    Full Text Available Background. Hemophilia is a rare recessive X-linked disease characterized by a deficiency of coagulation factor VIII or factor IX. Its current treatment is merely palliative. Advanced therapies are likely to become the treatment of choice for the disease as they could provide a curative treatment. Methods. The present study looks into the use of a safe non-viral transfection method based on nucleofection to express and secrete human clotting factor IX (hFIX where human adipose tissue derived mesenchymal stem cells were used as target cells in vitro studies and NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice were used to analyze factor IX expression in vivo studies. Previously, acute liver injury was induced by an injected intraperitoneal dose of 500 mg/kg body weight of acetaminophen. Results. Nucleofection showed a percentage of positive cells ranging between 30.7% and 41.9% and a cell viability rate of 29.8%, and cells were shown to secrete amounts of hFIX between 36.8 and 71.9 ng/mL. hFIX levels in the blood of NSG mice injected with ASCs transfected with this vector, were 2.7 ng/mL 48 h after injection. Expression and secretion of hFIX were achieved both in vitro cell culture media and in vivo in the plasma of mice treated with the transfected ASCs. Such cells are capable of eventually migrating to a previously damaged target tissue (the liver where they secrete hFIX, releasing it to the bloodstream over a period of at least five days from administration. Conclusions. The results obtained in the present study may form a preliminary basis for the establishment of a future ex vivo non-viral gene/cellular safe therapy protocol that may eventually contribute to advancing the treatment of hemophilia.

  17. Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

    Institute of Scientific and Technical Information of China (English)

    陆华中; 陈立; 王红卫; 伍志坚; 吴小兵; 王学峰; 王鸿利; 卢大儒; 邱信芳; 薛京伦

    2001-01-01

    A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ- ated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%± 5.23%, which is remarkably higher than that of (14.27% ± 3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors development in the treated mice; and (iv) no local or systemic side-effects and toxicity associated with the gene transfer were found. It demonstrated the potential use of treating hemophilia B by recombinant adeno-associated viral vectors with mutant hFIXR338A gene, an alternative strategy for hemophilia B gene therapy to wild-type human factor IX.

  18. A computer-based model to assess costs associated with the use of factor VIII and factor IX one-stage and chromogenic activity assays.

    Science.gov (United States)

    Kitchen, S; Blakemore, J; Friedman, K D; Hart, D P; Ko, R H; Perry, D; Platton, S; Tan-Castillo, D; Young, G; Luddington, R J

    2016-04-01

    Measurement of coagulation factor factor VIII (FVIII) and factor IX (FIX) activity can be associated with a high level of variability using one-stage assays based on activated partial thromboplastin time (APTT). Chromogenic assays show less variability, but are less commonly used in clinical laboratories. In addition, one-stage assay accuracy using certain reagent and instrument combinations is compromised by some modified recombinant factor concentrates. Reluctance among some in the hematology laboratory community to adopt the use of chromogenic assays may be partly attributable to lack of familiarity and perceived higher associated costs. To identify and characterize key cost parameters associated with one-stage APTT and chromogenic assays for FVIII and FIX activity using a computer-based cost analysis model. A cost model for FVIII and FIX chromogenic assays relative to APTT assays was generated using assumptions derived from interviews with hematologists and laboratory scientists, common clinical laboratory practise, manufacturer list prices and assay kit configurations. Key factors that contribute to costs are factor-deficient plasma and kit reagents for one-stage and chromogenic assays, respectively. The stability of chromogenic assay kit reagents also limits the cost efficiency compared with APTT testing. Costs for chromogenic assays might be reduced by 50-75% using batch testing, aliquoting and freezing of kit reagents. Both batch testing and aliquoting of chromogenic kit reagents might improve cost efficiency for FVIII and FIX chromogenic assays, but would require validation. Laboratory validation and regulatory approval as well as education and training in the use of chromogenic assays might facilitate wider adoption by clinical laboratories. © 2016 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

  19. Evolutionary pattern of mutation in the factor IX genes of great apes: How does it compare to the pattern of recent germline mutation in patients with hemophilia B?

    Energy Technology Data Exchange (ETDEWEB)

    Grouse, L.H.; Ketterling, R.P.; Sommer, S.S. [Mayo Clinic/Foundation, Rochester, MN (United States)

    1994-09-01

    Most mutations causing hemophilia B have arisen within the past 150 years. By correcting for multiple biases, the underlying rates of spontaneous germline mutation have been estimated in the factor IX gene. From these rates, an underlying pattern of mutation has emerged. To determine if this pattern compares to a underlying pattern found in the great apes, sequence changes were determined in intronic regions of the factor IX gene. The following species were studied: Gorilla gorilla, Pan troglodytes (chimpanzee), Pongo pygmacus (orangutan) and Homo sapiens. Intronic sequences at least 200 bp from a splice junction were randomly chosen, amplified by cross-species PCR, and sequenced. These regions are expected to be subject to little if any selective pressure. Early diverged species of Old World monkeys were also studied to help determine the direction of mutational changes. A total of 62 sequence changes were observed. Initial data suggest that the average pattern since evolution of the great apes has a paucity of transitions at CpG dinucleotides and an excess of microinsertions to microdeletions when compared to the pattern observed in humans during the past 150 years (p<.05). A larger study is in progress to confirm these results.

  20. Hybrid retroviral vector with MCK enhancers inserted in LTR for stable and specific expression of human factor IX in skeletal muscle

    Institute of Scientific and Technical Information of China (English)

    WANG Jian-min 王健民; HOU Jun 侯军; QIU Xin-fang 邱信芳; Kurachi Kotoku; XUE Jing-lun 薛京伦

    2004-01-01

    Background Retroviral vectors have been widely used to introduce foreign into various target cells in vitro, thus showing relatively high systemic delivery efficiency of various transgene products. The authors investigated the stability and efficiency of skeletal muscle-specific hybrid retroviral vectors in expression of human factor IX (FIX) in vitro and iv vivo. Methods FIX cDNA in LIXSN vector was replaced with a FIX minigene containing splicing donor and splicing acceptor sequence of first intron of human FIX gene. Two copies of muscle creatine kinase enhancer (MCK, Me2) were inserted in forward or reverse orientation at NheI site of 3' long terminal repeat (LTR), resulting in two hybrid vectors, which were designated as LMe2IXm2SN(F) and LMe2IXm2SN(R), respectively. The vectors were tested in vitro and in vivo for stability and muscle-specificity of factor IX expression with SCID mice. Results Muscle cells carrying vector with Me2 expressed significantly higher levels of FIX (up to 1800 ng/106.24h) than those without Me2, thus suggesting that Me2 could specifically increase expression level of FIX in muscle cells. Myoblasts transduced with LMe2IXm2SN(R) produced much less FIX in vivo in SCID mice than LMe2IXm2SN(F). One or two copies of Me2 sequence were deleted in myoblasts transduced with LMe2IXm2SN(R) without changing the orientation of Me2. Conclusions LTR inserted with MCK enhancers can specifically increase human FIX expression in skeletal muscle cells in vitro and in vivo, and MCK enhancer should be positioned in the same orientation as that of LTR promoter.

  1. Crystallization and preliminary X-ray analysis of coagulation factor IX-binding protein from habu snake venom at pH 6.5 and 4.6

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    Suzuki, Nobuhiro [Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572 (Japan); Department of Biochemisty, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602 (Japan); Shikamoto, Yasuo; Fujimoto, Zui [Department of Biochemisty, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602 (Japan); Morita, Takashi [Department of Biochemistry, Meiji Pharmaceutical University, Kiyose, Tokyo 204-8588 (Japan); Mizuno, Hiroshi, E-mail: mizuno@affrc.go.jp [Department of Biochemisty, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602 (Japan); Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572 (Japan)

    2005-01-01

    Crystals of habu coagulation factor IX-binding protein have been obtained at pH 6.5 and 4.6 and characterized by X-ray diffraction. Coagulation factor IX-binding protein isolated from Trimeresurus flavoviridis (IX-bp) is a C-type lectin-like protein. It is an anticoagulant protein consisting of homologous subunits A and B. The subunits both contain a Ca{sup 2+}-binding site with differing affinity (K{sub d} values of 14 and 130 µM at pH 7.5). These binding characteristics are pH-dependent; under acidic conditions, the affinity of the low-affinity site was reduced considerably. In order to identify which site has high affinity and also to investigate the Ca{sup 2+}-releasing mechanism, IX-bp was crystallized at pH 6.5 and 4.6. The crystals at pH 6.5 and 4.6 diffracted to 1.72 and 2.29 Å resolution, respectively; the former crystals belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 60.7, b = 63.5, c = 66.9 Å, β = 117.0°, while the latter belong to the monoclinic space group C2, with a = 134.1, b = 37.8, c = 55.8 Å, β = 110.4°.

  2. Preparation of a recombinant adeno-associated viral vector with a mutation of human factor IX in large scale and its expression in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A series of adeno-associated viral vectors conraining a mutation of human factor IX (hFIXR338A) with different regulation elements were constructed and used to transduce cell lines. The plasmids and the stable transduction cell clones with high expression level of hFIXR338Awere obtained by selecting and optimizing, and then, the recombinant adeno-associated viral vector with hFIXR338Awas prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for producing rAAV-hFIXR338A viral stocks on a large scale and higher fiter was established,which can be used for industrial purpose. The titer of rAAV-hFIXR338A was more than 1.25x1012 particle/mL, and then, a mammalian cell line, C2C12 and the factor IXknock-out mice were transfected with the rAAV-hFIXR338Ain vitro and in vivo. The results show that the high-level expression of rAAV-hFIXR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32±92.14) ng@ (106cells)-1 @ (24 h)-1 in C2C12 cell in vitro and had a peak concentration of 463.28 ng/mL in mice treated with rAAV-hFIX R338A, which was as high as the expression of rAAV-hFIX -wt (2565.76±64.36) ng@ (106 cells)-1@ (24 h)-1 in C2C12 and 453.92 ng/mL in the mice treated with rAAV-hFIX-wt) in vitro and in vivo, there is no any difference between two groups, but the clotting activity of hFIXR338A is about 2.46times higher than that of hFIX-wt. It was first reported that a mutation of human factor IX was used into gene therapy research for hemophilia B, meanwhile, a novel packaging system, rAAV/HSV was used for preparation of rAAV-hFIX R338A on a large scale, which laid the foundation of industrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated with rAAV-hFIX.``

  3. The Role of Hypoxia-Inducible Factor-1α, Glucose Transporter-1, (GLUT-1 and Carbon Anhydrase IX in Endometrial Cancer Patients

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    Pawel Sadlecki

    2014-01-01

    Full Text Available Hypoxia-inducible factor-1α (HIF-1α, glucose transporter-1 (GLUT-1, and carbon anhydrase IX (CAIX are important molecules that allow adaptation to hypoxic environments. The aim of our study was to investigate the correlation between HIF-1α, GLUT-1, and CAIX protein level with the clinicopathological features of endometrial cancer patients. Materials and Methods. 92 endometrial cancer patients, aged 37–84, were enrolled to our study. In all patients clinical stage, histologic grade, myometrial invasion, lymph node, and distant metastases were determined. Moreover, the survival time was assessed. Immunohistochemical analyses were performed on archive formalin fixed paraffin embedded tissue sections. Results. High significant differences (P=0.0115 were reported between HIF-1α expression and the histologic subtype of cancer. Higher HIF-1α expression was associated with the higher risk of recurrence (P=0.0434. The results of GLUT-1 and CAIX expression did not reveal any significant differences between the proteins expression in the primary tumor and the clinicopathological features. Conclusion. The important role of HIF-1α in the group of patients with the high risk of recurrence and the negative histologic subtype of the tumor suggest that the expression of this factor might be useful in the panel of accessory pathomorphological tests and could be helpful in establishing more accurate prognosis in endometrial cancer patients.

  4. Cell-matrix Interactions of Factor IX (FIX)-engineered human mesenchymal stromal cells encapsulated in RGD-alginate vs. fibrinogen-alginate microcapsules.

    Science.gov (United States)

    Sayyar, Bahareh; Dodd, Megan; Marquez-Curtis, Leah; Janowska-Wieczorek, Anna; Hortelano, Gonzalo

    2014-04-01

    The success of cell microencapsulation technology in tissue engineering and protein delivery applications depends on the viability and functionality of the encapsulated cells, which in turn are dependent upon cell/matrix interactions. In this work, we compared the viability of cord blood-derived mesenchymal stromal cells (CB MSCs), engineered to secrete factor IX (FIX) for hemophilia treatment, and encapsulated in arginine-glycine-aspartate (RGD)-alginate versus fibrinogen-alginate microcapsules. We evaluated the effect of the biomimetic matrix on cell attachment, proliferation, and secretion of FIX. Compared with nonsupplemented alginate matrix, RGD-alginate significantly enhanced the viability of the encapsulated MSCs. Further, cells in RGD-alginate displayed distinct attachment morphology, thus suggesting that RGD-alginate can potentially be used for the encapsulation of MSCs in tissue engineering applications that require enhanced cell attachment and viability. However, our data also showed that RGD-alginate microcapsules, in contrast to fibrinogen-alginate microcapsules, did not significantly improve cell proliferation of or FIX secretion by encapsulated MSCs. Our findings suggest that evidence of cell attachment alone may not accurately predict the functionality of cells in biomimetic microcapsules.

  5. Efficient detection of factor IX mutations by denaturing high-performance liquid chromatography in Taiwanese hemophilia B patients, and the identification of two novel mutations

    Directory of Open Access Journals (Sweden)

    Pei-Chin Lin

    2014-04-01

    Full Text Available Hemophilia B (HB is an X-linked recessive disorder characterized by mutations in the clotting factor IX (FIX gene that result in FIX deficiency. Previous studies have shown a wide variation of FIX gene mutations in HB. Although the quality of life in HB has greatly improved mainly because of prophylactic replacement therapy with FIX concentrates, there exists a significant burden on affected families and the medical care system. Accurate detection of FIX gene mutations is critical for genetic counseling and disease prevention in HB. In this study, we used denaturing high-performance liquid chromatography (DHPLC, which has proved to be a highly informative and practical means of detecting mutations, for the molecular diagnosis of our patients with HB. Ten Taiwanese families affected by HB were enrolled. We used the DHPLC technique followed by direct sequencing of suspected segments to detect FIX gene mutations. In all, 11 FIX gene mutations (8 point mutations, 2 small deletions/insertions, and 1 large deletion, including two novel mutations (exon6 c.687–695, del 9 mer and c.460–461, ins T were found. According to the HB pedigrees, 25% and 75% of our patients were defined as familial and sporadic HB cases, respectively. We show that DHPLC is a highly sensitive and cost-effective method for FIX gene analysis and can be used as a convenient system for disease prevention.

  6. The rates of G:C[yields]T:A and G:C[yields]C:G transversions at CpG dinucleotides in the human factor IX gene

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    Ketterling, R.P.; Vielhaber, E.; Sommer, S.S. (Mayo Clinic/Foundation, Rochester, MN (United States))

    1994-05-01

    The authors have identified eight independent transversions at CpG in 290 consecutive families with hemophilia B. These eight transversions account for 16.3% of all independent transversions in the sample, yet the expected frequency of CpG transversions at random in the factor IX gene is only 2.6% (P<0.1). The aggregate data suggest that the two types of CpG transversions (G:C[yields]T:A and G:C[yields]C:G) possess similar mutation rates (24.8 [times] 10[sup [minus]10] and 20.6 [times] 10[sup [minus]10], respectively), which are about fivefold greater than the comparable rates for transversions at non-CpG dinucleotides. The enhancement of transversions at CpG suggest that the model by which mutations occur at CpG may need to be reevaluated. The relationship, if any, between deamination of 5-methyl cytosine and enhancement of transversions at CpG remains to be defined. 28 refs., 2 tabs.

  7. Isorhamnetin Inhibits Reactive Oxygen Species-Dependent Hypoxia Inducible Factor (HIF)-1α Accumulation.

    Science.gov (United States)

    Seo, Suho; Seo, Kyuhwa; Ki, Sung Hwan; Shin, Sang Mi

    2016-01-01

    Isorhamnetin is a flavonoid metabolite of quercetin and isolated from water dropwort (Oenanthe javanica, Umbelliferae). It has been reported that isorhamnetin exerts beneficial effects including antioxidant, anti-inflammatory, and anti-proliferative activities. The present study investigated whether the antioxidant activity of isorhamnetin is correlated with its anti-cancer effects on colorectal cancer cells. Isorhamnetin significantly repressed cobalt chloride (CoCl2)- or hypoxia-induced hypoxia inducible factor-1α (HIF-1α) accumulation in HCT116 and HT29 cells. When compared with quercetin, isorhamnetin showed potent inhibition of HIF-1α. Moreover, it inhibited CoCl2-induced activity of hypoxia response element reporter gene and HIF-1α-dependent transcription of genes such as glucose transporter 1, lactate dehydrogenase A, carbonic anhydrase-IX, and pyruvate dehydrogenase kinase 1. Isorhamnetin also blocked hydrogen peroxide (H2O2)-induced HIF-1α accumulation. The antioxidant effects of isorhamnetin were confirmed by observation of CoCl2- or H2O2-induced reactive oxygen species (ROS) production. Consistently, overexpressed HIF-1α was decreased by isorhamnetin or N-acetyl-L-cysteine in HEK293 cells. In vitro migration and invasion assay further confirmed the inhibitory effects of isorhamnetin on cancer cells. Collectively, these results demonstrate that isorhamnetin inhibits ROS-mediated HIF-1α accumulation, which contributes to its anti-metastatic efficacy.

  8. Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

    Institute of Scientific and Technical Information of China (English)

    LU; Huazhong; (

    2001-01-01

    [1]Chang, J., Jin, J., Lollar, P. et al., Changing residue 338 in human factor IX from arginine to alanine causes an increase in catalytic activity, J. Bio. Chem., 1998, 273 (20): 12089-12094.[2]Lai, L., Chen, L., Zhou, H. et al., Clinical phenotype and genetic stability of factor IX gene knock out mice, J. Fudan Uni., 1999, 38 (4): 435-438.[3]Wu, Z. J., Wu, X. B., Hou, Y. D., Generation of a recombinant herps simplex virus which can provide packaging function for recombinant adeno-associated virus, Chinese Sci. Bull., 1999, 44 (8): 715-719.[4]Snyder, R. O., Miao, C. H., Patijn, G. A. et al., Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors, Nat. Genet., 1997, 16 (3): 270-276.[5]Lai, L. H., Chen, L., Wang, J. M. et al., Skeletal muscle-specific expression of human blood coagulation factor IX rescues factor IX deficiency mouse by AAV-mediated gene transfer, Science in China, Ser. C, 1999, 42 (6): 628-634.[6]Snyder, R. O., Miao, C., Meuse, L. et al., Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors, Nat. Med., 1999, 5 (1): 64-70.[7]Kung, S. H., Hagstrom, J. N., Cass, D. et al., Human factor IX corrects the bleeding diathesis of mice with hemophilia B, Blood, 1998, 91(3): 784-790.[8]Hirt, B., Selective extraction of polyoma DNA from infected mouse cell culture, J. Mol. Biol., 1967, 26: 365-369.[9]Sambrook, J., Fritsch, E., Maniatis, T., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989, 6, 20-21.[10]Chao, H., Samulski, R. J., Bellinger, D. A. et al., Persistent expression of canine factor IX in hemophilia B canines, Gene Ther., 1999, 6: 1695-1704.[11]Kaufman, R. J., Advances toward gene therapy for hemophilia at the millennium, Hum. Gene Ther., 1999, 10 (13): 2091-2107.[12]Lu, D. R., Zhou, J. M., Zheng, B. et al., Stage I clinical trial of gene

  9. Multicentre, randomized, open-label study of on-demand treatment with two prophylaxis regimens of recombinant coagulation factor IX in haemophilia B subjects.

    Science.gov (United States)

    Valentino, L A; Rusen, L; Elezovic, I; Smith, L M; Korth-Bradley, J M; Rendo, P

    2014-05-01

    Few randomized studies have reported on the use of factor IX (FIX) for secondary prophylaxis in haemophilia B patients. This study aimed to evaluate the efficacy and safety of two secondary prophylaxis regimens of recombinant coagulation FIX, nonacog alfa, compared with on-demand therapy. Male subjects aged 6-65 years with severe or moderately severe haemophilia B (FIX:C ≤ 2, n = 50) and ≥12 bleeding episodes (including ≥6 haemarthroses episodes) within 12 months of study participation were enrolled in this multicentre, randomized, open-label, four-period crossover trial. The primary measure was the annualized bleeding rate (ABR) of two prophylactic regimens vs. on-demand therapy. In the intent-to-treat group, mean ABR values were 35.1, 2.6 and 4.6 for the first on-demand period, the 50 IU kg(-1) twice-weekly period, and the 100 IU kg(-1) once-weekly period respectively. Differences in ABR between the first on-demand period and both prophylaxis regimens were significant (P < 0.0001); no significant differences were observed between prophylaxis regimens (P = 0.22). Seven serious adverse events occurred in five subjects, none related to study drug. Results demonstrated that secondary prophylaxis therapy with nonacog alfa 50 IU kg(-1) twice weekly or 100 IU kg(-1) once weekly reduced ABR by 89.4% relative to on-demand treatment. Both prophylaxis regimens demonstrated favourable safety profiles in subjects with haemophilia B.

  10. An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants

    Directory of Open Access Journals (Sweden)

    Dario Balestra

    2016-01-01

    Full Text Available In cellular models we have demonstrated that a unique U1snRNA targeting an intronic region downstream of a defective exon (Exon-specific U1snRNA, ExSpeU1 can rescue multiple exon-skipping mutations, a relevant cause of genetic disease. Here, we explored in mice the ExSpeU1 U1fix9 toward two model Hemophilia B-causing mutations at the 5′ (c.519A > G or 3′ (c.392-8T > G splice sites of F9 exon 5. Hydrodynamic injection of wt-BALB/C mice with plasmids expressing the wt and mutant (hFIX-2G5′ss and hFIX-8G3′ss splicing-competent human factor IX (hFIX cassettes resulted in the expression of hFIX transcripts lacking exon 5 in liver, and in low plasma levels of inactive hFIX. Coinjection of U1fix9, but not of U1wt, restored exon inclusion of variants and in the intrinsically weak FIXwt context. This resulted in appreciable circulating hFIX levels (mean ± SD; hFIX-2G5′ss, 1.0 ± 0.5 µg/ml; hFIX-8G3′ss, 1.2 ± 0.3 µg/ml; and hFIXwt, 1.9 ± 0.6 µg/ml, leading to a striking shortening (from ≃100 seconds of untreated mice to ≃80 seconds of FIX-dependent coagulation times, indicating a hFIX with normal specific activity. This is the first proof-of-concept in vivo that a unique ExSpeU1 can efficiently rescue gene expression impaired by distinct exon-skipping variants, which extends the applicability of ExSpeU1s to panels of mutations and thus cohort of patients.

  11. Beyond Behavioral Inhibition: Etiological Factors in Childhood Anxiety

    Science.gov (United States)

    Manassis, Katharina; Hudson, Jennifer L.; Webb, Alicia; Albano, Anne Marie

    2004-01-01

    Theoretical models of childhood anxiety have emphasized temperamental vulnerability, principally behavioral inhibition, and its interaction with various environmental factors promoting anxiety (for example, overprotective parenting, insecure attachment, life stress). Although clearly establishing the importance of both nature and nurture in…

  12. Saccharin: a lead compound for structure-based drug design of carbonic anhydrase IX inhibitors.

    Science.gov (United States)

    Mahon, Brian P; Hendon, Alex M; Driscoll, Jenna M; Rankin, Gregory M; Poulsen, Sally-Ann; Supuran, Claudiu T; McKenna, Robert

    2015-02-15

    Carbonic anhydrase IX (CA IX) is a key modulator of aggressive tumor behavior and a prognostic marker and target for several cancers. Saccharin (SAC) based compounds may provide an avenue to overcome CA isoform specificity, as they display both nanomolar affinity and preferential binding, for CA IX compared to CA II (>50-fold for SAC and >1000-fold when SAC is conjugated to a carbohydrate moiety). The X-ray crystal structures of SAC and a SAC-carbohydrate conjugate bound to a CA IX-mimic are presented and compared to CA II. The structures provide substantial new insight into the mechanism of SAC selective CA isoform inhibition.

  13. Oxygen Availability for Porphyrin Biosynthesis Enzymes Determines the Production of Protoporphyrin IX (PpIX during Hypoxia.

    Directory of Open Access Journals (Sweden)

    Shimpei Otsuka

    Full Text Available 5-Aminolevulinic acid (ALA, a precursor of porphyrin, is specifically converted to the fluorescent substance protoporphyrin IX (PpIX in tumors to be used as a prodrug for photodynamic therapy and diagnosis. Hypoxia, a common feature of solid tumors, decreases the efficacy of ALA-based photodynamic therapy and diagnosis. This decrease results from the excretion of porphyrin precursor coproporphyrinogen III (CPgenIII, an intermediate in the biosynthesis of PpIX. However, the mechanism of CPgenIII excretion during hypoxia remains unclear. In this study, we revealed the importance of mitochondrial respiration for the production of PpIX during hypoxia. Porphyrin concentrations were estimated in human gastric cancer cell lines by HPLC. Expression levels of porphyrin biosynthesis genes were measured by qRT-PCR and immunoblotting. Blockage of porphyrin biosynthesis was an oxygen-dependent phenomenon resulting from decreased PpIX production in mitochondria under hypoxic conditions. PpIX production was increased by the inhibition of mitochondrial respiration complexes, which indicates that the enzymes of porphyrin biosynthesis compete with respiration complexes for molecular oxygen. Our results indicate that targeting the respiration complexes is a rationale for enhancing the effect of ALA-mediated treatment and diagnosis.

  14. Physiological levels of blood coagulation factors IX and X control coagulation kinetics in an in vitro model of circulating tissue factor

    OpenAIRE

    Tormoen, Garth W.; Khader, Ayesha; Gruber, András; McCarty, Owen J. T.

    2013-01-01

    Thrombosis significantly contributes to cancer morbidity and mortality. The mechanism behind thrombosis in cancer may be circulating tissue factor (TF), as levels of circulating TF are associated with thrombosis. However, circulating TF antigen level alone has failed to predict thrombosis in patients with cancer. We hypothesize that coagulation factor levels regulate the kinetics of circulating TF-induced thrombosis. Coagulation kinetics were measured as a function of individual coagulation f...

  15. A common G10430A mutation (Gly 60 Ser) in the factor IX gene describes the presence of moderate and mild hemophilia B in the majority of the Gujarati population.

    Science.gov (United States)

    Quadros, Leera; Ghosh, Kanjaksha; Shetty, Shrimati

    2007-05-01

    Hemophilia B is an X-linked recessively inherited bleeding disorder afflicting humans across all socio-economic as well as racial groups. A wide range of mutations showing high heterogeneity has been reported in different populations. Thus, it has been difficult to adopt a cost-effective strategy for the genetic diagnosis of hemophilia B families. We report the presence of a common G10430A mutation in exon d of the factor IX gene, wherein the highly conserved Gly 60 residue of the first epidermal growth like domain was changed to Ser in 22 out of 22 moderately severe to mild hemophilia B patients originating from Gujarat. None of the eight Gujarati severe hemophilia B patients, 30 normal Gujarati men, and 20 moderately severe to mild hemophilia B patients belonging to other communities showed the presence of this mutation. This mutation occurred in the same haplotype background thereby suggesting a 'founder effect.' The direct detection of this G10430A mutation can be used for accurate carrier detection and prenatal diagnosis in mild to moderate factor-IX-deficient patients belonging to the Gujarat state of western India.

  16. Therapeutic approaches for tumor necrosis factor inhibition

    Directory of Open Access Journals (Sweden)

    Maria Letícia de Castro Barbosa

    2011-09-01

    Full Text Available Tumor necrosis factor (TNF consists of an inflammatory cytokine essential for homeostasis and organism defense. Despite its physiological relevance, both increased biosynthesis and release of TNF lead to the exacerbation of inflammatory and oxidative responses, which are related to the pathogenesis of a host of diseases of an inflammatory, autoimmune and/or infectious nature. In this context, effective therapeutic approaches for the modulation of TNF have been the focus of research efforts. Approximately one million individuals worldwide have been treated with biotechnological inhibitors of this cytokine, the so-called anti-TNF biopharmaceuticals. However, given the high risk of infection and the limitations related to cost and administration routes, new therapeutic approaches aimed at biological targets that directly or indirectly modulate the production and/or activation of TNF appear promising alternatives for the discovery of new anti-inflammatory and immunomodulatory orally active drugs and are therefore discussed in this paper.O fator de necrose tumoral (do inglês, tumor necrosis factor - TNF consiste em uma citocina inflamatória essencial para a homeostase e defesa do organismo. A despeito de sua relevância fisiológica, o aumento da biossíntese e liberação do TNF conduzem à exacerbação das respostas inflamatória e oxidativa, as quais estão relacionadas à patogênese de várias doenças de natureza inflamatória, auto-imune e/ou infecciosa. A busca por abordagens terapêuticas eficientes na modulação do TNF tem sido alvo de diversos esforços de pesquisa. Aproximadamente um milhão de pessoas ao redor do mundo já foi tratado com inibidores biotecnológicos desta citocina, os chamados biofármacos anti-TNF. Entretanto, em face ao elevado risco de infecções e as limitações relacionadas ao custo e a via de administração, novas abordagens terapêuticas com foco em alvos que modulem, de forma direta ou indireta, a produ

  17. The spectroscopy analyses of PpIX by ultrasound irradiation and its sonotoxicity in vitro.

    Science.gov (United States)

    Wang, Pan; Wang, Xiaobing; Zhang, Kun; Gao, Kaili; Song, Ming; Liu, Quanhong

    2013-07-01

    play an important role in the enhanced cell killing of PpIX-SDT. SDT induced obvious ROS generation in S180 cells, which could be mostly inhibited by the general ROS scavenge NAC (N-acetylcysteine). Other scavengers such as NaN3, histidine, mannitol all partially prevented the SDT induced cell viability loss of S180 cells, suggesting OH, (1)O2 might be involved during the process.

  18. Breakpoint of a balanced translocation (X:14) (q27.1;q32.3) in a girl with severe hemophilia B maps proximal to the factor IX gene.

    Science.gov (United States)

    Di Paola, J; Goldman, T; Qian, Q; Patil, S R; Schutte, B C; Schute, B C

    2004-03-01

    Hemophilia B is an X-linked bleeding disorder caused by the deficiency of coagulation factor (F)IX, with an estimated prevalence of 1 in 30 000 male births. It is almost exclusively seen in males with rare exceptions. We report a girl who was diagnosed with severe (PAC DNA probe, RP6-88D7 (which contains the FIX gene) hybridized only on the normal chromosome X as well as onto the derivative 14. Using a PAC DNA probe, RP11-963P9 that is located proximal to the FIX gene, we obtained signals on the normal and derivative X and also on the derivative 14. We conclude that the breakpoint is located within the DNA sequence of this clone mapping proximal to the FIX gene. Since the FIX gene seems to be intact in the derivative 14, the breakpoint may affect an upstream regulatory sequence that subjects the gene to position effect variegation (PEV).

  19. Epidermal growth factor inhibits cysteamine-induced duodenal ulcers

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier

    1983-01-01

    The effect of the duodenal ulcerogen cysteamine on secretion of epidermal growth factor from Brunner's gland pouches was studied in the rat. Total output of immunoreactive epidermal growth factor was reduced to approximately 55%, compared with controls, 5 h after administration of cysteamine (300...... mg/kg, s.c.). Furthermore, measurements on tissue extracts of the pouches revealed that 5 h after cysteamine treatment, Brunner's glands were depleted of epidermal growth factor. The effect on ulcer development of intraduodenally applied exogenous epidermal growth factor (1 micrograms/kg . h) also...... factor used, when tested on chronic fistula rats, had no effect on acid secretion and did not influence bicarbonate secretion from Brunner's gland pouches. These results demonstrate that epidermal growth factor has a cytoprotective effect on the duodenal mucosa, and it is suggested that inhibition...

  20. Competitive inhibition of transcription factors by small interfering peptides.

    Science.gov (United States)

    Seo, Pil Joon; Hong, Shin-Young; Kim, Sang-Gyu; Park, Chung-Mo

    2011-10-01

    Combinatorial assortment by dynamic dimer formation diversifies gene transcriptional specificities of transcription factors. A similar but biochemically distinct mechanism is competitive inhibition in which small proteins act as negative regulators by competitively forming nonfunctional heterodimers with specific transcription factors. The most extensively studied is the negative regulation of auxin response factors by AUXIN/INDOLE-3-ACETIC ACID repressors. Similarly, Arabidopsis thaliana (Arabidopsis) little zipper and mini finger proteins act as competitive inhibitors of target transcription factors. Competitive inhibitors are also generated by alternative splicing and controlled proteolytic processing. Because they provide a way of attenuating transcription factors we propose to call them small interfering peptides (siPEPs). The siPEP-mediated strategy could be applied to deactivate specific transcription factors in crop plants.

  1. Inhibition of coagulation factors by recombinant barley serpin BSZx

    DEFF Research Database (Denmark)

    Dahl, Søren Weis; Rasmussen, S.K.; Petersen, L..C.;

    1996-01-01

    Barley serpin BSZx is a potent inhibitor of trypsin and chymotrypsin at overlapping reactive sites (Dahl, S.W., Rasmussen, S.K. and Hejgaard, J. (1996) J. Biol, Chem., in press), We have now investigated the interactions of BSZx with a range of serine proteinases from human plasma, pancreas...... as substrate, Complexes of these proteinase with BSZx resisted boiling in SDS, and amino acid sequencing showed that cleavage in the reactive center loop only occurred after P-1 Arg. Activated protein C and leukocyte elastase were slowly inhibited by BSZx (k(ass) = 1-2 x 10(2) M(-1) s(-1)) whereas factor XIIa......, urokinase and tissue type plasminogen activator, plasmin and pancreas kallikrein and elastase were not or only weakly affected, The inhibition pattern with mammalian proteinases reveal a specificity of BSZx similar to that of antithrombin III. Trypsin from Fusarium was not inhibited while interaction...

  2. Inhibition of placenta growth factor with TB-403

    DEFF Research Database (Denmark)

    Nielsen, Dorte Lisbet; Sengeløv, Lisa

    2012-01-01

    targeting angiogenesis. AREAS COVERED: The data are obtained by searching in the PubMed database. The search terms used included antiangiogenic therapy, TB-403 (RO5323441), placenta growth factor (PlGF) and VEGFR-1 (Flt-1). We review preclinical data concerning the function and inhibition of Pl......GF and summarize data on expression of PlGF in cancer patients. Data from early-phase clinical trials of TB-403 (RO5323441), a monoclonal antibody inhibiting PlGF, are discussed. Future development strategies, therapeutic potentials and limitations of TB-403 are further evaluated. EXPERT OPINION: There are some...... conflicting data on the function of PlGF and the importance of its role in primary tumor growth. Data from some preclinical models of PlGF inhibition and early-phase clinical trials with TB-403 are, however, promising, although the true potential of the drug is yet to be determined. Further clinical...

  3. Optimization of a novel peptide ligand targeting human carbonic anhydrase IX.

    Directory of Open Access Journals (Sweden)

    Shoaib Rana

    Full Text Available BACKGROUND: Carbonic anhydrase IX (CA IX is a hypoxia-regulated transmembrane protein over-expressed in various types of human cancer. Recently, a new peptide with affinity for human carbonic anhydrase IX (CaIX-P1 was identified using the phage display technology. Aim of the present study is to characterize the binding site in the sequence of CaIX-P1, in order to optimize the binding and metabolic properties and use it for targeting purposes. METHODOLOGY/PRINCIPAL FINDINGS: Various fragments of CaIX-P1 were synthesized on solid support using Fmoc chemistry. Alanine scanning was performed for identification of the amino acids crucial for target binding. Derivatives with increased binding affinity were radiolabeled and in vitro studies were carried out on the CA IX positive human renal cell carcinoma cell line SKRC 52 and the CA IX negative human pancreatic carcinoma cell line BxPC3. Metabolic stability was investigated in cell culture medium and human serum. Organ distribution and planar scintigraphy studies were performed in Balb/c nu/nu mice carrying subcutaneously transplanted SKRC 52 tumors. The results of our studies clearly identified amino acids that are important for target binding. Among various fragments and derivatives the ligand CaIX-P1-4-10 (NHVPLSPy was found to possess increased binding potential in SKRC 52 cells, whereas no binding capacity for BxPC3 cells was observed. Binding of radiolabeled CaIX-P1-4-10 on CA IX positive cells could be inhibited by both the unlabeled and the native CaIX-P1 peptide but not by control peptides. Stability experiments indicated the degradation site in the sequence of CaIX-P1-4-10. Biodistribution studies showed a higher in vivo accumulation in the tumor than in most healthy tissues. CONCLUSIONS: Our data reveal modifications in the sequence of the CA IX affine ligand CaIX-P1 that might be favorable for improvement of target affinity and metabolic stability, which are necessary prior to the use of

  4. In vivo imaging and quantification of carbonic anhydrase IX expression as an endogenous biomarker of tumor hypoxia.

    Directory of Open Access Journals (Sweden)

    Bagna Bao

    Full Text Available Carbonic anhydrase IX (CA IX is a transmembrane protein that has been shown to be greatly upregulated under conditions of hypoxia in many tumor cell lines. Tumor hypoxia is associated with impaired efficacy of cancer therapies making CA IX a valuable target for preclinical and diagnostic imaging. We have developed a quantitative in vivo optical imaging method for detection of CA IX as a marker of tumor hypoxia based on a near-infrared (NIR fluorescent derivative of the CA IX inhibitor acetazolamide (AZ. The agent (HS680 showed single digit nanomolar inhibition of CA IX as well as selectivity over other CA isoforms and demonstrated up to 25-fold upregulation of fluorescent CA IX signal in hypoxic versus normoxic cells, which could be blocked by 60%-70% with unlabeled AZ. CA IX negative cell lines (HCT-116 and MDA-MB-231, as well as a non-binding control agent on CA IX positive cells, showed low fluorescent signal under both conditions. In vivo FMT imaging showed tumor accumulation and excellent tumor definition from 6-24 hours. In vivo selectivity was confirmed by pretreatment of the mice with unlabeled AZ resulting in >65% signal inhibition. HS680 tumor signal was further upregulated >2X in tumors by maintaining tumor-bearing mice in a low oxygen (8% atmosphere. Importantly, intravenously injected HS680 signal was co-localized specifically with both CA IX antibody and pimonidazole (Pimo, and was located away from non-hypoxic regions indicated by a Hoechst stain. Thus, we have established a spatial correlation of fluorescence signal obtained by non-invasive, tomographic imaging of HS680 with regions of hypoxia and CA IX expression. These results illustrate the potential of HS680 and combined with FMT imaging to non-invasively quantify CA IX expression as a hypoxia biomarker, crucial to the study of the underlying biology of hypoxic tumors and the development and monitoring of novel anti-cancer therapies.

  5. Inhibition of natriuretic factors increases blood pressure in rats.

    Science.gov (United States)

    Banday, Anees Ahmad; Lokhandwala, Mustafa F

    2009-08-01

    Renal dopamine and nitric oxide contribute to natriuresis during high-salt intake which maintains sodium and blood pressure homeostasis. We wanted to determine whether concurrent inhibition of these natriuretic factors increases blood pressure during high-sodium intake. Male Sprague-Dawley rats were divided into the following groups: 1) vehicle (V)-tap water, 2) NaCl-1% NaCl drinking water, 3) 30 mM l-buthionine sulfoximine (BSO), an oxidant, 4) BSO plus NaCl, and 5) BSO plus NaCl with 1 mM tempol (antioxidant). Compared with V, NaCl intake for 10 days doubled sodium intake and increased urinary dopamine level but reduced urinary nitric oxide content. NaCl intake also reduced basal renal proximal tubular Na-K-ATPase activity with no effect on blood pressure. However, NaCl intake in BSO-treated rats failed to reduce basal Na-K-ATPase activity despite higher urinary dopamine levels. Also, dopamine failed to inhibit proximal tubular Na-K-ATPase activity and these rats exhibited reduced urinary nitric oxide levels and high blood pressure. Tempol supplementation in NaCl plus BSO-treated rats reduced blood pressure. BSO treatment alone did not affect the urinary nitric oxide and dopamine levels or blood pressure. However, dopamine failed to inhibit proximal tubular Na-K-ATPase activity in BSO-treated rats. BSO treatment also increased basal protein kinase C activity, D1 receptor serine phosphorylation, and oxidative markers like malondialdehyde and 8-isoprostane. We suggest that NaCl-mediated reduction in nitric oxide does not increase blood pressure due to activation of D1 receptor signaling. Conversely, oxidative stress-provoked inhibition of D1 receptor signaling fails to elevate blood pressure due to presence of normal nitric oxide. However, simultaneously decreasing nitric oxide levels with NaCl and inhibiting D1 receptor signaling with BSO elevated blood pressure.

  6. Fatty Acid Biosynthesis IX

    DEFF Research Database (Denmark)

    Carey, E. M.; Hansen, Heinz Johs. Max; Dils, R.

    1972-01-01

    # 1. I. [I-14C]Acetate was covalently bound to rabbit mammary gland fatty acid synthetase by enzymic transacylation from [I-14C]acetyl-CoA. Per mole of enzyme 2 moles of acetate were bound to thiol groups and up to I mole of acetate was bound to non-thiol groups. # 2. 2. The acetyl-fatty acid...... synthetase complex was isolated free from acetyl-CoA. It was rapidly hydrolysed at 30°C, but hydrolysis was greatly diminished at o°C and triacetic lactone synthesis occurred. In the presence of malonyl-CoA and NADPH, all the acetate bound to fatty acid synthetase was incorporated into long-chain fatty acids....... Hydrolysis of bound acetate and incorporation of bound acetate into fatty acids were inhibited to the same extent by guanidine hydrochloride. # 3. 3. Acetate was also covalently bound to fatty acid synthetase by chemical acetylation with [I-14C]acetic anhydride in the absence of CoASH. A total of 60 moles...

  7. Inhibition of natriuretic factors increases blood pressure in rats

    OpenAIRE

    Banday, Anees Ahmad; Lokhandwala, Mustafa F.

    2009-01-01

    Renal dopamine and nitric oxide contribute to natriuresis during high-salt intake which maintains sodium and blood pressure homeostasis. We wanted to determine whether concurrent inhibition of these natriuretic factors increases blood pressure during high-sodium intake. Male Sprague-Dawley rats were divided into the following groups: 1) vehicle (V)-tap water, 2) NaCl-1% NaCl drinking water, 3) 30 mM l-buthionine sulfoximine (BSO), an oxidant, 4) BSO plus NaCl, and 5) BSO plus NaCl with 1 mM t...

  8. Allosteric Inhibition of Macrophage Migration Inhibitory Factor Revealed by Ibudilast

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Y.; Crichlow, G; Vermeire, J; Leng, L; Du, X; Hodsdon, M; Bucala, R; Cappello, M; Gross, M; et al.

    2010-01-01

    AV411 (ibudilast; 3-isobutyryl-2-isopropylpyrazolo-[1,5-a]pyridine) is an antiinflammatory drug that was initially developed for the treatment of bronchial asthma but which also has been used for cerebrovascular and ocular indications. It is a nonselective inhibitor of various phosphodiesterases (PDEs) and has varied antiinflammatory activity. More recently, AV411 has been studied as a possible therapeutic for the treatment of neuropathic pain and opioid withdrawal through its actions on glial cells. As described herein, the PDE inhibitor AV411 and its PDE-inhibition-compromised analog AV1013 inhibit the catalytic and chemotactic functions of the proinflammatory protein, macrophage migration inhibitory factor (MIF). Enzymatic analysis indicates that these compounds are noncompetitive inhibitors of the p-hydroxyphenylpyruvate (HPP) tautomerase activity of MIF and an allosteric binding site of AV411 and AV1013 is detected by NMR. The allosteric inhibition mechanism is further elucidated by X-ray crystallography based on the MIF/AV1013 binary and MIF/AV1013/HPP ternary complexes. In addition, our antibody experiments directed against MIF receptors indicate that CXCR2 is the major receptor for MIF-mediated chemotaxis of peripheral blood mononuclear cells.

  9. The Cyclomodulin Cycle Inhibiting Factor (CIF) Alters Cullin Neddylation Dynamics*

    Science.gov (United States)

    Toro, Tasha B.; Toth, Julia I.; Petroski, Matthew D.

    2013-01-01

    The bacterial effector protein cycle inhibiting factor (CIF) converts glutamine 40 of NEDD8 to glutamate (Q40E), causing cytopathic effects and inhibiting cell proliferation. Although these have been attributed to blocking the functions of cullin-RING ubiquitin ligases, how CIF modulates NEDD8-dependent signaling is unclear. Here we use conditional NEDD8-dependent yeast to explore the effects of CIF on cullin neddylation. Although CIF causes cullin deneddylation and the generation of free NEDD8 Q40E, inhibiting the COP9 signalosome (CSN) allows Q40E to form only on NEDD8 attached to cullins. In the presence of the CSN, NEDD8 Q40E is removed from cullins more rapidly than NEDD8, leading to a decrease in steady-state cullin neddylation. As NEDD8 Q40E is competent for cullin conjugation in the absence of functional CSN and with overexpression of the NEDD8 ligase Dcn1, our data are consistent with NEDD8 deamidation causing enhanced deneddylation of cullins by the CSN. This leads to a dramatic change in the extent of activated cullin-RING ubiquitin ligases. PMID:23589306

  10. The cyclomodulin cycle inhibiting factor (CIF) alters cullin neddylation dynamics.

    Science.gov (United States)

    Toro, Tasha B; Toth, Julia I; Petroski, Matthew D

    2013-05-24

    The bacterial effector protein cycle inhibiting factor (CIF) converts glutamine 40 of NEDD8 to glutamate (Q40E), causing cytopathic effects and inhibiting cell proliferation. Although these have been attributed to blocking the functions of cullin-RING ubiquitin ligases, how CIF modulates NEDD8-dependent signaling is unclear. Here we use conditional NEDD8-dependent yeast to explore the effects of CIF on cullin neddylation. Although CIF causes cullin deneddylation and the generation of free NEDD8 Q40E, inhibiting the COP9 signalosome (CSN) allows Q40E to form only on NEDD8 attached to cullins. In the presence of the CSN, NEDD8 Q40E is removed from cullins more rapidly than NEDD8, leading to a decrease in steady-state cullin neddylation. As NEDD8 Q40E is competent for cullin conjugation in the absence of functional CSN and with overexpression of the NEDD8 ligase Dcn1, our data are consistent with NEDD8 deamidation causing enhanced deneddylation of cullins by the CSN. This leads to a dramatic change in the extent of activated cullin-RING ubiquitin ligases.

  11. Inhibition of enterovirus 71 entry by transcription factor XBP1

    Energy Technology Data Exchange (ETDEWEB)

    Jheng, Jia-Rong; Lin, Chiou-Yan [Department of Biochemistry and Research Center for Emerging Viral Infections, Chang Gung University, 259 Wen-Hwa First Road, Kweishan, Taoyuan 333, Taiwan (China); Horng, Jim-Tong, E-mail: jimtong@mail.cgu.edu.tw [Department of Biochemistry and Research Center for Emerging Viral Infections, Chang Gung University, 259 Wen-Hwa First Road, Kweishan, Taoyuan 333, Taiwan (China); Lau, Kean Seng [Department of Biochemistry and Research Center for Emerging Viral Infections, Chang Gung University, 259 Wen-Hwa First Road, Kweishan, Taoyuan 333, Taiwan (China)

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer IRE1 was activated but no XBP1 splicing was detected during enterovirus 71 infection. Black-Right-Pointing-Pointer XBP1 was subject to translational shutoff by enterovirus 71-induced eIF4G cleavage. Black-Right-Pointing-Pointer The uptake of UV-irradiated virus was decreased in XBP1-overexpressing cells. -- Abstract: Inositol-requiring enzyme 1 (IRE1) plays an important role in the endoplasmic reticulum (ER), or unfolded protein, stress response by activating its downstream transcription factor X-box-binding protein 1 (XBP1). We demonstrated previously that enterovirus 71 (EV71) upregulated XBP1 mRNA levels but did not activate spliced XBP1 (XBP1s) mRNA or its downstream target genes, EDEM and chaperones. In this study, we investigated further this regulatory mechanism and found that IRE1 was phosphorylated and activated after EV71 infection, whereas its downstream XBP1s protein level decreased. We also found that XBP1s was not cleaved directly by 2A{sup pro}, but that cleavage of eukaryotic translation initiation factor 4G by the EV71 2A{sup pro} protein may contribute to the decrease in XBP1s expression. Knockdown of XBP1 increased viral protein expression, and the synthesis of EV71 viral protein and the production of EV71 viral particles were inhibited in XBP1-overexpressing RD cells. When incubated with replication-deficient and UV-irradiated EV71, XBP1-overexpressing RD cells exhibited reduced viral RNA levels, suggesting that the inhibition of XBP1s by viral infection may underlie viral entry, which is required for viral replication. Our findings are the first indication of the ability of XBP1 to inhibit viral entry, possibly via its transcriptional activity in regulating molecules in the endocytic machinery.

  12. Pirfenidone exerts antifibrotic effects through inhibition of GLI transcription factors.

    Science.gov (United States)

    Didiasova, Miroslava; Singh, Rajeev; Wilhelm, Jochen; Kwapiszewska, Grazyna; Wujak, Lukasz; Zakrzewicz, Dariusz; Schaefer, Liliana; Markart, Philipp; Seeger, Werner; Lauth, Matthias; Wygrecka, Malgorzata

    2017-02-01

    Pirfenidone is an antifibrotic drug, recently approved for the treatment of patients suffering from idiopathic pulmonary fibrosis (IPF). Although pirfenidone exhibits anti-inflammatory, antioxidant, and antifibrotic properties, the molecular mechanism underlying its protective effects remains unknown. Here, we link pirfenidone action with the regulation of the profibrotic hedgehog (Hh) signaling pathway. We demonstrate that pirfenidone selectively destabilizes the glioma-associated oncogene homolog (GLI)2 protein, the primary activator of Hh-mediated gene transcription. Consequently, pirfenidone decreases overall Hh pathway activity in patients with IPF and in patient-derived primary lung fibroblasts and leads to diminished levels of Hh target genes such as GLI1, Hh receptor Patched-1, α-smooth muscle actin, and fibronectin and to reduced cell migration and proliferation. Interestingly, Hh-triggered TGF-β1 expression potentiated Hh responsiveness of primary lung fibroblasts by elevating the available pool of glioma-associated oncogene homolog (GLI)1/GLI2, thus creating a vicious cycle of amplifying fibrotic processes. Because GLI transcription factors are not only crucial for Hh-mediated changes but are also required as mediators of TGF-β signaling, our findings suggest that pirfenidone exerts its clinically beneficial effects through dual Hh/TGF-β inhibition by targeting the GLI2 protein.-Didiasova, M., Singh, R., Wilhelm, J., Kwapiszewska, G., Wujak, L., Zakrzewicz, D., Schaefer, L., Markart, P., Seeger, W., Lauth, M., Wygrecka, M. Pirfenidone exerts antifibrotic effects through inhibition of GLI transcription factors.

  13. Student Research Projects Inhibiting Factors from the Students Perspective

    Directory of Open Access Journals (Sweden)

    Laila Nikrooz

    2012-09-01

    Full Text Available Background & Objective: Identifying the research barriers and assess the ability of students to use the university services and facilities is crucial to promote research activities. Present study was carried out to determine the inhibiting factors influencing the student's research projects from the view point of Yasuj University of Medical Sciences students in 2008. Materials & Methods: In this cross sectional study 96 students of Yasuj Medical University were selected by stratified random sampling. The data were collected by validate & reliable questionnaire, containing demographic information, inhibiting factors related to students (personal and organization. The data were analyzed by SPSS software. Results: The mean scores against the personal barriers and the organizational barriers questions were 43.23±12.96 and 62.58±12.08 respectively. There was a significant difference between personal and organizational barriers (P<0.001 and personal barriers were more important. According to the results, the student's inadequate skills & knowledge of research methodology and lack of awareness of research topics were the most prevalent personal barriers. The most prevalent organizational barriers were unavailability of research consulters, inadequate research skills of consulter, insufficient facilities & equipment and lack of motivating staff & faculties. Other variables such as gender, subject of study and research experience are mentioned in the full text. Conclusion: This study showed that the personal barriers were more important than organizational barriers which interfere with the student's research projects. This can be corrected and controlled by teachers, faculty members, university officials and students, themselves.

  14. Inhibition of vascular permeability by antisense-mediated inhibition of plasma kallikrein and coagulation factor 12.

    Science.gov (United States)

    Bhattacharjee, Gourab; Revenko, Alexey S; Crosby, Jeffrey R; May, Chris; Gao, Dacao; Zhao, Chenguang; Monia, Brett P; MacLeod, A Robert

    2013-06-01

    Hereditary angioedema (HAE) is a rare disorder characterized by recurrent, acute, and painful episodes of swelling involving multiple tissues. Deficiency or malfunction of the serine protease inhibitor C1 esterase inhibitor (C1-INH) results in HAE types 1 and 2, respectively, whereas mutations in coagulation factor 12 (f12) have been associated with HAE type 3. C1-INH is the primary inhibitor of multiple plasma cascade pathways known to be altered in HAE patients, including the complement, fibrinolytic, coagulation, and kinin-kallikrein pathways. We have selectively inhibited several components of both the kinin-kallikrein system and the coagulation cascades with potent and selective antisense oligonucleotides (ASOs) to investigate their relative contributions to vascular permeability. We have also developed ASO inhibitors of C1-INH and characterized their effects on vascular permeability in mice as an inducible model of HAE. Our studies demonstrate that ASO-mediated reduction in C1-INH plasma levels results in increased vascular permeability and that inhibition of proteases of the kinin-kallikrein system, either f12 or prekallikrein (PKK) reverse the effects of C1-INH depletion with similar effects on both basal and angiotensin converting enzyme (ACE) inhibitor-induced permeability. In contrast, inhibition of coagulation factors 11 (f11) or 7 (f7) had no effect. These results suggest that the vascular defects observed in C1-INH deficiency are dependent on the kinin-kallikrein system proteases f12 and PKK, and not mediated through the coagulation pathways. In addition, our results highlight a novel therapeutic modality that can potentially be employed prophylactically to prevent attacks in HAE patients.

  15. Design of a carbonic anhydrase IX active-site mimic to screen inhibitors for possible anticancer properties.

    Science.gov (United States)

    Genis, Caroli; Sippel, Katherine H; Case, Nicolette; Cao, Wengang; Avvaru, Balendu Sankara; Tartaglia, Lawrence J; Govindasamy, Lakshmanan; Tu, Chingkuang; Agbandje-McKenna, Mavis; Silverman, David N; Rosser, Charles J; McKenna, Robert

    2009-02-17

    Recently, a convincing body of evidence has accumulated suggesting that the overexpression of carbonic anhydrase isozyme IX (CA IX) in some cancers contributes to the acidification of the extracellular matrix, which in turn promotes the growth and metastasis of the tumor. These observations have made CA IX an attractive drug target for the selective treatment of certain cancers. Currently, there is no available X-ray crystal structure of CA IX, and this lack of availability has hampered the rational design of selective CA IX inhibitors. In light of these observations and on the basis of structural alignment homology, using the crystal structure of carbonic anhydrase II (CA II) and the sequence of CA IX, a double mutant of CA II with Ala65 replaced by Ser and Asn67 replaced by Gln has been constructed to resemble the active site of CA IX. This CA IX mimic has been characterized kinetically using (18)O-exchange and structurally using X-ray crystallography, alone and in complex with five CA sulfonamide-based inhibitors (acetazolamide, benzolamide, chlorzolamide, ethoxzolamide, and methazolamide), and compared to CA II. This structural information has been evaluated by both inhibition studies and in vitro cytotoxicity assays and shows a correlated structure-activity relationship. Kinetic and structural studies of CA II and CA IX mimic reveal chlorzolamide to be a more potent inhibitor of CA IX, inducing an active-site conformational change upon binding. Additionally, chlorzolamide appears to be cytotoxic to prostate cancer cells. This preliminary study demonstrates that the CA IX mimic may provide a useful model to design more isozyme-specific CA IX inhibitors, which may lead to development of new therapeutic treatments of some cancers.

  16. Celecoxib inhibits Helicobacter pylori colonization-related factors

    Science.gov (United States)

    Wang, Jing; Wang, Wei-Hong; Li, Jiang; Liu, Fang-Xun

    2010-01-01

    AIM: To investigate the effect of celecoxib, a selective COX-2 inhibitor, on Helicobacter pylori (H. pylori) colonization-related factors and its mechanism. METHODS: After co-incubation with celecoxib, morphology of H. pylori strain 26695 was observed under a transmission electron microscope. Flagella motility was assessed by stab agar motility test. Adherence of H. pylori to AGS cells was determined by enzyme linked immunosorbent assay. Levels of mRNA expression in flagellar genes (flaA, flaB), urease genes (ureA, ureB) and adhesin genes (babA, sabA, alpA, alpB, hpaA, hopZ) were measured by real-time polymerase chain reaction. RESULTS: Separation and non-integrity of bacterial cell wall, rarefaction and asymmetry of cytoplasm, and even lysis of H. pylori were observed in the presence of celecoxib. When H. pylori strains were incubated in the presence of celecoxib, their flagellar motility and adherence to AGS cells were inhibited. The expression of ureA, ureB, babA, sabA, alpA, alpB, hpaA, hopZ was up-regulated while the expression of flaA, flaB was down-regulated in the presence of celecoxib. CONCLUSION: Celecoxib inhibits flagellar motility and adherence of H. pylori to AGS cells, and destructs their normal structure in vitro. PMID:20143463

  17. p27{sup Kip1} inhibits tissue factor expression

    Energy Technology Data Exchange (ETDEWEB)

    Breitenstein, Alexander, E-mail: alexander.breitenstein@usz.ch [Cardiology, University Heart Center, University Hospital Zurich (Switzerland); Cardiovascular Research, Physiology Institute, University of Zurich (Switzerland); Center for Integrative Human Physiology (ZHIP), University of Zurich (Switzerland); Akhmedov, Alexander; Camici, Giovanni G.; Lüscher, Thomas F.; Tanner, Felix C. [Cardiology, University Heart Center, University Hospital Zurich (Switzerland); Cardiovascular Research, Physiology Institute, University of Zurich (Switzerland); Center for Integrative Human Physiology (ZHIP), University of Zurich (Switzerland)

    2013-10-04

    Highlights: •p27{sup Kip1}regulates the expression of tissue factor at the transcriptional level. •This inhibitory effect of p27{sup Kip1} is independently of its cell regulatory action. •The current study provides new insights into a pleiotrophic function of p27{sup Kip1}. -- Abstract: Background: The cyclin-dependent kinase inhibitor (CDKI) p27{sup Kip1} regulates cell proliferation and thus inhibits atherosclerosis and vascular remodeling. Expression of tissue factor (TF), the key initator of the coagulation cascade, is associated with atherosclerosis. Yet, it has not been studied whether p27{sup Kip1} influences the expression of TF. Methods and results: p27{sup Kip1} overexpression in human aortic endothelial cells was achieved by adenoviral transfection. Cells were rendered quiescent for 24 h in 0.5% fetal-calf serum. After stimulation with TNF-α (5 ng/ml), TF protein expression and activity was significantly reduced (n = 4; P < 0.001) in cells transfected with p27{sup Kip1}. In line with this, p27{sup Kip1} overexpression reduced cytokine-induced TF mRNA expression (n = 4; P < 0.01) and TF promotor activity (n = 4; P < 0.05). In contrast, activation of the MAP kinases p38, ERK and JNK was not affected by p27{sup Kip1} overexpression. Conclusion: This in vitro study suggests that p27{sup Kip1} inhibits TF expression at the transcriptional level. These data indicate an interaction between p27{sup Kip1} and TF in important pathological alterations such as atherosclerosis and vascular remodeling.

  18. Factor VII activating protease (FSAP) promotes the proteolysis and inhibition of tissue factor pathway inhibitor (TFPI)

    Science.gov (United States)

    Kanse, Sandip M.; Declerck, Paul J.; Ruf, Wolfram; Broze, George; Etscheid, Michael

    2013-01-01

    Objectives Factor VII activating protease (FSAP) activates FVII as well as pro-urokinase and inhibits platelet-derived growth factor-BB, thus regulating haemostasis- and remodeling-associated processes in the vasculature. A genetic variant of FSAP (Marburg I polymorphism) results in low enzymatic activity and is associated with an enhanced risk for carotid stenosis and stroke. We postulate that there are additional substrates for FSAP that will help to explain its role in vascular biology and have searched for such a substrate. Results and Methods Using screening procedures to determine the influence of FSAP on various haemostasis-related processes on endothelial cells we discovered that FSAP inhibited tissue factor pathway inhibitor (TFPI), a major anti-coagulant secreted by these cells. Proteolytic degradation of TFPI by FSAP could also be demonstrated by Western blotting and the exact cleavage sites were determined by N-terminal sequencing. The Marburg I variant of FSAP had a diminished ability to inhibit TFPI. A monoclonal antibody to FSAP, that specifically inhibited FSAP binding to TFPI, reversed the inhibitory effect of FSAP on TFPI. Conclusions The identification of TFPI as a sensitive substrate for FSAP increases our understanding of its role in regulating haemostasis and proliferative remodeling events in the vasculature. PMID:22116096

  19. Thioredoxin interacting protein inhibits hypoxia-inducible factor transcriptional activity

    Science.gov (United States)

    Farrell, Michael R; Rogers, Lynette K; Liu, Yusen; Welty, Stephen E; Tipple, Trent E

    2010-01-01

    Vascular endothelial growth factor (VEGF) is required for proper lung development and is transcriptionally regulated in alveolar epithelial cells by hypoxia inducible factor (HIF). Previous findings in a newborn mouse model of bronchopulmonary dysplasia (BPD) suggest that thioredoxin interacting protein (Txnip) is a novel regulator of VEGF expression. The present studies were designed to test the hypothesis that Txnip negatively regulates VEGF through effects on HIF-mediated gene expression. To test this hypothesis, we first examined the levels of VEGF and Txnip protein in the lungs of 1 day-old newborn and E19 embryos and detected a significant inverse correlation. To elucidate the mechanisms underlying this relationship, we studied the effects of Txnip overexpression on HIF-mediated transcription using murine lung epithelial (MLE-12) cells. Overexpression of Txnip inhibited HIF-mediated reporter activity in both hypoxia and room air. Suppression of HIF activity by Txnip appeared to be independent of the ability of Txnip to bind to thioredoxin. Thus, our studies support a model in which Txnip is a potentially critical regulator of HIF-mediated gene transcription in the murine lung. Alterations in Txnip expression could alter lung VEGF expression in prematurely born human infants and contribute to the development of BPD. PMID:20692333

  20. Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

    Directory of Open Access Journals (Sweden)

    Pangburn Heather A

    2005-09-01

    Full Text Available Abstract Background Regular use of nonsteroidal anti-inflammatory drugs (NSAIDs is associated with a decreased mortality from colorectal cancer (CRC. NSAIDs induce apoptotic cell death in colon cancer cells in vitro and inhibit growth of neoplastic colonic mucosa in vivo however, the biochemical mechanisms required for these growth inhibitory effects are not well defined. We previously reported that metabolites of the NSAID sulindac downregulate extracellular-signal regulated kinase 1/2 (ERK1/2 signaling and that this effect is both necessary and sufficient for the apoptotic effects of these drugs. The goal of this project was to specifically test the hypothesis that sulindac metabolites block activation and/or expression of the epidermal growth factor (EGF receptor (EGFR. Methods HT29 human colon cancer cells were treated with EGF, alone, or in the presence of sulindac sulfide or sulindac sulfone. Cells lysates were assayed by immunoblotting for phosphorylated EGFR (pEGFR, pY1068, total EGFR, phosphorylated ERK1/2 (pERK1/2, total ERK1/2, activated caspase-3, and α-tubulin. Results EGF treatment rapidly induced phosphorylation of both EGFR and ERK1/2 in HT29 colon cancer cells. Pretreatment with sulindac metabolites for 24 h blocked EGF-induced phosphorylation of both EGFR and ERK1/2 and decreased total EGFR protein expression. Under basal conditions, downregulation of pEGFR and total EGFR was detected as early as 12 h following sulindac sulfide treatment and persisted through at least 48 h. Sulindac sulfone induced downregulation of pEGFR and total EGFR was detected as early as 1 h and 24 h, respectively, following drug treatment, and persisted through at least 72 h. EGFR downregulation by sulindac metabolites was observed in three different CRC cell lines, occurred prior to the observed downregulation of pERK1/2 and induction of apoptosis by these drugs, and was not dependent of caspase activation. Conclusion These results suggest that

  1. Efficacy and safety of a recombinant factor IX (Bax326) in previously treated patients with severe or moderately severe haemophilia B undergoing surgical or other invasive procedures: a prospective, open-label, uncontrolled, multicentre, phase III study.

    Science.gov (United States)

    Windyga, J; Lissitchkov, T; Stasyshyn, O; Mamonov, V; Ghandehari, H; Chapman, M; Fritsch, S; Wong, W-Y; Pavlova, B G; Abbuehl, B E

    2014-09-01

    Haemostatic management of haemophilia B patients undergoing surgery is critical to patient safety. The aim of this ongoing prospective trial was to investigate the haemostatic efficacy and safety of a recombinant factor IX (rFIX) (Bax326) in previously treated subjects (12-65 years, without history of FIX inhibitors) with severe or moderately severe haemophilia B, undergoing surgical, dental or other invasive procedures. Haemostatic efficacy was assessed according to a predefined scale. Blood loss was compared to the average and maximum blood loss predicted preoperatively. Haemostatic FIX levels were achieved peri- and postoperatively in 100% of subjects (n = 14). Haemostasis was 'excellent' intraoperatively in all patients and postoperatively in those without a drain, and 'excellent' or 'good' at the time of drain removal and day of discharge in those with a drain employed. Following the initial dose, the mean FIX activity level rose from 6.55% to 107.58% for major surgeries and from 3.60% to 81.4% for minor surgeries. Actual vs. predicted blood loss matched predicted intraoperative blood loss but was equal to or higher than (but less than 150%) the maximum predicted postoperative blood loss reflecting the severity of procedure and FIX requirements. There were no related adverse events, severe allergic reactions or thrombotic events. There was no evidence that BAX326 increased the risk of inhibitor or binding antibody development to FIX. BAX326 was safe and effective for peri-operative management of 14 subjects with severe and moderately severe haemophilia B.

  2. Pharmacokinetics, efficacy and safety of BAX326, a novel recombinant factor IX: a prospective, controlled, multicentre phase I/III study in previously treated patients with severe (FIX level <1%) or moderately severe (FIX level ≤2%) haemophilia B.

    Science.gov (United States)

    Windyga, J; Lissitchkov, T; Stasyshyn, O; Mamonov, V; Rusen, L; Lamas, J L; Oh, M-S; Chapman, M; Fritsch, S; Pavlova, B G; Wong, W-Y; Abbuehl, B E

    2014-01-01

    BAX326 is a recombinant factor IX (rFIX; nonacog gamma) manufactured without the addition of any materials of human or animal origin, and with two viral inactivation steps (solvent/detergent treatment and 15 nm nanofiltration). The aim of this prospective trial was to investigate the pharmacokinetics, haemostatic efficacy and safety of BAX326 in previously treated patients aged 12-65 years with severe or moderately severe haemophilia B. BAX326 was safe and well tolerated in all 73 treated subjects; adverse events considered related to treatment (2.7% incidence, all non-serious) were transient and mild, and no hypersensitivity reactions, inhibitor formation or thrombotic events were observed. Pharmacokinetic (PK) equivalence (n = 28) between BAX326 and a licensed rFIX was confirmed in terms of the ratio of geometric mean AUC(0-72) h per dose. Twice-weekly prophylaxis [mean duration 6.2 (±0.7) months; 1.8 (±0.1) infusions per week, 49.5 (±4.8) IU kg(-1) per infusion] was effective in preventing bleeding episodes, with a significantly lower (79%, P < 0.001) annualized bleed rate (4.2) compared to an on-demand treatment in a historical control group (20.0); 24 of 56 subjects on prophylaxis (43%) did not bleed throughout the study observation period. Of 249 total acute bleeds, 211 (84.7%) were controlled with one to two infusions of BAX326. Haemostatic efficacy at resolution of bleed was rated excellent or good in 96.0% of all treated bleeding episodes. The results of this study indicate that BAX326 is safe and efficacious in treating bleeds and routine prophylaxis in patients aged 12 years and older with haemophilia B.

  3. Thermodynamics of mixtures containing amines. IX. Application of the concentration-concentration structure factor to the study of binary mixtures containing pyridines

    Energy Technology Data Exchange (ETDEWEB)

    Gonzalez, Juan Antonio, E-mail: jagl@termo.uva.es [G.E.T.E.F. Dpto Termodinamica y Fisica Aplicada, Facultad de Ciencias, Universidad de Valladolid, Valladolid 47071 (Spain); Cobos, Jose Carlos; Garcia de la Fuente, Isaias; Mozo, Ismael [G.E.T.E.F. Dpto Termodinamica y Fisica Aplicada, Facultad de Ciencias, Universidad de Valladolid, Valladolid 47071 (Spain)

    2009-10-10

    Binary mixtures formed by a pyridine base and an alkane, or an aromatic hydrocarbon, or a 1-alkanol have been studied in the framework of the concentration-concentration structure factor, S{sub CC}(0), formalism. Deviations between experimental data and those provided by the DISQUAC model are discussed. Systems containing alkanes are characterized by homocoordination. In pyridine + alkane mixtures, S{sub CC}(0) decreases with the chain length of the longer alkanes, due to size effects. For a given alkane, S{sub CC}(0) also decreases with the number of CH{sub 3}- groups in the pyridine base. This has been interpreted assuming that the number of amine-amine interactions available to be broken upon mixing also decreases similarly, probably as steric hindrances exerted by the methyl groups of the aromatic amine increase with the number of these groups. Homocoordination is higher in mixtures with 3,5-dimethylpyridine than in those with 2,6-dimethylpyridine. That is, steric effects exerted by methyl groups in positions 3 and 5 are stronger than when they are in positions 2 and 6. Similarly, from the application of the DISQUAC (dispersive-quasichemical) model, it is possible to conclude that homocoordination is higher in systems with 3- or 4-methylpyridine than in those involving 2-methylpyridine. Systems including aromatic hydrocarbons are nearly ideal, which seems to indicate that there is no specific interaction in such solutions. Mixtures with 1-alkanols show heterocoordination. This reveals the existence of interactions between unlike molecules, characteristic of alkanol + amine mixtures. Methanol systems show the lowest S{sub CC}(0) values due, partially, to size effects. This explains the observed decrease of homocoordination in such solutions in the order: pyridine > 2-methylpyridine > 2,6-dimethylpyridine. Moreover, as the energies of the OH-N hydrogen bonds are practically independent of the pyridine base considered when mixed with methanol, it suggests that

  4. Clearance of protoporphyrin IX induced by 5-aminolevulinic acid from WiDr human colon carcinoma cells

    Science.gov (United States)

    Juzeniene, Asta; Kaliszewski, Miron; Bugaj, Andrzej; Moan, Johan

    2009-06-01

    5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) is the most widely practiced form of PDT in dermatology. One of the advantages of ALA-PDT is that undesirable photosensitization lasts only for 24-48 h. In order to optimize ALA-PDT it is necessary to understand the mechanisms controlling intracellular PpIX clearance (efflux and transformation into heme) in order to decrease protoporphyrin IX (PpIX) clearance rates in the early stages of its production. The aim of this study was to investigate the factors controlling the clearance of intracellular PpIX. Fluorescence spectroscopy was used to study PpIX kinetics in WiDr cells initially treated with ALA. The clearance rate of PpIX in WiDr cells was faster after application of a low concentration of ALA (0.1 mM) than after application of high concentration of ALA (1 mM). PpIX was cleared faster from cells which initially were seeded at low densities than cells seeded at higher densities. The presence of the iron chelator deferoxamine reduced the clearance rate of PpIX, while the presence of ferrous sulfate acted oppositely. The decay rate of PpIX in WiDr cells was faster at higher temperature than at lower. The ferrochelatase activity at pH 7.2 was significantly greater than that at pH 6.7. ALA concentration, application time, cell density, temperature, pH, intracellular iron content, intracellular amount and localization of PpIX are factors controlling PpIX clearance.

  5. Quantum Supersymmetric Bianchi IX Cosmology

    CERN Document Server

    Damour, Thibault

    2014-01-01

    We study the quantum dynamics of a supersymmetric squashed three-sphere by dimensionally reducing to one timelike dimension the action of D=4 simple supergravity for a Bianchi IX cosmological model. After imposition of the diffeomorphism constraints, the wave function of the Universe becomes a spinor of Spin(8,4) depending on the three squashing parameters, which satisfies Dirac, and Klein-Gordon-like, wave equations describing the propagation of a quantum spinning particle reflecting off spin-dependent potential walls. The algebra of the susy constraints and of the Hamiltonian one is found to close. One finds that the quantum Hamiltonian is built from operators that generate a 64-dimensional representation of the maximally compact sub-algebra of the rank-3 hyperbolic Kac-Moody algebra AE3. The (quartic-in-fermions) squared-mass term entering the Klein-Gordon-like equation has several remarkable properties: 1)it commutes with all the other (Kac-Moody-related) building blocks of the Hamiltonian; 2)it is a quad...

  6. Inhibition of cullin RING ligases by cycle inhibiting factor: evidence for interference with Nedd8-induced conformational control.

    Science.gov (United States)

    Boh, Boon Kim; Ng, Mei Ying; Leck, Yee Chin; Shaw, Barry; Long, Jed; Sun, Guang Wen; Gan, Yunn Hwen; Searle, Mark S; Layfield, Robert; Hagen, Thilo

    2011-10-21

    Cycle inhibiting factor (Cif) is produced by pathogenic intracellular bacteria and injected into the host cells via a type III secretion system. Cif is known to interfere with the eukaryotic cell cycle by inhibiting the function of cullin RING E3 ubiquitin ligases (CRLs). Cullin proteins form the scaffold protein of CRLs and are modified with the ubiquitin-like protein Nedd8, which exerts important conformational control required for CRL activity. Cif has recently been shown to catalyze the deamidation of Gln40 in Nedd8 to Glu. Here, we addressed how Nedd8 deamidation inhibits CRL activity. Our results indicate that Burkholderia pseudomallei Cif (also known as CHBP) inhibits the deconjugation of Nedd8 in vivo by inhibiting binding of the deneddylating COP9 signalosome (CSN) complex. We provide evidence that the reduced binding of CSN and the inhibition of CRL activity by Cif are due to interference with Nedd8-induced conformational control, which is dependent on the interaction between the Nedd8 hydrophobic patch and the cullin winged-helix B subdomain. Of note, mutation of Gln40 to Glu in ubiquitin, an additional target of Cif, inhibits the interaction between the hydrophobic surface of ubiquitin and the ubiquitin-binding protein p62/SQSTM1, showing conceptually that Cif activity can impair ubiquitin/ubiquitin-like protein non-covalent interactions. Our results also suggest that Cif may exert additional cellular effects by interfering with the association between ubiquitin and ubiquitin-binding proteins.

  7. El Titulo IX y La Discriminacion por Sexo (Title IX and Sex Discrimination).

    Science.gov (United States)

    Office for Civil Rights (ED), Washington, DC.

    Title IX of the Education Amendments of 1972 protects people from discrimination based on sex in education programs or activities that receive Federal financial assistance. This brochure outlines the responsibilities of education programs and activities covered by Title IX, the responsibilities of the Office for Civil Rights (OCR) in enforcing…

  8. A new peptide ligand for targeting human carbonic anhydrase IX, identified through the phage display technology.

    Directory of Open Access Journals (Sweden)

    Vasileios Askoxylakis

    Full Text Available UNLABELLED: Carbonic anhydrase IX (CAIX is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy. METHODS: Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC. Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR. RESULTS: In vitro binding experiments of (125I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney. CONCLUSIONS: These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.

  9. Quantum supersymmetric Bianchi IX cosmology

    Science.gov (United States)

    Damour, Thibault; Spindel, Philippe

    2014-11-01

    We study the quantum dynamics of a supersymmetric squashed three-sphere by dimensionally reducing (to one timelike dimension) the action of D =4 simple supergravity for a S U (2 ) -homogeneous (Bianchi IX) cosmological model. The quantization of the homogeneous gravitino field leads to a 64-dimensional fermionic Hilbert space. After imposition of the diffeomorphism constraints, the wave function of the Universe becomes a 64-component spinor of spin(8,4) depending on the three squashing parameters, which satisfies Dirac-like, and Klein-Gordon-like, wave equations describing the propagation of a "quantum spinning particle" reflecting off spin-dependent potential walls. The algebra of the supersymmetry constraints and of the Hamiltonian one is found to close. One finds that the quantum Hamiltonian is built from operators that generate a 64-dimensional representation of the (infinite-dimensional) maximally compact subalgebra of the rank-3 hyperbolic Kac-Moody algebra A E3 . The (quartic-in-fermions) squared-mass term μ^ 2 entering the Klein-Gordon-like equation has several remarkable properties: (i) it commutes with all the other (Kac-Moody-related) building blocks of the Hamiltonian; (ii) it is a quadratic function of the fermion number NF; and (iii) it is negative in most of the Hilbert space. The latter property leads to a possible quantum avoidance of the singularity ("cosmological bounce"), and suggests imposing the boundary condition that the wave function of the Universe vanish when the volume of space tends to zero (a type of boundary condition which looks like a final-state condition when considering the big crunch inside a black hole). The space of solutions is a mixture of "discrete-spectrum states" (parametrized by a few constant parameters, and known in explicit form) and of continuous-spectrum states (parametrized by arbitrary functions entering some initial-value problem). The predominantly negative values of the squared-mass term lead to a "bottle

  10. Behavioral Inhibition as a Risk Factor for the Development of Childhood Anxiety Disorders: A Longitudinal Study

    OpenAIRE

    Muris, Peter; Brakel, Anna; Arntz, Arnoud; Schouten, Erik

    2010-01-01

    textabstractThis longitudinal study examined the additive and interactive effects of behavioral inhibition and a wide range of other vulnerability factors in the development of anxiety problems in youths. A sample of 261 children, aged 5 to 8 years, 124 behaviorally inhibited and 137 control children, were followed during a 3-year period. Assessments took place on three occasions to measure children's level of behavioral inhibition, anxiety disorder symptoms, other psychopathological symptoms...

  11. Targeting Notch1 inhibits invasion and angiogenesis of human breast cancer cells via inhibition Nuclear Factor-κB signaling.

    Science.gov (United States)

    Liu, Yuan; Su, Chuanfu; Shan, Yuqing; Yang, Shouxiang; Ma, Guifeng

    2016-01-01

    Notch-1, a type-1 transmembrane protein, plays critical roles in the pathogenesis and progression of human malignancies, including breast cancer; however, the precise mechanism by which Notch-1 causes tumor cell invasion and angiogenesis remain unclear. Nuclear factor-κB (NF-κB), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMP) are critically involved in the processes of tumor cell invasion and metastasis, we investigated whether targeting Notch-1 could be mechanistically associated with the down-regulation of NF-κB, IL-8, VEGF, and MMP-9, resulting in the inhibition of invasion and angiogenesis of breast cancer cells. Our data showed that down-regulation of Notch-1 leads to the inactivation of NF-κB activity and inhibits the expression of its target genes, such as IL-8, VEGF and MMP-9. We also found that down-regulation of Notch-1 decreased cell invasion, and vice versa Consistent with these results, we also found that the down-regulation of Notch-1 not only decreased MMP-9 mRNA and its protein expression but also inhibited MMP-9 active form. Moreover, conditioned medium from Notch-1 siRNA-transfected breast cancer cells showed reduced levels of IL-8 and VEGF and, in turn, inhibited the tube formation of HUVECs, suggesting that down-regulation of Notch-1 leads to the inhibition of angiogenesis. Furthermore, conditioned medium from Notch-1 cDNA-transfected breast cancer cells showed increased levels of IL-8 and VEGF and, in turn, promoted the tube formation of HUVECs, suggesting that Notch-1 overexpression leads to the promotion of angiogenesis.We therefore concluded that down-regulation of Notch-1 leads to the inactivation NF-κB and its target genes (IL-8, MMP-9 and VEGF), resulting in the inhibition of invasion and angiogenesis.

  12. Lack of prognostic and predictive value of CA IX in radiotherapy of squamous cell carcinoma of the head and neck with known modifiable hypoxia: An evaluation of the DAHANCA 5 study

    DEFF Research Database (Denmark)

    Eriksen, Jesper Grau; Overgaard, Jens

    2007-01-01

    BACKGROUND AND PURPOSE: CA IX is suggested to be an endogenous marker of hypoxia in tumours like squamous cell carcinomas of the head and neck (HNSCC). The aim of the present study was to investigate whether CA IX served as a prognostic factor for outcome in a large population of HNSCC and if CA IX......+/-the hypoxic radiosensitizer nimorazole. CA IX was measured using immunohistochemistry and results were divided into four groups of CA IX expression: 30% of the tumour area with positive membrane staining. Locoregional control and disease-specific survival were used as endpoints...

  13. Behavioral Inhibition as a Risk Factor for the Development of Childhood Anxiety Disorders: A Longitudinal Study

    NARCIS (Netherlands)

    P.E.H.M. Muris (Peter); A.M.L. van Brakel (Anna); A. Arntz (Arnoud); E. Schouten (Erik)

    2011-01-01

    textabstractThis longitudinal study examined the additive and interactive effects of behavioral inhibition and a wide range of other vulnerability factors in the development of anxiety problems in youths. A sample of 261 children, aged 5 to 8 years, 124 behaviorally inhibited and 137 control childre

  14. Fibroblast growth factor 21 as a possible endogenous factor inhibits apoptosis in cardiac endothelial cells

    Institute of Scientific and Technical Information of China (English)

    L(U) Yun; ZHANG Ying-chuan; LIU Jing-hua; ZHANG Li-ke; DU Jie; ZENG Xiang-jun; HAO Gang; HUANG Ji; ZHAO Dong-hui; WANG Guo-zhong

    2010-01-01

    Background Fibroblast growth factor 21 (FGF21) is a new member of FGF super family that is an important endogenous regulator for systemic glucose and lipid metabolism. This study aimed to explore whether FGF21 reduces atherosclerotic injury and prevents endothelial dysfunction as an independent protection factor.Methods The present study was designed to investigate the changes of FGF21 levels induced by oxidized-low density lipoprotein (ox-LDL), and the changes of apoptosis affected by regulating FGF21 expression. The FGF21 mRNA levels of cultured cardiac microvascular endothelial cells (CMECs) were determined by real time-PCR and the protein concentration in culture media was detected by enzyme-linked immunosorbent assay. We analyzed the different expression levels of untreated controls and CMFCs incubated with ox-LDL, and the changes of CMECs apoptosis initiated by the enhancement or suppression of FGF21 levels.Results The secretion levels of FGF21 mRNA and protein were significantly upregulated in CMECs incubated with ox-LDL. Furthermore, FGF21 levels increased by 200 μmol/L bezafibrate could reduce CMECs apoptosis, and inhibit FGF21 expression by shRNA induced apoptosis (P <0.05).Conclusions FGF21 may be a signal of injured target tissue, and may play physiological roles in improving the endothelial function at an early stage of atherosclerosis and in stopping the development of coronary heart disease.

  15. Synthesis and characterization of 18F-labeled active site inhibited factor VII (ASIS)

    DEFF Research Database (Denmark)

    Erlandsson, Maria; Nielsen, Carsten Haagen; Jeppesen, Troels Elmer

    2015-01-01

    Activated factor VII blocked in the active site with Phe-Phe-Arg-chloromethyl ketone (active site inhibited factor VII (ASIS)) is a 50-kDa protein that binds with high affinity to its receptor, tissue factor (TF). TF is a transmembrane glycoprotein that plays an important role in, for example, th...

  16. Coagulation factor V mediates inhibition of tissue factor signaling by activated protein C in mice.

    Science.gov (United States)

    Liang, Hai Po H; Kerschen, Edward J; Basu, Sreemanti; Hernandez, Irene; Zogg, Mark; Jia, Shuang; Hessner, Martin J; Toso, Raffaella; Rezaie, Alireza R; Fernández, José A; Camire, Rodney M; Ruf, Wolfram; Griffin, John H; Weiler, Hartmut

    2015-11-19

    The key effector molecule of the natural protein C pathway, activated protein C (aPC), exerts pleiotropic effects on coagulation, fibrinolysis, and inflammation. Coagulation-independent cell signaling by aPC appears to be the predominant mechanism underlying its highly reproducible therapeutic efficacy in most animal models of injury and infection. In this study, using a mouse model of Staphylococcus aureus sepsis, we demonstrate marked disease stage-specific effects of the anticoagulant and cell signaling functions of aPC. aPC resistance of factor (f)V due to the R506Q Leiden mutation protected against detrimental anticoagulant effects of aPC therapy but also abrogated the anti-inflammatory and mortality-reducing effects of the signaling-selective 5A-aPC variant that has minimal anticoagulant function. We found that procofactor V (cleaved by aPC at R506) and protein S were necessary cofactors for the aPC-mediated inhibition of inflammatory tissue-factor signaling. The anti-inflammatory cofactor function of fV involved the same structural features that govern its cofactor function for the anticoagulant effects of aPC, yet its anti-inflammatory activities did not involve proteolysis of activated coagulation factors Va and VIIIa. These findings reveal a novel biological function and mechanism of the protein C pathway in which protein S and the aPC-cleaved form of fV are cofactors for anti-inflammatory cell signaling by aPC in the context of endotoxemia and infection.

  17. The scorpion toxin Bot IX is a potent member of the α-like family and has a unique N-terminal sequence extension.

    Science.gov (United States)

    Martin-Eauclaire, Marie-France; Salvatierra, Juan; Bosmans, Frank; Bougis, Pierre E

    2016-09-01

    We report the detailed chemical, immunological and pharmacological characterization of the α-toxin Bot IX from the Moroccan scorpion Buthus occitanus tunetanus venom. Bot IX, which consists of 70 amino acids, is a highly atypical toxin. It carries a unique N-terminal sequence extension and is highly lethal in mice. Voltage clamp recordings on oocytes expressing rat Nav1.2 or insect BgNav1 reveal that, similar to other α-like toxins, Bot IX inhibits fast inactivation of both variants. Moreover, Bot IX belongs to the same structural/immunological group as the α-like toxin Bot I. Remarkably, radioiodinated Bot IX competes efficiently with the classical α-toxin AaH II from Androctonus australis, and displays one of the highest affinities for Nav channels.

  18. The RNA splicing factor ASF/SF2 inhibits human topoisomerase I mediated DNA relaxation

    DEFF Research Database (Denmark)

    Andersen, Félicie Faucon; Tange, Thomas Ø.; Sinnathamby, Thayaline

    2002-01-01

    with the SR-proteins controlling topoisomerase I DNA activity. We demonstrate that the splicing factor ASF/SF2 inhibits relaxation by interfering with the DNA cleavage and/or DNA binding steps of human topoisomerase I catalysis. The inhibition of relaxation correlated with the ability of various deletion...... extract reduced the inhibition of relaxation activity. Taken together with the previously published studies of the topoisomerase I kinase activity, these observations suggest that topoisomerase I activity is shifted from relaxation to kinasing by specific interaction with SR-splicing factors....

  19. Psychological Factors that Promote and Inhibit Pathological Gambling

    Science.gov (United States)

    Morasco, Benjamin J.; Weinstock, Jeremiah; Ledgerwood, David M.; Petry, Nancy M.

    2007-01-01

    This paper describes qualitative data regarding psychological factors that may affect gambling behavior among treatment-seeking pathological gamblers. Participants (n = 84) diagnosed with pathological gambling were treated in a clinical trial examining the efficacy of cognitive behavioral therapy (CBT). Qualitative data were collected from…

  20. [Inhibiting the pro-tumor and transcription factor FACT: Mechanisms].

    Science.gov (United States)

    Maluchenko, N V; Chang, H W; Kozinova, M T; Valieva, M E; Gerasimova, N S; Kitashov, A V; Kirpichnikov, M P; Georgiev, P G; Studitsky, V M

    2016-01-01

    Conventional antitumor therapy is often complicated by the emergence of the so-called cancer stem cells (CSCs), which are characterized by low metabolic rates and high resistance to almost all existing therapies. Many problems of clinical oncology and a poor efficacy of current treatments in particular are ascribed to CSCs. Therefore, it is important to develop new compounds capable of eliminating both rapidly proliferating tumor cells and standard treatment-resistant CSCs. Curaxins have been demonstrated to manifest various types of antitumor activity. Curaxins simultaneously affect at least three key molecular cascades involved in tumor development, including the p53, NF-κB, and HSF1 metabolic pathways. In addition, studies of some curaxins indicate that they can inhibit the transcriptional induction of the genes for matrix metalloproteinases 1 and 8 (MMP1 and MMP8); the PI3K/AKT/mTOR signaling cascades; cIAP-1 (apoptosis protein 1) inhibitor activity; topoisomerase II; and a number of oncogenes, such as c-MYC and others. In vivo experiments have shown that the CSC population increases on gemcitabine monotherapy and is reduced on treatment with curaxin CBL0137. The data support the prospective use of FACT inhibitors as new anticancer drugs with multiple effects on cell metabolism.

  1. Behavioral Inhibition as a Risk Factor for the Development of Childhood Anxiety Disorders: A Longitudinal Study.

    Science.gov (United States)

    Muris, Peter; van Brakel, Anna M L; Arntz, Arnoud; Schouten, Erik

    2011-04-01

    This longitudinal study examined the additive and interactive effects of behavioral inhibition and a wide range of other vulnerability factors in the development of anxiety problems in youths. A sample of 261 children, aged 5 to 8 years, 124 behaviorally inhibited and 137 control children, were followed during a 3-year period. Assessments took place on three occasions to measure children's level of behavioral inhibition, anxiety disorder symptoms, other psychopathological symptoms, and a number of other vulnerability factors such as insecure attachment, negative parenting styles, adverse life events, and parental anxiety. Results obtained with Structural Equation Modeling indicated that behavioral inhibition primarily acted as a specific risk factor for the development of social anxiety symptoms. Furthermore, the longitudinal model showed additive as well as interactive effects for various vulnerability factors on the development of anxiety symptoms. That is, main effects of anxious rearing and parental trait anxiety were found, whereas behavioral inhibition and attachment had an interactive effect on anxiety symptomatology. Moreover, behavioral inhibition itself was also influenced by some of the vulnerability factors. These results provide support for dynamic, multifactorial models for the etiology of child anxiety problems.

  2. The role of factor inhibiting HIF (FIH-1 in inhibiting HIF-1 transcriptional activity in glioblastoma multiforme.

    Directory of Open Access Journals (Sweden)

    Enfeng Wang

    Full Text Available Glioblastoma multiforme (GBM accounts for about 38% of primary brain tumors in the United States. GBM is characterized by extensive angiogenesis induced by vascular growth factors and cytokines. The transcription of these growth factors and cytokines is regulated by the Hypoxia-Inducible-Factor-1(HIF-1, which is a key regulator mediating the cellular response to hypoxia. It is known that Factor Inhibiting HIF-1, or FIH-1, is also involved in the cellular response to hypoxia and has the capability to physically interact with HIF-1 and block its transcriptional activity under normoxic conditions. Delineation of the regulatory role of FIH-1 will help us to better understand the molecular mechanism responsible for tumor growth and progression and may lead to the design of new therapies targeting cellular pathways in response to hypoxia. Previous studies have shown that the chromosomal region of 10q24 containing the FIH-1 gene is often deleted in GBM, suggesting a role for the FIH-1 in GBM tumorigenesis and progression. In the current study, we found that FIH-1 is able to inhibit HIF-mediated transcription of GLUT1 and VEGF-A, even under hypoxic conditions in human glioblastoma cells. FIH-1 has been found to be more potent in inhibiting HIF function than PTEN. This observation points to the possibility that deletion of 10q23-24 and loss or decreased expression of FIH-1 gene may lead to a constitutive activation of HIF-1 activity, an alteration of HIF-1 targets such as GLUT-1 and VEGF-A, and may contribute to the survival of cancer cells in hypoxia and the development of hypervascularization observed in GBM. Therefore FIH-1 can be potential therapeutic target for the treatment of GBM patients with poor prognosis.

  3. Celecoxib inhibits Helicobacter pylori colonization-related factors

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM:To investigate the effect of celecoxib,a selective COX-2 inhibitor,on Helicobacter pylori(H.pylori) colonization-related factors and its mechanism.METHODS:After co-incubation with celecoxib,morphology of H.pylori strain 26695 was observed under a transmission electron microscope.Flagella motility was assessed by stab agar motility test.Adherence of H.pylori to AGS cells was determined by enzyme linked immunosorbent assay.Levels of mRNA expression in flagellar genes(flaA,flaB),urease genes(ureA,ureB)and ...

  4. Inhibition of Cronobacter sakazakii Virulence Factors by Citral

    Science.gov (United States)

    Shi, Chao; Sun, Yi; Liu, Zhiyuan; Guo, Du; Sun, Huihui; Sun, Zheng; Chen, Shan; Zhang, Wenting; Wen, Qiwu; Peng, Xiaoli; Xia, Xiaodong

    2017-01-01

    Cronobacter sakazakii is a foodborne pathogen associated with fatal forms of necrotizing enterocolitis, meningitis and sepsis in neonates and infants. The aim of this study was to determine whether citral, a major component of lemongrass oil, could suppress putative virulence factors of C. sakazakii that contribute to infection. Sub-inhibitory concentrations of citral significantly decreased motility, quorum sensing, biofilm formation and endotoxin production. Citral substantially reduced the adhesion and invasion of C. sakazakii to Caco-2 cells and decreased bacterial survival and replication within the RAW 264.7 macrophage cells. Citral also repressed the expression of eighteen genes involved in the virulence. These findings suggest that citral has potential to be developed as an alternative or supplemental agent to mitigate the infections caused by C. sakazakii. PMID:28233814

  5. Homoclinic Chaos in Axisymmetric Bianchi-IX cosmological models with an "ad hoc" quantum potential

    OpenAIRE

    Corrêa, G. C.; Stuchi, T. J.; Jorás, S. E.

    2010-01-01

    In this work we study the dynamics of the axisymmetric Bianchi IX cosmological model with a term of quantum potential added. As it is well known this class of Bianchi IX models are homogeneous and anisotropic with two scale factors, $A(t)$ and $B(t)$, derived from the solution of Einstein's equation for General Relativity. The model we use in this work has a cosmological constant and the matter content is dust. To this model we add a quantum-inspired potential that is intended to represent sh...

  6. Carbonic anhydrase IX, a hypoxia-induced catalytic component of the pH regulating machinery in tumors.

    Science.gov (United States)

    Sedlakova, Olga; Svastova, Eliska; Takacova, Martina; Kopacek, Juraj; Pastorek, Jaromir; Pastorekova, Silvia

    2014-01-08

    Acidic tissue microenvironment contributes to tumor progression via multiple effects including the activation of angiogenic factors and proteases, reduced cell-cell adhesion, increased migration and invasion, etc. In addition, intratumoral acidosis can influence the uptake of anticancer drugs and modulate the response of tumors to conventional therapy. Acidification of the tumor microenvironment often develops due to hypoxia-triggered oncogenic metabolism, which leads to the extensive production of lactate, protons, and carbon dioxide. In order to avoid intracellular accumulation of the acidic metabolic products, which is incompatible with the survival and proliferation, tumor cells activate molecular machinery that regulates pH by driving transmembrane inside-out and outside-in ion fluxes. Carbonic anhydrase IX (CA IX) is a hypoxia-induced catalytic component of the bicarbonate import arm of this machinery. Through its catalytic activity, CA IX directly participates in many acidosis-induced features of tumor phenotype as demonstrated by manipulating its expression and/or by in vitro mutagenesis. CA IX can function as a survival factor protecting tumor cells from hypoxia and acidosis, as a pro-migratory factor facilitating cell movement and invasion, as a signaling molecule transducing extracellular signals to intracellular pathways (including major signaling and metabolic cascades) and converting intracellular signals to extracellular effects on adhesion, proteolysis, and other processes. These functional implications of CA IX in cancer are supported by numerous clinical studies demonstrating the association of CA IX with various clinical correlates and markers of aggressive tumor behavior. Although our understanding of the many faces of CA IX is still incomplete, existing knowledge supports the view that CA IX is a biologically and clinically relevant molecule, exploitable in anticancer strategies aimed at targeting adaptive responses to hypoxia and/or acidosis.

  7. Complement Factor H Inhibits CD47-Mediated Resolution of Inflammation.

    Science.gov (United States)

    Calippe, Bertrand; Augustin, Sebastien; Beguier, Fanny; Charles-Messance, Hugo; Poupel, Lucie; Conart, Jean-Baptiste; Hu, Shulong J; Lavalette, Sophie; Fauvet, Alexandre; Rayes, Julie; Levy, Olivier; Raoul, William; Fitting, Catherine; Denèfle, Thomas; Pickering, Matthew C; Harris, Claire; Jorieux, Sylvie; Sullivan, Patrick M; Sahel, José-Alain; Karoyan, Philippe; Sapieha, Przemyslaw; Guillonneau, Xavier; Gautier, Emmanuel L; Sennlaub, Florian

    2017-02-21

    Variants of the CFH gene, encoding complement factor H (CFH), show strong association with age-related macular degeneration (AMD), a major cause of blindness. Here, we used murine models of AMD to examine the contribution of CFH to disease etiology. Cfh deletion protected the mice from the pathogenic subretinal accumulation of mononuclear phagocytes (MP) that characterize AMD and showed accelerated resolution of inflammation. MP persistence arose secondary to binding of CFH to CD11b, which obstructed the homeostatic elimination of MPs from the subretinal space mediated by thrombospsondin-1 (TSP-1) activation of CD47. The AMD-associated CFH(H402) variant markedly increased this inhibitory effect on microglial cells, supporting a causal link to disease etiology. This mechanism is not restricted to the eye, as similar results were observed in a model of acute sterile peritonitis. Pharmacological activation of CD47 accelerated resolution of both subretinal and peritoneal inflammation, with implications for the treatment of chronic inflammatory disease.

  8. Tumor necrosis factor-alpha inhibits pre-osteoblast differentiation through its type-1 receptor.

    Science.gov (United States)

    Abbas, Sabiha; Zhang, Yan-Hong; Clohisy, John C; Abu-Amer, Yousef

    2003-04-01

    Tumor necrosis factor-alpha (TNF) is a pro-inflammatory cytokine with a profound role in many skeletal diseases. The cytokine has been described as a mediator of bone loss in osteolysis and other inflammatory bone diseases. In addition to its known bone resorptive action, TNF reduces bone formation by inhibiting osteoblast differentiation. Using primary and transformed osteoblastic cells, we first document that TNF inhibits expression of alkaline phosphatase and matrix deposition, both considered markers of osteoblast differentiation. The effects are dose- and time-dependent. Core-binding factor A1 (cbfa1) is a transcription factor critical for osteoblast differentiation, and we show here that it is activated by the osteoblast differentiation agent, beta-glycerophosphate. Therefore, we investigated whether the inhibitory effects of TNF were associated with altered activity of this transcription factor. Using retardation assays, we show that TNF significantly inhibits cbfal activation by beta-glycerophosphate, manifested by reduced DNA-binding activity. Next, we turned to determine the signaling pathway by which TNF inhibits osteoblast differentiation. Utilizing animals lacking individual TNF receptors, we document that TNFr1 is required for transmitting the cytokine's inhibitory effect. In the absence of this receptor, TNF failed to impact all osteoblast differentiation markers tested. In summary, TNF blocks expression of osteoblast differentiation markers and inhibits beta-glycerophosphate-induced activation of the osteoblast differentiation factor cbfa1. Importantly, these effects are mediated via a mechanism requiring the TNF type-1 receptor.

  9. The RNA splicing factor ASF/SF2 inhibits human topoisomerase I mediated DNA relaxation

    DEFF Research Database (Denmark)

    Andersen, Félicie Faucon; Tange, Thomas Ø.; Sinnathamby, Thayaline;

    2002-01-01

    Human topoisomerase I interacts with and phosphorylates the SR-family of RNA splicing factors, including ASF/SF2, and has been suggested to play an important role in the regulation of RNA splicing. Here we present evidence to support the theory that the regulation can go the other way around...... with the SR-proteins controlling topoisomerase I DNA activity. We demonstrate that the splicing factor ASF/SF2 inhibits relaxation by interfering with the DNA cleavage and/or DNA binding steps of human topoisomerase I catalysis. The inhibition of relaxation correlated with the ability of various deletion...... extract reduced the inhibition of relaxation activity. Taken together with the previously published studies of the topoisomerase I kinase activity, these observations suggest that topoisomerase I activity is shifted from relaxation to kinasing by specific interaction with SR-splicing factors....

  10. The Broadband Spectral Variability of Holmberg IX X-1

    CERN Document Server

    Walton, D J; Harrison, F A; Middleton, M J; Fabian, A C; Bachetti, M; Barret, D; Miller, J M; Ptak, A; Rana, V; Stern, D; Tao, L

    2016-01-01

    We present the results from four new broadband X-ray observations of the extreme ultraluminous X-ray source Holmberg IX X-1 ($L_{\\rm{X}} > 10^{40}$ erg s$^{-1}$), performed by the $Suzaku$ and $NuSTAR$ observatories in coordination. Combined with the two prior observations coordinating $XMM$-$Newton$, $Suzaku$ and $NuSTAR$, we now have broadband observations of this remarkable source from six separate epochs. Two of these new observations probe lower fluxes than seen previously, allowing us to extend our knowledge of the broadband spectral variability exhibited by Holmberg IX X-1. The broadband spectra are well fit by two thermal blackbody components, which dominate the emission below 10 keV, as well as a steep ($\\Gamma \\sim 3.5$) powerlaw tail which dominates above $\\sim$15 keV. Remarkably, while the 0.3-10.0 keV flux varies by a factor of $\\sim$3 between all these epochs, the 15-40 keV flux varies by only $\\sim$20%. Although the spectral variability is strongest in the $\\sim$1-10 keV band, the broadband var...

  11. Heat stable cell growth inhibiting factor isolated from rat liver microsomes.

    Directory of Open Access Journals (Sweden)

    Inaba,Kozo

    1979-08-01

    Full Text Available A heat stable cell growth inhibiting factor was isolated from rat liver microsomes by hot salt extraction, ethanol fractionation and the hot phenol method. The factor was contained in the RNA fraction (designated as mhRNA. mhRNA inhibited the growth of mouse fibroblast (L-929 cells at a relatively low concentration (55 microgram/ml of culture medium. The molecular weight of mhRNA was about 27,000 and the base composition was guanine and cytosine rich.

  12. Epidermal growth factor inhibits hormone- and fibroblast growth factor-induced activation of phospholipase C in rat pancreatic acini.

    Science.gov (United States)

    Stryjek-Kaminska, D; Piiper, A; Caspary, W F; Zeuzem, S

    1995-01-01

    Epidermal growth factor (EGF) inhibits cholecystokinin-octapeptide-stimulated amylase release and inositol 1,4,5-trisphosphate (1,4,5-IP3) production in isolated rat pancreatic acini. In the present study, pancreatic acini were used to investigate the effect of EGF on amylase release and 1,4,5-IP3 production induced by secretagogues that activate either phospholipase C-beta (carbachol, bombesin) or phospholipase C-gamma [basic fibroblast growth factor (bFGF)]. The results show that EGF (100 ng/ml) inhibited bombesin (0.1 nM-1 microM)-induced amylase release almost completely. Similarly, the effect of EGF on carbachol-stimulated amylase release was substantial at submaximal (0.1 microM: 44% inhibition), maximal (1 microM: 75% inhibition), and supramaximal (100 microM: 33% inhibition) carbachol concentrations. EGF reduced amylase release at submaximal bFGF concentrations (0.1 nM: 40% inhibition), but not at supramaximal bFGF concentrations (1 and 10 nM). EGF decreased the peak increase of 1,4,5-IP3 in response to bombesin and carbachol (5 s after beginning of the incubation) and bFGF (15 s after beginning of the incubation) by 81 +/- 19%, 65 +/- 15%, and 56 +/- 18%, respectively. Receptor binding characteristics for secretagogues that activate phospholipase C were not influenced by coincubation with EGF excluding heterologous transmembrane receptor modulation. These results suggest that EGF inhibits the action of phospholipase C-beta- and gamma-isoenzyme-activating secretagogues in the exocrine pancreas by a postreceptor mechanism.

  13. Synthesis and biological evaluation of a {sup 99m}Tc-labelled sulfonamide conjugate for in vivo visualization of carbonic anhydrase IX expression in tumor hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Akurathi, Vamsidhar [Laboratory for Radiopharmacy, Katholieke Universiteit Leuven, 3000 Leuven (Belgium); Dubois, Ludwig; Lieuwes, Natasja G. [Department of Radiation Oncology (MAASTRO), GROW School for Oncology and Developmental Biology, University of Maastricht, 6200 Maastricht (Netherlands); Chitneni, Satish K. [Department of Radiology, Duke University Medical Centre, Durham, NC 27710 (United States); Cleynhens, Bernard J. [Laboratory for Radiopharmacy, Katholieke Universiteit Leuven, 3000 Leuven (Belgium); Vullo, Daniela; Supuran, Claudiu T. [Universita degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, 50019 Sesto Fiorentino, Florence (Italy); Verbruggen, Alfons M. [Laboratory for Radiopharmacy, Katholieke Universiteit Leuven, 3000 Leuven (Belgium); Lambin, Philippe [Department of Radiation Oncology (MAASTRO), GROW School for Oncology and Developmental Biology, University of Maastricht, 6200 Maastricht (Netherlands); Bormans, Guy M., E-mail: guy.bormans@pharm.kuleuven.b [Laboratory for Radiopharmacy, Katholieke Universiteit Leuven, 3000 Leuven (Belgium)

    2010-07-15

    Introduction: Carbonic anhydrase (CA) IX is a transmembrane protein overexpressed in many frequently occurring tumors associated with tumor hypoxia. Sulfonamides and their bioisosteres are known to inhibit CA IX activity. In this study, 4-(2-aminoethyl)benzenesulfonamide was conjugated to a tridentate ligand, N-2-picolyl-N-acetic acid and labeled with a {sup 99m}Tc(I)-tricarbonyl moiety resulting in [{sup 99m}Tc(CO){sub 3} (L)] (L=N-(pyridin-2-yl-methyl)-N[2-(4-sulfamoylphenyl)-ethyl]aminoethyl acetate) complex, [{sup 99m}Tc]-5. Similarly the corresponding rhenium congener (Re-4) was synthesized. The in vitro CA IX affinity and inhibitory activity of Re-4 were determined and [{sup 99m}Tc]-5 was evaluated as a tracer for in vivo visualisation of CA IX expression. Methods: Evaluation of the in vitro affinity (inhibition constant, K{sub i}) of Re-4 for CA isozymes I, II, IX and XII was carried out by assaying the CA catalyzed CO{sub 2} hydration activity and efficacy studies were performed in HT 29 cell lines expressing CA IX under normoxia or hypoxia. Biodistribution studies of [{sup 99m}Tc]-5 were performed in xenograft mice bearing CA IX expressing tumors. Results: The in vitro affinity of Re-4 for CA IX was 58 nM and CA IX induced acidification of extracellular medium was efficiently reduced (P<.05) in the presence of 1 mM Re-4. Biodistribution studies indicated a maximal tumor uptake of [{sup 99m}Tc]-5 of 0.1% ID/g at 30 min post injection. Conclusion: [{sup 99m}Tc]-5 and its rhenium congener were synthesized and characterized. In vitro studies showed that the rhenium compound has a high affinity for CA IX and effectively inhibits CA IX activity. In vivo studies revealed a limited tracer accumulation in a CA IX expressing tumor but with increasing tumor-to-blood activity ratios as a function of time.

  14. Glycoprotein Ib-IX-V Complex Transmits Cytoskeletal Forces That Enhance Platelet Adhesion.

    Science.gov (United States)

    Feghhi, Shirin; Munday, Adam D; Tooley, Wes W; Rajsekar, Shreya; Fura, Adriane M; Kulman, John D; López, Jose A; Sniadecki, Nathan J

    2016-08-09

    Platelets bind to exposed vascular matrix at a wound site through a highly specialized surface receptor, glycoprotein (GP) Ib-IX-V complex, which recognizes von Willebrand factor (VWF) in the matrix. GPIb-IX-V is a catch bond for it becomes more stable as force is applied to it. After attaching to the wound site, platelets generate cytoskeletal forces to compact and reinforce the hemostatic plug. Here, we evaluated the role of the GPIb-IX-V complex in the transmission of cytoskeletal forces. We used arrays of flexible, silicone nanoposts to measure the contractility of individual platelets on VWF. We found that a significant proportion of cytoskeletal forces were transmitted to VWF through GPIb-IX-V, an unexpected finding given the widely held notion that platelet forces are transmitted exclusively through its integrins. In particular, we found that the interaction between GPIbα and the A1 domain of VWF mediates this force transmission. We also demonstrate that the binding interaction between GPIbα and filamin A is involved in force transmission. Furthermore, our studies suggest that cytoskeletal forces acting through GPIbα are involved in maintaining platelet adhesion when external forces are absent. Thus, the GPIb-IX-V/VWF bond is able to transmit force, and uses this force to strengthen the bond through a catch-bond mechanism. This finding expands our understanding of how platelets attach to sites of vascular injury, describing a new, to the best of our knowledge, mechanism in which the catch bonds of GPIb-IX-V/VWF can be supported by internal forces produced by cytoskeletal tension.

  15. Protein disulfide isomerase inhibition blocks thrombin generation in humans by interfering with platelet factor V activation

    Science.gov (United States)

    Stopa, Jack D.; Neuberg, Donna; Puligandla, Maneka; Furie, Bruce; Zwicker, Jeffrey I.

    2017-01-01

    BACKGROUND: Protein disulfide isomerase (PDI) is required for thrombus formation. We previously demonstrated that glycosylated quercetin flavonoids such as isoquercetin inhibit PDI activity and thrombus formation in animal models, but whether extracellular PDI represents a viable anticoagulant target in humans and how its inhibition affects blood coagulation remain unknown. METHODS: We evaluated effects of oral administration of isoquercetin on platelet-dependent thrombin generation in healthy subjects and patients with persistently elevated anti-phospholipid antibodies. RESULTS: Following oral administration of 1,000 mg isoquercetin to healthy adults, the measured peak plasma quercetin concentration (9.2 μM) exceeded its IC50 for inhibition of PDI by isoquercetin in vitro (2.5 ± 0.4 μM). Platelet-dependent thrombin generation decreased by 51% in the healthy volunteers compared with baseline (P = 0.0004) and by 64% in the anti-phospholipid antibody cohort (P = 0.015) following isoquercetin ingestion. To understand how PDI affects thrombin generation, we evaluated substrates of PDI identified using an unbiased mechanistic-based substrate trapping approach. These studies identified platelet factor V as a PDI substrate. Isoquercetin blocked both platelet factor Va and thrombin generation with an IC50 of ~5 μM. Inhibition of PDI by isoquercetin ingestion resulted in a 53% decrease in the generation of platelet factor Va (P = 0.001). Isoquercetin-mediated inhibition was reversed with addition of exogenous factor Va. CONCLUSION: These studies show that oral administration of isoquercetin inhibits PDI activity in plasma and diminishes platelet-dependent thrombin generation predominantly by blocking the generation of platelet factor Va. These pharmacodynamic and mechanistic observations represent an important step in the development of a novel class of antithrombotic agents targeting PDI. TRIAL REGISTRATION: Clinicaltrials.gov (NCT01722669) FUNDING: National Heart

  16. Carbonic anhydrase IX as a specific biomarker for clear cell renal cell carcinoma: comparative study of Western blot and immunohistochemistry and implications for diagnosis.

    Science.gov (United States)

    Giménez-Bachs, José M; Salinas-Sánchez, Antonio S; Serrano-Oviedo, Leticia; Nam-Cha, Syong H; Rubio-Del Campo, Antonio; Sánchez-Prieto, Ricardo

    2012-10-01

    This study aimed to evaluate the usefulness of carbonic anhydrase IX (CA-IX) expression in clear cell renal cell carcinoma (CCRCC) using two different techniques to detect protein expression. An experimental, cross-sectional, analytical study was conducted to analyse proteins in renal tumour and healthy tissue specimens from 38 consecutive patients who underwent nephrectomy for renal cancer. CA-IX protein expression was measured by immunohistochemistry and Western blot analysis and quantified. Statistical analysis was performed with the positive and negative specific agreements and kappa coefficient. The sensitivity and specificity of both techniques were assessed. Statistical tests were conducted to analyse the association between CA-IX expression quantitation and normal prognosis factors (TNM stage and Fuhrman nuclear grade), only in CCRCC. The mean patient age was 65 years, 78.9% of patients were men and 57.9% of tumours were CCRCC. CA-IX protein expression was positive in 63.2% of tumours by immunohistochemistry and in 60.5% by Western blot. Both techniques detected CA-IX expression only in CCRCC and unclassifiable tumours. High concordance indices were observed for CCRCC diagnosis. Western blot and immunohistochemistry had a sensitivity of 95.5% and 100%, respectively; the specificity was 100% in both techniques. CA-IX expression quantitation did not correlate with tumour stage or Fuhrman nuclear grade. Immunochemistry and Western blot techniques can be used to detect abnormal CA-IX protein expression in CCRCC and to support morphology-based diagnostic techniques.

  17. The Role of Factor XIa (FXIa) Catalytic Domain Exosite Residues in Substrate Catalysis and Inhibition by the Kunitz Protease Inhibitor Domain of Protease Nexin 2*

    Science.gov (United States)

    Su, Ya-Chi; Miller, Tara N.; Navaneetham, Duraiswamy; Schoonmaker, Robert T.; Sinha, Dipali; Walsh, Peter N.

    2011-01-01

    To select residues in coagulation factor XIa (FXIa) potentially important for substrate and inhibitor interactions, we examined the crystal structure of the complex between the catalytic domain of FXIa and the Kunitz protease inhibitor (KPI) domain of a physiologically relevant FXIa inhibitor, protease nexin 2 (PN2). Six FXIa catalytic domain residues (Glu98, Tyr143, Ile151, Arg3704, Lys192, and Tyr5901) were subjected to mutational analysis to investigate the molecular interactions between FXIa and the small synthetic substrate (S-2366), the macromolecular substrate (factor IX (FIX)) and inhibitor PN2KPI. Analysis of all six Ala mutants demonstrated normal Km values for S-2366 hydrolysis, indicating normal substrate binding compared with plasma FXIa; however, all except E98A and K192A had impaired values of kcat for S-2366 hydrolysis. All six Ala mutants displayed deficient kcat values for FIX hydrolysis, and all were inhibited by PN2KPI with normal values of Ki except for K192A, and Y5901A, which displayed increased values of Ki. The integrity of the S1 binding site residue, Asp189, utilizing p-aminobenzamidine, was intact for all FXIa mutants. Thus, whereas all six residues are essential for catalysis of the macromolecular substrate (FIX), only four (Tyr143, Ile151, Arg3704, and Tyr5901) are important for S-2366 hydrolysis; Glu98 and Lys192 are essential for FIX but not S-2366 hydrolysis; and Lys192 and Tyr5901 are required for both inhibitor and macromolecular substrate interactions. PMID:21778227

  18. Carbonic anhydrase IX is a marker of hypoxia and correlates with higher Gleason scores and ISUP grading in prostate cancer.

    Science.gov (United States)

    Ambrosio, Maria Raffaella; Di Serio, Claudia; Danza, Giovanna; Rocca, Bruno Jim; Ginori, Alessandro; Prudovsky, Igor; Marchionni, Niccolò; Del Vecchio, Maria Teresa; Tarantini, Francesca

    2016-05-25

    Carbonic anhydrase IX is a member of α-carbonic anhydrases that is preferentially expressed in solid tumors. It enables bicarbonate transport across the plasma membrane, neutralizing intracellular pH and conferring to cancer cells a survival advantage in hypoxic/acidic microenvironments. Overexpression of carbonic anhydrase IX in cancer tissues is regulated by hypoxia inducible factor 1α - mediated transcription and the enzyme is considered a marker of tumor hypoxia and poor outcome. The role of carbonic anhydrase IX in prostate cancer has not been fully clarified and controversy has arisen on whether this enzyme is overexpressed in hypoxic prostate cancer tissues. We analyzed the expression of carbonic anhydrase IX and hypoxia inducible factor 1α in two prostate cancer cell lines, LNCaP and PC-3, and in 110 cancer biopsies, by western blotting and immunocyto/histochemistry. In LNCaP and PC-3 cells, carbonic anhydrase IX was mostly cytoplasmic/nuclear, with very limited membrane localization. Nuclear staining became stronger under hypoxia. When we analyzed carbonic anhydrase IX expression in human prostate cancer biopsies, we found that protein staining positively correlated with hypoxia inducible factor 1α and with Gleason pattern and score, as well as with the novel grading system proposed by the International Society of Urological Pathology for prostate cancer. Once more, carbonic anhydrase IX was mainly cytoplasmic in low grade carcinomas, whereas in high grade tumors was strongly expressed in the nucleus of the neoplastic cell. An association between carbonic anhydrase IX expression level and the main clinic-pathological features involved in prostate cancer aggressiveness was identified. There was a statistically significant association between carbonic anhydrase IX and hypoxia inducible factor 1α in prostate cancer tissues, that identifies the enzyme as a reliable marker of tumor hypoxia. In addition, carbonic anhydrase IX expression positively

  19. Lyso-Sulfatide Binds Factor Xa and Inhibits Thrombin Generation by the Prothrombinase Complex.

    Directory of Open Access Journals (Sweden)

    Subramanian Yegneswaran

    Full Text Available Blood coagulation reactions are strongly influenced by phospholipids, but little is known about the influence of sphingolipids on coagulation mechanisms. Lysosulfatide (lyso-SF (sulfogalactosyl sphingosine prolonged factor Xa (fXa 1-stage plasma clotting assays, showing it had robust anticoagulant activity. In studies using purified clotting factors, lyso-SF inhibited >90% of prothrombin (II activation for reaction mixtures containing fXa/factor Va (fVa/II, and also inhibited II activation generation by fXa/ phospholipids and by Gla-domainless-fXa/fVa/phospholipids. When lyso-SF analogs were tested, results showed that N-acetyl-sulfatide was not anticoagulant, implying that the free amine group was essential for the anticoagulant effects of lyso-SF. Lyso-SF did not inhibit fXa enzymatic hydrolysis of small peptide substrates, showing it did not directly inhibit the fXa activity. In surface plasmon resonance studies, lyso-SF bound to immobilized inactivated fXa as well as inactivated Gla-domainless-fXa. Confirming this lyso-SF:fXa interaction, fluorescence studies showed that fluorescently-labeled-fXa in solution bound to lyso-SF. Thus, lyso-SF is an anticoagulant lipid that inhibits fXa when this enzyme is bound to either phospholipids or to fVa. Mechanisms for inhibition of procoagulant activity are likely to involve lyso-SF binding to fXa domain(s that are distinct from the fXa Gla domain. This suggests that certain sphingolipids, including lyso-SF and some of its analogs, may down-regulate fXa activity without inhibiting the enzyme's active site or binding to the fXa Gla domain.

  20. MiR-34a inhibits colon cancer proliferation and metastasis by inhibiting platelet-derived growth factor receptor α.

    Science.gov (United States)

    Li, Chunyan; Wang, Yulin; Lu, Shuming; Zhang, Zhuqing; Meng, Hua; Liang, Lina; Zhang, Yan; Song, Bo

    2015-11-01

    The microRNA (miRNA), miR‑34a is significant in colon cancer progression. In the present study, the role of miR‑34a in colon cancer cell proliferation and metastasis was investigated. It was found that the expression of miR‑34a in colon cancer tissues and cell lines was lower when compared with that of normal tissues and cells. Further research demonstrated that miR‑34a inhibited cell proliferation, induced G1 phase arrest, and suppressed metastasis and epithelial mesenchymal transition in colon cancer cells. Bioinformatic prediction indicated that platelet‑derived growth factor receptor α (PDGFRA) was a potential target gene of miR‑34a and a luciferase assay identified that PDGFRA was a novel direct target gene of miR‑34a. In addition, assays of western blot analyses and quantitative reverse‑transcription polymerase chain reaction confirmed that miR‑34a decreased PDGFRA mRNA expression and protein levels in colon cancer cells. Assessment of cellular function indicated that miR‑34a inhibited colon cancer progression via PDGFRA. These findings demonstrate that miR‑34a may act as a negative regulator in colon cancer by targeting PDGFRA.

  1. Tumor necrosis factor-alpha inhibits myogenesis through redox-dependent and -independent pathways

    NARCIS (Netherlands)

    Langen, R.C.J.; Schols, A.M.W.J.; Kelders, M.C.J.M.; van der Velden, A.L.J.; Wouters, E.F.M.; Janssen, Y.M.W.

    2002-01-01

    Tumor necrosis factor-alpha inhibits myogenesis through redox-dependent and -independent pathways. Langen RC, Schols AM, Kelders MC, Van Der Velden JL, Wouters EF, Janssen-Heininger YM. Department of Pulmonology, Maastricht University, The Netherlands. Muscle wasting accompanies diseases that are as

  2. RNA interference inhibits expression of vascular endothelial growth factor (VEGF) in human retinal pigment epithelial cells

    Institute of Scientific and Technical Information of China (English)

    CAI Chun-mei; SUN Bao-chen; LIU Xu-yang; WANG Jin-jin; LI Jun-fa; HAN Song; WANG Ning-li; LU Qing-jun

    2005-01-01

    @@ Choroidal neovascularization (CNV), a major cause of vision loss, is the result of the increased vascular endothelial growth factor (VEGF) expression in human retinal pigment epithelial (RPE) cells. It is important to inhibit the expression of VEGF protein in RPE cells.

  3. Faculty as Mentors in Undergraduate Research, Scholarship, and Creative Work: Motivating and Inhibiting Factors

    Science.gov (United States)

    Baker, Vicki L.; Pifer, Meghan J.; Lunsford, Laura G.; Greer, Jane; Ihas, Dijana

    2015-01-01

    In this study, we sought to contribute to research about the high-impact practice of undergraduate research from the understudied faculty perspective. We relied on focus group data from faculty members (N = 41) across five institutions to better understand the supporting and inhibiting factors that contribute to faculty members' engagement in…

  4. Shikonin Promotes Skin Cell Proliferation and Inhibits Nuclear Factor-κB Translocation via Proteasome Inhibition In Vitro

    Institute of Scientific and Technical Information of China (English)

    Yan Yan; Minao Furumura; Takako Gouya; Atsufumi Iwanaga; Kwesi Teye; Sanae Numata; Tadashi Karashima

    2015-01-01

    Background:Shikonin is a major active chemical component extracted from Lithospermi Radix,an effective traditional herb in various types of wound healing.Shikonin can accelerate granulomatous tissue formation by the rat cotton pellet method and induce neovascularization in granulomatous tissue.The purpose of the study was to investigate its mechanism of action in human skin cells.Methods:MTS assay was used to measure cell growth.The collagen type Ⅰ (COL1) mRNA expression and procollagen type Ⅰ C-peptide (PIP) production were detected by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay,respectively.Immunofluorescence and western blot analyses were carried out to investigate nuclear factor-κB (NF-κB) signaling pathway.Cell-based proteasome activity assay was used to determine proteasome activity.Results:In this study,we found that 10 μmol/L shikonin stimulated the growth of normal human keratinocytes and 1 μmol/L shikonin promoted growth of human dermal fibroblasts.However,shikonin did not directly induce COLI mRNA expression and PIP production in dermal fibroblasts in vitro.In addition,1 μmol/L shikonin inhibited translocation of NF-κB p65 from cytoplasm to nucleus induced by tumor necrosis factor-α stimulation in dermal fibroblasts.Furthermore,shikonin inhibited chymotrypsin-like activity of proteasome and was associated with accumulation ofphosphorylated inhibitor κB-α in dermal fibroblasts.Conclusions:These results suggested that shikonin may promote wound healing via its cell growth promoting activity and suppress skin inflammation via inhibitory activity on proteasome.Thus,shikonin may be a potential therapeutic reagent both in wound healing and inflammatory skin diseases.

  5. Purification and Characterization of a Sperm Motility Inhibiting Factor from Caprine Epididymal Plasma

    Science.gov (United States)

    Das, Sujoy; Saha, Sudipta; Majumder, Gopal Chandra; Dungdung, Sandhya Rekha

    2010-01-01

    Several studies have been reported on the occurrence of sperm motility inhibiting factors in the male reproductive fluids of different mammalian species, but these proteins have not been adequately purified and characterized. A novel sperm motility inhibiting factor (MIF-II) has been purified from caprine epididymal plasma (EP) by Hydroxylapatite gel adsorption chromatography, DEAE-Cellulose ion-exchange chromatography and chromatofocusing. The MIF-II has been purified to apparent homogeneity and the molecular weight estimated by Sephacryl S-300 gel filtration is 160 kDa. MIF-II is a dimeric protein, made up of two subunits each having a molecular mass of 80 kDa as shown by SDS-PAGE. The isoelectric point of MIF-II is 5.1 as determined by chromatofocusing and isoelectric focusing. It is a heat labile protein and maximal active at the pH 6.9 to 7.5. The sperm motility inhibiting protein factor at 2 µg/ml (12.5 nM) level showed maximal motility-inhibiting activity. The observation that the epididymal plasma factor lowered the intracellular cAMP level of spermatozoa in a concentration-dependent manner suggests that it may block the motility of caprine cauda spermatozoa by interfering the cAMP dependent motility function. The results revealed that the purified protein factor has the potential of sperm motility inhibition and may serve as a vaginal contraceptive. The antibody raised against the MIF-II has the potential for enhancement of forward motility of cauda-spermatozoa. This antibody may thus be useful for solving some of the problems of male infertility due to low sperm motility. PMID:20706623

  6. Clopidogrel inhibits angiogenesis of gastric ulcer healing via downregulation of vascular endothelial growth factor receptor 2.

    Science.gov (United States)

    Luo, Jiing-Chyuan; Peng, Yen-Ling; Chen, Tseng-Shing; Huo, Teh-Ia; Hou, Ming-Chih; Huang, Hui-Chun; Lin, Han-Chieh; Lee, Fa-Yauh

    2016-09-01

    Although clopidogrel does not cause gastric mucosal injury, it does not prevent peptic ulcer recurrence in high-risk patients. We explored whether clopidogrel delays gastric ulcer healing via inhibiting angiogenesis and to elucidate the possible mechanisms. Gastric ulcers were induced in Sprague Dawley rats, and ulcer healing and angiogenesis of ulcer margin were compared between clopidogrel-treated rats and controls. The expressions of the proangiogenic growth factors and their receptors including basic fibroblast growth factor (bFGF), bFGF receptor (FGFR), vascular endothelial growth factor (VEGF), VEGFR1, VEGFR2, platelet-derived growth factor (PDGF)A, PDGFB, PDGFR A, PDGFR B, and phosphorylated form of mitogenic activated protein kinase pathways over the ulcer margin were compared via western blot and reverse transcription polymerase chain reaction. In vitro, human umbilical vein endothelial cells (HUVECs) were used to elucidate how clopidogrel inhibited growth factors-stimulated HUVEC proliferation. The ulcer sizes were significantly larger and the angiogenesis of ulcer margin was significantly diminished in the clopidogrel (2 and 10 mg/kg/d) treated groups. Ulcer induction markedly increased the expression of phosphorylated form of extracellular signal-regulated kinase (pERK), FGFR2, VEGF, VEGFR2, and PDGFRA when compared with those of normal mucosa. Clopidogrel treatment significantly decreased pERK, FGFR2, VEGF, VEGFR2, and PDGFRA expression at the ulcer margin when compared with those of the respective control group. In vitro, clopidogrel (10(-6)M) inhibited VEGF-stimulated (20 ng/mL) HUVEC proliferation, at least, via downregulation of VEGFR2 and pERK. Clopidogrel inhibits the angiogenesis of gastric ulcer healing at least partially by the inhibition of the VEGF-VEGFR2-ERK signal transduction pathway. Copyright © 2015. Published by Elsevier B.V.

  7. Purification and characterization of a sperm motility inhibiting factor from caprine epididymal plasma.

    Directory of Open Access Journals (Sweden)

    Sujoy Das

    Full Text Available Several studies have been reported on the occurrence of sperm motility inhibiting factors in the male reproductive fluids of different mammalian species, but these proteins have not been adequately purified and characterized. A novel sperm motility inhibiting factor (MIF-II has been purified from caprine epididymal plasma (EP by Hydroxylapatite gel adsorption chromatography, DEAE-Cellulose ion-exchange chromatography and chromatofocusing. The MIF-II has been purified to apparent homogeneity and the molecular weight estimated by Sephacryl S-300 gel filtration is 160 kDa. MIF-II is a dimeric protein, made up of two subunits each having a molecular mass of 80 kDa as shown by SDS-PAGE. The isoelectric point of MIF-II is 5.1 as determined by chromatofocusing and isoelectric focusing. It is a heat labile protein and maximal active at the pH 6.9 to 7.5. The sperm motility inhibiting protein factor at 2 microg/ml (12.5 nM level showed maximal motility-inhibiting activity. The observation that the epididymal plasma factor lowered the intracellular cAMP level of spermatozoa in a concentration-dependent manner suggests that it may block the motility of caprine cauda spermatozoa by interfering the cAMP dependent motility function. The results revealed that the purified protein factor has the potential of sperm motility inhibition and may serve as a vaginal contraceptive. The antibody raised against the MIF-II has the potential for enhancement of forward motility of cauda-spermatozoa. This antibody may thus be useful for solving some of the problems of male infertility due to low sperm motility.

  8. Kruppel-like factor-9 (KLF9) inhibits glioblastoma stemness through global transcription repression and integrin α6 inhibition.

    Science.gov (United States)

    Ying, Mingyao; Tilghman, Jessica; Wei, Yingying; Guerrero-Cazares, Hugo; Quinones-Hinojosa, Alfredo; Ji, Hongkai; Laterra, John

    2014-11-21

    It is increasingly important to understand the molecular basis for the plasticity of neoplastic cells and their capacity to transition between differentiated and stemlike phenotypes. Kruppel-like factor-9 (KLF9), a member of the large KLF transcription factor family, has emerged as a regulator of oncogenesis, cell differentiation, and neural development; however, the molecular basis for the diverse contextual functions of KLF9 remains unclear. This study focused on the functions of KLF9 in human glioblastoma stemlike cells. We established for the first time a genome-wide map of KLF9-regulated targets in human glioblastoma stemlike cells and show that KLF9 functions as a transcriptional repressor and thereby regulates multiple signaling pathways involved in oncogenesis and stem cell regulation. A detailed analysis of one such pathway, integrin signaling, showed that the capacity of KLF9 to inhibit glioblastoma cell stemness and tumorigenicity requires ITGA6 repression. These findings enhance our understanding of the transcriptional networks underlying cancer cell stemness and differentiation and identify KLF9-regulated molecular targets applicable to cancer therapeutics.

  9. Bianchi IX Cosmologies and the Golden Ratio

    CERN Document Server

    Bryant, M S

    2016-01-01

    Solutions to the Einstein equations for Bianchi IX cosmologies are examined through the use of Ellis MacCallum Wainwright (expansion-normalized) variables. Using an iterative map derived from the Einstein equations one can construct an infinite number of periodic solutions. The simplest periodic solutions consist of 3-cycles. It is shown that for 3-cycles the time series of the logarithms of the expansion-normalized spatial curvature components vs normalized time (which is runs backwards towards the initial singularity), generates a set of self-similar golden rectangles. In addition the golden ratio appears in other aspects of the same time series representation.

  10. MiR-125a TNF receptor-associated factor 6 to inhibit osteoclastogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Li-Juan; Liao, Lan [Department of Endocrinology, Xiangya Hospital of Central South University, 87 Xiangya Road, Changsha, Hunan 410008 (China); Yang, Li [Department of Endocrinology, Hunan Province Geriatric Hospital, Changsha, Hunan 410001 (China); Li, Yu [Department of Endocrinology, Xiangya Hospital of Central South University, 87 Xiangya Road, Changsha, Hunan 410008 (China); Jiang, Tie-Jian, E-mail: jiangtiejian@gmail.com [Department of Endocrinology, Xiangya Hospital of Central South University, 87 Xiangya Road, Changsha, Hunan 410008 (China)

    2014-02-15

    MicroRNAs (miRNAs) play important roles in osteoclastogenesis and bone resorption. In the present study, we found that miR-125a was dramatically down-regulated during macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) induced osteoclastogenesis of circulating CD14+ peripheral blood mononuclear cells (PBMCs). Overexpression of miR-125a in CD14+ PBMCs inhibited osteoclastogenesis, while inhibition of miR-125a promoted osteoclastogenesis. TNF receptor-associated factor 6 (TRAF6), a transduction factor for RANKL/RANK/NFATc1 signal, was confirmed to be a target of miR-125a. EMSA and ChIP assays confirmed that NFATc1 bound to the promoter of the miR-125a. Overexpression of NFATc1 inhibited miR-125a transcription, and block of NFATc1 expression attenuated RANKL-regulated miR-125a transcription. Here, we reported that miR-125a played a biological function in osteoclastogenesis through a novel TRAF6/ NFATc1/miR-125a regulatory feedback loop. It suggests that regulation of miR-125a expression may be a potential strategy for ameliorating metabolic disease. - Highlights: • MiR-125a was significantly down-regulated in osteoclastogenesis of CD14+ PBMCs. • MiR-125a inhibited osteoclast differentiation by targeting TRAF6. • NFATc1 inhibited miR-125a transciption by binding to the promoter of miR-125a. • TRAF6/NFATc1 and miR-125a form a regulatory feedback loop in osteoclastogenesis.

  11. A bacteriophage transcription regulator inhibits bacterial transcription initiation by σ-factor displacement.

    Science.gov (United States)

    Liu, Bing; Shadrin, Andrey; Sheppard, Carol; Mekler, Vladimir; Xu, Yingqi; Severinov, Konstantin; Matthews, Steve; Wigneshweraraj, Sivaramesh

    2014-04-01

    Bacteriophages (phages) appropriate essential processes of bacterial hosts to benefit their own development. The multisubunit bacterial RNA polymerase (RNAp) enzyme, which catalyses DNA transcription, is targeted by phage-encoded transcription regulators that selectively modulate its activity. Here, we describe the structural and mechanistic basis for the inhibition of bacterial RNAp by the transcription regulator P7 encoded by Xanthomonas oryzae phage Xp10. We reveal that P7 uses a two-step mechanism to simultaneously interact with the catalytic β and β' subunits of the bacterial RNAp and inhibits transcription initiation by inducing the displacement of the σ(70)-factor on initial engagement of RNAp with promoter DNA. The new mode of interaction with and inhibition mechanism of bacterial RNAp by P7 underscore the remarkable variety of mechanisms evolved by phages to interfere with host transcription.

  12. A synthetic manassantin a derivative inhibits hypoxia-inducible factor 1 and tumor growth.

    Science.gov (United States)

    Lang, Liwei; Liu, Xiaoyu; Li, Yan; Zhou, Qing; Xie, Ping; Yan, Chunhong; Chen, Xiaoguang

    2014-01-01

    The dineolignan manassantin A from Saururaceae was recently identified as a hypoxia-inducible factor 1 (HIF-1) inhibitor, but its in-vivo anti-tumor effect has not been explored. We synthesized a series of manassantin A derivatives, and found that replacing the central tetrahydrofuran moiety with a cyclopentane ring yielded a compound (LXY6006) with increased HIF-1-inhibitory activity yet decreased stereochemically complexity amenable to a simplified synthesis scheme. LXY6006 inhibited HIF-1α nuclear accumulation induced by hypoxia, and inhibited cancer cell growth as a consequence of G2/M arrest. Oral administration of LXY6006 significantly inhibited growth of breast, lung, and pancreatic tumors implanted in nude mice. These results indicate that LXY6006 represents a novel class of agents targeting a broad range of human cancers.

  13. A synthetic manassantin a derivative inhibits hypoxia-inducible factor 1 and tumor growth.

    Directory of Open Access Journals (Sweden)

    Liwei Lang

    Full Text Available The dineolignan manassantin A from Saururaceae was recently identified as a hypoxia-inducible factor 1 (HIF-1 inhibitor, but its in-vivo anti-tumor effect has not been explored. We synthesized a series of manassantin A derivatives, and found that replacing the central tetrahydrofuran moiety with a cyclopentane ring yielded a compound (LXY6006 with increased HIF-1-inhibitory activity yet decreased stereochemically complexity amenable to a simplified synthesis scheme. LXY6006 inhibited HIF-1α nuclear accumulation induced by hypoxia, and inhibited cancer cell growth as a consequence of G2/M arrest. Oral administration of LXY6006 significantly inhibited growth of breast, lung, and pancreatic tumors implanted in nude mice. These results indicate that LXY6006 represents a novel class of agents targeting a broad range of human cancers.

  14. Cyclooxygenase-2 inhibitor, celecoxib, inhibits leiomyoma cell proliferation through the nuclear factor κB pathway.

    Science.gov (United States)

    Park, Seung Bin; Jee, Byung Chul; Kim, Seok Hyun; Cho, Yeon Jean; Han, Myoungseok

    2014-09-01

    Our aim was to investigate whether celecoxib, a cyclooxygenase 2 (COX-2) inhibitor, decreases the in vitro proliferation of leiomyoma cells if the inflammatory pathway is blocked. Menstruation is an inflammation of uterus that produces cytokines and prostanoids, but the inflammatory mechanism underlying the growth of leiomyoma remains unexplained. Using in vitro cultures of leiomyoma cells obtained from 5 patients who underwent hysterectomy, cell proliferation, inflammatory signaling, transcription factors, growth factors, and extracellular matrix were examined by (4,5-dimethylthiaxol-2-yi)-2,5-diphenyltetraxolium bromide assay, immunoblotting, and quantitative polymerase chain reaction. Prostaglandin E2 was used to induce menstruation-like condition in the cells. We found that celecoxib inhibited COX-2 through the expression of nuclear factor κB in the cells. Celcoxib also decreased the gene expression of interleukin 6, tumor necrosis factor α, collagen A, fibronectin, platelet-derived growth factor, epidermal growth factor, and transforming growth factor β. In conclusion, the present study indicated that celecoxib could inhibit leiomyoma cell proliferation through blocking the inflammatory pathway that is probably one of the mechanisms underlying its pathogenesis.

  15. The M358R variant of α(1)-proteinase inhibitor inhibits coagulation factor VIIa.

    Science.gov (United States)

    Sheffield, William P; Bhakta, Varsha

    2016-02-12

    The naturally occurring M358R mutation of the plasma serpin α1-proteinase inhibitor (API) changes both its cleavable reactive centre bond to Arg-Ser and the efficacy with which it inhibits different proteases, reducing the rate of inhibition of neutrophil elastase, and enhancing that of thrombin, factor XIa, and kallikrein, by several orders of magnitude. Although another plasma serpin with an Arg-Ser reactive centre, antithrombin (AT), has been shown to inhibit factor VIIa (FVIIa), no published data are available with respect to FVIIa inhibition by API M358R. Recombinant bacterially-expressed API M358R and plasma-derived AT were therefore compared using gel-based and kinetic assays of FVIIa integrity and activity. Under pseudo-first order conditions of excess serpin over protease, both AT and API M358R formed denaturation-resistant inhibitory complexes with FVIIa in reactions accelerated by TF; AT, but not API M358R, also required heparin for maximal activity. The second order rate constant for heparin-independent API M358R-mediated FVIIa inhibition was determined to be 7.8 ± 0.8 × 10(2) M(-1)sec(-1). We conclude that API M358R inhibits FVIIa by forming inhibitory complexes of the serpin type more rapidly than AT in the absence of heparin. The likely 20-fold excess of API M358R over AT in patient plasma during inflammation raises the possibility that it could contribute to the hemorrhagic tendencies manifested by rare individuals expressing this mutant serpin.

  16. Resveratrol inhibits growth of orthotopic pancreatic tumors through activation of FOXO transcription factors.

    Directory of Open Access Journals (Sweden)

    Sanjit K Roy

    Full Text Available BACKGROUND: The forkhead transcription factors of the O class (FOXO play a direct role in cellular proliferation, oxidative stress response, and tumorigenesis. The objectives of this study were to examine whether FOXOs regulate antitumor activities of resveratrol in pancreatic cancer cells in vitro and in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Pancreatic cancer cell lines were treated with resveratrol. Cell viability, colony formation, apoptosis and cell cycle were measured by XTT, soft agar, TUNEL and flow cytometry assays, respectively. FOXO nuclear translocation, DNA binding and transcriptional activities were measured by fluorescence technique, gelshift and luciferase assay, respectively. Mice were orthotopically implanted with PANC1 cells and orally gavaged with resveratrol. The components of PI3K and ERK pathways, FOXOs and their target gene expressions were measured by the Western blot analysis. Resveratrol inhibited cell viability and colony formations, and induced apoptosis through caspase-3 activation in four pancreatic cancer cell lines (PANC-1, MIA PaCa-2, Hs766T, and AsPC-1. Resveratrol induced cell cycle arrest by up-regulating the expression of p21/CIP1, p27/KIP1 and inhibiting the expression of cyclin D1. Resveratrol induced apoptosis by up-regulating Bim and activating caspase-3. Resveratrol inhibited phosphorylation of FOXOs, and enhanced their nuclear translocation, FOXO-DNA binding and transcriptional activities. The inhibition of PI3K/AKT and MEK/ERK pathways induced FOXO transcriptional activity and apoptosis. Furthermore, deletion of FOXO genes abrogated resveratrol-induced cell cycle arrest and apoptosis. Finally, resveratrol-treated mice showed significant inhibition in tumor growth which was associated with reduced phosphorylation of ERK, PI3K, AKT, FOXO1 and FOXO3a, and induction of apoptosis and FOXO target genes. CONCLUSIONS: These data suggest that inhibition of ERK and AKT pathways act together to activate FOXO

  17. Ares I-X Ground Diagnostic Prototype

    Science.gov (United States)

    Schwabacher, Mark A.; Martin, Rodney Alexander; Waterman, Robert D.; Oostdyk, Rebecca Lynn; Ossenfort, John P.; Matthews, Bryan

    2010-01-01

    The automation of pre-launch diagnostics for launch vehicles offers three potential benefits: improving safety, reducing cost, and reducing launch delays. The Ares I-X Ground Diagnostic Prototype demonstrated anomaly detection, fault detection, fault isolation, and diagnostics for the Ares I-X first-stage Thrust Vector Control and for the associated ground hydraulics while the vehicle was in the Vehicle Assembly Building at Kennedy Space Center (KSC) and while it was on the launch pad. The prototype combines three existing tools. The first tool, TEAMS (Testability Engineering and Maintenance System), is a model-based tool from Qualtech Systems Inc. for fault isolation and diagnostics. The second tool, SHINE (Spacecraft Health Inference Engine), is a rule-based expert system that was developed at the NASA Jet Propulsion Laboratory. We developed SHINE rules for fault detection and mode identification, and used the outputs of SHINE as inputs to TEAMS. The third tool, IMS (Inductive Monitoring System), is an anomaly detection tool that was developed at NASA Ames Research Center. The three tools were integrated and deployed to KSC, where they were interfaced with live data. This paper describes how the prototype performed during the period of time before the launch, including accuracy and computer resource usage. The paper concludes with some of the lessons that we learned from the experience of developing and deploying the prototype.

  18. Fibroblast growth factor receptors as therapeutic targets in human melanoma: synergism with BRAF inhibition.

    Science.gov (United States)

    Metzner, Thomas; Bedeir, Alexandra; Held, Gerlinde; Peter-Vörösmarty, Barbara; Ghassemi, Sara; Heinzle, Christine; Spiegl-Kreinecker, Sabine; Marian, Brigitte; Holzmann, Klaus; Grasl-Kraupp, Bettina; Pirker, Christine; Micksche, Michael; Berger, Walter; Heffeter, Petra; Grusch, Michael

    2011-10-01

    Cutaneous melanoma is a tumor with rising incidence and a very poor prognosis at the disseminated stage. Melanomas are characterized by frequent mutations in BRAF and also by overexpression of fibroblast growth factor 2 (FGF2), offering opportunities for therapeutic intervention. We investigated inhibition of FGF signaling and its combination with dacarbazine or BRAF inhibitors as an antitumor strategy in melanoma. The majority of melanoma cell lines displayed overexpression of FGF2 but also FGF5 and FGF18 together with different isoforms of FGF receptors (FGFRs) 1-4. Blockade of FGF signals with dominant-negative receptor constructs (dnFGFR1, 3, or 4) or small-molecule inhibitors (SU5402 and PD166866) reduced melanoma cell proliferation, colony formation, as well as anchorage-independent growth, and increased apoptosis. DnFGFR constructs also significantly inhibited tumor growth in vivo. Combination of FGF inhibitors with dacarbazine showed additive or antagonistic effects, whereas synergistic drug interaction was observed when combining FGFR inhibition with the multikinase/BRAF inhibitor sorafenib or the V600E mutant-specific BRAF inhibitor RG7204. In conclusion, FGFR inhibition has antitumor effects against melanoma cells in vitro and in vivo. Combination with BRAF inhibition offers a potential for synergistic antimelanoma effects and represents a promising therapeutic strategy against advanced melanoma.

  19. Neuronal growth inhibitory factor inhibits pheochromo-cytoma PC12 in vitro

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Neuronal growth inhibitory factor (GIF),named Metaliothioneins-Ⅲ (MT-Ⅲ), is the first protein validated to be capable of inhibiting the growth of neurons in nervous system. We have detected the effects of recombinant GIF on the growth of neuroblastoma SY5Y and pheochromocytoma PC12 by the MTT (Thiazolyl blue) reduction assay. Recombinant GIF inhibited PC12 in vitro; the inhibitory rate was about 25% when GIF was at 100 mg/L; and the inhibitory rate was about 50% when GIF was at 300 mg/L. It is shown that PC12 could serve as a proper model for detecting neuronal growth inhibitory activity of GIF. Recombinant GIF did not inhibit neuroblastoma SY5Y in vitro, a common model of neuroma; it is also shown that GIF could not inhibit neuromata extensively. The reason for GIF inhibiting PC12 may be that PC12 have some properties of cholinergic neuron. It must play an important role in discovering the mechanism of GIF's neuronal growth inhibitory activity.``

  20. Ares I-X Ground Diagnostic Prototype

    Science.gov (United States)

    Schwabacher, Mark; Martin, Rodney; Waterman, Robert; Oostdyk, Rebecca; Ossenfort, John; Matthews, Bryan

    2010-01-01

    Automating prelaunch diagnostics for launch vehicles offers three potential benefits. First, it potentially improves safety by detecting faults that might otherwise have been missed so that they can be corrected before launch. Second, it potentially reduces launch delays by more quickly diagnosing the cause of anomalies that occur during prelaunch processing. Reducing launch delays will be critical to the success of NASA's planned future missions that require in-orbit rendezvous. Third, it potentially reduces costs by reducing both launch delays and the number of people needed to monitor the prelaunch process. NASA is currently developing the Ares I launch vehicle to bring the Orion capsule and its crew of four astronauts to low-earth orbit on their way to the moon. Ares I-X will be the first unmanned test flight of Ares I. It is scheduled to launch on October 27, 2009. The Ares I-X Ground Diagnostic Prototype is a prototype ground diagnostic system that will provide anomaly detection, fault detection, fault isolation, and diagnostics for the Ares I-X first-stage thrust vector control (TVC) and for the associated ground hydraulics while it is in the Vehicle Assembly Building (VAB) at John F. Kennedy Space Center (KSC) and on the launch pad. It will serve as a prototype for a future operational ground diagnostic system for Ares I. The prototype combines three existing diagnostic tools. The first tool, TEAMS (Testability Engineering and Maintenance System), is a model-based tool that is commercially produced by Qualtech Systems, Inc. It uses a qualitative model of failure propagation to perform fault isolation and diagnostics. We adapted an existing TEAMS model of the TVC to use for diagnostics and developed a TEAMS model of the ground hydraulics. The second tool, Spacecraft Health Inference Engine (SHINE), is a rule-based expert system developed at the NASA Jet Propulsion Laboratory. We developed SHINE rules for fault detection and mode identification. The prototype

  1. Factors facilitating and inhibiting value stream mapping processes at hospital units in three Nordic countries - a Nordic multicenter study

    DEFF Research Database (Denmark)

    Winkel, Jørgen; Birgisdóttir, Birna Dröfn; Dudas, Kerstin

    2014-01-01

    The aim of this paper is to present observations that may indicate facilitating and inhibiting factors for the VSM process.......The aim of this paper is to present observations that may indicate facilitating and inhibiting factors for the VSM process....

  2. Pretreatment to enhance protoporphyrin IX accumulation in photodynamic therapy.

    NARCIS (Netherlands)

    Gerritsen, M.J.P.; Smits, T.; Kleinpenning, M.M.; Kerkhof, P.C.M. van de; Erp, P.E.J. van

    2009-01-01

    The response rates of photodynamic therapy (PDT) vary widely. Limited uptake of topically applied 5-aminolaevulinic acid (ALA), or its methyl ester (MAL), and suboptimal production of protoporphyrin IX (PpIX) may account for these differences. Recently, we demonstrated that hyperkeratosis is an impo

  3. ARES I-X USS Fracture Analysis Loads Spectra Development

    Science.gov (United States)

    Larsen, Curtis; Mackey, Alden

    2008-01-01

    This report describes the development of a set of bounding load spectra for the ARES I-X launch vehicle. These load spectra are used in the determination of the critical initial flaw size (CIFS) of the welds in the ARES I-X upper stage simulator (USS).

  4. Transcription factor TnrA inhibits the biosynthetic activity of glutamine synthetase in Bacillus subtilis.

    Science.gov (United States)

    Fedorova, Ksenia; Kayumov, Airat; Woyda, Kathrin; Ilinskaja, Olga; Forchhammer, Karl

    2013-05-02

    The Bacillus subtilis glutamine synthetase (GS) plays a dual role in cell metabolism by functioning as catalyst and regulator. GS catalyses the ATP-dependent synthesis of glutamine from glutamate and ammonium. Under nitrogen-rich conditions, GS becomes feedback-inhibited by high intracellular glutamine levels and then binds transcription factors GlnR and TnrA, which control the genes of nitrogen assimilation. While GS-bound TnrA is no longer able to interact with DNA, GlnR-DNA binding is shown to be stimulated by GS complex formation. In this paper we show a new physiological feature of the interaction between glutamine synthetase and TnrA. The transcription factor TnrA inhibits the biosynthetic activity of glutamine synthetase in vivo and in vitro, while the GlnR protein does not affect the activity of the enzyme.

  5. The inhibiting factors that principal investigators experience in leading publicly funded research projects

    OpenAIRE

    Cunningham, James; O'Reilly, Paul; O'Kane, Conor; Mangematin, Vincent

    2014-01-01

    International audience; Securing public funding to conduct research and leading it by being a principal investigator (PI) is seen as significant career development step. Such a role brings professional prestige but also new responsibilities beyond research leadership to research management. If public funding brings financial and infrastructure support, little is understood about the inhibiting factors that publicly funded PIs face given the research autonomy offered by publicly funded researc...

  6. A Synthetic Manassantin A Derivative Inhibits Hypoxia-Inducible Factor 1 and Tumor Growth

    OpenAIRE

    Liwei Lang; Xiaoyu Liu; Yan Li; Qing Zhou; Ping Xie; Chunhong Yan; Xiaoguang Chen

    2014-01-01

    The dineolignan manassantin A from Saururaceae was recently identified as a hypoxia-inducible factor 1 (HIF-1) inhibitor, but its in-vivo anti-tumor effect has not been explored. We synthesized a series of manassantin A derivatives, and found that replacing the central tetrahydrofuran moiety with a cyclopentane ring yielded a compound (LXY6006) with increased HIF-1-inhibitory activity yet decreased stereochemically complexity amenable to a simplified synthesis scheme. LXY6006 inhibited HIF-1α...

  7. Vasohibin-1 expression inhibits advancement of ovarian cancer producing various angiogenic factors.

    Science.gov (United States)

    Takahashi, Yoshifumi; Saga, Yasushi; Koyanagi, Takahiro; Takei, Yuji; Machida, Shizuo; Taneichi, Akiyo; Mizukami, Hiroaki; Sato, Yasufumi; Matsubara, Shigeki; Fujiwara, Hiroyuki

    2016-05-01

    Vasohibin-1 (VASH1) is a negative feedback regulator of angiogenesis, the first to be discovered, and was identified in vascular endothelial growth factor (VEGF)-stimulated vascular endothelial cells. Vasohibin-1 inhibits abnormal vascularization induced by various angiogenic factors including fibroblast growth factor and platelet-derived growth factor (PDGF), in addition to VEGF. By focusing on this characteristic of VASH1, we investigated the antitumor effects of VASH1 expression on ovarian cancer cells that produce different angiogenic factors. By using a high VEGF-producing ovarian cancer cell line, SHIN-3, and a high PDGF-producing ovarian cancer cell line, KOC-2S, the cells were transfected with either a VEGF antagonist, soluble VEGF receptor-1 (sVEGFR-1, or sFlt-1), or VASH1 genes to establish their respective cellular expression. The characteristics of these transfectants were compared with controls. We previously reported that the expression of sFlt-1 inhibited tumor vascularization and growth of high VEGF-producing ovarian cancer cells, reduced peritoneal dissemination and ascites development, and prolonged the survival time of the host. However, in the current study, the expression of sFlt-1 had no such effect on the high PDGF-producing ovarian cancer cells used here, whereas VASH1 expression inhibited tumor vascularization and growth, not only in high VEGF-producing cells, but also in high PDGF-producing cells, reduced their peritoneal dissemination and ascites, and prolonged the survival time of the host. These results suggest that VASH1 is an effective treatment for ovarian cancer cells that produce different angiogenic factors.

  8. Anti-tumor necrosis factor therapy inhibits lung metastasis in an osteosarcoma cell line.

    Science.gov (United States)

    Kato, Hiroaki; Wakabayashi, Hiroki; Naito, Yohei; Kato, Sho; Nakagawa, Taro; Matsumine, Akihiko; Sudo, Akihiro

    2015-01-01

    Osteosarcoma is the most common primary malignancy of bone, and patients often develop pulmonary metastases. In a previous study, tumor necrosis factor (TNF)-α treatment of human osteosarcoma cells increases their metastatic ability in an animal model. TNF-α can act as a tumor necrosis factor and also as a tumor-promoting factor. In the present study, the effect of a TNF-α inhibitor on osteosarcoma aggressiveness and pulmonary metastases was investigated in vitro and in vivo. The effect of infliximab, a TNF-α inhibitor, on a metastatic osteosarcoma 143B cell growth and motility was investigated in vitro. An orthotopic xenograft model of 143B cell growth and spontaneous metastasis in SCID mice was used to assess the in vivo effect of infliximab. Infliximab greatly reduced cell motility and pulmonary metastases in 143B cells. The mechanism of pulmonary metastasis inhibition involved decreased expression of CXC chemokine receptor 4 (CXCR4), Rho (small GTPase protein), and its effector. These results suggest a novel role for TNF-α inhibition in the reduction or prevention of pulmonary metastases of osteosarcoma in this animal model. TNF-α inhibition may become a preventive therapeutic option for the pulmonary metastases of osteosarcoma. © 2014 S. Karger AG, Basel.

  9. Colony stimulating factor 1 receptor inhibition eliminates microglia and attenuates brain injury after intracerebral hemorrhage.

    Science.gov (United States)

    Li, Minshu; Li, Zhiguo; Ren, Honglei; Jin, Wei-Na; Wood, Kristofer; Liu, Qiang; Sheth, Kevin N; Shi, Fu-Dong

    2017-07-01

    Microglia are the first responders to intracerebral hemorrhage, but their precise role in intracerebral hemorrhage remains to be defined. Microglia are the only type of brain cells expressing the colony-stimulating factor 1 receptor, a key regulator for myeloid lineage cells. Here, we determined the effects of a colony-stimulating factor 1 receptor inhibitor (PLX3397) on microglia and the outcome in the context of experimental mouse intracerebral hemorrhage. We show that PLX3397 effectively depleted microglia, and the depletion of microglia was sustained after intracerebral hemorrhage. Importantly, colony-stimulating factor 1 receptor inhibition attenuated neurodeficits and brain edema in two experimental models of intracerebral hemorrhage induced by injection of collagenase or autologous blood. The benefit of colony-stimulating factor 1 receptor inhibition was associated with reduced leukocyte infiltration in the brain and improved blood-brain barrier integrity after intracerebral hemorrhage, and each observation was independent of lesion size or hematoma volume. These results demonstrate that suppression of colony-stimulating factor 1 receptor signaling ablates microglia and confers protection after intracerebral hemorrhage.

  10. rNAPc2 inhibits colorectal cancer in mice through tissue factor.

    Science.gov (United States)

    Zhao, Jingsong; Aguilar, Gerard; Palencia, Servando; Newton, Elizabeth; Abo, Arie

    2009-01-01

    Recombinant nematode anticoagulant protein c2 (rNAPc2) is a specific inhibitor of tissue factor (TF)/factor VIIa complex with novel antithrombotic activity. TF is highly expressed in human colorectal tumors, and levels are positively correlated with disease progression. To explore the therapeutic potential and mechanism of action of rNAPc2 during tumor growth and metastasis, we tested rNAPc2 in several experimental colorectal cancer models in mice. Administration of rNAPc2 inhibited pulmonary metastasis in mice systemically disseminated with CT26 murine colon carcinoma cells in a dose-dependent fashion. Combining rNAPc2 with the cytotoxic agent 5-fluorouracil or bevacizumab (humanized anti-vascular endothelial growth factor monoclonal antibody) resulted in additive growth inhibition and simultaneous reduction of microvessel density in HCT116 human colorectal tumor xenografts in nude mice. Furthermore, rNAPc2 potentiated CPT-11 in inhibiting hepatic metastasis in nude mice with portal vein injection of HCT116 tumor cells. Long-term administration of rNAPc2 significantly suppressed spontaneous formation of intestinal tumors in Apc(Min/+) mice. Using a RNA interference approach, we showed that TF expression is necessary for rNAPc2-mediated inhibition of HCT116 human colorectal tumor xenograft growth in nude mice, indicating that the antitumor effect of rNAPc2 may be transduced through TF that is expressed on tumor cells. rNAPc2 is a potent anticancer agent when used in combination with chemotherapy or antiangiogenic therapy in mouse models of colorectal cancer, and TF positivity appears to be required for its activity.

  11. Ares I-X Flight Test Philosophy

    Science.gov (United States)

    Davis, S. R.; Tuma, M. L.; Heitzman, K.

    2007-01-01

    In response to the Vision for Space Exploration, the National Aeronautics and Space Administration (NASA) has defined a new space exploration architecture to return humans to the Moon and prepare for human exploration of Mars. One of the first new developments will be the Ares I Crew Launch Vehicle (CLV), which will carry the Orion Crew Exploration Vehicle (CEV), into Low Earth Orbit (LEO) to support International Space Station (ISS) missions and, later, support lunar missions. As part of Ares I development, NASA will perform a series of Ares I flight tests. The tests will provide data that will inform the engineering and design process and verify the flight hardware and software. The data gained from the flight tests will be used to certify the new Ares/Orion vehicle for human space flight. The primary objectives of this first flight test (Ares I-X) are the following: Demonstrate control of a dynamically similar integrated Ares CLV/Orion CEV using Ares CLV ascent control algorithms; Perform an in-flight separation/staging event between an Ares I-similar First Stage and a representative Upper Stage; Demonstrate assembly and recovery of a new Ares CLV-like First Stage element at Kennedy Space Center (KSC); Demonstrate First Stage separation sequencing, and quantify First Stage atmospheric entry dynamics and parachute performance; and Characterize the magnitude of the integrated vehicle roll torque throughout the First Stage (powered) flight. This paper will provide an overview of the Ares I-X flight test process and details of the individual flight tests.

  12. Blockade of nonhormonal fibroblast growth factors by FP-1039 inhibits growth of multiple types of cancer.

    Science.gov (United States)

    Harding, Thomas C; Long, Li; Palencia, Servando; Zhang, Hongbing; Sadra, Ali; Hestir, Kevin; Patil, Namrata; Levin, Anita; Hsu, Amy W; Charych, Deborah; Brennan, Thomas; Zanghi, James; Halenbeck, Robert; Marshall, Shannon A; Qin, Minmin; Doberstein, Stephen K; Hollenbaugh, Diane; Kavanaugh, W Michael; Williams, Lewis T; Baker, Kevin P

    2013-03-27

    The fibroblast growth factor (FGF) pathway promotes tumor growth and angiogenesis in many solid tumors. Although there has long been interest in FGF pathway inhibitors, development has been complicated: An effective FGF inhibitor must block the activity of multiple mitogenic FGF ligands but must spare the metabolic hormone FGFs (FGF-19, FGF-21, and FGF-23) to avoid unacceptable toxicity. To achieve these design requirements, we engineered a soluble FGF receptor 1 Fc fusion protein, FP-1039. FP-1039 binds tightly to all of the mitogenic FGF ligands, inhibits FGF-stimulated cell proliferation in vitro, blocks FGF- and vascular endothelial growth factor (VEGF)-induced angiogenesis in vivo, and inhibits in vivo growth of a broad range of tumor types. FP-1039 antitumor response is positively correlated with RNA levels of FGF2, FGF18, FGFR1c, FGFR3c, and ETV4; models with genetic aberrations in the FGF pathway, including FGFR1-amplified lung cancer and FGFR2-mutated endometrial cancer, are particularly sensitive to FP-1039-mediated tumor inhibition. FP-1039 does not appreciably bind the hormonal FGFs, because these ligands require a cell surface co-receptor, klotho or β-klotho, for high-affinity binding and signaling. Serum calcium and phosphate levels, which are regulated by FGF-23, are not altered by administration of FP-1039. By selectively blocking nonhormonal FGFs, FP-1039 treatment confers antitumor efficacy without the toxicities associated with other FGF pathway inhibitors.

  13. ALA-induced PpIX fluorescence in epileptogenic tissue

    Science.gov (United States)

    Kleen, Jonathan K.; Valdes, Pablo A.; Harris, Brent T.; Holmes, Gregory L.; Paulsen, Keith D.; Roberts, David W.

    2011-03-01

    Astrogliotic tissue displays markedly increased levels of ALA-induced PpIX fluorescence, making it useful for fluorescence-guided resection in glioma surgery. In patients with temporal lobe epilepsy (TLE) and corresponding animal models, there are areas of astrogliosis that often co-localize with the epileptic focus, which can be resected to eliminate seizures in the majority of treated patients. If this epileptogenic tissue can exhibit PpIX fluorescence that is sufficiently localized, it could potentially help identify margins in epilepsy surgery. We tested the hypothesis that ALA-induced PpIX fluorescence could visually accentuate epileptogenic tissue, using an established animal model of chronic TLE. An acute dose of pilocarpine was used to induce chronic seizure activity in a rat. This rat and a normal control were given ALA, euthanized, and brains examined post-mortem for PpIX fluorescence and neuropathology. Preliminary evidence indicates increased PpIX fluorescence in areas associated with chronic epileptic changes and seizure generation in TLE, including the hippocampus and parahippocampal areas. In addition, strong PpIX fluorescence was clearly observed in layer II of the piriform cortex, a region known for epileptic reorganization and involvement in the generation of seizures in animal studies. We are further investigating whether ALA-induced PpIX fluorescence can consistently identify epileptogenic zones, which could warrant the extension of this technique to clinical studies for use as an adjuvant guidance technology in the resection of epileptic tissue.

  14. Quercetin induces human colon cancer cells apoptosis by inhibiting the nuclear factor-kappa B Pathway.

    Science.gov (United States)

    Zhang, Xiang-An; Zhang, Shuangxi; Yin, Qing; Zhang, Jing

    2015-01-01

    Quercetin can inhibit the growth of cancer cells with the ability to act as chemopreventers. Its cancer-preventive effect has been attributed to various mechanisms, including the induction of cell-cycle arrest and/or apoptosis as well as the antioxidant functions. Nuclear factor kappa-B (NF-κB) is a signaling pathway that controls transcriptional activation of genes important for tight regulation of many cellular processes and is aberrantly expressed in many types of cancer. Inhibitors of NF-κB pathway have shown potential anti-tumor activities. However, it is not fully elucidated in colon cancer. In this study, we demonstrate that quercetin induces apoptosis in human colon cancer CACO-2 and SW-620 cells through inhibiting NF-κB pathway, as well as down-regulation of B-cell lymphoma 2 and up-regulation of Bax, thus providing basis for clinical application of quercetin in colon cancer cases.

  15. Cycle Inhibiting Factors (Cifs): Cyclomodulins That Usurp the Ubiquitin-Dependent Degradation Pathway of Host Cells

    Science.gov (United States)

    Taieb, Frédéric; Nougayrède, Jean-Philippe; Oswald, Eric

    2011-01-01

    Cycle inhibiting factors (Cifs) are type III secreted effectors produced by diverse pathogenic bacteria. Cifs are “cyclomodulins” that inhibit the eukaryotic host cell cycle and also hijack other key cellular processes such as those controlling the actin network and apoptosis. This review summarizes current knowledge on Cif since its first characterization in enteropathogenic Escherichia coli, the identification of several xenologues in distant pathogenic bacteria, to its structure elucidation and the recent deciphering of its mode of action. Cif impairs the host ubiquitin proteasome system through deamidation of ubiquitin or the ubiquitin-like protein NEDD8 that regulates Cullin-Ring-ubiquitin Ligase (CRL) complexes. The hijacking of the ubiquitin-dependent degradation pathway of host cells results in the modulation of various cellular functions such as epithelium renewal, apoptosis and immune response. Cif is therefore a powerful weapon in the continuous arm race that characterizes host-bacteria interactions. PMID:22069713

  16. Ramucirumab (IMC-1121B): Monoclonal antibody inhibition of vascular endothelial growth factor receptor-2.

    Science.gov (United States)

    Spratlin, Jennifer

    2011-04-01

    Angiogenesis, a well-recognized characteristic of malignancy, has been exploited more than any other pathway targeted by biologic anti-neoplastic therapies. Vascular endothelial growth factor receptor-2 (VEGFR-2) is the critical receptor involved in malignant angiogenesis with its activation inducing a number of other cellular modifications resulting in tumor growth and metastases. Ramucirumab (IMC-1121B; ImClone Systems Corporation, Branchburg, NJ) is a fully human monoclonal antibody developed to specifically inhibit VEGFR-2. Ramucirumab is currently being investigated in multiple clinical trials across a variety of tumor types. Herein, angiogenesis inhibition in cancer is reviewed and up-to-date information on the clinical development of ramucirumab is presented.

  17. Matrine inhibits proliferation of mouse skin fibroblasts induced by platelet-derived growth factor-BB

    Institute of Scientific and Technical Information of China (English)

    WU Yan-an; GAO Chun-fang; WANG Hao; HUANG Chao; KONG Xian-tao

    2001-01-01

    To study the effect of matrine on proliferation of mouse skin fibroblasts induced by platelet-derived growth factor-BB (PDGF-BB). Methods: Mouse skin fibroblasts were obtained from newborn ⅠCR mice and propagated in vitro. Proliferation of cell was analyzed by mitochondrial reduction of tetrazolium salt MTT and actual cell count. Results: Matrine (50 to 500 μg/ml) caused dose-dependent reduction of serum-stimulated cell growth. Growth inhibition was totally reversed after removal of the drug. Matrine also inhibited PDGF-BB induced cell growth dose-dependently. Conclusion: Matrine exhibits potent anti-proliferation effect on mouse skin fibroblast. This effect appears to be mediated by decrease of PDGF-induced growth. These results suggest that matrine might have preventive and therapeutic implication in skin fibrosis.

  18. Cycle inhibiting factors (cifs): cyclomodulins that usurp the ubiquitin-dependent degradation pathway of host cells.

    Science.gov (United States)

    Taieb, Frédéric; Nougayrède, Jean-Philippe; Oswald, Eric

    2011-04-01

    Cycle inhibiting factors (Cifs) are type III secreted effectors produced by diverse pathogenic bacteria. Cifs are "cyclomodulins" that inhibit the eukaryotic host cell cycle and also hijack other key cellular processes such as those controlling the actin network and apoptosis. This review summarizes current knowledge on Cif since its first characterization in enteropathogenic Escherichia coli, the identification of several xenologues in distant pathogenic bacteria, to its structure elucidation and the recent deciphering of its mode of action. Cif impairs the host ubiquitin proteasome system through deamidation of ubiquitin or the ubiquitin-like protein NEDD8 that regulates Cullin-Ring-ubiquitin Ligase (CRL) complexes. The hijacking of the ubiquitin-dependent degradation pathway of host cells results in the modulation of various cellular functions such as epithelium renewal, apoptosis and immune response. Cif is therefore a powerful weapon in the continuous arm race that characterizes host-bacteria interactions.

  19. Cycle Inhibiting Factors (Cifs: Cyclomodulins That Usurp the Ubiquitin-Dependent Degradation Pathway of Host Cells

    Directory of Open Access Journals (Sweden)

    Eric Oswald

    2011-03-01

    Full Text Available Cycle inhibiting factors (Cifs are type III secreted effectors produced by diverse pathogenic bacteria. Cifs are “cyclomodulins” that inhibit the eukaryotic host cell cycle and also hijack other key cellular processes such as those controlling the actin network and apoptosis. This review summarizes current knowledge on Cif since its first characterization in enteropathogenic Escherichia coli, the identification of several xenologues in distant pathogenic bacteria, to its structure elucidation and the recent deciphering of its mode of action. Cif impairs the host ubiquitin proteasome system through deamidation of ubiquitin or the ubiquitin-like protein NEDD8 that regulates Cullin-Ring-ubiquitin Ligase (CRL complexes. The hijacking of the ubiquitin-dependent degradation pathway of host cells results in the modulation of various cellular functions such as epithelium renewal, apoptosis and immune response. Cif is therefore a powerful weapon in the continuous arm race that characterizes host-bacteria interactions.

  20. Activation of MAPK/ERK signaling by Burkholderia pseudomallei cycle inhibiting factor (Cif)

    Science.gov (United States)

    Ng, Mei Ying; Wang, Mei; Casey, Patrick J.; Gan, Yunn-Hwen; Hagen, Thilo

    2017-01-01

    Cycle inhibiting factors (Cifs) are virulence proteins secreted by the type III secretion system of some Gram-negative pathogenic bacteria including Burkholderia pseudomallei. Cif is known to function to deamidate Nedd8, leading to inhibition of Cullin E3 ubiquitin ligases (CRL) and consequently induction of cell cycle arrest. Here we show that Cif can function as a potent activator of MAPK/ERK signaling without significant activation of other signaling pathways downstream of receptor tyrosine kinases. Importantly, we found that the ability of Cif to activate ERK is dependent on its deamidase activity, but independent of Cullin E3 ligase inhibition. This suggests that apart from Nedd8, other cellular targets of Cif-dependent deamidation exist. We provide evidence that the mechanism involved in Cif-mediated ERK activation is dependent on recruitment of the Grb2-SOS1 complex to the plasma membrane. Further investigation revealed that Cif appears to modify the phosphorylation status of SOS1 in a region containing the CDC25-H and proline-rich domains. It is known that prolonged Cullin E3 ligase inhibition leads to cellular apoptosis. Therefore, we hypothesize that ERK activation is an important mechanism to counter the pro-apoptotic effects of Cif. Indeed, we show that Cif dependent ERK activation promotes phosphorylation of the proapoptotic protein Bim, thereby potentially conferring a pro-survival signal. In summary, we identified a novel deamidation-dependent mechanism of action of the B. pseudomallei virulence factor Cif/CHBP to activate MAPK/ERK signaling. Our study demonstrates that bacterial proteins such as Cif can serve as useful molecular tools to uncover novel aspects of mammalian signaling pathways. PMID:28166272

  1. Grape seed extract inhibits angiogenesis via suppression of the vascular endothelial growth factor receptor signaling pathway.

    Science.gov (United States)

    Wen, Wei; Lu, Jianming; Zhang, Keqiang; Chen, Shiuan

    2008-12-01

    Blockade of angiogenesis is an important approach for cancer treatment and prevention. Vascular endothelial growth factor (VEGF) is one of the most critical factors that induce angiogenesis and has thus become an attractive target for antiangiogenesis treatment. However, most current anti-VEGF agents often cause some side effects when given chronically. Identification of naturally occurring VEGF inhibitors derived from diet would be one alternative approach with an advantage of known safety. Grape seed extract (GSE), a widely used dietary supplement, is known to have antitumor activity. In this study, we have explored the activity of GSE on VEGF receptor and angiogenesis. We found that GSE could directly inhibit the kinase activity of purified VEGF receptor 2, a novel activity of GSE that has not been characterized. GSE could also inhibit the VEGF receptor/mitogen-activated protein kinase-mediated signaling pathway in endothelial cells. As a result, GSE could inhibit VEGF-induced endothelial cell proliferation and migration as well as sprout formation from aorta ring. In vivo assay further showed that GSE could inhibit tumor growth and tumor angiogenesis of MDA-MB-231 breast cancer cells in mice. Consistent with the in vitro data, GSE treatment of tumor-bearing mice led to concomitant reduction of blood vessel density and phosphorylation of mitogen-activated protein kinase. Depletion of polyphenol with polyvinylpyrrolidone abolished the antiangiogenic activity of GSE, suggesting a water-soluble fraction of polyphenol in GSE is responsible for the antiangiogenic activity. Taken together, this study indicates that GSE is a well-tolerated and inexpensive natural VEGF inhibitor and could potentially be useful in cancer prevention or treatment.

  2. Activation of MAPK/ERK signaling by Burkholderia pseudomallei cycle inhibiting factor (Cif).

    Science.gov (United States)

    Ng, Mei Ying; Wang, Mei; Casey, Patrick J; Gan, Yunn-Hwen; Hagen, Thilo

    2017-01-01

    Cycle inhibiting factors (Cifs) are virulence proteins secreted by the type III secretion system of some Gram-negative pathogenic bacteria including Burkholderia pseudomallei. Cif is known to function to deamidate Nedd8, leading to inhibition of Cullin E3 ubiquitin ligases (CRL) and consequently induction of cell cycle arrest. Here we show that Cif can function as a potent activator of MAPK/ERK signaling without significant activation of other signaling pathways downstream of receptor tyrosine kinases. Importantly, we found that the ability of Cif to activate ERK is dependent on its deamidase activity, but independent of Cullin E3 ligase inhibition. This suggests that apart from Nedd8, other cellular targets of Cif-dependent deamidation exist. We provide evidence that the mechanism involved in Cif-mediated ERK activation is dependent on recruitment of the Grb2-SOS1 complex to the plasma membrane. Further investigation revealed that Cif appears to modify the phosphorylation status of SOS1 in a region containing the CDC25-H and proline-rich domains. It is known that prolonged Cullin E3 ligase inhibition leads to cellular apoptosis. Therefore, we hypothesize that ERK activation is an important mechanism to counter the pro-apoptotic effects of Cif. Indeed, we show that Cif dependent ERK activation promotes phosphorylation of the proapoptotic protein Bim, thereby potentially conferring a pro-survival signal. In summary, we identified a novel deamidation-dependent mechanism of action of the B. pseudomallei virulence factor Cif/CHBP to activate MAPK/ERK signaling. Our study demonstrates that bacterial proteins such as Cif can serve as useful molecular tools to uncover novel aspects of mammalian signaling pathways.

  3. Brain-derived neurotrophic factor mediates the activity-dependent regulation of inhibition in neocortical cultures.

    Science.gov (United States)

    Rutherford, L C; DeWan, A; Lauer, H M; Turrigiano, G G

    1997-06-15

    The excitability of cortical circuits is modulated by interneurons that release the inhibitory neurotransmitter GABA. In primate and rodent visual cortex, activity deprivation leads to a decrease in the expression of GABA. This suggests that activity is able to adjust the strength of cortical inhibition, but this has not been demonstrated directly. In addition, the nature of the signal linking activity to GABA expression has not been determined. Activity is known to regulate the expression of the neurotrophin brain-derived neurotrophic factor (BDNF), and BDNF has been shown to influence the phenotype of GABAergic interneurons. We use a culture system from postnatal rat visual cortex to test the hypothesis that activity is regulating the strength of cortical inhibition through the regulation of BDNF. Cultures were double-labeled against GABA and the neuronal marker MAP2, and the percentage of neurons that were GABA-positive was determined. Blocking spontaneous activity in these cultures reversibly decreased the number of GABA-positive neurons without affecting neuronal survival. Voltage-clamp analysis of inhibitory currents demonstrated that activity blockade also decreased GABA-mediated inhibition onto pyramidal neurons and raised pyramidal neuron firing rates. All of these effects were prevented by incubation with BDNF during activity blockade, but not by neurotrophin 3 or nerve growth factor. Additionally, blockade of neurotrophin signaling mimicked the effects of activity blockade on GABA expression. These data suggest that activity regulates cortical inhibition through a BDNF-dependent mechanism and that this neurotrophin plays an important role in the control of cortical excitability.

  4. Expression of Smad7 inhibits fibrogenic responses of keratocytes to transforming growth factor β2

    Institute of Scientific and Technical Information of China (English)

    WANG Ti; ZHOU Xing-tao; YU Yan; DAI Jin-hui; QU Xiao-mei; LE Qi-hua; CHU Ren-yuan

    2011-01-01

    Background Transforming growth factor β (TGFβ) is one of the most important growth factors in the development of fibrosis and scarring on cornea. Smad7, an inhibitory Smad, can inhibit TGFβ signal transaction. In recent years, effects of lentiviral-mediated Smad7 on inhibition of fibrosis on some organs have been studied, while little is known about the effects on cornea. This study aimed to determine the effects of lentiviral-mediated Smad7 gene expression on keratocyte proliferation and fibrosis induced by TGF β2 in vitro.Methods Keratocytes were cultured from comeal tissue isolated from Sprague-Dawley (SD) rats and transfected with Smad7 expressing lentiviral vector (Lv-Smad7) or non-functioning control vector (Lv-blank). Following the exposure to TGFβ2, keratocytes were processed for immunoblotting to assess the phosphorylation of Smad2 as down-stream event of TGFβ/Smad signaling. Expression of fibrotic markers α-smooth muscle actin (α-SMA), type Ⅲ collagen (collagen Ⅲ) were measured by Western blotting and quantitative real time polymerase chain reaction (PCR). Overall cell proliferation was determined by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-dipheny1tetrazolium bromide (MTT) assay and the expression of cell cycle-related marker Ki67 at both mRNA and protein levels.Results The Smad7 gene transfer suppressed TGFβ/Smad signaling in keratocytes by down-regulating phosphorylation of Smad2. Markers of cell proliferation and fibrosis including Ki67, α-SMA, collagen Ⅲ were inhibited by introduction of Smad 7 into TGFβ exposed keratocytes. Consequently, the rate of cell proliferation was attenuated.Conclusion Smad7 gene transfer inhibited fibrogenic responses of keratocytes to TGFβ2.

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

    Science.gov (United States)

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

    2016-01-01

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

  6. Overexpression of ERβ is sufficient to inhibit hypoxia-inducible factor-1 transactivation

    Energy Technology Data Exchange (ETDEWEB)

    Park, Choa; Lee, YoungJoo, E-mail: yjlee@sejong.ac.kr

    2014-07-18

    Highlights: • We examined the effect of ERβ specific ligand on HIF-1 inhibition. • DPN down-regulates the ARNT protein levels in PC3 cells. • DPN did not show additional effect in ERβ transfected MCF-7 cells. • Our study shows that unliganded ERβ is sufficient to inhibit HIF-1 in systems of overexpression. - Abstract: Estrogen receptor (ER) β is predicted to play an important role in the prevention of breast cancer development and progression. We have previously shown that ERβ suppresses hypoxia inducible factor (HIF)-1-mediated transcription through aryl hydrocarbon receptor nuclear translocator (ARNT) degradation via ubiquitination processes. In this study, we attempted to examine the effect of ERβ specific ligand on HIF-1 inhibition in ERβ positive PC3 cells and ERβ transfected MCF-7 cells. ERβ specific agonist diarylpropionitrile (DPN) stimulated estrogen response element (ERE)-luciferase activity in a similar fashion to estradiol in PC3 cells. We observed that DPN down-regulates the ARNT protein levels leading to an attenuation of hypoxia-induced hypoxia response element (HRE)-driven luciferase reporter gene activation in PC3 cells. Treatment of DPN reduced vascular endothelial growth factor (VEGF) expression and co-treatment with ERβ specific antagonist PHTPP abrogated the effect in PC3 cells. We then examined the effect of DPN in ERβ transfected MCF-7 cells. HIF-1 transcriptional activity repression by ERβ was not further reduced by DPN, as examined by HRE-driven luciferase assays. Expression of ERβ significantly decreased VEGF secretion and ARNT expression under hypoxic conditions. However, DPN did not additionally affect this suppression in MCF-7 cells transfected with ERβ. This result shows that unliganded ERβ is sufficient to inhibit HIF-1 in systems of overexpression.

  7. Atrial natriuretic factor inhibits mitogen-induced growth in aortic smooth muscle cells.

    Science.gov (United States)

    Baldini, P M; De Vito, P; Fraziano, M; Mattioli, P; Luly, P; Di Nardo, P

    2002-10-01

    Atrial natriuretic factor (ANF) is a polypeptide able to affect cardiovascular homeostasis exhibiting diuretic, natriuretic, and vasorelaxant activities. ANF shows antimitogenic effects in different cell types acting through R(2) receptor. Excessive proliferation of smooth muscle cells is a common phenomenon in diseases such as atherosclerosis, but the role of growth factors in the mechanism which modulate this process has yet to be clarified. The potential antimitogenic role of ANF on the cell growth induced by growth factors appears very intriguing. Aim of the present study was to investigate the possible involvement of ANF on rat aortic smooth muscle (RASM) cells proliferation induced by known mitogens and the mechanism involved. Our data show that ANF, at physiological concentration range, inhibits RASM cell proliferation induced by known mitogens such as PDGF and insulin, and the effect seems to be elicited through the modulation of phosphatidic acid (PA) production and MAP kinases involvement.

  8. Zinc inhibits nuclear factor-kappa B activation and sensitizes prostate cancer cells to cytotoxic agents.

    Science.gov (United States)

    Uzzo, Robert G; Leavis, Paul; Hatch, William; Gabai, Vladimir L; Dulin, Nickolai; Zvartau, Nadezhda; Kolenko, Vladimir M

    2002-11-01

    Prostate carcinogenesis involves transformation of zinc-accumulating normal epithelial cells to malignant cells, which do not accumulate zinc. In this study, we demonstrate by immunoblotting and immunohistochemistry that physiological levels of zinc inhibit activation of nuclear factor (NF)-kappa B transcription factor in PC-3 and DU-145 human prostate cancer cells, reduce expression of NF-kappa B-controlled antiapoptotic protein c-IAP2, and activate c-Jun NH(2)-terminal kinases. Preincubation of PC-3 cells with physiological concentrations of zinc sensitized tumor cells to tumor necrosis factor (TNF)-alpha, and paclitaxel mediated cell death as defined by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. These results suggest one possible mechanism for the inhibitory effect of zinc on the development and progression of prostate malignancy and might have important consequences for the prevention and treatment of prostate cancer.

  9. Inhibition of tubulointerstitial fibrosis by pentoxifylline is associated with improvement of vascular endothelial growth factor expression

    Institute of Scientific and Technical Information of China (English)

    Qiu-gen ZHOU; Fa-lei ZHENG; Fan-fan HOU

    2009-01-01

    Aim: Recent information indicates that pentoxifylline (PTX) has the ability to suppress inflammation and profibrotic cell proliferation. In this study, we investigated the effect of PTX on tubulointerstitial fibrosis and the expression of vascular endothelial growth factor (VEGF) in a rat model of obstructive nephropathy. Methods: Wistar rats with left ureteral ligation were divided into control and PTX-treated groups. The histopathologic degree of tubulointerstitial fibrosis was scored with PAS and Masson-stained sections. The protein and mRNA for vascular endothelial growth factor (VEGF) were semiquantitatively measured with immunohistochemistry and RT-PCR. The pro-tein for transforming growth factor β1 (TGFβ1) and hypoxia-induced factor 1 alpha (HIF-1α) was determined by Western blot. Results: Compared with the control group, PTX treatment reduced fibrosis scores at d 7 and d 14 (P<0.05). The reduction was accompanied by inhibited expression of transforming growth factor-beta 1 (TGFβ1), a key cytokine in tubulointerstitial fibrogenesis (P<0.01). Meanwhile, VEGF protein and mRNA in the kidney were increased in the PTX-treated group com-pared with the control group (P<0.01). PTX up-regulated expression of VEGF mRNA in a dose- and time-dependent man-ner in cultured HK-2 cells (P<0.01). However, expression of HIF-1α (a key transcription factor for VEGF gene expression) was unchanged by PTX treatment. PTX prolonged the half-life of VEGF mRNA by a 1.07-fold increase. Conclusions: PTX inhibited tubulointerstitial fibrosis in a rat model of obstructive nephropathy while preventing loss of VEGF. PTX up-regulated expression of VEGF mRNA through stabilization of its mRNA in cultured renal tubular epithelial cells.

  10. IBM PC/IX operating system evaluation plan

    Science.gov (United States)

    Dominick, Wayne D. (Editor); Granier, Martin; Hall, Philip P.; Triantafyllopoulos, Spiros

    1984-01-01

    An evaluation plan for the IBM PC/IX Operating System designed for IBM PC/XT computers is discussed. The evaluation plan covers the areas of performance measurement and evaluation, software facilities available, man-machine interface considerations, networking, and the suitability of PC/IX as a development environment within the University of Southwestern Louisiana NASA PC Research and Development project. In order to compare and evaluate the PC/IX system, comparisons with other available UNIX-based systems are also included.

  11. Genome-wide RNAi screen identifies broadly-acting host factors that inhibit arbovirus infection.

    Directory of Open Access Journals (Sweden)

    Ari Yasunaga

    2014-02-01

    Full Text Available Vector-borne viruses are an important class of emerging and re-emerging pathogens; thus, an improved understanding of the cellular factors that modulate infection in their respective vertebrate and insect hosts may aid control efforts. In particular, cell-intrinsic antiviral pathways restrict vector-borne viruses including the type I interferon response in vertebrates and the RNA interference (RNAi pathway in insects. However, it is likely that additional cell-intrinsic mechanisms exist to limit these viruses. Since insects rely on innate immune mechanisms to inhibit virus infections, we used Drosophila as a model insect to identify cellular factors that restrict West Nile virus (WNV, a flavivirus with a broad and expanding geographical host range. Our genome-wide RNAi screen identified 50 genes that inhibited WNV infection. Further screening revealed that 17 of these genes were antiviral against additional flaviviruses, and seven of these were antiviral against other vector-borne viruses, expanding our knowledge of invertebrate cell-intrinsic immunity. Investigation of two newly identified factors that restrict diverse viruses, dXPO1 and dRUVBL1, in the Tip60 complex, demonstrated they contributed to antiviral defense at the organismal level in adult flies, in mosquito cells, and in mammalian cells. These data suggest the existence of broadly acting and functionally conserved antiviral genes and pathways that restrict virus infections in evolutionarily divergent hosts.

  12. FACTORS WHICH INHIBIT PROFESSORIAL CREATIVITY. PSYCHO PEDAGOGICAL ANALYSIS / FACTORES QUE INHIBEN LA CREATIVIDAD PROFESORAL. ANÁLISIS PSICOPEDAGÓGICO

    Directory of Open Access Journals (Sweden)

    Odiel Estrada Molina

    2012-07-01

    Full Text Available Several researches exposed some factors that inhibit creative thinking, but generally without psychological or pedagogical argument. In this paper is shown from a psycho pedagogical perspective some factors that hinder the development of creativity, and its final execution to students. It presents four types of inhibitory factors of creativity as: a Disorders in the affective process of teachers; b The burnout syndrome, c The modeling, and d the application ineffective communication styles. It refers to the disturbances in the processes of psychological phenomena; outlines the main theories of emotion, burnout, and modeling, and its negative impact on creativity, finally makes a pedagogical reflection on the different communication styles and their relationship with creativity.RESUMENDiversas investigaciones exponen algunos factores que inhiben el pensamiento creativo, pero generalmente sin una previa argumentación psicológica o pedagógica, es por ello que en este trabajo se presenta desde una perspectiva psicopedagógica cuatro factores que impiden el desarrollo de la creatividad, así como su ejecución final ante los alumnos. Los factores que se exponen en este trabajo son a los trastornos en el proceso afectivo del profesor; b el síndrome de desgaste profesional; c la imitación o el modelado; y d la aplicación inefectiva de los estilos de comunicación. En el desarrollo del artículo se alude a los trastornos en los procesos de los fenómenos psicológicos; se exponen las principales teorías sobre las emociones, el burnout y el modelado, y sus consecuencias negativas en la creatividad, por último se realiza una reflexión pedagógica sobre los diferentes estilos de comunicación y su relación con la creatividad.

  13. Glial Tumor Necrosis Factor Alpha (TNFα) Generates Metaplastic Inhibition of Spinal Learning

    Science.gov (United States)

    Huie, J. Russell; Baumbauer, Kyle M.; Lee, Kuan H.; Bresnahan, Jacqueline C.; Beattie, Michael S.; Ferguson, Adam R.; Grau, James W.

    2012-01-01

    Injury-induced overexpression of tumor necrosis factor alpha (TNFα) in the spinal cord can induce chronic neuroinflammation and excitotoxicity that ultimately undermines functional recovery. Here we investigate how TNFα might also act to upset spinal function by modulating spinal plasticity. Using a model of instrumental learning in the injured spinal cord, we have previously shown that peripheral intermittent stimulation can produce a plastic change in spinal plasticity (metaplasticity), resulting in the prolonged inhibition of spinal learning. We hypothesized that spinal metaplasticity may be mediated by TNFα. We found that intermittent stimulation increased protein levels in the spinal cord. Using intrathecal pharmacological manipulations, we showed TNFα to be both necessary and sufficient for the long-term inhibition of a spinal instrumental learning task. These effects were found to be dependent on glial production of TNFα and involved downstream alterations in calcium-permeable AMPA receptors. These findings suggest a crucial role for glial TNFα in undermining spinal learning, and demonstrate the therapeutic potential of inhibiting TNFα activity to rescue and restore adaptive spinal plasticity to the injured spinal cord. TNFα modulation represents a novel therapeutic target for improving rehabilitation after spinal cord injury. PMID:22745823

  14. SNS-032 Prevents Tumor Cell-Induced Angiogenesis By Inhibiting Vascular Endothelial Growth Factor

    Directory of Open Access Journals (Sweden)

    M. Aktar Ali

    2007-05-01

    Full Text Available Cell proliferation, migration, and capillary network formation of endothelial cells are the fundamental steps for angiogenesis, which involves the formation of new blood vessels. The purpose of this study is to investigate the effect of a novel aminothiazole SNS-032 on these critical steps for in vitro angiogenesis using a coculture system consisting of human umbilical vein endothelial cells (HUVECs and human glioblastoma cells (U87MG. SNS-032 is a potent selective inhibitor of cyclin-dependent kinases 2, 7, and 9, and inhibits both transcription and cell cycle. In this study, we examined the proliferation and viability of HUVECs and U87MG cells in the presence of SNS-032 and observed a dose-dependent inhibition of cellular proliferation in both cell lines. SNS-032 inhibited threedimensional capillary network formations of endothelial cells. In a coculture study, SNS-032 completely prevented U87MG cell-mediated capillary formation of HUVECs. This inhibitor also prevented the migration of HUVECs when cultured alone or cocultured with U87MG cells. In addition, SNS-032 significantly prevented the production of vascular endothelial growth factor (VEGF in both cell lines, whereas SNS-032 was less effective in preventing capillary network formation and migration of endothelial cells when an active recombinant VEGF was added to the medium. In conclusion, SNS-032 prevents in vitro angiogenesis, and this action is attributable to blocking of VEGF.

  15. Inhibition of epidermal growth factor receptor expression by RNA interference in A549 cells

    Institute of Scientific and Technical Information of China (English)

    MinZHANG; XinZHANG; Chun-xueBAI; JieCHEN; MinQWEI

    2004-01-01

    AIM: To investigate the biological features of A549 cells in which epidermal growth factor (EGF) receptors expression were suppressed by RNA interference (RNAi). METHODS: A549 cells were transfected using short small interfering RNAs (siRNAs) formulated with Lipofectamine 2000. The EGF receptor numbers were determined by Western blotting and flowcytometry. The antiproliferative effects of sequence specific double stranded RNA (dsRNA) were assessed using cell count, colony assay and scratch assay. The chemosensitivity of transfected cells to cisplatin was measured by MTT. RESULTS: Sequence specific dsRNA-EGFR down-regulated EGF receptor expression dramatically. Compared with the control group, dsRNA-EGFR reduced the cell number by 85.0 %, decreased the colonies by 63.3 %, inhibited the migration by 87.2 %, and increased the sensitivity of A549 to cisplatin by four-fold. CONCLUSION: Sequence specific dsRNA-EGFR were capable of suppressing EGF receptor expression, hence significantly inhibiting cellular proliferation and motility, and enhancing chemosensitivity of A549 cells to cisplatin. The successful application of dsRNA-EGFR for inhibition of proliferation in EGF receptor overexpressing cells can help extend the list of available therapeutic modalities in the treatment of non-small-cell lung carcinoma (NSCLC).

  16. Tumour necrosis factorinhibition can stabilize disease in progressive vitiligo.

    Science.gov (United States)

    Webb, K C; Tung, R; Winterfield, L S; Gottlieb, A B; Eby, J M; Henning, S W; Le Poole, I C

    2015-09-01

    Tumour necrosis factor (TNF)-α, a proinflammatory cytokine central to many autoimmune diseases, has been implicated in the depigmentation process in vitiligo. We review its role in vitiligo by exploring its pro- and anti-inflammatory properties and examine the effects of blocking its actions with TNF-α antagonist therapeutics in reports available in the literature. We found that TNF-α inhibition halts disease progression in patients with progressive vitiligo but that, paradoxically, treatment can be associated with de novo vitiligo development in some patients when used for other autoimmune conditions, particularly when using adalimumab and infliximab. These studies reinforce the importance of stating appropriate outcomes measures, as most pilot trials propose to measure repigmentation, whereas halting depigmentation is commonly overlooked as a measure of success. We conclude that TNF-α inhibition has proven useful for patients with progressive vitiligo, where TNF-α inhibition is able to quash cytotoxic T-cell-mediated melanocyte destruction. However, a lingering concern for initiating de novo disease will likely prevent more widespread application of TNF inhibitors to treat vitiligo.

  17. Protoporphyrin IX in the skin measured noninvasively predicts photosensitivity in patients with erythropoietic protoporphyria

    DEFF Research Database (Denmark)

    Heerfordt, I M; Wulf, H C

    2016-01-01

    BACKGROUND: Erythropoietic protoporphyria (EPP) is a rare genetic disease that causes severe sensitivity to visible light as a result of protoporphyrin IX (PpIX) accumulation in the skin. OBJECTIVES: To establish a noninvasive method to measure PpIX in the skin of patients with EPP...... and to investigate how skin PpIX relates to erythrocyte PpIX and photosensitivity. METHODS: Skin PpIX was measured in 25 patients with EPP by calculating the difference in PpIX fluorescence before and after complete photobleaching of PpIX using controlled illumination. The patients reported symptoms during...... the illumination and skin erythema was measured before and after illumination. Confirmation of the presence of PpIX was obtained in seven patients by measuring the in vivo fluorescence emission spectrum. This method was used to examine skin PpIX during the hours after an illumination in seven patients. RESULTS: We...

  18. Inhibition of nuclear factor-kappa B differentially affects thyroid cancer cell growth, apoptosis, and invasion

    Directory of Open Access Journals (Sweden)

    Schweppe Rebecca E

    2010-05-01

    Full Text Available Abstract Background Nuclear factor-κB (NF-κB is constitutively activated in many cancers and plays a key role in promoting cell proliferation, survival, and invasion. Our understanding of NF-κB signaling in thyroid cancer, however, is limited. In this study, we have investigated the role of NF-κB signaling in thyroid cancer cell proliferation, invasion, and apoptosis using selective genetic inhibition of NF-κB in advanced thyroid cancer cell lines. Results Three pharmacologic inhibitors of NF-κB differentially inhibited growth in a panel of advanced thyroid cancer cell lines, suggesting that these NF-κB inhibitors may have off-target effects. We therefore used a selective genetic approach to inhibit NF-κB signaling by overexpression of a dominant-negative IκBα (mIκBα. These studies revealed decreased cell growth in only one of five thyroid cancer cell lines (8505C, which occurred through a block in the S-G2/M transition. Resistance to TNFα-induced apoptosis was observed in all cell lines, likely through an NF-κB-dependent mechanism. Inhibition of NF-κB by mIκBα sensitized a subset of cell lines to TNFα-induced apoptosis. Sensitive cell lines displayed sustained activation of the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK pathway, defining a potential mechanism of response. Finally, NF-κB inhibition by mIκBα expression differentially reduced thyroid cancer cell invasion in these thyroid cancer cell lines. Sensitive cell lines demonstrated approximately a two-fold decrease in invasion, which was associated with differential expression of MMP-13. MMP-9 was reduced by mIκBα expression in all cell lines tested. Conclusions These data indicate that selective inhibition of NF-κB represents an attractive therapeutic target for the treatment of advanced thyroid. However, it is apparent that global regulation of thyroid cancer cell growth and invasion is not achieved by NF-κB signaling alone. Instead, our

  19. Bianchi-IX string cosmological model in Lyra geometry

    Indian Academy of Sciences (India)

    F Rahaman; S Chakraborty; N Begum; M Hossain; M Kalam

    2003-06-01

    A class of cosmological solutions of massive strings for the Bianchi-IX space-time are obtained within the framework of Lyra geometry. Various physical and kinematical properties of the models are discussed.

  20. Heterogeneous nuclear ribonucleoprotein K inhibits heat shock-induced transcriptional activity of heat shock factor 1.

    Science.gov (United States)

    Kim, Hee-Jung; Lee, Jae-Jin; Cho, Jin-Hwan; Jeong, Jaeho; Park, A Young; Kang, Wonmo; Lee, Kong-Joo

    2017-08-04

    When cells are exposed to heat shock and various other stresses, heat shock factor 1 (HSF1) is activated, and the heat shock response (HSR) is elicited. To better understand the molecular regulation of the HSR, we used 2D-PAGE-based proteome analysis to screen for heat shock-induced post-translationally modified cellular proteins. Our analysis revealed that two protein spots typically present on 2D-PAGE gels and containing heterogeneous nuclear ribonucleoprotein K (hnRNP K) with trioxidized Cys(132) disappeared after the heat shock treatment and reappeared during recovery, but the total amount of hnRNP K protein remained unchanged. We next tested whether hnRNP K plays a role in HSR by regulating HSF1 and found that hnRNP K inhibits HSF1 activity, resulting in reduced expression of hsp70 and hsp27 mRNAs. hnRNP K also reduced binding affinity of HSF1 to the heat shock element by directly interacting with HSF1 but did not affect HSF1 phosphorylation-dependent activation or nuclear localization. hnRNP K lost its ability to induce these effects when its Cys(132) was substituted with Ser, Asp, or Glu. These findings suggest that hnRNP K inhibits transcriptional activity of HSF1 by inhibiting its binding to heat shock element and that the oxidation status of Cys(132) in hnRNP K is critical for this inhibition. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Luteolin inhibits human prostate tumor growth by suppressing vascular endothelial growth factor receptor 2-mediated angiogenesis.

    Directory of Open Access Journals (Sweden)

    Poyil Pratheeshkumar

    Full Text Available Angiogenesis, the formation of new blood vessels from pre-existing vascular beds, is essential for tumor growth, invasion, and metastasis. Luteolin is a common dietary flavonoid found in fruits and vegetables. We studied the antiangiogenic activity of luteolin using in vitro, ex vivo, and in vivo models. In vitro studies using rat aortic ring assay showed that luteolin at non-toxic concentrations significantly inhibited microvessel sprouting and proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Luteolin also inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM and matrigel plug assay. Gelatin zymographic analysis demonstrated the inhibitory effect of luteolin on the activation of matrix metalloproteinases MMP-2 and MMP-9. Western blot analysis showed that luteolin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 in HUVECs. Proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α level were significantly reduced by the treatment of luteolin in PC-3 cells. Luteolin (10 mg/kg/d significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that luteolin inhibited tumorigenesis by targeting angiogenesis. CD31 and CD34 immunohistochemical staining further revealed that the microvessel density could be remarkably suppressed by luteolin. Moreover, luteolin reduced cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 expressions. Taken together, our findings demonstrate that luteolin inhibits human prostate tumor growth by suppressing vascular endothelial growth factor receptor 2-mediated angiogenesis.

  2. Olive oil compounds inhibit vascular endothelial growth factor receptor-2 phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Lamy, Sylvie, E-mail: lamy.sylvie@uqam.ca; Ouanouki, Amira; Béliveau, Richard; Desrosiers, Richard R.

    2014-03-10

    Vascular endothelial growth factor (VEGF) triggers crucial signaling processes that regulate tumor angiogenesis and, therefore, represents an attractive target for the development of novel anticancer therapeutics. Several epidemiological studies have confirmed that abundant consumption of foods from plant origin is associated with reduced risk of developing cancers. In the Mediterranean basin, the consumption of extra virgin olive oil is an important constituent of the diet. Compared to other vegetable oils, the presence of several phenolic antioxidants in olive oil is believed to prevent the occurrence of a variety of pathological processes, such as cancer. While the strong antioxidant potential of these molecules is well characterized, their antiangiogenic activities remain unknown. The aim of this study is to investigate whether tyrosol (Tyr), hydroxytyrosol (HT), taxifolin (Tax), oleuropein (OL) and oleic acid (OA), five compounds contained in extra virgin olive oil, can affect in vitro angiogenesis. We found that HT, Tax and OA were the most potent angiogenesis inhibitors through their inhibitory effect on specific autophosphorylation sites of VEGFR-2 (Tyr951, Tyr1059, Tyr1175 and Tyr1214) leading to the inhibition of endothelial cell (EC) signaling. Inhibition of VEGFR-2 by these olive oil compounds significantly reduced VEGF-induced EC proliferation and migration as well as their morphogenic differentiation into capillary-like tubular structures in Matrigel. Our study demonstrates that HT, Tax and OA are novel and potent inhibitors of the VEGFR-2 signaling pathway. These findings emphasize the chemopreventive properties of olive oil and highlight the importance of nutrition in cancer prevention. - Highlights: • We investigated five compounds contained in extra virgin olive oil on angiogenesis. • Hydroxytyrosol, taxifolin and oleic acid are the best angiogenesis inhibitors. • Olive oil compounds affect endothelial cell functions essential for

  3. Dauricine inhibits insulin-like growth factor-Ⅰ-induced hypoxia inducible factor 1α protein accumulation and vascular endothelial growth factor expression in human breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    Xu-dong TANG; Xin ZHOU; Ke-yuan ZHOU

    2009-01-01

    Aim: To investigate the effects of dauricine (Dau) on insulin-like growth factor-Ⅰ (IGF-Ⅰ)-induced hypoxia inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression in human breast cancer cells (MCF-7).Methods: Serum-starved MCF-7 cells were pretreated for 1 h with different concentrations of Dau, followed by incubation with IGF-Ⅰ for 6 h. HIF-1α and VEGF protein expression levels were analyzed by Western blotting and ELISA, respectively.HIF-1α and VEGF mRNA levels were determined by real-time PCR. In vitro angiogenesis was observed via the human umbilical vein endothelial cell (HUVEC) tube formation assay. An in vitro invasion assay on HUVECs was performed.Results: Dau significantly inhibited IGF-Ⅰ-induced HIF-1α protein expression but had no effect on HIF-1α mRNA expression. However, Dau remarkably suppressed VEGF expression at both protein and mRNA levels in response to IGF-Ⅰ.Mechanistically, Dau suppressed IGF-Ⅰ-induced HIF-1α and VEGF protein expression mainly by blocking the activation of PI-3K/AKT/mTOR signaling pathway. In addition, Dan reduced IGF-Ⅰ-induced HIF-1α protein accumulation by inhibiting its synthesis as well as by promoting its degradation. Functionally, Dau inhibited angiogenesis in vitro. Moreover, Dau had a direct effect on IGF-Ⅰ-induced invasion of HUVECs.Conclusion: Dau inhibits human breast cancer angiogenesis by suppressing HIF-1α protein accumulation and VEGF expression, which may provide a novel potential mechanism for the anticancer activities of Dau in human breast cancer.

  4. Recent advances in the structural molecular biology of Ets transcription factors: interactions, interfaces and inhibition.

    Science.gov (United States)

    Cooper, Christopher D O; Newman, Joseph A; Gileadi, Opher

    2014-02-01

    The Ets family of eukaryotic transcription factors is based around the conserved Ets DNA-binding domain. Although their DNA-binding selectivity is biochemically and structurally well characterized, structures of homodimeric and ternary complexes point to Ets domains functioning as versatile protein-interaction modules. In the present paper, we review the progress made over the last decade to elucidate the structural mechanisms involved in modulation of DNA binding and protein partner selection during dimerization. We see that Ets domains, although conserved around a core architecture, have evolved to utilize a variety of interaction surfaces and binding mechanisms, reflecting Ets domains as dynamic interfaces for both DNA and protein interaction. Furthermore, we discuss recent advances in drug development for inhibition of Ets factors, and the roles structural biology can play in their future.

  5. Insulin like growth factor-1/insulin bypasses Pref-1/FA1-mediated inhibition of adipocyte differentiation

    DEFF Research Database (Denmark)

    Zhang, Hongbin; Nøhr, Jane; Jensen, Charlotte Harken;

    2003-01-01

    of Pref-1/FA1 in 3T3-L1 or 3T3-F442A cells inhibited adipocyte differentiation when insulin or insulin-like growth factor-1 (IGF-1) was omitted from the differentiation mixture. We demonstrate that the level of the mature form of the IGF-1 receptor is reduced and that IGF-1-dependent activation of p42/p44......, and adipocyte differentiation in a dose-dependent manner. Udgivelsesdato: 2003-Jun-6......Pref-1 is a highly glycosylated Delta-like transmembrane protein containing six epidermal growth factor-like repeats in the extracellular domain. Pref-1 is abundantly expressed in preadipocytes, but expression is down-regulated during adipocyte differentiation. Forced expression of Pref-1 in 3T3-L1...

  6. Protamine sulfate down-regulates thrombin generation by inhibiting factor V activation.

    LENUS (Irish Health Repository)

    Ni Ainle, Fionnuala

    2009-08-20

    Protamine sulfate is a positively charged polypeptide widely used to reverse heparin-induced anticoagulation. Paradoxically, prospective randomized trials have shown that protamine administration for heparin neutralization is associated with increased bleeding, particularly after cardiothoracic surgery with cardiopulmonary bypass. The molecular mechanism(s) through which protamine mediates this anticoagulant effect has not been defined. In vivo administration of pharmacologic doses of protamine to BALB\\/c mice significantly reduced plasma thrombin generation and prolonged tail-bleeding time (from 120 to 199 seconds). Similarly, in pooled normal human plasma, protamine caused significant dose-dependent prolongations of both prothrombin time and activated partial thromboplastin time. Protamine also markedly attenuated tissue factor-initiated thrombin generation in human plasma, causing a significant decrease in endogenous thrombin potential (41% +\\/- 7%). As expected, low-dose protamine effectively reversed the anticoagulant activity of unfractionated heparin in plasma. However, elevated protamine concentrations were associated with progressive dose-dependent reduction in thrombin generation. To assess the mechanism by which protamine mediates down-regulation of thrombin generation, the effect of protamine on factor V activation was assessed. Protamine was found to significantly reduce the rate of factor V activation by both thrombin and factor Xa. Protamine mediates its anticoagulant activity in plasma by down-regulation of thrombin generation via a novel mechanism, specifically inhibition of factor V activation.

  7. Inhibition of the TEF/TEAD transcription factor activity by nuclear calcium and distinct kinase pathways.

    Science.gov (United States)

    Thompson, M; Andrade, V A; Andrade, S J; Pusl, T; Ortega, J M; Goes, A M; Leite, M F

    2003-02-07

    Transcription enhancer factor (TEF/TEAD) is a family of four transcription factors that share a common TEA-DNA binding domain and are involved in similar cellular functions, such as cell differentiation and proliferation. All adult tissues express at least one of the four TEAD genes, so this family of transcription factors may be of widespread importance, yet little is known about their regulation. Here we examine the factors that regulate TEAD activity in CHO cells. RT-PCR indicated the presence of TEAD-1, TEAD-3, and both isoforms of TEAD-4, but not TEAD-2. Quantitative measurements showed that TEAD-4 is most abundant, followed by TEAD-3, then TEAD-1. We examined the relative effects of nuclear and cytosolic Ca(2+) on TEAD activity, since TEAD proteins are localized to the nucleus and since free Ca(2+) within the nucleus selectively regulates transcription in some systems. Chelation of nuclear but not cytosolic Ca(2+) increased TEAD activity two times above control. Inhibition of mitogen-activated protein kinase (MAPK) also increased TEAD activity, while cAMP decreased TEAD activity, and protein kinase C had no effect. Together, these results show that nuclear Ca(2+), MAPK, and cAMP each negatively regulate the activity of the TEAD transcription factor.

  8. 硬汉 现代ix45

    Institute of Scientific and Technical Information of China (English)

    TNT

    2012-01-01

    在纽约车展亮相的现代圣达菲虽然采用了ix45的全新家族命名,但是在美国市场上还依旧傈留着Santa Fe的名字,预计年内在北京现代三工厂投产后也将采用ix45这一命名。

  9. Potassium inhibits dietary salt-induced transforming growth factor-beta production.

    Science.gov (United States)

    Ying, Wei-Zhong; Aaron, Kristal; Wang, Pei-Xuan; Sanders, Paul W

    2009-11-01

    Human and animal studies demonstrate an untoward effect of excess dietary NaCl (salt) intake on cardiovascular function and life span. The endothelium in particular augments the production of transforming growth factor (TGF)-beta, a fibrogenic growth factor, in response to excess dietary salt intake. This study explored the initiating mechanism that regulates salt-induced endothelial cell production of TGF-beta. Male Sprague-Dawley rats were given diets containing different amounts of NaCl and potassium for 4 days. A bioassay for TGF-beta demonstrated increased (35.2%) amounts of active TGF-beta in the medium of aortic ring segments from rats on the high-salt diet compared with rats maintained on a 0.3% NaCl diet. Inhibition of the large-conductance, calcium-activated potassium channel inhibited dietary salt-induced vascular production of TGF-beta but did not affect production of TGF-beta by ring segments from rats on the low-salt diet. Immunohistochemical and Western analyses demonstrated the alpha subunit of the calcium-activated potassium channel in endothelial cells. Increasing medium [K+] inhibited production of dietary salt-induced vascular production levels of total and active TGF-beta but did not alter TGF-beta production by aortic rings from rats on the 0.3% NaCl diet. Increasing dietary potassium content decreased urinary active TGF-beta in animals receiving the high-salt diet but did not change urinary active TGF-beta in animals receiving the low-salt diet. The findings demonstrated an interesting interaction between the dietary intake of potassium and excess NaCl and further showed the fundamental role of the endothelial calcium-activated potassium channel in the vascular response to excess salt intake.

  10. Ghrelin Attenuated Lipotoxicity via Autophagy Induction and Nuclear Factor-κB Inhibition

    Directory of Open Access Journals (Sweden)

    Yuqing Mao

    2015-09-01

    Full Text Available Background/Aims: Nonalcoholic fatty liver disease (NAFLD is the most common chronic liver disease worldwide. Autophagy is associated with NAFLD. Ghrelin is a gut hormone with various functions including energy metabolism and inflammation inhibition. We investigated the therapeutic effect of ghrelin on NAFLD and its association with autophagy. Methods: C57bl/6 mice were fed a high-fat diet for 8 weeks to induce a model of chronic NAFLD, with ghrelin (10 µg/kg administrated subcutaneously twice weekly from weeks 6 to 8. LO2 cells were pretreated with ghrelin (10-8 M before stimulation with free fatty acid (palmitic and oleic acids; 1 mM. Lipid droplets were identified by hematoxylin and eosin and Red O staining and quantified by triglyceride test kits. LC3I/II, an important biomarker protein of autophagy was detected by western blotting, real-time polymerase chain reaction, immunohistochemistry and immunofluorescence. Tumor necrosis factor (TNF-a and interleukin (IL-6 were detected by ELISA and immunohistochemistry. Nuclear factor (NF-κB p65 was detected by western blotting and immunofluorescence. AMP-activated protein kinase (AMPK and mammalian target of rapamycin (mTOR were detected by western blotting. Results: Ghrelin reduced the triglyceride content in high fat diet (HFD group in vivo and free fatty acid (FFA group in vitro. TNF-a and IL-6 were significantly reduced in the ghrelin-treated mice compared with the control group. Autophagy induction was accompanied with intracellular lipid reduction in ghrelin-treated mice. Ghrelin upregulated autophagy via AMPK/mTOR restoration and inhibited translocation of NF-κB into the nucleus. Conclusions: The results indicate that ghrelin attenuates lipotoxicity by autophagy stimulation and NF-κB inhibition.

  11. Effects of epidermal growth factor receptor kinase inhibition on radiation response in canine osteosarcoma cells.

    Science.gov (United States)

    Mantovani, Fernanda B; Morrison, Jodi A; Mutsaers, Anthony J

    2016-05-31

    Radiation therapy is a palliative treatment modality for canine osteosarcoma, with transient improvement in analgesia observed in many cases. However there is room for improvement in outcome for these patients. It is possible that the addition of sensitizing agents may increase tumor response to radiation therapy and prolong quality of life. Epidermal growth factor receptor (EGFR) expression has been documented in canine osteosarcoma and higher EGFR levels have been correlated to a worse prognosis. However, effects of EGFR inhibition on radiation responsiveness in canine osteosarcoma have not been previously characterized. This study examined the effects of the small molecule EGFR inhibitor erlotinib on canine osteosarcoma radiation responses, target and downstream protein expression in vitro. Additionally, to assess the potential impact of treatment on tumor angiogenesis, vascular endothelial growth factor (VEGF) levels in conditioned media were measured. Erlotinib as a single agent reduced clonogenic survival in two canine osteosarcoma cell lines and enhanced the impact of radiation in one out of three cell lines investigated. In cell viability assays, erlotinib enhanced radiation effects and demonstrated single agent effects. Erlotinib did not alter total levels of EGFR, nor inhibit downstream protein kinase B (PKB/Akt) activation. On the contrary, erlotinib treatment increased phosphorylated Akt in these osteosarcoma cell lines. VEGF levels in conditioned media increased after erlotinib treatment as a single agent and in combination with radiation in two out of three cell lines investigated. However, VEGF levels decreased with erlotinib treatment in the third cell line. Erlotinib treatment promoted modest enhancement of radiation effects in canine osteosarcoma cells, and possessed activity as a single agent in some cell lines, indicating a potential role for EGFR inhibition in the treatment of a subset of osteosarcoma patients. The relative radioresistance of

  12. Memory extinction entails the inhibition of the transcription factor NF-kappaB.

    Directory of Open Access Journals (Sweden)

    Emiliano Merlo

    Full Text Available In contextual memories, an association between a positive or negative reinforcement and the contextual cues where the reinforcement occurs is formed. The re-exposure to the context without reinforcement can lead to memory extinction or reconsolidation, depending on the number of events or duration of a single event of context re-exposure. Extinction involves the temporary waning of the previously acquired conditioned response. The molecular processes underlying extinction and the mechanisms which determine if memory will reconsolidate or extinguish after retrieval are not well characterized, particularly the role of transcription factors and gene expression. Here we studied the participation of a transcription factor, NF-kappaB, in memory extinction. In the crab context-signal memory, the activation of NF-kappaB plays a critical role in consolidation and reconsolidation, memory processes that are well characterized in this model. The administration of a NF-kappaB inhibitor, sulfasalazine prior to extinction session impeded spontaneous recovery. Moreover, reinstatement experiments showed that the original memory was not affected and that NF-kappaB inhibition by sulfasalazine impaired spontaneous recovery strengthening the ongoing memory extinction process. Interestingly, in animals with fully consolidated memory, a brief re-exposure to the training context induced neuronal NF-kappaB activation and reconsolidation, while prolonged re-exposure induced NF-kappaB inhibition and memory extinction. These data constitutes a novel insight into the molecular mechanisms involved in the switch between memory reconsolidation and extinction. Moreover, we propose the inhibition of NF-kappaB as the engaged mechanism underlying extinction, supporting a novel approach for the pharmacological enhancement of this memory process. The accurate description of the molecular mechanisms that support memory extinction is potentially useful for developing new strategies

  13. AZD5363 inhibits inflammatory synergy between interleukin-17 and insulin/insulin-like growth factor 1

    Directory of Open Access Journals (Sweden)

    Chong eChen

    2014-12-01

    Full Text Available In the United States, one third of population is affected by obesity and almost 29 million people are suffering from type 2 diabetes. Obese people have elevated serum levels of insulin, insulin-like growth factor 1 (IGF1 and interleukin-17 (IL-17. Insulin and IGF1 are known to enhance IL-17-induced expression of inflammatory cytokines and chemokines, which may contribute to the chronic inflammatory status observed in obese people. We have previously demonstrated that insulin/IGF1 signaling pathway crosstalks with IL-17-activated nuclear factor-kappa B (NF-κB pathway through inhibiting glycogen synthase kinase 3β (GSK3β activity. However, it is unclear whether GSK3α also plays a role and whether this crosstalk can be manipulated by AZD5363, a novel pan-Akt inhibitor that has been shown to increase GSK3 activity through reducing phosphorylation of GSK3α and GSK3β. In this study, we investigated IL-17-induced expression of C-X-C motif ligand 1 (Cxcl1, C-C motif ligand 20 (Ccl20 and interleukin-6 (Il-6 in wild-type, GSK3α-/-, and GSK3β-/- mouse embryonic fibroblast (MEF cells as well as in mouse prostate tissues by real-time quantitative PCR. We examined the proteins involved in the signaling pathways by Western blot analysis. We found that insulin and IGF1 enhanced IL-17- induced expression of Cxcl1, Ccl20 and Il-6, which was associated with increased phosphorylation of GSK3α and GSK3β in the presence of insulin and IGF1. AZD5363 inhibited the synergy between IL-17 and insulin/IGF1 through reducing phosphorylation of GSK3α and GSK3β by inhibiting Akt function. These findings imply that the cooperative crosstalk of IL-17 and insulin/IGF1 in initiating inflammatory responses may be alleviated by AZD5363.

  14. Niacin inhibits vascular inflammation via downregulating nuclear transcription factor-κB signaling pathway.

    Science.gov (United States)

    Si, Yanhong; Zhang, Ying; Zhao, Jilong; Guo, Shoudong; Zhai, Lei; Yao, Shutong; Sang, Hui; Yang, Nana; Song, Guohua; Gu, Jue; Qin, Shucun

    2014-01-01

    The study aimed to investigate the effect of niacin on vascular inflammatory lesions in vivo and in vitro as well as its lipid-regulating mechanism. In vivo study revealed that niacin downregulated the levels of inflammatory factors (IL-6 and TNF-α) in plasma, suppressed protein expression of CD68 and NF-κB p65 in arterial wall, and attenuated oxidative stress in guinea pigs that have been fed high fat diet. In vitro study further confirmed that niacin decreased the secretion of IL-6 and TNF-α and inhibited NF-κB p65 and notch1 protein expression in oxLDL-stimulated HUVECs and THP-1 macrophages. Moreover, niacin attenuated oxLDL-induced apoptosis of HUVECs as well. In addition, niacin significantly lessened lipid deposition in arterial wall, increased HDL-C and apoA levels and decreased TG and non-HDL-C levels in plasma, and upregulated the mRNA amount of cholesterol 7 α-hydroxylase A1 in liver of guinea pigs. These data suggest for the first time that niacin inhibits vascular inflammation in vivo and in vitro via downregulating NF-κB signaling pathway. Furthermore, niacin also modulates plasma lipid by upregulating the expression of factors involved in the process of reverse cholesterol transport.

  15. Ellagic acid induces apoptosis through inhibition of nuclear factor in pancreatic cancer cells

    Institute of Scientific and Technical Information of China (English)

    Mouad Edderkaoui; Irina Odinokova; Izumi Ohno; Ilya Gukovsky; Vay Liang W Go; Stephen J Pandol; Anna S Gukovskaya

    2008-01-01

    AIM: To determine the effect of ellagic acid on apoptosis and proliferation in pancreatic cancer cells and to determine the mechanism of the pro-survival effects of ellagic acid.METHODS: The effect of ellagic acid on apoptosis was assessed by measuring Phosphatidylserine externalization, caspase activity, mitochondrial membrane potential and DNA fragmentation; and proliferation by measuring DNA thymidine incorporation. Mitochondrial membrane potential was measured in permeabilized cells, and in isolated mitochondria. Nuclear factor kB (NF-kB) activity was measured by electromobility shift assay (EMSA).RESULTS: We show that ellagic acid, a polyphenolic compound in fruits and berries, at concentrations 10 to 50 mmol/L stimulates apoptosis in human pancreatic adenocarcinoma cells. Further, ellagic acid decreases proliferation by up to 20-fold at 50 mmol/L Ellagic acid stimulates the mitochondrial pathway of apoptosis associated with mitochondrial depolarization, cytochrome C release, and the downstream caspase activation. Ellagic acid does not directly affect mitochondria. Ellagic acid dose-dependently decreased NF-kB binding activity. Furthermore, inhibition of NF-kB activity using IkB wild type plasmid prevented the effect of ellagic acid on apoptosis.CONCLUSION: Our data indicate that ellagic acid stimulates apoptosis through inhibition of the prosurvival transcription factor NF-kB.

  16. Value-added and Supporting - Inhibiting Factors for the Wet Processing of Coffee

    Directory of Open Access Journals (Sweden)

    Yuli Hariyati

    2014-01-01

    Full Text Available Coffee is one of the annual crops which are widely favored by coffee enjoyers. SidomulyoVillage is one of the fourth largest coffee producing villages in District of Silo with a land area of 180 ha in 2009. Coffee experiences a process of harvest and post harvest; one of the activities of post-harvest is coffee processing. Coffee processing is divided into two; wet processing and dry processing. The majority of farmers in SidomulyoVillage do dry processing; about 75% of farmers do dry processing and 25% of farmers do wet processing. This research was intended to: (1 to find out the value added coffee processed,(2 to identify supporting and inhibiting factors the farmers to do wet processing, and (3 to identify the income differences of farmers undertakingthe wet and dry processing. This research was carried out on purpose (purposive method in the Sidomulyo Village, District of Silo, by taking samples; that is the total sampling of farmer group of Sidomulyo 1. Data analysis used including value added, Force Field and financial analysis. The research results showed that: (1 value added of coffee beans processing turn to HS coffee was IDR 975,- whereas coffee beans processing turn to ose coffee was IDR 529,-. (2 The strongest supporting factor of wet processing was the ability to absorb workers, while the strongest inhibiting factor of wet processing was less adequate water facilities; (3 The coffee farmer incomescarrying out wet processing and dry processing were different. PerHa coffee income of wet processing was IDR 11,228,805,- and that of dry processing per ha was IDR 7,901,249,-

  17. Aspirin inhibits tumor necrosis factor-α-stimulated fractalkine expression in human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    JIANG De-qian; LIU Hong; ZHANG She-bing; ZHANG Xiao-lian

    2009-01-01

    Background Fractalkine is an important chemokine mediating local monocyte accumulation and inflammatory reactions in the vascular wall. Aspirin inhibits inflammatory cytokine expression closely related to atherosclerosis through the way independent of platelet and cyclooxygenase (COX). There has been no report about the effect of aspirin on fractalkine expression. We aimed to determine the fractalkine expression in human umbilical vein endothelial cell (HUVEC) stimulated by tumor necrosis factor (TNF)-α and the effect of aspirin intervention.Methods Six of 8 HUVEC groups received either different concentrations of aspirin (0.02, 0.2, 1.0, 5.0 mmol/L) or 40 μmol/L pyrrolidinecarbodithioc acid (PDTC) or 0.5 μmol/L NS-398. The other two groups were negative control and positive control (TNF-α-stimulated). After being incubated for 24 hours, cells of the 8 groups except the negative control one were stimulated with TNF-a (4 ng/ml) for another 24 hours. After that, the cells were collected for RNA isolation and protein extraction.Results Both mRNA and protein expressions of fractalkine in HUVEC were upregulated by 4 ng/ml TNF-α stimulation,Aspirin inhibited fractalkine expression in a dose-dependent manner at mRNA and protein levels. Nuclear factor-kappa B inhibitor, PDTC, effectively decreased the fractalkine expression. Fractalkine expression was not influenced by COX-2 selective inhibitor NS-398. COX-1 protein expression was not changed by either TNF-α stimulation or aspirin, PDTC,NS-398 intervention. Both mRNA and protein expression of COX-2 in HUVEC were upregulated by 4 ng/ml TNF-α stimulation. Aspirin decreased COX-2 expression in a dose-dependent manner at mRNA and protein levels.Conclusions TNF-α-stimulated fractalkine expression is suppressed by aspirin in a dose-dependent manner through the nuclear factor-kappa B p65 pathway.

  18. Inhibition of tissue factor pathway inhibitor increases the sensitivity of thrombin generation assay to procoagulant microvesicles.

    Science.gov (United States)

    Gheldof, Damien; Mullier, François; Chatelain, Bernard; Dogné, Jean-Michel; Chatelain, Christian

    2013-07-01

    Patients with cancer have a seven-fold to 10-fold increased risk of developing venous thromboembolism (VTE). Circulating microvesicles could be a predictive biomarker for VTE in cancer. Thrombin generation assay (TGA) is a useful technique to detect procoagulant activity of microvesicles. However, TGA suffers from a lack of sensitivity due to the presence of tissue factor pathway inhibitor (TFPI) in plasma. The aim of the study was to improve the sensitivity of TGA to tissue factor by limiting the interference of TFPI. Serial dilutions of MDA-MB231 cells were incubated for 45 min at 37°C to generate microvesicles. Samples were then centrifuged and supernatants that contain microvesicles were used for TGA. Normal pooled plasma was incubated with inhibitor of TFPI or was diluted twice to decrease plasma level of TFPI. Lagtime was used as a surrogate marker of TGA to detect procoagulant activity of microvesicles. Inhibition of TFPI decreased twice the cell concentration needed for a significant reduction of lagtime and decreased 2.4-fold the intraassay variability. Plasma dilution had no impact on the TGA sensitivity when TGA was triggered by microvesicles derived from MDA-MB-231. Thrombin generation is a very sensitive method to study the procoagulant activity of tissue factor bearing microvesicles. The sensitivity can be increased by inhibition of TFPI with specific monoclonal antibody against its Kunitz domain I. A two times plasma dilution is an interesting cheaper alternative to study the procoagulant activity of microvesicles by TGA with a good sensitivity, especially when low plasma quantities are available.

  19. Coleusin factor, a novel anticancer diterpenoid, inhibits osteosarcoma growth by inducing bone morphogenetic protein-2-dependent differentiation.

    Science.gov (United States)

    Geng, Shuo; Sun, Bo; Lu, Ran; Wang, Jingze

    2014-06-01

    Coleusin factor is a diterpenoid compound isolated from the root of a tropical plant, Coleus forskohlii. Although Coleusin factor has been reported to suppress proliferation of and induce apoptosis in several types of cancer cells, the effects of Coleusin factor on osteosarcoma and the underlying mechanism are still not fully understood. In this study, we show that Coleusin factor treatment potently inhibits the growth of osteosarcoma cells associated with G(1) cell-cycle arrest. Interestingly, apoptosis and cell death are not induced. Instead, Coleusin factor causes osteosarcoma cells to exhibit typical properties of differentiated osteoblasts, including a morphologic alteration resembling osteoblasts, the expression of osteoblast differentiation markers, elevated alkaline phosphatase activity, and increased cellular mineralization. Coleusin factor treatment significantly increases the expression of bone morphogenetic protein-2 (BMP-2), a crucial osteogenic regulator, and runt-related transcription factor 2 (RUNX2), one of the key transcription factors of the BMP pathway. When BMP-2 signaling is blocked, Coleusin factor fails to inhibit cell proliferation and to induce osteoblast differentiation. Thus, upregulation of BMP-2 autocrine is critical for Coleusin factor to induce osteoblast differentiation and exert its anticancer effects on osteosarcoma. Importantly, administration of Coleusin factor inhibits the growth of osteosarcoma xenografted in nude mice without systemic or immunologic toxicity. Osteosarcoma is a highly aggressive cancer marked by the loss of normal differentiation. Coleusin factor represents a new type of BMP-2 inducer that restores differentiation in osteosarcoma cells. It may provide a promising therapeutic strategy against osteosarcoma with minimal side effects.

  20. Antagonism of CD11b with neutrophil inhibitory factor (NIF) inhibits vascular lesions in diabetic retinopathy.

    Science.gov (United States)

    Veenstra, Alexander A; Tang, Jie; Kern, Timothy S

    2013-01-01

    Leukocytes and proteins that govern leukocyte adhesion to endothelial cells play a causal role in retinal abnormalities characteristic of the early stages of diabetic retinopathy, including diabetes-induced degeneration of retinal capillaries. Leukocyte integrin αmβ2 (CD11b/CD18, MAC1), a protein mediating adhesion, has been shown to mediate damage to endothelial cells by activated leukocytes in vitro. We hypothesized that Neutrophil Inhibitory Factor (NIF), a selective antagonist of integrin αmβ2, would inhibit the diabetes-induced degeneration of retinal capillaries by inhibiting the excessive interaction between leukocytes and retinal endothelial cells in diabetes. Wild type animals and transgenic animals expressing NIF were made diabetic with streptozotocin and assessed for diabetes-induced retinal vascular abnormalities and leukocyte activation. To assess if the leukocyte blocking therapy compromised the immune system, animals were challenged with bacteria. Retinal superoxide production, leukostasis and leukocyte superoxide production were increased in wild type mice diabetic for 10 weeks, as was the ability of leukocytes isolated from diabetic animals to kill retinal endothelial cells in vitro. Retinal capillary degeneration was significantly increased in wild type mice diabetic 40 weeks. In contrast, mice expressing NIF did not develop any of these abnormalities, with the exception that non-diabetic and diabetic mice expressing NIF generated greater amounts of superoxide than did similar mice not expressing NIF. Importantly, NIF did not significantly impair the ability of mice to clear an opportunistic bacterial challenge, suggesting that NIF did not compromise immune surveillance. We conclude that antagonism of CD11b (integrin αmβ2) by NIF is sufficient to inhibit early stages of diabetic retinopathy, while not compromising the basic immune response.

  1. Antagonism of CD11b with neutrophil inhibitory factor (NIF inhibits vascular lesions in diabetic retinopathy.

    Directory of Open Access Journals (Sweden)

    Alexander A Veenstra

    Full Text Available Leukocytes and proteins that govern leukocyte adhesion to endothelial cells play a causal role in retinal abnormalities characteristic of the early stages of diabetic retinopathy, including diabetes-induced degeneration of retinal capillaries. Leukocyte integrin αmβ2 (CD11b/CD18, MAC1, a protein mediating adhesion, has been shown to mediate damage to endothelial cells by activated leukocytes in vitro. We hypothesized that Neutrophil Inhibitory Factor (NIF, a selective antagonist of integrin αmβ2, would inhibit the diabetes-induced degeneration of retinal capillaries by inhibiting the excessive interaction between leukocytes and retinal endothelial cells in diabetes. Wild type animals and transgenic animals expressing NIF were made diabetic with streptozotocin and assessed for diabetes-induced retinal vascular abnormalities and leukocyte activation. To assess if the leukocyte blocking therapy compromised the immune system, animals were challenged with bacteria. Retinal superoxide production, leukostasis and leukocyte superoxide production were increased in wild type mice diabetic for 10 weeks, as was the ability of leukocytes isolated from diabetic animals to kill retinal endothelial cells in vitro. Retinal capillary degeneration was significantly increased in wild type mice diabetic 40 weeks. In contrast, mice expressing NIF did not develop any of these abnormalities, with the exception that non-diabetic and diabetic mice expressing NIF generated greater amounts of superoxide than did similar mice not expressing NIF. Importantly, NIF did not significantly impair the ability of mice to clear an opportunistic bacterial challenge, suggesting that NIF did not compromise immune surveillance. We conclude that antagonism of CD11b (integrin αmβ2 by NIF is sufficient to inhibit early stages of diabetic retinopathy, while not compromising the basic immune response.

  2. Bivalirudin inhibits periprocedural platelet function and tissue factor expression of human smooth muscle cells.

    Science.gov (United States)

    Pepke, Wojciech; Eisenreich, Andreas; Jaster, Markus; Ayral, Yunus; Bobbert, Peter; Mayer, Alexander; Schultheiss, Heinz-Peter; Rauch, Ursula

    2013-04-01

    A major concern of stent implantation after percutaneous coronary intervention (PCI) is acute stent thrombosis. Effective inhibition of periprocedural platelet function in patients with coronary artery disease (CAD) leads to an improved outcome. In this study, we examined the periprocedural platelet reactivity after administrating bivalirudin during PCI compared to unfractionated heparin (UFH) administration. Further, the effect of bivalirudin on induced tissue factor (TF) expression in smooth muscle cells (SMC) was determined. Patients with CAD (n = 58) and double antithrombotic medication were treated intraprocedural with UFH (n = 30) or bivalirudin (n = 28). Platelet activation markers were flow cytometrically measured before and after stenting. The expression of TF in SMC was determined by real-time PCR and Western blotting. The thrombogenicity of platelet-derived microparticles and SMC was assessed via a TF activity assay. Bivalirudin significantly diminished the agonist-induced platelet reactivity post-PCI. Compared to UFH treatment, the adenosine diphosphate (ADP) and thrombin receptor-activating peptide (TRAP)-induced thrombospondin expression post-PCI was reduced when bivalirudin was administrated during intervention. In contrast to UFH, bivalirudin reduced the P-selectin expression of unstimulated and ADP-induced platelets post-PCI. Moreover, bivalirudin inhibited the thrombin-, but not FVIIa- or FVIIa/FX-induced TF expression and pro-coagulant TF activity of SMC. Moreover, bivalirudin reduced the TF activity of platelet-derived microparticles postinduction with TRAP or ADP. Bivalirudin is better than UFH in reducing periprocedural platelet activation. Moreover, thrombin-induced TF expression is inhibited by bivalirudin. Thus, bivalirudin seems to be a better anticoagulant during PCI than UFH. © 2011 Blackwell Publishing Ltd.

  3. Fibroblast growth factor 7 inhibits cholesterol 7{alpha}-hydroxylase gene expression in hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Zhichao [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Yu, Xuemei [Department of Endocrinology, Fengxian Central Hospital, Shanghai (China); Wu, Weibin; Jia, Dongwei; Chen, Yinle; Ji, Lingling; Liu, Xijun; Peng, Xiaomin [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Li, Yintao [Institute of Endocrinology and Diabetology, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai (China); Yang, Lili [Department of Endocrinology, Fengxian Central Hospital, Shanghai (China); Ruan, Yuanyuan; Gu, Jianxin [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Ren, Shifang, E-mail: renshifang@fudan.edu.cn [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Zhang, Songwen, E-mail: songwenzhang@fudan.edu.cn [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China)

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer FGF7 strongly and rapidly down-regulates the expression of CYP7A1 in hepatocytes. Black-Right-Pointing-Pointer FGF7 suppresses the expression of CYP7A1 via FGFR2 and downstream JNK activation. Black-Right-Pointing-Pointer Blocking FGF7 abrogates HSC-induced inhibition of CYP7A1 expression in hepatocytes. -- Abstract: Cholesterol 7{alpha}-hydroxylase (CYP7A1) is the initial and rate-limiting enzyme for bile acid synthesis. Transcription of the CYP7A1 gene is regulated by bile acids, nuclear receptors and cytokines. Fibroblast growth factor 7 (FGF7) secreted from activated hepatic stellate cells (HSC) during chronic liver fibrosis regulates hepatocyte survival and liver regeneration. In the carbon tetrachloride (CCl{sub 4})-induced fibrotic mouse liver, we demonstrated that the expression of CYP7A1 was largely decreased while the expression of FGF7 was significantly increased. We further demonstrated that FGF7 inhibited CYP7A1 gene expression in hepatocytes. Knockdown study by short interfering RNA, kinase inhibition and phosphorylation assays revealed that the suppression of CYP7A1 expression by FGF7 was mediated by FGFR2 and its downstream JNK signaling cascade. The FGF7 neutralizing antibody restored CYP7A1 expression in Hep3B cells treated with conditioned medium from HSC. In summary, the data suggest that FGF7 is a novel regulator of CYP7A1 expression in hepatocytes and may prevent hepatocytes from accumulating toxic bile acids during liver injury and fibrosis.

  4. Adipose stem cells can secrete angiogenic factors that inhibit hyaline cartilage regeneration.

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    Lee, Christopher Sd; Burnsed, Olivia A; Raghuram, Vineeth; Kalisvaart, Jonathan; Boyan, Barbara D; Schwartz, Zvi

    2012-08-24

    Adipose stem cells (ASCs) secrete many trophic factors that can stimulate tissue repair, including angiogenic factors, but little is known about how ASCs and their secreted factors influence cartilage regeneration. Therefore, the aim of this study was to determine the effects ASC-secreted factors have in repairing chondral defects. ASCs isolated from male Sprague Dawley rats were cultured in monolayer or alginate microbeads supplemented with growth (GM) or chondrogenic medium (CM). Subsequent co-culture, conditioned media, and in vivo cartilage defect studies were performed. ASC monolayers and microbeads cultured in CM had decreased FGF-2 gene expression and VEGF-A secretion compared to ASCs cultured in GM. Chondrocytes co-cultured with GM-cultured ASCs for 7 days had decreased mRNAs for col2, comp, and runx2. Chondrocytes treated for 12 or 24 hours with conditioned medium from GM-cultured ASCs had reduced sox9, acan, and col2 mRNAs; reduced proliferation and proteoglycan synthesis; and increased apoptosis. ASC-conditioned medium also increased endothelial cell tube lengthening whereas conditioned medium from CM-cultured ASCs had no effect. Treating ASCs with CM reduced or abolished these deleterious effects while adding a neutralizing antibody for VEGF-A eliminated ASC-conditioned medium induced chondrocyte apoptosis and restored proteoglycan synthesis. FGF-2 also mitigated the deleterious effects VEGF-A had on chondrocyte apoptosis and phenotype. When GM-grown ASC pellets were implanted in 1 mm non-critical hyaline cartilage defects in vivo, cartilage regeneration was inhibited as evaluated by radiographic and equilibrium partitioning of an ionic contrast agent via microCT imaging. Histology revealed that defects with GM-cultured ASCs had no tissue ingrowth from the edges of the defect whereas empty defects and defects with CM-grown ASCs had similar amounts of neocartilage formation. ASCs must be treated to reduce the secretion of VEGF-A and other factors that

  5. Human Immunodeficiency Virus Type 1 Nef Inhibits Autophagy through Transcription Factor EB Sequestration.

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    Grant R Campbell

    2015-06-01

    Full Text Available HIV Nef acts as an anti-autophagic maturation factor through interaction with beclin-1 (BECN1. We report that exposure of macrophages to infectious or non-infectious purified HIV induces toll-like receptor 8 (TLR8 and BECN1 dependent dephosphorylation and nuclear translocation of TFEB and that this correlates with an increase in autophagy markers. RNA interference for ATG13, TFEB, TLR8, or BECN1 inhibits this HIV-induced autophagy. However, once HIV establishes a productive infection, TFEB phosphorylation and cytoplasmic sequestration are increased resulting in decreased autophagy markers. Moreover, by 7 d post-infection, autophagy levels are similar to mock infected controls. Conversely, although Nef deleted HIV similarly induces TFEB dephosphorylation and nuclear localization, and increases autophagy, these levels remain elevated during continued productive infection. Thus, the interaction between HIV and TLR8 serves as a signal for autophagy induction that is dependent upon the dephosphorylation and nuclear translocation of TFEB. During permissive infection, Nef binds BECN1 resulting in mammalian target of rapamycin (MTOR activation, TFEB phosphorylation and cytosolic sequestration, and the inhibition of autophagy. To our knowledge, this is the first report of a virus modulating TFEB localization and helps to explain how HIV modulates autophagy to promote its own replication and cell survival.

  6. Melatonin inhibits the expression of vascular endothelial growth factor in pancreatic cancer cells

    Institute of Scientific and Technical Information of China (English)

    Dong Lv; Pei-Lin Cui; Shi-Wei Yao; You-Qing Xu; Zhao-Xu Yang

    2012-01-01

    Objective:To investigate the effects of melatonin on cellular proliferation and endogenous vascular endothelial growth factor (VEGF) expression in pancreatic carcinoma cells (PANC-1).Methods:PANC-1 cells were cultured for this study.The secreted VEGF concentration in the culture medium was determined using ELISA method,VEGF production in the tumor cells was detected by immunocytochemistry,and VEGF mRNA expression was determined by RT-PCR.Results:Higher melatonin concentrations significantly inhibited cellular proliferation,with 1 mmol/L concentration exhibiting the highest inhibitory effect (P<0.01).VEGF concentrations in the cell culture supernatants and intra-cellules were all significantly reduced after melatonin (1 mmol/L) incubation (P<0.05).VEGF mRNA expression decreased markedly in a time-dependent manner during the observation period (P<0.05).Conclusions:High melatonin concentrations markedly inhibited the proliferation of pancreatic carcinoma cells.The endogenous VEGF expression was also suppressed by melatonin incubation.

  7. The Arabidopsis transcription factor ABIG1 relays ABA signaled growth inhibition and drought induced senescence

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    Liu, Tie; Longhurst, Adam D; Talavera-Rauh, Franklin; Hokin, Samuel A; Barton, M Kathryn

    2016-01-01

    Drought inhibits plant growth and can also induce premature senescence. Here we identify a transcription factor, ABA INSENSITIVE GROWTH 1 (ABIG1) required for abscisic acid (ABA) mediated growth inhibition, but not for stomatal closure. ABIG1 mRNA levels are increased both in response to drought and in response to ABA treatment. When treated with ABA, abig1 mutants remain greener and produce more leaves than comparable wild-type plants. When challenged with drought, abig1 mutants have fewer yellow, senesced leaves than wild-type. Induction of ABIG1 transcription mimics ABA treatment and regulates a set of genes implicated in stress responses. We propose a model in which drought acts through ABA to increase ABIG1 transcription which in turn restricts new shoot growth and promotes leaf senescence. The results have implications for plant breeding: the existence of a mutant that is both ABA resistant and drought resistant points to new strategies for isolating drought resistant genetic varieties. DOI: http://dx.doi.org/10.7554/eLife.13768.001 PMID:27697148

  8. Five Transcription Factors and FGF Pathway Inhibition Efficiently Induce Erythroid Differentiation in the Epiblast

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    Wei Weng

    2014-03-01

    Full Text Available Primitive erythropoiesis follows a stereotypic developmental program of mesoderm ventralization and internalization, hemangioblast formation and migration, and erythroid lineage specification. Induction of erythropoiesis is inefficient in either ES/iPS cells in vitro or nonhemangioblast cell populations in vivo. Using the chick model, we report that epiblast cells can be directly and efficiently differentiated into the erythroid lineage by expressing five hematopoietic transcription regulators (SCL+LMO2+GATA2+LDB1+E2A and inhibiting the FGF pathway. We show that these five genes are expressed with temporal specificity during normal erythropoiesis. Initiation of SCL and LMO2 expression requires FGF activity, whereas erythroid differentiation is enhanced by FGF inhibition. The lag between hematopoiesis and erythropoiesis is attributed to sequential coregulator expression and hemangioblast migration. Globin gene transcription can be ectopically and prematurely induced by manipulating the availability of these factors and the FGF pathway activity. We propose that similar approaches can be taken for efficient erythroid differentiation in vitro.

  9. Inhibition of nuclear factor kappa B activation reduces Coxsackievirus B3 replication in lymphoid cells.

    Science.gov (United States)

    Sobotta, Katharina; Wilsky, Steffi; Althof, Nadine; Wiesener, Nadine; Wutzler, Peter; Henke, Andreas

    2012-02-01

    Interactions between viral replication machineries and host cell metabolism display interesting information how certain viruses capitalize cellular pathways to support progeny production. Among those pathogens, Coxsackievirus B3 (CVB3) has been identified to manipulate intracellular signaling very comprehensively. Next to others, this human pathogenic virus causes acute and chronic forms of myocarditis, pancreatitis, and meningitis. Here, activation of nuclear factor kappa B (NFκB) signaling appears to be involved in successful infection. Viral replication is not restricted to solid organs but involves susceptible immune cells as well. In the present study, p65 phosphorylation as one aspect of NFκB activation and inhibition via BAY 11-7085 administration was analyzed in the context of CVB3 replication in lymphoid cells. During CVB3 infection, an up-regulation of p65 translation is detectable, which is accompanied by noticeable phosphorylation. Inhibition of NFκB signaling reduces viral replication in a dose- and time-dependent manner. Taken together, these results indicate that during CVB3 replication in human and murine lymphoid cells, NFκB signaling is activated and facilitates viral replication. Therefore, antiviral strategies to target such central cellular signaling pathways may represent potential possibilities for the development of new virostatica. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Women’s experiences of factors that facilitate or inhibit gestational diabetes self-management

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    Carolan Mary

    2012-09-01

    Full Text Available Abstract Background Gestational diabetes rates have increased dramatically in the past two decades and this pattern of increase appears to relate primarily to the obesity epidemic, older maternal age and migration from world areas of high GDM risk. Women from disadvantaged and migrant backgrounds are most at risk of developing and of mismanaging this condition. The aim of the study was to explore the factors that facilitated or inhibited gestational diabetes self-management among women in a socially deprived area. Methods Fifteen pregnant women, with a diagnosis of gestational diabetes, were purposively recruited for this study. Qualitative semi structured interviews and 1 focus group were conducted when participants were approximately 28–38 weeks gestation. The study’s theoretical framework was based on interpretative phenomenology and data was analysed using a thematic analysis approach. Results Women in this study identified a number of factors that complicated their task of GDM self-management. Barriers included: (1 time pressures; (2 physical constraints; (3 social constraints; (4 limited comprehension of requirements, and (5 insulin as an easier option. Factors facilitating GDM self-management included: thinking about the baby and psychological support from partners and families. Conclusion Women from low socio economic and migrant backgrounds often struggle to comprehend GDM self-management requirements. To improve adherence to management plans, these women require educational and supportive services that are culturally appropriate and aimed at a low level of literacy.

  11. Regulation of neural gene transcription by optogenetic inhibition of the RE1-silencing transcription factor.

    Science.gov (United States)

    Paonessa, Francesco; Criscuolo, Stefania; Sacchetti, Silvio; Amoroso, Davide; Scarongella, Helena; Pecoraro Bisogni, Federico; Carminati, Emanuele; Pruzzo, Giacomo; Maragliano, Luca; Cesca, Fabrizia; Benfenati, Fabio

    2016-01-01

    Optogenetics provides new ways to activate gene transcription; however, no attempts have been made as yet to modulate mammalian transcription factors. We report the light-mediated regulation of the repressor element 1 (RE1)-silencing transcription factor (REST), a master regulator of neural genes. To tune REST activity, we selected two protein domains that impair REST-DNA binding or recruitment of the cofactor mSin3a. Computational modeling guided the fusion of the inhibitory domains to the light-sensitive Avena sativa light-oxygen-voltage-sensing (LOV) 2-phototrophin 1 (AsLOV2). By expressing AsLOV2 chimeras in Neuro2a cells, we achieved light-dependent modulation of REST target genes that was associated with an improved neural differentiation. In primary neurons, light-mediated REST inhibition increased Na(+)-channel 1.2 and brain-derived neurotrophic factor transcription and boosted Na(+) currents and neuronal firing. This optogenetic approach allows the coordinated expression of a cluster of genes impinging on neuronal activity, providing a tool for studying neuronal physiology and correcting gene expression changes taking place in brain diseases.

  12. Cripto Binds Transforming Growth Factor β (TGF-β) and Inhibits TGF-β Signaling▿

    Science.gov (United States)

    Gray, Peter C.; Shani, Gidi; Aung, Kevin; Kelber, Jonathan; Vale, Wylie

    2006-01-01

    Cripto is a developmental oncoprotein and a member of the epidermal growth factor-Cripto, FRL-1, Cryptic family of extracellular signaling molecules. In addition to having essential functions during embryogenesis, Cripto is highly expressed in tumors and promotes tumorigenesis. During development, Cripto acts as an obligate coreceptor for transforming growth factor β (TGF-β) ligands, including nodals, growth and differentiation factor 1 (GDF1), and GDF3. As an oncogene, Cripto is thought to promote tumor growth via mechanisms including activation of mitogenic signaling pathways and antagonism of activin signaling. Here, we provide evidence supporting a novel mechanism in which Cripto inhibits the tumor suppressor function of TGF-β. Cripto bound TGF-β and reduced the association of TGF-β with its type I receptor, TβRI. Consistent with its ability to block receptor assembly, Cripto suppressed TGF-β signaling in multiple cell types and diminished the cytostatic effects of TGF-β in mammary epithelial cells. Furthermore, targeted disruption of Cripto expression by use of small inhibitory RNA enhanced TGF-β signaling, indicating that endogenous Cripto plays a role in restraining TGF-β responses. PMID:17030617

  13. Sumoylation delays the ATF7 transcription factor subcellular localization and inhibits its transcriptional activity.

    Science.gov (United States)

    Hamard, Pierre-Jacques; Boyer-Guittaut, Michaël; Camuzeaux, Barbara; Dujardin, Denis; Hauss, Charlotte; Oelgeschläger, Thomas; Vigneron, Marc; Kedinger, Claude; Chatton, Bruno

    2007-01-01

    Over the past few years, small ubiquitin-like modifier (SUMO) modification has emerged as an important regulator of diverse pathways and activities including protein localization and transcriptional regulation. We identified a consensus sumoylation motif (IKEE), located within the N-terminal activation domain of the ATF7 transcription factor and thus investigated the role of this modification. ATF7 is a ubiquitously expressed transcription factor, homologous to ATF2, that binds to CRE elements within specific promoters. This protein is able to heterodimerize with Jun or Fos proteins and its transcriptional activity is mediated by interaction with TAF12, a subunit of the general transcription factor TFIID. In the present article, we demonstrate that ATF7 is sumoylated in vitro (using RanBP2 as a E3-specific ligase) and in vivo. Moreover, we show that ATF7 sumoylation affects its intranuclear localization by delaying its entry into the nucleus. Furthermore, SUMO conjugation inhibits ATF7 transactivation activity by (i) impairing its association with TAF12 and (ii) blocking its binding-to-specific sequences within target promoters.

  14. Factor IXa as a target for pharmacologic inhibition in acute coronary syndrome.

    Science.gov (United States)

    Roser-Jones, Christopher; Chan, Mark; Howard, Emily L; Becker, Kristian C D; Rusconi, Christopher P; Becker, Richard C

    2011-08-01

    Anticoagulant therapy, combined with platelet-directed inhibitors, represents a standard-of-care in the management of patients with acute coronary syndrome, particularly those who require percutaneous coronary interventions. While a vast clinical experience, coupled with large clinical trials have collectively provided guidance, an optimal anticoagulant drug and applied strategy, defined as one that reduces thrombotic and hemorrhagic events consistently, with minimal off-target effects and active control of systemic anticoagulation according to patient and clinical-setting specific need, remains at large. An advancing knowledge of coagulation, hemostasis, and thrombosis suggests that factor IXa, a protease that governs thrombin generation in common thrombotic disorders may represent a prime target for pharmacologic inhibition.

  15. Mechanism of inhibition of HSV-1 replication by tumor necrosis factor and interferon gamma.

    Science.gov (United States)

    Feduchi, E; Carrasco, L

    1991-02-01

    Tumor necrosis factor (TNF) synergizes with interferon (IFN gamma) in the blockade of HSV-1 replication. Antibodies against IFN beta block this synergism, implying a role of IFN beta in the antiviral activity of TNF plus IFN gamma. IFN beta 1 added exogenously to Hep-2 cells shows antiviral activity against HSV-1 only at high concentrations, whereas IFN beta 2 (also known as IL-6) alone has no effect on the replication of VSV or HSV-1 even when 1,000 U/ml are present. Our results are in accordance with the idea that TNF induces IFN beta 1 and that both cytokines must be present in the culture medium to synergize with IFN gamma in order to inhibit HSV-1 replication.

  16. Inhibition of platelet-derived growth factor signaling prevents muscle fiber growth during skeletal muscle hypertrophy.

    Science.gov (United States)

    Sugg, Kristoffer B; Korn, Michael A; Sarver, Dylan C; Markworth, James F; Mendias, Christopher L

    2017-03-01

    The platelet-derived growth factor receptors alpha and beta (PDGFRα and PDGFRβ) mark fibroadipogenic progenitor cells/fibroblasts and pericytes in skeletal muscle, respectively. While the role that these cells play in muscle growth and development has been evaluated, it was not known whether the PDGF receptors activate signaling pathways that control transcriptional and functional changes during skeletal muscle hypertrophy. To evaluate this, we inhibited PDGFR signaling in mice subjected to a synergist ablation muscle growth procedure, and performed analyses 3 and 10 days after induction of hypertrophy. The results from this study indicate that PDGF signaling is required for fiber hypertrophy, extracellular matrix production, and angiogenesis that occur during muscle growth. © 2017 Federation of European Biochemical Societies.

  17. ATXN1L, CIC, and ETS Transcription Factors Modulate Sensitivity to MAPK Pathway Inhibition

    Science.gov (United States)

    Wang, Belinda; Krall, Elsa Beyer; Aguirre, Andrew James; Kim, Miju; Widlund, Hans Ragnar; Doshi, Mihir Bhavik; Sicinska, Ewa; Sulahian, Rita; Goodale, Amy; Cowley, Glenn Spencer; Piccioni, Federica; Doench, John Gerard; Root, David Edward; Hahn, William Chun

    2017-01-01

    SUMMARY Intrinsic resistance and RTK-RAS-MAPK pathway reactivation has limited the effectiveness of MEK and RAF inhibitors (MAPKi) in RAS- and RAF-mutant cancers. To identify genes that modulate sensitivity to MAPKi, we performed genome scale CRISPR-Cas9 loss-of-function screens in two KRAS-mutant pancreatic cancer cell lines treated with the MEK1/2 inhibitor trametinib. Loss of CIC, a transcriptional repressor of ETV1, 4, and 5, promoted survival in the setting of MAPKi in cancer cells derived from several lineages. ATXN1L deletion, which reduces CIC protein, or ectopic expression of ETV1, 4, or 5 also modulated sensitivity to trametinib. ATXN1L expression inversely correlates with response to MAPKi inhibition in clinical studies. These observations identify the ATXN1L-CIC-ETS transcription factor axis as a mediator of resistance to MAPKi. PMID:28178529

  18. Inhibition of nuclear factor kappa-B signaling reduces growth in medulloblastoma in vivo

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    Deckard Lindsey A

    2011-04-01

    Full Text Available Abstract Background Medulloblastoma is a highly malignant pediatric brain tumor that requires surgery, whole brain and spine irradiation, and intense chemotherapy for treatment. A more sophisticated understanding of the pathophysiology of medulloblastoma is needed to successfully reduce the intensity of treatment and improve outcomes. Nuclear factor kappa-B (NFκB is a signaling pathway that controls transcriptional activation of genes important for tight regulation of many cellular processes and is aberrantly expressed in many types of cancer. Methods To test the importance of NFκB to medulloblastoma cell growth, the effects of multiple drugs that inhibit NFκB, pyrrolidine dithiocarbamate, diethyldithiocarbamate, sulfasalazine, curcumin and bortezomib, were studied in medulloblastoma cell lines compared to a malignant glioma cell line and normal neurons. Expression of endogenous NFκB was investigated in cultured cells, xenograft flank tumors, and primary human tumor samples. A dominant negative construct for the endogenous inhibitor of NFκB, IκB, was prepared from medulloblastoma cell lines and flank tumors were established to allow specific pathway inhibition. Results We report high constitutive activity of the canonical NFκB pathway, as seen by Western analysis of the NFκB subunit p65, in medulloblastoma tumors compared to normal brain. The p65 subunit of NFκB is extremely highly expressed in xenograft tumors from human medulloblastoma cell lines; though, conversely, the same cells in culture have minimal expression without specific stimulation. We demonstrate that pharmacological inhibition of NFκB in cell lines halts proliferation and leads to apoptosis. We show by immunohistochemical stain that phosphorylated p65 is found in the majority of primary tumor cells examined. Finally, expression of a dominant negative form of the endogenous inhibitor of NFκB, dnIκB, resulted in poor xenograft tumor growth, with average tumor volumes

  19. Transforming growth factor-β1 short hairpin RNA inhibits renal allograft fibrosis

    Institute of Scientific and Technical Information of China (English)

    YIN Zhi-kang; WU Xiao-hou; XIA Yu-guo; LUO Chun-li

    2011-01-01

    Background Transforming growth factor-β1 (TGF-β1) is known to be a key fibrogenic cytokine in a number of chronic fibrotic diseases, including chronic allograft nephropathy. We examined the effects of inhibition of TGF-β1 expression by RNA interference on renal allograft fibrosis, and explored the mechanisms responsible for these effects.Methods A Sprague-Dawley-to-Wistar rat model of accelerated kidney transplant fibrosis was used. Sixty recipient adult Wistar rats were randomly divided into four groups: group T (sham-operated group), group T (plasmid-transfected group), group H (control plasmid group), and group Y (transplant only group). Rats in group T were transfected with 200μg of TGF-β1 short hairpin RNA (shRNA). Reverse transcription-polymerase chain reaction and Western blotting were used to examine the expression of TGF-β1, Smad3/7, E-cadherin, and type I collagen. The distribution of type I collagen was measured by immunohistochemistry. The pathologic changes and extent of fibrosis were assessed by hematoxylin and eosin and Masson staining. E-cadherin and α-smooth muscle actin immunohistochemical staining were used to label tubular epithelial cells and fibroblasts, respectively.Results Plasmid transfection significantly inhibited the expression of TGF-β1, as well as that of its target gene, type I collagen (P <0.05 and P <0.01, respectively). In addition, the degree of fibrosis was mild, and its development was delayed in plasmid-transfected rats. In contrast, TGF-β1-shRNA transfection maintained the expression of E-cadherin in tubular epithelial cells while it inhibited the transformation from epithelial cells to fibroblasts. Blood urea nitrogen and serum creatinine were lower in the plasmid group than in the control groups (P <0.05 and P<0.01, respectively).Conclusions This study suggests that transfection of a TGF-β1-shRNA plasmid could inhibit the fibrosis of renal allografts. The mechanism may be associated with the downregulation

  20. Inhibition of peripubertal sheep mammary gland development by cysteamine through reducing progesterone and growth factor production.

    Science.gov (United States)

    Zhao, Yong; Feng, Yanni; Zhang, Hongfu; Kou, Xin; Li, Lan; Liu, Xinqi; Zhang, Pengfei; Cui, Liantao; Chu, Meiqiang; Shen, Wei; Min, Lingjiang

    2017-02-01

    Cysteamine has been used for treating cystinosis for many years, and furthermore it has also been used as a therapeutic agent for different diseases including Huntington's disease, Parkinson's disease (PD), nonalcoholic fatty liver disease, malaria, cancer, and others. Although cysteamine has many potential applications, its use may also be problematic. The effects of low doses of cysteamine on the reproductive system, especially the mammary glands are currently unknown. In the current investigation, low dose (10 mg/kg BW/day) of cysteamine did not affect sheep body weight gain or organ index of the liver, spleen, or heart; it did, however, increase the levels of blood lymphocytes, monocytes, and platelets. Most interestingly, it inhibited mammary gland development after 2 or 5 months of treatment by reducing the organ index and the number of mammary gland ducts. Plasma growth hormone and estradiol remained unchanged; however, plasma progesterone levels and the protein level of HSD3β1 in sheep ovaries were decreased by cysteamine. In addition to steroid hormones, growth factors produced in the mammary glands also play crucial roles in mammary gland development. Results showed that protein levels of HGF, GHR, and IGF1R were decreased after 5 months of cysteamine treatment. These findings together suggest that progesterone and local growth factors in mammary glands might be involved in cysteamine initiated inhibition of pubertal ovine mammary gland development. Furthermore, it may lead to a reduction in fertility. Therefore, cysteamine should be used with great caution until its actions have been further investigated and its limitations overcome.

  1. Mangiferin inhibits macrophage classical activation via downregulating interferon regulatory factor 5 expression.

    Science.gov (United States)

    Wei, Zhiquan; Yan, Li; Chen, Yixin; Bao, Chuanhong; Deng, Jing; Deng, Jiagang

    2016-08-01

    Mangiferin is a natural polyphenol and the predominant effective component of Mangifera indica Linn. leaves. For hundreds of years, Mangifera indica Linn. leaf has been used as an ingredient in numerous traditional Chinese medicine preparations for the treatment of bronchitis. However, the pharmacological mechanism of mangiferin in the treatment of bronchitis remains to be elucidated. Macrophage classical activation is important role in the process of bronchial airway inflammation, and interferon regulatory factor 5 (IRF5) has been identified as a key regulatory factor for macrophage classical activation. The present study used the THP‑1 human monocyte cell line to investigate whether mangiferin inhibits macrophage classical activation via suppressing IRF5 expression in vitro. THP‑1 cells were differentiated to macrophages by phorbol 12‑myristate 13‑acetate. Macrophages were polarized to M1 macrophages following stimulation with lipopolysaccharide (LPS)/interferon‑γ (IFN‑γ). Flow cytometric analysis was conducted to detect the M1 macrophages. Reverse transcription‑quantitative polymerase chain reaction was used to investigate cellular IRF5 gene expression. Levels of proinflammatory cytokines and IRF5 were assessed following cell culture and cellular homogenization using enzyme‑linked immunosorbent assay. IRF5 protein and nuclei co‑localization was performed in macrophages with laser scanning confocal microscope immunofluorescence analysis. The results of the present study demonstrated that mangiferin significantly inhibits LPS/IFN‑γ stimulation‑induced classical activation of macrophages in vitro and markedly decreases proinflammatory cytokine release. In addition, cellular IRF5 expression was markedly downregulated. These results suggest that the inhibitory effect of mangiferin on classical activation of macrophages may be exerted via downregulation of cellular IRF5 expression levels.

  2. Endocannabinoids inhibit release of nerve growth factor by inflammation-activated mast cells.

    Science.gov (United States)

    Cantarella, Giuseppina; Scollo, Mimmo; Lempereur, Laurence; Saccani-Jotti, Gloria; Basile, Francesco; Bernardini, Renato

    2011-08-15

    Nerve growth factor (NGF) is a pleiotropic member of the neurotrophin family. Beside its neuronal effects, NGF plays a role in various processes, including angiogenesis. Mast cells release NGF and are among elements contributing to angiogenesis, a process regulated by arrays of factors, including the inhibitory cannabinoids. The possible inhibitory role of cannabinoids on mast cell-related NGF mitogenic effect on endothelial cells was then investigated. Human mastocytic cells HMC-1, challenged with PMA to yield release of NGF, were preincubated with the endocannabinoid PEA. Then, conditioned media were added to HUVEC cultures. PMA-activated HMC-1 cells released substantial amounts of NGF, whereas PEA inhibited PMA-induced NGF release. HUVEC proliferation increased after treatment with media from activated HMC-1 cells, while was reduced with media from HMC-1 cells treated with PEA. To characterize receptors mediating such effects of PEA, RT-PCR and western blot analysis were performed on HMC-1 cells. None of the two cannabinoid CB1 and CB2 receptors was expressed by HMC-1 cells, which on the other hand expressed the orphan receptor GPR55. PEA was ineffective in inhibiting NGF release from HMC-1 cells treated with PMA and transfected with positive GPR55 RNAi, whereas it induced significant reduction of NGF in cells transfected with the corresponding negative control RNAi. Results indicate that NGF released from inflammatory mast cells induces angiogenesis. Cannabinoids attenuate such pro-angiogenic effects of NGF. Finally, cannabinoids could be considered for antiangiogenic treatment in disorders characterized by prominent inflammation.

  3. Coffee inhibits nuclear factor-kappa B in prostate cancer cells and xenografts.

    Science.gov (United States)

    Kolberg, Marit; Pedersen, Sigrid; Mitake, Maiko; Holm, Kristine Lillebø; Bøhn, Siv Kjølsrud; Blomhoff, Heidi Kiil; Carlsen, Harald; Blomhoff, Rune; Paur, Ingvild

    2016-01-01

    Chronic inflammation contributes to prostate cancer and the transcription factor Nuclear Factor-kappa B (NF-κB) is constitutively active in most such cancers. We examine the effects of coffee on NF-κB and on the regulation of selected genes in human-derived prostate cancer cells (PC3) and in PC3 xenografts in athymic nude mice. PC3 cells stably transduced with an NF-κB-luciferase reporter were used both in vitro and for xenografts. NF-κB activity was measured by reporter assays, DNA binding and in vivo imaging. Gene expression was measured in PC3 cells, xenografts and tumor microenvironment by low-density arrays. Western blotting of activated caspases was used to quantify apoptosis. Coffee inhibited TNFα-induced NF-κB activity and DNA-binding in PC3 cells. Furthermore, coffee increased apoptosis and modulated expression of a number of inflammation- and cancer-related genes in TNFα-treated PC3 cells. In vivo imaging revealed a 31% lower NF-κB-luciferase activation in the xenografts of the mice receiving 5% coffee compared to control mice. Interestingly, we observed major changes in gene expression in the PC3 cells in xenografts as compared to PC3 cells in vitro. In PC3 xenografts, genes related to inflammation, apoptosis and cytoprotection were down-regulated in mice receiving coffee, and coffee also affected the gene expression in the xenograft microenvironment. Our data demonstrate that coffee inhibits NF-κB activity in PC3 cells in vitro and in xenografts. Furthermore, coffee modulates transcription of genes related to prostate cancer and inflammation. Our results are the first to suggest mechanistic links between coffee consumption and prostate cancer in an experimental mouse model.

  4. Mangiferin inhibits macrophage classical activation via downregulating interferon regulatory factor 5 expression

    Science.gov (United States)

    Wei, Zhiquan; Yan, Li; Chen, Yixin; Bao, Chuanhong; Deng, Jing; Deng, Jiagang

    2016-01-01

    Mangiferin is a natural polyphenol and the predominant effective component of Mangifera indica Linn. leaves. For hundreds of years, Mangifera indica Linn. leaf has been used as an ingredient in numerous traditional Chinese medicine preparations for the treatment of bronchitis. However, the pharmacological mechanism of mangiferin in the treatment of bronchitis remains to be elucidated. Macrophage classical activation is important role in the process of bronchial airway inflammation, and interferon regulatory factor 5 (IRF5) has been identified as a key regulatory factor for macrophage classical activation. The present study used the THP-1 human monocyte cell line to investigate whether mangiferin inhibits macrophage classical activation via suppressing IRF5 expression in vitro. THP-1 cells were differentiated to macrophages by phorbol 12-myristate 13-acetate. Macrophages were polarized to M1 macrophages following stimulation with lipopolysaccharide (LPS)/interferon-γ (IFN-γ). Flow cytometric analysis was conducted to detect the M1 macrophages. Reverse transcription-quantitative polymerase chain reaction was used to investigate cellular IRF5 gene expression. Levels of proinflammatory cytokines and IRF5 were assessed following cell culture and cellular homogenization using enzyme-linked immunosorbent assay. IRF5 protein and nuclei co-localization was performed in macrophages with laser scanning confocal microscope immunofluorescence analysis. The results of the present study demonstrated that mangiferin significantly inhibits LPS/IFN-γ stimulation-induced classical activation of macrophages in vitro and markedly decreases proinflammatory cytokine release. In addition, cellular IRF5 expression was markedly downregulated. These results suggest that the inhibitory effect of mangiferin on classical activation of macrophages may be exerted via downregulation of cellular IRF5 expression levels. PMID:27277156

  5. Inhibition of CRM1-mediated nuclear export of transcription factors by leukemogenic NUP98 fusion proteins.

    Science.gov (United States)

    Takeda, Akiko; Sarma, Nayan J; Abdul-Nabi, Anmaar M; Yaseen, Nabeel R

    2010-05-21

    NUP98 is a nucleoporin that plays complex roles in the nucleocytoplasmic trafficking of macromolecules. Rearrangements of the NUP98 gene in human leukemia result in the expression of numerous fusion oncoproteins whose effect on nucleocytoplasmic trafficking is poorly understood. The present study was undertaken to determine the effects of leukemogenic NUP98 fusion proteins on CRM1-mediated nuclear export. NUP98-HOXA9, a prototypic NUP98 fusion, inhibited the nuclear export of two known CRM1 substrates: mutated cytoplasmic nucleophosmin and HIV-1 Rev. In vitro binding assays revealed that NUP98-HOXA9 binds CRM1 through the FG repeat motif in a Ran-GTP-dependent manner similar to but stronger than the interaction between CRM1 and its export substrates. Two NUP98 fusions, NUP98-HOXA9 and NUP98-DDX10, whose fusion partners are structurally and functionally unrelated, interacted with endogenous CRM1 in myeloid cells as shown by co-immunoprecipitation. These leukemogenic NUP98 fusion proteins interacted with CRM1, Ran, and the nucleoporin NUP214 in a manner fundamentally different from that of wild-type NUP98. NUP98-HOXA9 and NUP98-DDX10 formed characteristic aggregates within the nuclei of a myeloid cell line and primary human CD34+ cells and caused aberrant localization of CRM1 to these aggregates. These NUP98 fusions caused nuclear accumulation of two transcription factors, NFAT and NFkappaB, that are regulated by CRM1-mediated export. The nuclear entrapment of NFAT and NFkappaB correlated with enhanced transcription from promoters responsive to these transcription factors. Taken together, the results suggest a new mechanism by which NUP98 fusions dysregulate transcription and cause leukemia, namely, inhibition of CRM1-mediated nuclear export with aberrant nuclear retention of transcriptional regulators.

  6. Tumor necrosis factor-alpha inhibits insulin's stimulating effect on glucose uptake and endothelium-dependent vasodilation in humans

    DEFF Research Database (Denmark)

    Rask-Madsen, Christian; Domínguez, Helena; Ihlemann, Nikolaj

    2003-01-01

    -alpha was smaller (PTNF-alpha had no effect on the SNP response without insulin infusion. Thus, TNF-alpha inhibition of the combined response to insulin and ACh was likely mediated through inhibition of NO production. CONCLUSIONS: These results support the concept that TNF-alpha could play a role......BACKGROUND: Inflammatory mechanisms could be involved in the pathogenesis of both insulin resistance and atherosclerosis. Therefore, we aimed at examining whether the proinflammatory cytokine tumor necrosis factor (TNF)-alpha inhibits insulin-stimulated glucose uptake and insulin....../or TNF-alpha were coinfused. During infusion of insulin alone for 20 minutes, forearm glucose uptake increased by 220+/-44%. This increase was completely inhibited during coinfusion of TNF-alpha (started 10 min before insulin) with a more pronounced inhibition of glucose extraction than of blood flow...

  7. Suramin inhibits growth and transforming growth factor-beta 1 (TGF-beta 1) binding in osteosarcoma cell lines.

    Science.gov (United States)

    Kloen, P; Jennings, C L; Gebhardt, M C; Springfield, D S; Mankin, H J

    1994-01-01

    Autocrine production of growth factors has been shown to be involved in the multistep process of tumorigenesis. The ability of suramin, a polyanionic anti-parasitic drug, to block growth factor-induced cell proliferation makes it a potential antineoplastic drug. We studied the effects of suramin on seven osteosarcoma cell lines. Using clinically achievable concentrations of suramin (50-400 micrograms/ml), we found a time- and dose-dependent inhibition of [3H]thymidine incorporation. We also showed that suramin is able, dose-dependently, to prevent binding of transforming growth factor (TGF)-beta 1 to its receptors. DNA synthesis inhibition by suramin was attenuated by TGF-beta 1 in some cell lines. Two cell lines that were inhibited by TGF-beta 1 were affected similarly by suramin as cell lines that were stimulated by TGF-beta 1. In conclusion, in five out of seven osteosarcoma cell lines, we showed a correlation between inhibition of growth factor-stimulated mitogenesis and binding of TGF-beta 1 to its receptor. Similar effects in TGF-beta 1-inhibited osteosarcoma cell lines suggest involvement of other mechanisms and/or growth factors. However, suramin proves to be a potent inhibitor of osteosarcoma cell proliferation in vitro.

  8. The Structure of Carbonic Anhydrase IX Is Adapted for Low-pH Catalysis

    OpenAIRE

    Mahon, Brian P.; Bhatt, Avni; Socorro, Lilien; Driscoll, Jenna M.; Okoh, Cynthia; Lomelino, Carrie L.; Mboge, Mam Y.; Kurian, Justin J.; Tu, Chingkuang; Agbandje-McKenna, Mavis; Frost, Susan C; McKenna, Robert

    2016-01-01

    Human carbonic anhydrase IX (hCA IX) expression in many cancers is associated with hypoxic tumors and poor patient outcome. Inhibitors of hCA IX have been used as anticancer agents with some entering Phase I clinical trials. hCA IX is transmembrane protein whose catalytic domain faces the extracellular tumor milieu, which is typically associated with an acidic microenvironment. Here, we show that the catalytic domain of hCA IX (hCA IX-c) exhibits the necessary biochemical and biophysical prop...

  9. Rapamycin Inhibits Lymphatic Endothelial Cell Tube Formation by Downregulating Vascular Endothelial Growth Factor Receptor 3 Protein Expression

    Directory of Open Access Journals (Sweden)

    Yan Luo

    2012-03-01

    Full Text Available Mammalian target of rapamycin (mTOR controls lymphangiogenesis. However, the underlying mechanism is not clear. Here we show that rapamycin suppressed insulin-like growth factor 1 (IGF-1- or fetal bovine serum (FBS-stimulated lymphatic endothelial cell (LEC tube formation, an in vitro model of lymphangiogenesis. Expression of a rapamycin-resistant and kinase-active mTOR (S2035T, mTOR-T, but not a rapamycin-resistant and kinase-dead mTOR (S2035T/D2357E, mTOR-TE, conferred resistance to rapamycin inhibition of LEC tube formation, suggesting that rapamycin inhibition of LEC tube formation is mTOR kinase activity dependent. Also, rapamycin inhibited proliferation and motility in the LECs. Furthermore, we found that rapamycin inhibited protein expression of VEGF receptor 3 (VEGFR-3 by inhibiting protein synthesis and promoting protein degradation of VEGFR-3 in the cells. Down-regulation of VEGFR-3 mimicked the effect of rapamycin, inhibiting IGF-1- or FBS-stimulated tube formation, whereas over-expression of VEGFR-3 conferred high resistance to rapamycin inhibition of LEC tube formation. The results indicate that rapamycin inhibits LEC tube formation at least in part by downregulating VEGFR-3 protein expression.

  10. Pentoxifylline inhibits the fibrogenic activity of pleural effusions and transforming growth factor

    Directory of Open Access Journals (Sweden)

    P. Entzian

    1997-01-01

    Full Text Available Physiopathology of organ fibrosis is far from being completely understood, and the efficacy of the available therapeutic strategies is disappointing. We chose pleural disease for further studies and addressed the questions of which cytokines are relevant in pleural fibrosis and which drugs might interrupt its development. We screened pleural effusions for mediators thought to interfere with fibrogenesis (transforming growth factor-β (TGF-β, tumour necrosis factor α (TNFα, soluble TNF-receptor p55 (sTNF-R and correlated the results with patient clinical outcome in terms of extent of pleural thickenings. We found pleural thickenings correlated with TGF-β (p<0.005 whereas no correlations could be observed with TNFα and sTNF-R. Further, we were interested in finding out how TGF-β effects on fibroblast growth could be modulated. We found that pentoxifylline is able to inhibit both fibroblast proliferation and collagen synthesis independently of the stimulus. We conclude that, judging from in vitro studies, pentoxifylline might offer a new approach in the therapy of pleural as well as pulmonary fibrosis.

  11. Ghrelin Inhibition Restores Glucose Homeostasis in Hepatocyte Nuclear Factor-1α (MODY3)-Deficient Mice.

    Science.gov (United States)

    Brial, François; Lussier, Carine R; Belleville, Karine; Sarret, Philippe; Boudreau, François

    2015-09-01

    Hepatocyte nuclear factor-1α (HNF1α) is a transcription factor expressed in tissues of endoderm origin. Mutations in HNF1A are associated with maturity-onset diabetes of the young 3 (MODY3). Mice deficient for Hnf1α are hyperglycemic, with their pancreatic β-cells being defective in glucose-sensing insulin secretion. The specific mechanisms involved in this defect are unclear. Gut hormones control glucose homeostasis. Our objective was to explore whether changes in these hormones play a role in glucose homeostasis in the absence of Hnf1α. An increase in ghrelin gene transcript and a decrease in glucose-dependent insulinotropic polypeptide (GIP) gene transcripts were observed in the gut of Hnf1α-null mice. These changes correlated with an increase of ghrelin and a decrease of GIP-labeled cells. Ghrelin serological levels were significantly induced in Hnf1α-null mice. Paradoxically, GIP levels were also induced in these mice. Treatment of Hnf1α-null mice with a ghrelin antagonist led to a recovery of the diabetic symptoms. We conclude that upregulation of ghrelin in the absence of Hnf1α impairs insulin secretion and can be reversed by pharmacological inhibition of ghrelin/GHS-R interaction. These observations open up on future strategies to counteract ghrelin action in a program that could become beneficial in controlling non-insulin-dependent diabetes.

  12. Synthesis and Biological Evaluation of Manassantin Analogues for Hypoxia-Inducible FactorInhibition.

    Science.gov (United States)

    Kwon, Do-Yeon; Lee, Hye Eun; Weitzel, Douglas H; Park, Kyunghye; Lee, Sun Hee; Lee, Chen-Ting; Stephenson, Tesia N; Park, Hyeri; Fitzgerald, Michael C; Chi, Jen-Tsan; Mook, Robert A; Dewhirst, Mark W; Lee, You Mie; Hong, Jiyong

    2015-10-08

    To cope with hypoxia, tumor cells have developed a number of adaptive mechanisms mediated by hypoxia-inducible factor 1 (HIF-1) to promote angiogenesis and cell survival. Due to significant roles of HIF-1 in the initiation, progression, metastasis, and resistance to treatment of most solid tumors, a considerable amount of effort has been made to identify HIF-1 inhibitors for treatment of cancer. Isolated from Saururus cernuus, manassantins A (1) and B (2) are potent inhibitors of HIF-1 activity. To define the structural requirements of manassantins for HIF-1 inhibition, we prepared and evaluated a series of manassantin analogues. Our SAR studies examined key regions of manassantin's structure in order to understand the impact of these regions on biological activity and to define modifications that can lead to improved performance and drug-like properties. Our efforts identified several manassantin analogues with reduced structural complexity as potential lead compounds for further development. Analogues MA04, MA07, and MA11 down-regulated hypoxia-induced expression of the HIF-1α protein and reduced the levels of HIF-1 target genes, including cyclin-dependent kinase 6 (Cdk6) and vascular endothelial growth factor (VEGF). These findings provide an important framework to design potent and selective HIF-1α inhibitors, which is necessary to aid translation of manassantin-derived natural products to the clinic as novel therapeutics for cancers.

  13. Transforming growth factor β1 inhibition protects from noise-induced hearing loss

    Directory of Open Access Journals (Sweden)

    Silvia eMurillo-Cuesta

    2015-03-01

    Full Text Available Excessive exposure to noise damages the principal cochlear structures leading to hearing impairment. Inflammatory and immune responses are central mechanisms in cochlear defensive response to noise but, if unregulated, they contribute to inner ear damage and hearing loss. Transforming growth factor ß (TGF-ß is a key regulator of both responses and high levels of this factor have been associated with cochlear injury in hearing loss animal models. To evaluate the potential of targeting TGF-ß as a therapeutic strategy for preventing or ameliorating noise-induced hearing loss, we studied the auditory function, cochlear morphology, gene expression and oxidative stress markers in mice exposed to noise and treated with TGF-ß1 peptidic inhibitors P17 and P144, just before or immediately after noise insult. Our results indicate that systemic administration of both peptides significantly improved both the evolution of hearing thresholds and the degenerative changes induced by noise-exposure in lateral wall structures. Moreover, treatments ameliorated the inflammatory state and redox balance. These therapeutic effects were dose-dependent and more effective if the TGF-ß1 inhibitors were administered prior to inducing the injury. In conclusion, inhibition of TGF-ß1 actions with antagonistic peptides represents a new, promising therapeutic strategy for the prevention and repair of noise-induced cochlear damage.

  14. Identification of factors in human urine that inhibit the binding of Escherichia coli adhesins.

    Science.gov (United States)

    Parkkinen, J; Virkola, R; Korhonen, T K

    1988-10-01

    Earlier studies on the binding of Escherichia coli adhesins to the human urinary tract have indicated that the ability to recognize binding sites on the urinary tract epithelial cells is not a characteristic for P fimbriae only, but is also shared by some other adhesins that are not associated with pyelonephritis, especially S fimbriae. In the present study we have investigated whether human urine contains inhibitors of the binding of E. coli adhesins. Normal human urine was found to inhibit hemagglutination by S and type 1 fimbriae but not P fimbriae. The major inhibitor of S fimbriae in normal urine was identified as Tamm-Horsfall glycoprotein, and the interaction with S fimbriae is probably mediated by its sialyloligosaccharide chains. No significant variation was observed in the inhibitory effect of T-H glycoprotein preparations originating from different individuals. In contrast to S fimbriae, the major inhibitors of type 1 fimbriae in urine were identified as low-molecular-weight compounds. Gel filtration and ion-exchange chromatography and alpha-mannosidase treatment indicated that they were neutral alpha-mannosides, probably manno-oligosaccharides with three to five saccharides. Studies of urine samples collected from several individuals indicated the common occurrence of these inhibitory alpha-mannosides. Type 1 fimbriae bound to immobilized T-H glycoprotein, but, unlike S fimbriae, their binding was poorly inhibited by soluble T-H glycoprotein. Some urine samples were also found to contain low-molecular-weight inhibitors for the O75X adhesin of E. coli. These results emphasize that to function as a virulence factor in human urinary tract infections, an adhesin must evidently recognize such receptor structures at the infection sites that are not excreted in soluble form in urine. This prerequisite is filled by P fimbriae but not by type 1 or S fimbriae.

  15. Blocking brain-derived neurotrophic factor inhibits injury-induced hyperexcitability of hippocampal CA3 neurons.

    Science.gov (United States)

    Gill, Raminder; Chang, Philip K-Y; Prenosil, George A; Deane, Emily C; McKinney, Rebecca A

    2013-12-01

    Brain trauma can disrupt synaptic connections, and this in turn can prompt axons to sprout and form new connections. If these new axonal connections are aberrant, hyperexcitability can result. It has been shown that ablating tropomyosin-related kinase B (TrkB), a receptor for brain-derived neurotrophic factor (BDNF), can reduce axonal sprouting after hippocampal injury. However, it is unknown whether inhibiting BDNF-mediated axonal sprouting will reduce hyperexcitability. Given this, our purpose here was to determine whether pharmacologically blocking BDNF inhibits hyperexcitability after injury-induced axonal sprouting in the hippocampus. To induce injury, we made Schaffer collateral lesions in organotypic hippocampal slice cultures. As reported by others, we observed a 50% reduction in axonal sprouting in cultures treated with a BDNF blocker (TrkB-Fc) 14 days after injury. Furthermore, lesioned cultures treated with TrkB-Fc were less hyperexcitable than lesioned untreated cultures. Using electrophysiology, we observed a two-fold decrease in the number of CA3 neurons that showed bursting responses after lesion with TrkB-Fc treatment, whereas we found no change in intrinsic neuronal firing properties. Finally, evoked field excitatory postsynaptic potential recordings indicated an increase in network activity within area CA3 after lesion, which was prevented with chronic TrkB-Fc treatment. Taken together, our results demonstrate that blocking BDNF attenuates injury-induced hyperexcitability of hippocampal CA3 neurons. Axonal sprouting has been found in patients with post-traumatic epilepsy. Therefore, our data suggest that blocking the BDNF-TrkB signaling cascade shortly after injury may be a potential therapeutic target for the treatment of post-traumatic epilepsy.

  16. Small interference RNA targeting tissue factor inhibits human lung adenocarcinoma growth in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Wang Jianing

    2011-05-01

    Full Text Available Abstract Background The human coagulation trigger tissue factor (TF is overexpressed in several types of cancer and involved in tumor growth, vascularization, and metastasis. To explore the role of TF in biological processes of lung adenocarcinoma, we used RNA interference (RNAi technology to silence TF in a lung adenocarcinoma cell line A549 with high-level expression of TF and evaluate its antitumor effects in vitro and in vivo. Methods The specific small interfering RNA (siRNA designed for targeting human TF was transfected into A549 cells. The expression of TF was detected by reverse transcription-PCR and Western blot. Cell proliferation was measured by MTT and clonogenic assays. Cell apoptosis was assessed by flow cytometry. The metastatic potential of A549 cells was determined by wound healing, the mobility and Matrigel invasion assays. Expressions of PI3K/Akt, Erk1/2, VEGF and MMP-2/-9 in transfected cells were detected by Western blot. In vivo, the effect of TF-siRNA on the growth of A549 lung adenocarcinoma xenografts in nude mice was investigated. Results TF -siRNA significantly reduced the expression of TF in the mRNA and protein levels. The down-regulation of TF in A549 cells resulted in the suppression of cell proliferation, invasion and metastasis and induced cell apoptosis in dose-dependent manner. Erk MAPK, PI3K/Akt pathways as well as VEGF and MMP-2/-9 expressions were inhibited in TF-siRNA transfected cells. Moreover, intratumoral injection of siRNA targeting TF suppressed the tumor growth of A549 cells in vivo model of lung adenocarcinoma. Conclusions Down-regulation of TF using siRNA could provide a potential approach for gene therapy against lung adenocarcinoma, and the antitumor effects may be associated with inhibition of Erk MAPK, PI3K/Akt pathways.

  17. Signaling pathways involved in the inhibition of epidermal growth factor receptor by erlotinib in hepatocellular cancer

    Institute of Scientific and Technical Information of China (English)

    Alexander Huether; Michael H(o)pfner; Andreas P Sutter; Viola Baradari; Detlef Schuppan; Hans Scherübl

    2006-01-01

    AIM: To examine the underlying mechanisms of erlotinib-induced growth inhibition in hepatocellular carcinoma (HCC).METHODS: Erlotinib-induced alterations in gene expression were evaluated using cDNA array technology;changes in protein expression and/or protein activation due to erlotinib treatment as well as IGF-1-induced EGFR transactivation were investigated using Western blotting. RESULTS: Erlotinib treatment inhibited the mitogen activated protein (MAP)-kinase pathway and signal transducer of activation and transcription (STAT)mediated signaling which led to an altered expression of apoptosis and cell cycle regulating genes as demonstrated by cDNA array technology. Overexpression of proapoptotic factors like caspases and gadds associated with a down-regulation of antiapoptoticfactors like Bcl-2, Bcl-XL or jun D accounted for erlotinib's potency to induce apoptosis. Downregulation of cell cycle regulators promoting the G1/S-transition and overexpression of cyclin-dependent kinase inhibitors and gadds contributed to the induction of a G1/Go-arrest in response to erlotinib. Furthermore, we displayed the transactivation of EGFR-mediated signaling by the IGF-1-receptor and showed erlotinib's inhibitory effects on the receptor-receptor cross talk. CONCLUSION: Our study sheds light on the understanding of the mechanisms of action of EGFR-TKinhibition in HCC-cells and thus might facilitate the design of combination therapies that act additively or synergistically. Moreover, our data on the pathways responding to erlotinib treatment could be helpful in predicting the responsiveness of tumors to EGFR-TKIs in the future.

  18. Nickel ion inhibits nuclear factor-kappa B activity in human oral squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Takashi Shionome

    Full Text Available BACKGROUND: The spontaneous IL-8 secretion observed in OSCC is partially dependent on the disregulated activity of transcription factor NF-κB. Nickel compounds are well established human carcinogens, however, little is known about the influence of nickel on the spontaneous secretion of IL-8 in oral squamous cell carcinoma (OSCC cells. The aim of the present study was to investigate whether Ni(2+ ions can influence on IL-8 secretion by OSCC. METHODS AND RESULTS: The IL-8 secretion was measured by ELISA. The expression of IL-8 mRNA was examined by real-time PCR. The NF-κB activity was measured by luciferase assay. The phosphorylation status and nuclear localization of NF-κB subunits were examined by Western blotting or Transfactor kit and immunofluorescence staining, respectively. The interaction of NF-κB p50 subunit and Ni(2+ ions was examined by Ni(2+-column pull down assay. The site-directed mutagenesis was used to generate a series of p50 mutants. Scratch motility assay was used to monitor the cell mobility. Our results demonstrated that, on the contrary to our expectations, Ni(2+ ions inhibited the spontaneous secretion of IL-8. As IL-8 reduction was observed in a transcriptional level, we performed the luciferase assay and the data indicated that Ni(2+ ions reduced the NF-κB activity. Measurement of p50 subunit in the nucleus and the immunofluorescence staining revealed that the inhibitory effect of Ni(2+ ions was attributed to the prevention of p50 subunit accumulation to the nucleus. By Ni(2+-column pull down assay, Ni(2+ ions were shown to interact directly with His cluster in the N-terminus of p50 subunit. The inhibitory effect of Ni(2+ ions was reverted in the transfectant expressing the His cluster-deleted p50 mutant. Moreover, Ni(2+ ions inhibited the OSCC mobility in a dose dependent fashion. CONCLUSIONS: Taken together, inhibition of NF-κB activity by Ni(2+ ion might be a novel therapeutic strategy for the treatment of oral

  19. Nickel ion inhibits nuclear factor-kappa B activity in human oral squamous cell carcinoma.

    Science.gov (United States)

    Shionome, Takashi; Endo, Shigeki; Omagari, Daisuke; Asano, Masatake; Toyoma, Hitoshi; Ishigami, Tomohiko; Komiyama, Kazuo

    2013-01-01

    The spontaneous IL-8 secretion observed in OSCC is partially dependent on the disregulated activity of transcription factor NF-κB. Nickel compounds are well established human carcinogens, however, little is known about the influence of nickel on the spontaneous secretion of IL-8 in oral squamous cell carcinoma (OSCC) cells. The aim of the present study was to investigate whether Ni(2+) ions can influence on IL-8 secretion by OSCC. The IL-8 secretion was measured by ELISA. The expression of IL-8 mRNA was examined by real-time PCR. The NF-κB activity was measured by luciferase assay. The phosphorylation status and nuclear localization of NF-κB subunits were examined by Western blotting or Transfactor kit and immunofluorescence staining, respectively. The interaction of NF-κB p50 subunit and Ni(2+) ions was examined by Ni(2+)-column pull down assay. The site-directed mutagenesis was used to generate a series of p50 mutants. Scratch motility assay was used to monitor the cell mobility. Our results demonstrated that, on the contrary to our expectations, Ni(2+) ions inhibited the spontaneous secretion of IL-8. As IL-8 reduction was observed in a transcriptional level, we performed the luciferase assay and the data indicated that Ni(2+) ions reduced the NF-κB activity. Measurement of p50 subunit in the nucleus and the immunofluorescence staining revealed that the inhibitory effect of Ni(2+) ions was attributed to the prevention of p50 subunit accumulation to the nucleus. By Ni(2+)-column pull down assay, Ni(2+) ions were shown to interact directly with His cluster in the N-terminus of p50 subunit. The inhibitory effect of Ni(2+) ions was reverted in the transfectant expressing the His cluster-deleted p50 mutant. Moreover, Ni(2+) ions inhibited the OSCC mobility in a dose dependent fashion. Taken together, inhibition of NF-κB activity by Ni(2+) ion might be a novel therapeutic strategy for the treatment of oral cancer.

  20. Interpain A, a cysteine proteinase from Prevotella intermedia, inhibits complement by degrading complement factor C3.

    Directory of Open Access Journals (Sweden)

    Michal Potempa

    2009-02-01

    Full Text Available Periodontitis is an inflammatory disease of the supporting structures of the teeth caused by, among other pathogens, Prevotella intermedia. Many strains of P. intermedia are resistant to killing by the human complement system, which is present at up to 70% of serum concentration in gingival crevicular fluid. Incubation of human serum with recombinant cysteine protease of P. intermedia (interpain A resulted in a drastic decrease in bactericidal activity of the serum. Furthermore, a clinical strain 59 expressing interpain A was more serum-resistant than another clinical strain 57, which did not express interpain A, as determined by Western blotting. Moreover, in the presence of the cysteine protease inhibitor E64, the killing of strain 59 by human serum was enhanced. Importantly, we found that the majority of P. intermedia strains isolated from chronic and aggressive periodontitis carry and express the interpain A gene. The protective effect of interpain A against serum bactericidal activity was found to be attributable to its ability to inhibit all three complement pathways through the efficient degradation of the alpha-chain of C3 -- the major complement factor common to all three pathways. P. intermedia has been known to co-aggregate with P. gingivalis, which produce gingipains to efficiently degrade complement factors. Here, interpain A was found to have a synergistic effect with gingipains on complement degradation. In addition, interpain A was able to activate the C1 complex in serum, causing deposition of C1q on inert and bacterial surfaces, which may be important at initial stages of infection when local inflammatory reaction may be beneficial for a pathogen. Taken together, the newly characterized interpain A proteinase appears to be an important virulence factor of P. intermedia.

  1. Personality Factors Predicting Smartphone Addiction Predisposition: Behavioral Inhibition and Activation Systems, Impulsivity, and Self-Control.

    Science.gov (United States)

    Kim, Yejin; Jeong, Jo-Eun; Cho, Hyun; Jung, Dong-Jin; Kwak, Minjung; Rho, Mi Jung; Yu, Hwanjo; Kim, Dai-Jin; Choi, In Young

    2016-01-01

    The purpose of this study was to identify personality factor-associated predictors of smartphone addiction predisposition (SAP). Participants were 2,573 men and 2,281 women (n = 4,854) aged 20-49 years (Mean ± SD: 33.47 ± 7.52); participants completed the following questionnaires: the Korean Smartphone Addiction Proneness Scale (K-SAPS) for adults, the Behavioral Inhibition System/Behavioral Activation System questionnaire (BIS/BAS), the Dickman Dysfunctional Impulsivity Instrument (DDII), and the Brief Self-Control Scale (BSCS). In addition, participants reported their demographic information and smartphone usage pattern (weekday or weekend average usage hours and main use). We analyzed the data in three steps: (1) identifying predictors with logistic regression, (2) deriving causal relationships between SAP and its predictors using a Bayesian belief network (BN), and (3) computing optimal cut-off points for the identified predictors using the Youden index. Identified predictors of SAP were as follows: gender (female), weekend average usage hours, and scores on BAS-Drive, BAS-Reward Responsiveness, DDII, and BSCS. Female gender and scores on BAS-Drive and BSCS directly increased SAP. BAS-Reward Responsiveness and DDII indirectly increased SAP. We found that SAP was defined with maximal sensitivity as follows: weekend average usage hours > 4.45, BAS-Drive > 10.0, BAS-Reward Responsiveness > 13.8, DDII > 4.5, and BSCS > 37.4. This study raises the possibility that personality factors contribute to SAP. And, we calculated cut-off points for key predictors. These findings may assist clinicians screening for SAP using cut-off points, and further the understanding of SA risk factors.

  2. Understanding the Factors That Enable and Inhibit Value Creation in Buyer-Supplier Relationships Within the Outsourcing of IT Services

    DEFF Research Database (Denmark)

    Hald, Kim Sundtoft; Kinra, Aseem; Das, Ajay

    This study contributes to literature on value creation in buyer-supplier relationships as well as literature on IT service provision. It makes a first attempt at building a comprehensive model of the factors that enable and inhibit value creation within buyersupplier relationships. A distinction...... is made between value drivers with a direct influence on value creation, operational- and strategic performance, and moderating contextual factors that may enable or inhibit value creation from taking place. Initial application of the model to an IT service provision context is provided. Further case...

  3. Modes of action of ADP-ribosylated elongation factor 2 in inhibiting the polypeptide elongation cycle: a modeling study.

    Directory of Open Access Journals (Sweden)

    Kevin C Chen

    Full Text Available Despite the fact that ADP-ribosylation of eukaryotic elongation factor 2 (EF2 leads to inhibition of protein synthesis, the mechanism by which ADP-ribosylated EF2 (ADPR•EF2 causes this inhibition remains controversial. Here, we applied modeling approaches to investigate the consequences of various modes of ADPR•EF2 inhibitory actions on the two coupled processes, the polypeptide chain elongation and ADP-ribosylation of EF2. Modeling of experimental data indicates that ADPR•EF2 fully blocks the late-phase translocation of tRNAs; but the impairment in the translocation upstream process, mainly the GTP-dependent factor binding with the pretranslocation ribosome and/or the guanine nucleotide exchange in EF2, is responsible for the overall inhibition kinetics. The reduced ADPR•EF2-ribosome association spares the ribosome to bind and shield native EF2 against toxin attack, thereby deferring the inhibition of protein synthesis inhibition and inactivation of EF2. Minimum association with the ribosome also keeps ADPR•EF2 in an accessible state for toxins to catalyze the reverse reaction when nicotinamide becomes available. Our work underscores the importance of unveiling the interactions between ADPR•EF2 and the ribosome, and argues against that toxins inhibit protein synthesis through converting native EF2 to a competitive inhibitor to actively disable the ribosome.

  4. 20. IX saab Briti Nõukogus avatud uste päevade raames...

    Index Scriptorium Estoniae

    2005-01-01

    21. IX tutvustab kunstiteadlane Anneli Porri kunstnik Tracey Eminist valmistatud portreefilmi ja teisi Briti Nõukogu raamatukogus leiduvaid Briti kaasaegset kunsti käsitlevaid dokumentaalfilme. Tallinna Kunstihoones kuni 18. IX Briti kaasaegset abstraktsionismi tutvustav näitus "Supernoova"

  5. Inhibition of Vascular Endothelial Growth Factor A and Hypoxia-Inducible Factor 1α Maximizes the Effects of Radiation in Sarcoma Mouse Models Through Destruction of Tumor Vasculature

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hae-June [Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts (United States); Division of Radiation Effects, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Yoon, Changhwan [Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York (United States); Park, Do Joong [Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York (United States); Department of Surgery, Seoul National University Bundang Hospital, Sungnam (Korea, Republic of); Kim, Yeo-Jung [Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (United States); Schmidt, Benjamin [Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts (United States); Lee, Yoon-Jin [Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts (United States); Division of Radiation Effects, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Tap, William D. [Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York (United States); Eisinger-Mathason, T.S. Karin [Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (United States); Choy, Edwin [Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts (United States); Kirsch, David G. [Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina (United States); Department of Radiation Oncology, Duke University Medical Center, Durham, North Carolina (United States); Simon, M. Celeste [Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (United States); Howard Hughes Medical Institute (United States); and others

    2015-03-01

    Purpose: To examine the addition of genetic or pharmacologic inhibition of hypoxia-inducible factor 1α (HIF-1α) to radiation therapy (RT) and vascular endothelial growth factor A (VEGF-A) inhibition (ie trimodality therapy) for soft-tissue sarcoma. Methods and Materials: Hypoxia-inducible factor 1α was inhibited using short hairpin RNA or low metronomic doses of doxorubicin, which blocks HIF-1α binding to DNA. Trimodality therapy was examined in a mouse xenograft model and a genetically engineered mouse model of sarcoma, as well as in vitro in tumor endothelial cells (ECs) and 4 sarcoma cell lines. Results: In both mouse models, any monotherapy or bimodality therapy resulted in tumor growth beyond 250 mm{sup 3} within the 12-day treatment period, but trimodality therapy with RT, VEGF-A inhibition, and HIF-1α inhibition kept tumors at <250 mm{sup 3} for up to 30 days. Trimodality therapy on tumors reduced HIF-1α activity as measured by expression of nuclear HIF-1α by 87% to 95% compared with RT alone, and cytoplasmic carbonic anhydrase 9 by 79% to 82%. Trimodality therapy also increased EC-specific apoptosis 2- to 4-fold more than RT alone and reduced microvessel density by 75% to 82%. When tumor ECs were treated in vitro with trimodality therapy under hypoxia, there were significant decreases in proliferation and colony formation and increases in DNA damage (as measured by Comet assay and γH2AX expression) and apoptosis (as measured by cleaved caspase 3 expression). Trimodality therapy had much less pronounced effects when 4 sarcoma cell lines were examined in these same assays. Conclusions: Inhibition of HIF-1α is highly effective when combined with RT and VEGF-A inhibition in blocking sarcoma growth by maximizing DNA damage and apoptosis in tumor ECs, leading to loss of tumor vasculature.

  6. [Modified tests basophil degranulation and leukocyte migration inhibition factor in drug allergy. Study 2009-2014].

    Science.gov (United States)

    Irigoyen-Coria, María Lourdes; Rojo-Gutiérrez, María Isabel; Meyer-Gómez, Rubén Humberto; Leyva-Carmona, Isabel; Zendejas-Buitrón, Víctor Manuel; García-Ruiz, Angélica Dinorah; Mellado-Ábrego, Jaime; García-Ortega, Ilayalid; Castillo-Narváez, Gloria

    2016-01-01

    Antecedentes: Existe incremento de reacciones adversas a medicamentos y pocos estudios para el diagnóstico. Objetivo: Determinar la utilidad de pruebas modificadas de degranulación de basófilos (DB) y del factor inhibidor de la migración de leucocitos (LIF, leukocyte migration inhibition factor) para comprobar la hipersensibilidad a medicamentos. Métodos: Se estudiaron 177 pacientes, de uno y otro sexo, con diagnóstico de hipersensibilidad a medicamentos, en quienes se determinó pruebas modificadas de DB, LIF, o ambas entre 2009 y 2014. Se parearon con controles positivos, negativos y población no alérgica. Las solicitudes se emitieron de acuerdo con el tipo de hipersensibilidad, considerando tipo I a DB y tipo IV a LIF Resultados: 170 pacientes (96.04%) fueron positivos al menos a un medicamento (RR, 4.71). Se realizaron 561 pruebas modificadas de DB (73.62%) y 201 de LIF (26.37%). El sexo femenino fue más frecuente (64.41%); la edad promedio fue de 38.5 años. La prueba modificada de DB resultó positiva en 70.23% y la de LIF en 67.16%. La sensibilidad de las pruebas se incrementó en forma complementaria y a dos diluciones. La correlación de las pruebas fue altamente significativa. Conclusiones: Las mujeres presentan más reacciones a fármacos. La prueba modificada de DB es útil en cualquier edad. Como los medicamentos pueden activar uno u otro mecanismo de hipersensibilidad es importante solicitar las pruebas simultáneamente.

  7. Covalent Targeting of Fibroblast Growth Factor Receptor Inhibits Metastatic Breast Cancer.

    Science.gov (United States)

    Brown, Wells S; Tan, Li; Smith, Andrew; Gray, Nathanael S; Wendt, Michael K

    2016-09-01

    Therapeutic targeting of late-stage breast cancer is limited by an inadequate understanding of how tumor cell signaling evolves during metastatic progression and by the currently available small molecule inhibitors capable of targeting these processes. Herein, we demonstrate that both β3 integrin and fibroblast growth factor receptor-1 (FGFR1) are part of an epithelial-mesenchymal transition (EMT) program that is required to facilitate metastatic outgrowth in response to fibroblast growth factor-2 (FGF2). Mechanistically, β3 integrin physically disrupts an interaction between FGFR1 and E-cadherin, leading to a dramatic redistribution of FGFR1 subcellular localization, enhanced FGF2 signaling and increased three-dimensional (3D) outgrowth of metastatic breast cancer cells. This ability of β3 integrin to drive FGFR signaling requires the enzymatic activity of focal adhesion kinase (FAK). Consistent with these mechanistic data, we demonstrate that FGFR, β3 integrin, and FAK constitute a molecular signature capable of predicting decreased survival of patients with the basal-like subtype of breast cancer. Importantly, covalent targeting of a conserved cysteine in the P-loop of FGFR1-4 with our newly developed small molecule, FIIN-4, more effectively blocks 3D metastatic outgrowth as compared with currently available FGFR inhibitors. In vivo application of FIIN-4 potently inhibited the growth of metastatic, patient-derived breast cancer xenografts and murine-derived metastases growing within the pulmonary microenvironment. Overall, the current studies demonstrate that FGFR1 works in concert with other EMT effector molecules to drive aberrant downstream signaling, and that these events can be effectively targeted using our novel therapeutics for the treatment of the most aggressive forms of breast cancer. Mol Cancer Ther; 15(9); 2096-106. ©2016 AACR.

  8. Pimenta pseudocaryophyllus inhibits virulence factors and promotes metabolic changes in Candida yeast

    Directory of Open Access Journals (Sweden)

    Flávio Ezeddinne El Assal

    2014-10-01

    Full Text Available Introduction This is the first study to examine the in vitro susceptibility and the expression of virulence factors in Candida species in the presence of Pimenta pseudocaryophyllus (Gomes L.R. Landrum (Myrtaceae, a Brazilian plant known as paucravo. Additionally, the mechanisms of action of the crude ethanol extract and the ethyl acetate and aqueous fractions of this plant were investigated. Methods The in vitro susceptibility of Candida was tested using the broth microdilution method, whereas an XTT reduction assay was used for biofilms. Adherence was determined by counting the number of yeast cells that adhered to 100 oral epithelial cells, and hyphal formation was verified in the hyphal induction medium M199. Flow cytometry with propidium iodide and FUN-1 was performed to assess the mechanism of action. Results The results revealed that the crude ethanol extract and the ethyl acetate and aqueous fractions of P. pseudocaryophyllus inhibited the growth of Candida isolates at a minimal inhibitory concentration (MIC ranging from 64 to 256µg/mL, whereas the 50% sessile minimal inhibitory concentration (SMIC50 ranged from 512 to >1,024µg/mL. Adherence and hyphal formation were significantly reduced in the presence of the crude ethanol extract and both fractions. Although cell membrane injury was detected, the predominant mechanism of action appeared to be the alteration of yeast metabolism, as demonstrated by flow cytometry. Conclusions Our results indicated that antifungal activity reduced the expression of virulence factors in yeast via the alteration of yeast metabolism, suggesting that the crude extract of P. pseudocaryophyllus and its fractions may contain novel antifungal agents.

  9. Flavonoid glycosides from Hosta longipes, their inhibition on NO production, and nerve growth factor inductive effects

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Chung Sub; Lee, Kang Ro, E-mail: krlee@skku.edu [Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University (Korea, Republic of); Kwon, Oh Wook [Graduate School of East-West Medical Science, Kyung Hee University Global Campus (Korea, Republic of); Kim, Sun Yeou [College of Pharmacy, Gachon University (Korea, Republic of)

    2014-05-15

    An extended phytochemical investigation of the leaves of Hosta longipes identified the new flavonoid glycoside, kaempferol-3-O-β-D-glucopyranosyl-(1→2)- [6{sup '}-O-acetyl-β-D-glucopyranoside]-7-O-β-D-glucopyranoside and five known flavonoid derivatives. The structures of two compounds were revealed by extensive NMR methods ({sup 1}H and {sup 13}C NMR, {sup 1}H-{sup 1}H COSY, HMQC and HMBC) and chemical hydrolysis. NMR data of one of them are published for the first time. Bioactivities of six compounds revealed that five strongly inhibited the production of nitric oxide (NO) with IC{sub 50} values of 11.56-15.97 μm in lipopolysaccharide (LPS)-stimulated BV-2 cells without cell toxicity. Two compounds showed moderate induction of secretion of nerve growth factor (NGF) in C6 glioma cells (124.70 ± 7.71% and 117.02 ± 3.60%, respectively). (author)

  10. The AP-1 transcription factor Fra1 inhibits follicular B cell differentiation into plasma cells.

    Science.gov (United States)

    Grötsch, Bettina; Brachs, Sebastian; Lang, Christiane; Luther, Julia; Derer, Anja; Schlötzer-Schrehardt, Ursula; Bozec, Aline; Fillatreau, Simon; Berberich, Ingolf; Hobeika, Elias; Reth, Michael; Wagner, Erwin F; Schett, Georg; Mielenz, Dirk; David, Jean-Pierre

    2014-10-20

    The cornerstone of humoral immunity is the differentiation of B cells into antibody-secreting plasma cells. This process is tightly controlled by a regulatory gene network centered on the transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp1). Proliferation of activated B cells is required to foster Blimp1 expression but needs to be terminated to avoid overshooting immune reactions. Activator protein 1 (AP-1) transcription factors become quickly up-regulated upon B cell activation. We demonstrate that Fra1, a Fos member of AP-1, enhances activation-induced cell death upon induction in activated B cells. Moreover, mice with B cell-specific deletion of Fra1 show enhanced plasma cell differentiation and exacerbated antibody responses. In contrast, transgenic overexpression of Fra1 blocks plasma cell differentiation and immunoglobulin production, which cannot be rescued by Bcl2. On the molecular level, Fra1 represses Blimp1 expression and interferes with binding of the activating AP-1 member c-Fos to the Blimp1 promoter. Conversely, overexpression of c-Fos in Fra1 transgenic B cells releases Blimp1 repression. As Fra1 lacks transcriptional transactivation domains, we propose that Fra1 inhibits Blimp1 expression and negatively controls plasma cell differentiation through binding to the Blimp1 promoter. In summary, we demonstrate that Fra1 negatively controls plasma cell differentiation by repressing Blimp1 expression.

  11. Decreased Expression of SRSF2 Splicing Factor Inhibits Apoptotic Pathways in Renal Cancer

    Directory of Open Access Journals (Sweden)

    Hanna Kędzierska

    2016-09-01

    Full Text Available Serine and arginine rich splicing factor 2(SRSF2 belongs to the serine/arginine (SR-rich family of proteins that regulate alternative splicing. Previous studies suggested that SRSF2 can contribute to carcinogenic processes. Clear cell renal cell carcinoma (ccRCC is the most common subtype of kidney cancer, highly aggressive and difficult to treat, mainly due to resistance to apoptosis. In this study we hypothesized that SRSF2 contributes to the regulation of apoptosis in ccRCC. Using tissue samples obtained from ccRCC patients, as well as independent validation on The Cancer Genome Atlas (TCGA data, we demonstrate for the first time that expression of SRSF2 is decreased in ccRCC tumours when compared to non-tumorous control tissues. Furthermore, by employing a panel of ccRCC-derived cell lines with silenced SRSF2 expression and qPCR arrays we show that SRSF2 contributes not only to splicing patterns but also to expression of multiple apoptotic genes, including new SRSF2 targets: DIABLO, BIRC5/survivin, TRAIL, BIM, MCL1, TNFRSF9, TNFRSF1B, CRADD, BCL2L2, BCL2A1, and TP53. We also identified a new splice variant of CFLAR, an inhibitor of caspase activity. These changes culminate in diminished caspase-9 activity and inhibition of apoptosis. In summary, we show for the first time that decreased expression of SRSF2 in ccRCC contributes to protection of cancer cells viability.

  12. Von Willebrand factor inhibits mature smooth muscle gene expression through impairment of Notch signaling.

    Directory of Open Access Journals (Sweden)

    He Meng

    Full Text Available Von Willebrand factor (vWF, a hemostatic protein normally synthesized and stored by endothelial cells and platelets, has been localized beyond the endothelium in vascular disease states. Previous studies have implicated potential non-hemostatic functions of vWF, but signaling mechanisms underlying its effects are currently undefined. We present evidence that vWF breaches the endothelium and is expressed in a transmural distribution pattern in cerebral small vessel disease (SVD. To determine the potential molecular consequences of vWF permeation into the vessel wall, we also tested whether vWF impairs Notch regulation of key smooth muscle marker genes. In a co-culture system using Notch ligand expressing cells to stimulate Notch in A7R5 cells, vWF strongly inhibited both the Notch pathway and the activation of mature smooth muscle gene promoters. Similar repressive effects were observed in primary human cerebral vascular smooth muscle cells. Expression of the intracellular domain of NOTCH3 allowed cells to bypass the inhibitory effects of vWF. Moreover, vWF forms molecular complexes with all four mammalian Notch ectodomains, suggesting a novel function of vWF as an extracellular inhibitor of Notch signaling. In sum, these studies demonstrate vWF in the vessel wall as a common feature of cerebral SVD; furthermore, we provide a plausible mechanism by which non-hemostatic vWF may play a novel role in the promotion of vascular disease.

  13. Inhibition of soluble tumor necrosis factor ameliorates synaptic alterations and Ca2+ dysregulation in aged rats.

    Directory of Open Access Journals (Sweden)

    Diana M Sama

    Full Text Available The role of tumor necrosis factor α (TNF in neural function has been investigated extensively in several neurodegenerative conditions, but rarely in brain aging, where cognitive and physiologic changes are milder and more variable. Here, we show that protein levels for TNF receptor 1 (TNFR1 are significantly elevated in the hippocampus relative to TNF receptor 2 (TNFR2 in aged (22 months but not young adult (6 months Fischer 344 rats. To determine if altered TNF/TNFR1 interactions contribute to key brain aging biomarkers, aged rats received chronic (4-6 week intracranial infusions of XPro1595: a soluble dominant negative TNF that preferentially inhibits TNFR1 signaling. Aged rats treated with XPro1595 showed improved Morris Water Maze performance, reduced microglial activation, reduced susceptibility to hippocampal long-term depression, increased protein levels for the GluR1 type glutamate receptor, and lower L-type voltage sensitive Ca(2+ channel (VSCC activity in hippocampal CA1 neurons. The results suggest that diverse functional changes associated with brain aging may arise, in part, from selective alterations in TNF signaling.

  14. Intrahypothalamic corticotropin-releasing factor elevates gastric bicarbonate and inhibits stress ulcers in rats.

    Science.gov (United States)

    Gunion, M W; Kauffman, G L; Taché, Y

    1990-01-01

    The effects of intrahyopthalamic microinfusions of corticotropin-releasing factor (CRF) on gastric bicarbonate, acid, and pepsin content and on cold restraint-induced gastric lesion formation were tested in three experiments. Bilateral microinfusions of CRF into the hypothalamic ventromedial nucleus (0.86 nmol/rat) significantly increased both gastric bicarbonate concentration and total bicarbonate output. These effects were observed irrespective of whether rats were pretreated with the acid antisecretory drug omeprazole. In nonomeprazole-pretreated rats, CRF microinfusions also significantly reduced acid secretion and raised pH. The increase in bicarbonate content accounted for half of the observed decrease in acid output, suggesting that CRF microinfusions activated separable bicarbonate-stimulating and acid-inhibiting hypothalamic systems. In non-omeprazole-pretreated rats, CRF microinfusions significantly increased serum gastrin, whereas pepsin output was unchanged. Gastric mucosal damage produced by 4 h of cold restraint was significantly diminished by CRF microinfusion into the ventromedial hypothalamus. These data demonstrate that ventromedial hypothalamic microinfusions of CRF increase bicarbonate content, decrease gastric acid content, and confer protection against cold restraint-induced gastric mucosal damage. Hypothalamic CRF neuronal terminals and receptors may be involved in the central regulation of gastric bicarbonate secretion as well as acid secretion.

  15. Epidermal growth factor receptor inhibition with erlotinib partially prevents cisplatin-induced nephrotoxicity in rats.

    Directory of Open Access Journals (Sweden)

    Yukihiro Wada

    Full Text Available The effects of blocking the epidermal growth factor receptor (EGFR in acute kidney injury (AKI are controversial. Here we investigated the renoprotective effect of erlotinib, a selective tyrosine kinase inhibitor that can block EGFR activity, on cisplatin (CP-induced AKI. Groups of animals were given either erlotinib or vehicle from one day before up to Day 3 following induction of CP-nephrotoxicity (CP-N. In addition, we analyzed the effects of erlotinib on signaling pathways involved in CP-N by using human renal proximal tubular cells (HK-2. Compared to controls, rats treated with erlotinib exhibited significant improvement of renal function and attenuation of tubulointerstitial injury, and reduced the number of apoptotic and proliferating cells. Erlotinib-treated rats had a significant reduction of renal cortical mRNA for profibrogenic genes. The Bax/Bcl-2 mRNA and protein ratios were significantly reduced by erlotinib treatment. In vitro, we observed that erlotinib significantly reduced the phosphorylation of MEK1 and Akt, processes that were induced by CP in HK-2. Taken together, these data indicate that erlotinib has renoprotective properties that are likely mediated through decreases in the apoptosis and proliferation of tubular cells, effects that reflect inhibition of downstream signaling pathways of EGFR. These results suggest that erlotinib may be useful for preventing AKI in patients receiving CP chemotherapy.

  16. Decreased Expression of SRSF2 Splicing Factor Inhibits Apoptotic Pathways in Renal Cancer

    Science.gov (United States)

    Kędzierska, Hanna; Popławski, Piotr; Hoser, Grażyna; Rybicka, Beata; Rodzik, Katarzyna; Sokół, Elżbieta; Bogusławska, Joanna; Tański, Zbigniew; Fogtman, Anna; Koblowska, Marta; Piekiełko-Witkowska, Agnieszka

    2016-01-01

    Serine and arginine rich splicing factor 2(SRSF2) belongs to the serine/arginine (SR)-rich family of proteins that regulate alternative splicing. Previous studies suggested that SRSF2 can contribute to carcinogenic processes. Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer, highly aggressive and difficult to treat, mainly due to resistance to apoptosis. In this study we hypothesized that SRSF2 contributes to the regulation of apoptosis in ccRCC. Using tissue samples obtained from ccRCC patients, as well as independent validation on The Cancer Genome Atlas (TCGA) data, we demonstrate for the first time that expression of SRSF2 is decreased in ccRCC tumours when compared to non-tumorous control tissues. Furthermore, by employing a panel of ccRCC-derived cell lines with silenced SRSF2 expression and qPCR arrays we show that SRSF2 contributes not only to splicing patterns but also to expression of multiple apoptotic genes, including new SRSF2 targets: DIABLO, BIRC5/survivin, TRAIL, BIM, MCL1, TNFRSF9, TNFRSF1B, CRADD, BCL2L2, BCL2A1, and TP53. We also identified a new splice variant of CFLAR, an inhibitor of caspase activity. These changes culminate in diminished caspase-9 activity and inhibition of apoptosis. In summary, we show for the first time that decreased expression of SRSF2 in ccRCC contributes to protection of cancer cells viability. PMID:27690003

  17. Effect of environmental factors on allelopathic inhibition of Microcystis aeruginosa by berberine.

    Science.gov (United States)

    Zhang, Shulin; Dai, Wei; Bi, Xiangdong; Zhang, Dajuan; Xing, Kezhi

    2013-01-01

    To understand how environmental conditions affect the allelopathic inhibition of toxic Microcystis aeruginosa by berberine, the independent effects of some environmental factors, including temperature, light, and aeration, on the growth and extracellular microcystin (MC) content of M. aeruginosa (FACHB 905) treated with 0.000 and 0.001% (w/v) berberine were investigated. The results showed that higher temperature and light density, and aeration in daytime were beneficial for the growth of M. aeruginosa under the measured environmental conditions. The allelopathic effects of berberine on M. aeruginosa were closely associated with the environmental conditions. Berberine had the best inhibitory effects when temperature, light and aeration were more optimal for growth. In darkness, no changes in the density of M. aeruginosa were observed with the prolongation of culture time and berberine could hardly exhibit algicidal effects. Disturbance in the photosynthesis process might be one of the main reasons responsible for algicidal function. Berberine could increase extracellular MC contents significantly via killing and lyzing algal cells. Other treatments coupled with berberine needed to be carried out to degrade or remove MC released from berberine-killed algal cells.

  18. Eicosapentaenoic acid inhibits tumour necrosis factor-α-induced lipolysis in murine cultured adipocytes.

    Science.gov (United States)

    Lorente-Cebrián, Silvia; Bustos, Matilde; Marti, Amelia; Fernández-Galilea, Marta; Martinez, J Alfredo; Moreno-Aliaga, Maria J

    2012-03-01

    Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid with beneficial effects in obesity and insulin resistance. High levels of proinflammatory cytokine tumour necrosis factor-α (TNF-α) in obesity promote lipolysis in adipocytes, leading to the development of insulin resistance. Thus, the aims of the present study were to analyze the potential antilipolytic properties of EPA on cytokine-induced lipolysis and to investigate the possible mechanisms involved. The EPA effects on basal and TNF-α-induced lipolysis were determined in both primary rat and 3T3-L1 adipocytes. Treatment of primary rat adipocytes with EPA (100 and 200 μM) significantly decreased basal glycerol release (Plipolysis in a dose-dependent manner (Padipocytes. However, oral supplementation with EPA for 35 days was able to partially reverse the down-regulation of HSL and ATGL messenger RNA observed in retroperitoneal adipose tissue of high-fat-diet-fed rats. These findings suggest that EPA inhibits proinflammatory cytokine-induced lipolysis in adipocytes. This effect might contribute to explain the insulin-sensitizing properties of EPA. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Brain-derived neurotrophic factor inhibits osmotic swelling of rat retinal glial (Müller) and bipolar cells by activation of basic fibroblast growth factor signaling.

    Science.gov (United States)

    Berk, B-A; Vogler, S; Pannicke, T; Kuhrt, H; Garcia, T B; Wiedemann, P; Reichenbach, A; Seeger, J; Bringmann, A

    2015-06-04

    Water accumulation in retinal glial (Müller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Intravitreal administration of neurotrophins such as brain-derived neurotrophic factor (BDNF) is known to promote survival of retinal neurons. Here, we show that exogenous BDNF inhibits the osmotic swelling of Müller cell somata induced by superfusion of rat retinal slices or freshly isolated cells with a hypoosmotic solution containing barium ions. BDNF also inhibited the osmotic swelling of bipolar cell somata in retinal slices, but failed to inhibit the osmotic soma swelling of freshly isolated bipolar cells. The inhibitory effect of BDNF on Müller cell swelling was mediated by activation of tropomyosin-related kinase B (TrkB) and transactivation of fibroblast growth factor receptors. Exogenous basic fibroblast growth factor (bFGF) fully inhibited the osmotic swelling of Müller cell somata while it partially inhibited the osmotic swelling of bipolar cell somata. Isolated Müller cells displayed immunoreactivity of truncated TrkB, but not full-length TrkB. Isolated rod bipolar cells displayed immunoreactivities of both TrkB isoforms. Data suggest that the neuroprotective effect of exogenous BDNF in the retina is in part mediated by prevention of the cytotoxic swelling of retinal glial and bipolar cells. While BDNF directly acts on Müller cells by activation of TrkB, BDNF indirectly acts on bipolar cells by inducing glial release of factors like bFGF that inhibit bipolar cell swelling.

  20. 40 CFR Appendix Ix to Part 264 - Ground-Water Monitoring List

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 25 2010-07-01 2010-07-01 false Ground-Water Monitoring List IX... Pt. 264, App. IX Appendix IX to Part 264—Ground-Water Monitoring List Ground-Water Monitoring List... species in the ground water that contain this element are included. 3 CAS index names are those used in...

  1. 46 CFR 57.02-2 - Adoption of section IX of the ASME Code.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Adoption of section IX of the ASME Code. 57.02-2 Section... AND BRAZING General Requirements § 57.02-2 Adoption of section IX of the ASME Code. (a) The... accordance with section IX of the ASME (American Society of Mechanical Engineers) Code, as limited,...

  2. Understanding the Factors That Enable and Inhibit Value Creation in Buyer-Supplier Relationships Within the Outsourcing of IT Services

    DEFF Research Database (Denmark)

    Hald, Kim Sundtoft; Kinra, Aseem; Das, Ajay

    This study contributes to literature on value creation in buyer-supplier relationships as well as literature on IT service provision. It makes a first attempt at building a comprehensive model of the factors that enable and inhibit value creation within buyersupplier relationships. A distinction ...

  3. Rituximab inhibits structural joint damage in patients with rheumatoid arthritis with an inadequate response to tumour necrosis factor inhibitor therapies

    NARCIS (Netherlands)

    Keystone, E.; Emery, P.; Peterfy, C.G.; Tak, P.P.; Cohen, S.; Genovese, M.C.; Dougados, M.; Burmester, G.R.; Greenwald, M.; Kvien, T.K.; Williams, S.; Hagerty, D.; Cravets, M.W.; Shaw, T.

    2009-01-01

    OBJECTIVE: To determine if treatment with a B cell-targeted therapy can inhibit the progression of structural joint damage in patients with rheumatoid arthritis (RA), exhibiting an inadequate response to tumour necrosis factor (TNF) inhibitors. METHODS: In this phase III study, patients with an inad

  4. Selective inhibition of Erwinia amylovora by the herbicidally-active Germination-Arrest Factor (GAF) produced by Pseudomonas bacteria

    Science.gov (United States)

    Aims: The Germination-Arrest Factor (GAF) produced by Pseudomonas fluorescens WH6, and identified as 4-formylaminooxyvinylglycine, specifically inhibits the germination of a wide range of grassy weeds. The present study was undertaken to determine if GAF has antimicrobial activity in addition to it...

  5. Prostaglandin E-2 inhibits transforming growth factor beta 1-mediated induction of collagen alpha(1)(I) in hepatic stellate cells

    NARCIS (Netherlands)

    Hui, AY; Dannenberg, AJ; Sung, JJY; Subbaramaiah, K; Du, BH; Olinga, P; Friedman, SL

    Background/Aims: Cyclooxygenase-2 (COX-2) has been implicated in a number of hepatic stellate cell (HSC) functions but its relationship to transforming growth factor-beta1 (TGF-beta1)-mediated fibrogenesis is unknown. We assessed the impact of COX-2 inhibition and PGE(2) on the regulation of

  6. Inhibiting Factors in the Prevention of Overweight in Infants: An Explorative Qualitative Study among Child Healthcare Practitioners in the Netherlands

    Science.gov (United States)

    Dera de Bie, Eveliene; Jansen, Maria; Gerver, Willem Jan

    2012-01-01

    The aim of this study was to explore inhibiting factors in the prevention of overweight in infants younger than one year, among practitioners working for municipal child healthcare organisations in the Netherlands. Twelve in-depth interviews with child healthcare physicians and nurses were conducted. All interviews were tape-recorded, after which…

  7. Potential targets of transforming growth factor-beta1 during inhibition of oocyte maturation in zebrafish

    Directory of Open Access Journals (Sweden)

    Clelland Eric

    2005-09-01

    Full Text Available Abstract Background TGF-beta is a multifunctional growth factor involved in regulating a variety of cellular activities. Unlike mammals, the function of TGF-beta in the reproduction of lower vertebrates, such as fish, is not clear. Recently, we showed that TGF-beta1 inhibits gonadotropin- and 17alpha, 20beta-dihydroxyprogesterone (DHP-induced maturation in zebrafish. The aim of the present study was to investigate the mechanisms underlying this action. Method To determine if the effect of TGF-beta1 on oocyte maturation involves transcription and/or translation, ovarian follicles were pre-treated with actinomycin D, a blocker of transcription, and cyclohexamide, an inhibitor of translation, and incubated with hCG or DHP, either alone or in combination with TGF-beta1 and oocyte maturation scored. To determine the effect of TGF-beta1 on mRNA levels of several key effectors of oocyte maturation, three sets of experiments were performed. First, follicles were treated with control medium or TGF-beta1 for 2, 6, 12, and 24 h. Second, follicles were treated with different concentrations of TGF-beta1 (0 to 10 ng/ml for 18 h. Third, follicles were incubated with hCG in the absence or presence of TGF-beta1 for 18 h. At the end of each experiment, total RNA was extracted and reverse transcribed. PCR using primers specific for 20beta-hydroxysteroid dehydrogenase (20beta-HSD which is involved in DHP production, follicle stimulating hormone receptor (FSHR, luteinizing hormone receptor (LHR, the two forms of membrane progestin receptor: mPR-alpha and mPR-beta, as well as GAPDH (control, were performed. Results Treatment with actinomycin D, a blocker of transcription, reduced the inhibitory effect of TGF-beta1 on DHP-induced oocyte maturation, indicating that the inhibitory action of TGF-beta1 is in part due to regulation of gene transcription. Treatment with TGF-beta1 caused a dose and time-dependent decrease in mRNA levels of 20beta-HSD, LHR and mPR-beta in

  8. Treatment of metastatic colorectal carcinomas by systemic inhibition of vascular endothelial growth factor signaling in mice

    Institute of Scientific and Technical Information of China (English)

    Volker Schmitz; Miroslaw Kornek; Tobias Hilbert; Christian Dzienisowicz; Esther Raskopf; Christian Rabe; Tilman Sauerbruch; Cheng Qian; Wolfgang H Caselmann

    2005-01-01

    AIM: Tumor angiogenesis has been shown to be promoted by vascular endothelial growth factor (VEGF) via stimulating endothelial cell proliferation, migration, and survival.Blockade of VEGF signaling by different means has been demonstrated to result in reduced tumor growth and suppression of tumor angiogenesis in distinct tumor entities.Here, we tested a recombinant adenovirus, AdsFlt1-3,that encodes an antagonistically acting fragment of the VEGF receptor 1 (Flt-1), for systemic antitumor effects in pre-established subcutaneous CRC tumors in mice.METHODS: Murine colorectal carcinoma cells (CT26) were inoculated subcutaneously into Balb/c mice forin vivo studies. Tumor size and survival were determined. 293cell line was used for propagation of the adenoviral vectors.Human lung cancer line 4549 and human umbilical vein endothelial cells were transfected forin vitro experiments.RESULTS: Infection of tumor cells with AdsFlt1-3 resulted in protein secretion into cell supernatant, demonstrating correct vector function. As expected, the secreted sFlt1-3 protein had no direct effect on CT26 tumor cell proliferation in vitro, but endothelial cell function was inhibited by about 46% as compared to the AdLacZ control in a tube formation assay. When AdsFlt1-3 (5×109 PFU/animal) was applied to tumor bearing mice, we found a tumor inhibition by 72% at d 12 after treatment initiation. In spite of these antitumoral effects, the survival time was not improved.According to reduced intratumoral microvessel density in AdsFlt1-3-treated mice, the antitumor mechanism can be attributed to angiostatic vector effects. We did not detect increased systemic VEGF levels after AdsFlt1-3 treatment and liver toxicity was low as judged by serum alanine aminotransferase determination.CONCLUSION: In this study we confirmed the value of a systemic administration of AdsFlt1-3 to block VEGF signaling as antitumor therapy in an experimental metastatic colorectal carcinoma model in mice.

  9. Methods of producing protoporphyrin IX and bacterial mutants therefor

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Jizhong; Qiu, Dongru; He, Zhili; Xie, Ming

    2016-03-01

    The presently disclosed inventive concepts are directed in certain embodiments to a method of producing protoporphyrin IX by (1) cultivating a strain of Shewanella bacteria in a culture medium under conditions suitable for growth thereof, and (2) recovering the protoporphyrin IX from the culture medium. The strain of Shewanella bacteria comprises at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX. In certain embodiments of the method, the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or of shew_1140. In other embodiments, the presently disclosed inventive concepts are directed to mutant strains of Shewanella bacteria having at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX during cultivation of the bacteria. In certain embodiments the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or shew_1140.

  10. Spectroscopy of Ferric Heme and Protoporphyrin IX Ions In Vacuo

    DEFF Research Database (Denmark)

    Wyer, Jean; Nielsen, Steen Brøndsted

    2013-01-01

    This chapter deals with gas-phase spectroscopy of protoporphyrin IX and heme ions, two important biochromophores in nature. These ions strongly absorb blue and green light, which accounts for e.g. the red colour of blood. We present absorption spectra of four-coordinate ferric heme cations at room...

  11. Bianchi type IX string cosmological model in general relativity

    Indian Academy of Sciences (India)

    Raj Bali; Shuchi Dave

    2001-04-01

    We have investigated Bianchi type IX string cosmological models in general relativity. To get a determinate solution, we have assumed a condition ρ= i.e. rest energy density for a cloud of strings is equal to the string tension density. The various physical and geometrical aspects of the models are also discussed.

  12. IX : An OS for datacenter applications with aggressive networking requirements

    CERN Document Server

    CERN. Geneva

    2014-01-01

    The conventional wisdom is that aggressive networking requirements, such as high packet rates for small messages and microsecond-scale tail latency, are best addressed outside the kernel, in a user-level networking stack. We present IX, a dataplane operating system designed to support low-latency, high-throughput and high-connection count applications.  Like classic operating systems such as Linux, IX provides strong protection guarantees to the networking stack.  However, and unlike classic operating systems, IX is designed for the ground up to support applications with aggressive networking requirements on dense multi-core platforms with 10GbE and 40GbE Ethernet NICs.  IX outperforms Linux by an order of magnitude on micro benchmarks, and by up to 3.6x when running an unmodified memcached, a popular key-value store. The presentation is based on the joint work with Adam Belay, George Prekas, Ana Klimovic, Sam Grossman and Christos Kozyrakis, published at OSDI 2014; Best P...

  13. A Model Program for Statewide Title IX Capacity Building.

    Science.gov (United States)

    Berard, Barbara K.; Huppertz, Nancy

    This manual is intended to increase awareness of Title IX and related equity issues at the local school district level by providing materials and resources to specialists in school districts. The manual: (1) describes a model traveling equity resource display; and (2) provides instructions, agendas, and participant materials for a two-day training…

  14. Secondary Athletic Administrators' Perceptions of Title IX Policy Changes

    Science.gov (United States)

    Dahl, Gabriel Grawe

    2012-01-01

    The purpose of this study was to investigate North Dakota's Normal Competitive Region (NDNCR) high school athletic administrators' perceptions of 2010 Title IX policy changes respective to their athletic programs. Quantitative and qualitative data were collected to investigate the perceptions. Quantitatively, perception data were gathered from a…

  15. 23. IX korraldab Vaala galerii kunstioksjoni "Väliseesti eri"

    Index Scriptorium Estoniae

    2004-01-01

    Oksjonil on esindatud Eerik Haamer, Jaan Grünberg, Arno Vihalemm, Eduard Wiiralt, Ruth Tulving, Endel Kõks, Harald Jürissaar, Otto Paas, Ville Tops, Otto Puusta. Töödega saab tutvuda alates 18. IX, traditsiooniline sügisoksjon toimub 18. XI

  16. Alates 24. IX on Rotermanni soolalao suures saalis...

    Index Scriptorium Estoniae

    2005-01-01

    Arhitektuurifoto näitus "Urbanistlik antoloogia: Stockholmi portreed". Eksponeeritud 15 rootsi fotograafi tööd alates 1860. aastatest kuni tänapäevani. Näituse kuraator Lars Westberg. 23. IX pidas rootsi fotograaf ja näituse idee autor Bruno Ehrs loengu kunstist pildistada arhitektuuri ja inimesi linnamaastikul

  17. Gender Equity in Intercollegiate Athletics: Determinants of Title IX Compliance

    Science.gov (United States)

    Anderson, Deborah J.; Cheslock, John Jesse; Ehrenberg, Ronald G.

    2006-01-01

    Using new data on intercollegiate athletes, this article shows that recent improvement in Title IX compliance among NCAA Division I institutions was previously overestimated, and provides the first estimates of compliance in Divisions II and III. In addition, regression analyses investigate how institutional characteristics relate to the extent of…

  18. Ares I-X Malfunction Turn Range Safety Analysis

    Science.gov (United States)

    Beaty, J. R.

    2011-01-01

    Ares I-X was the designation given to the flight test version of the Ares I rocket which was developed by NASA (also known as the Crew Launch Vehicle (CLV) component of the Constellation Program). The Ares I-X flight test vehicle achieved a successful flight test on October 28, 2009, from Pad LC-39B at Kennedy Space Center, Florida (KSC). As part of the flight plan approval for the test vehicle, a range safety malfunction turn analysis was performed to support the risk assessment and vehicle destruct criteria development processes. Several vehicle failure scenarios were identified which could have caused the vehicle trajectory to deviate from its normal flight path. The effects of these failures were evaluated with an Ares I-X 6 degrees-of-freedom (6-DOF) digital simulation, using the Program to Optimize Simulated Trajectories Version II (POST2) simulation tool. The Ares I-X simulation analysis provided output files containing vehicle trajectory state information. These were used by other risk assessment and vehicle debris trajectory simulation tools to determine the risk to personnel and facilities in the vicinity of the launch area at KSC, and to develop the vehicle destruct criteria used by the flight test range safety officer in the event of a flight test anomaly of the vehicle. The simulation analysis approach used for this study is described, including descriptions of the failure modes which were considered and the underlying assumptions and ground rules of the study.

  19. Delphinidin, a dietary anthocyanidin, inhibits platelet-derived growth factor ligand/receptor (PDGF/PDGFR) signaling.

    Science.gov (United States)

    Lamy, Sylvie; Beaulieu, Edith; Labbé, David; Bédard, Valérie; Moghrabi, Albert; Barrette, Stéphane; Gingras, Denis; Béliveau, Richard

    2008-05-01

    Most cancers are dependent on the growth of tumor blood vessels and inhibition of tumor angiogenesis may thus provide an efficient strategy to retard or block tumor growth. Recently, tumor vascular targeting has expanded to include not only endothelial cells (ECs) but also smooth muscle cells (SMCs), which contribute to a mature and functional vasculature. We have reported previously that delphinidin, a major biologically active constituent of berries, inhibits the vascular endothelial growth factor-induced phosphorylation of vascular endothelial growth factor receptor-2 and blocks angiogenesis in vitro and in vivo. In the present study, we show that delphinidin also inhibits activation of the platelet-derived growth factor (PDGF)-BB receptor-beta [platelet-derived growth factor receptor-beta (PDGFR-beta)] in SMC and that this inhibition may contribute to its antitumor effect. The inhibitory effect of delphinidin on PDGFR-beta was very rapid and led to the inhibition of PDGF-BB-induced activation of extracellular signal-regulated kinase (ERK)-1/2 signaling and of the chemotactic motility of SMC, as well as the differentiation and stabilization of EC and SMC into capillary-like tubular structures in a three-dimensional coculture system. Using an anthocyan-rich extract of berries, we show that berry extracts were able to suppress the synergistic induction of vessel formation by basic fibroblast growth factor-2 and PDGF-BB in the mouse Matrigel plug assay. Oral administration of the berry extract also significantly retarded tumor growth in a lung carcinoma xenograft model. Taken together, these results provide new insight into the molecular mechanisms underlying the antiangiogenic activity of delphinidin that will be helpful for the development of dietary-based chemopreventive strategies.

  20. Porphyrin analogues as novel antagonists of fibroblast growth factor and vascular endothelial growth factor receptor binding that inhibit endothelial cell proliferation, tumor progression, and metastasis.

    Science.gov (United States)

    Aviezer, D; Cotton, S; David, M; Segev, A; Khaselev, N; Galili, N; Gross, Z; Yayon, A

    2000-06-01

    Fibroblast growth factors (FGFs) and vascular endothelial growth factor (VEGF) play a pivotal role in the multistep pathway of tumor progression, metastasis, and angiogenesis. We have identified a porphyrin analogue, 5,10,15,20-tetrakis(methyl-4-pyridyl)-21H,23H-porphine-tetra -p-tosylate salt (TMPP), as a potent inhibitor of FGF2 and VEGF receptor binding and activation. TMPP demonstrated potent inhibition of binding of soluble FGF receptor 1 (FGFR1) to FGF2 immobilized on heparin at submicromolar concentrations. TMPP inhibits binding of radiolabeled FGF2 to FGFR in a cell-free system as well as to cells genetically engineered to express FGFR1. Furthermore, TMPP also inhibits the binding of VEGF to its tyrosine kinase receptor in a dose-dependent manner. In an in vitro angiogenic assay measuring the extent of endothelial cell growth, tube formation, and sprouting, TMPP dramatically reduced the extent of the FGF2-induced endothelial cell outgrowth and differentiation. In a Lewis lung carcinoma model, mice receiving TMPP showed a marked inhibition of both primary tumor progression and lung metastases development, with nearly total inhibition of the metastatic phenotype upon alternate daily injections of TMPP at 25 microg/g of body mass. Finally, novel meso-pyridylium-substituted, nonsymmetric porphyrins, as well as a novel corrole-based derivative, with >50-fold increase in activity in vitro, had a significantly improved efficacy in blocking tumor progression and metastasis in vivo.

  1. Genetic damage induced by CrO3 can be reduced by low doses of Protoporphyrin-IX in somatic cells of Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Luz M. Vidal E.

    2014-01-01

    Full Text Available Several epidemiological studies have reported the relation between chromium exposure (used in different industrial processes and cancer risk. Evidence indicates that the hexavalent form is mutagenic and carcinogenic. Chemoprevention has emerged as a good strategy for reducing the risk from exposure to heavy metals. There is evidence that some tetrapyrrols such as protoporphyrin IX (PP-IX, a porphyrin without a metal center and which is a precursor of hemoglobin and cytochrome, acts as an antioxidant modulating the induction of antioxidant enzymes. The present study was performed to evaluate their antimutagenic potential of PP-IX against genetic damage induced by chromium trioxide (CrO3. The wing spot test was used. Groups of 48 h-old larvae were pretreated for 24 h with 0, 0.69, 6.9, or 69 mM of PP-IX, after which groups of larvae were fed 0.025–2.5 mM CrO3 solution in Drosophila instant medium. The results indicated that the lower PP-IX concentration (0.69 mM significantly reduced the genetic damage induced by all CrO3 concentrations tested. In contrast, 6.9 and 69 mM only inhibited the damage induced by CrO3 2.5 mM. Absence of an inhibitor effect of PP-IX against 20 Gy gamma rays suggested that this porphyrin acted primarily by forming complexes with chromium at low doses, inactivating its genotoxic action rather than capturing or inactivating the reactive oxygen species generated by the chromium.

  2. Genetic damage induced by CrO3 can be reduced by low doses of Protoporphyrin-IX in somatic cells of Drosophila melanogaster.

    Science.gov (United States)

    Vidal E, Luz M; Pimentel P, Emilio; Cruces M, M Patricia; Sánchez M, Juan C

    2014-01-01

    Several epidemiological studies have reported the relation between chromium exposure (used in different industrial processes) and cancer risk. Evidence indicates that the hexavalent form is mutagenic and carcinogenic. Chemoprevention has emerged as a good strategy for reducing the risk from exposure to heavy metals. There is evidence that some tetrapyrrols such as protoporphyrin IX (PP-IX), a porphyrin without a metal center and which is a precursor of hemoglobin and cytochrome, acts as an antioxidant modulating the induction of antioxidant enzymes. The present study was performed to evaluate their antimutagenic potential of PP-IX against genetic damage induced by chromium trioxide (CrO3). The wing spot test was used. Groups of 48 h-old larvae were pretreated for 24 h with 0, 0.69, 6.9, or 69 mM of PP-IX, after which groups of larvae were fed 0.025-2.5 mM CrO3 solution in Drosophila instant medium. The results indicated that the lower PP-IX concentration (0.69 mM) significantly reduced the genetic damage induced by all CrO3 concentrations tested. In contrast, 6.9 and 69 mM only inhibited the damage induced by CrO3 2.5 mM. Absence of an inhibitor effect of PP-IX against 20 Gy gamma rays suggested that this porphyrin acted primarily by forming complexes with chromium at low doses, inactivating its genotoxic action rather than capturing or inactivating the reactive oxygen species generated by the chromium.

  3. Diesel exhaust particulate extracts inhibit transcription of nuclear respiratory factor-1 and cell viability in human umbilical vein endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Mattingly, Kathleen A.; Klinge, Carolyn M. [University of Louisville School of Medicine, Department of Biochemistry and Molecular Biology, Center for Genetics and Molecular Medicine, Louisville, KY (United States)

    2012-04-15

    Endothelial dysfunction precedes cardiovascular disease and is accompanied by mitochondrial dysfunction. Here we tested the hypothesis that diesel exhaust particulate extracts (DEPEs), prepared from a truck run at different speeds and engine loads, would inhibit genomic estrogen receptor activation of nuclear respiratory factor-1 (NRF-1) transcription in human umbilical vein endothelial cells (HUVECs). Additionally, we examined how DEPEs affect NRF-1-regulated TFAM expression and, in turn, Tfam-regulated mtDNA-encoded cytochrome c oxidase subunit I (COI, MTCO1) and NADH dehydrogenase subunit I (NDI) expression as well as cell proliferation and viability. We report that 17{beta}-estradiol (E{sub 2}), 4-hydroxytamoxifen (4-OHT), and raloxifene increased NRF-1 transcription in HUVECs in an ER-dependent manner. DEPEs inhibited NRF-1 transcription, and this suppression was not ablated by concomitant treatment with E{sub 2}, 4-OHT, or raloxifene, indicating that the effect was not due to inhibition of ER activity. While E{sub 2} increased HUVEC proliferation and viability, DEPEs inhibited viability but not proliferation. Resveratrol increased NRF-1 transcription in an ER-dependent manner in HUVECs, and ablated DEPE inhibition of basal NRF-1 expression. Given that NRF-1 is a key nuclear transcription factor regulating genes involved in mitochondrial activity and biogenesis, these data suggest that DEPEs may adversely affect mitochondrial function leading to endothelial dysfunction and resveratrol may block these effects. (orig.)

  4. Andrographolide inhibits hypoxia-inducible factor-1 through phosphatidylinositol 3-kinase/AKT pathway and suppresses breast cancer growth

    Directory of Open Access Journals (Sweden)

    Li J

    2015-02-01

    Full Text Available Jie Li,1 Chao Zhang,1 Hongchuan Jiang,1 Jiao Cheng21Department of General Surgery, 2Department of Gynaecology and Obstetrics, Beijing Chao-Yang Hospital, Beijing, People’s Republic of ChinaAbstract: Hypoxia-inducible factor-1 (HIF-1 is a master regulator of the transcriptional response to hypoxia. HIF-1α is one of the most compelling anticancer targets. Andrographolide (Andro was newly identified to inhibit HIF-1 in T47D cells (a half maximal effective concentration [EC50] of 1.03×10-7 mol/L, by a dual-luciferase reporter assay. It suppressed HIF-1α protein and gene accumulation, which was dependent on the inhibition of upstream phosphatidylinositol 3-kinase (PI3K/AKT pathway. It also abrogated the expression of HIF-1 target vascular endothelial growth factor (VEGF gene and protein. Further, Andro inhibited T47D and MDA-MB-231 cell proliferation and colony formation. In addition, it exhibited significant in vivo efficacy and antitumor potential against the MDA-MB-231 xenograft in nude mice. In conclusion, these results highlighted the potential effects of Andro, which inhibits HIF-1, and hence may be developed as an antitumor agent for breast cancer therapy in future.Keywords: Andrographolide (Andro, HIF-1α, inhibit, breast cancer, hypoxia, PI3k/AKT/mTOR pathway

  5. Citrobacter rodentium NleB Protein Inhibits Tumor Necrosis Factor (TNF) Receptor-associated Factor 3 (TRAF3) Ubiquitination to Reduce Host Type I Interferon Production.

    Science.gov (United States)

    Gao, Xiaofei; Pham, Thanh H; Feuerbacher, Leigh Ann; Chen, Kangming; Hays, Michael P; Singh, Gyanendra; Rueter, Christian; Hurtado-Guerrero, Ramon; Hardwidge, Philip R

    2016-08-26

    Interferon signaling plays important roles in both intestinal homeostasis and in the host response to pathogen infection. The extent to which bacterial pathogens inhibit this host pathway is an understudied area of investigation. We characterized Citrobacter rodentium strains bearing deletions in individual type III secretion system effector genes to determine whether this pathogen inhibits the host type I IFN response and which effector is responsible. The NleB effector limited host IFN-β production by inhibiting Lys(63)-linked ubiquitination of TNF receptor-associated factor 3 (TRAF3). Inhibition was dependent on the glycosyltransferase activity of NleB. GAPDH, a target of NleB during infection, bound to TRAF3 and was required for maximal TRAF3 ubiquitination. NleB glycosyltransferase activity inhibited GAPDH-TRAF3 binding, resulting in reduced TRAF3 ubiquitination. Collectively, our data reveal important interplay between GAPDH and TRAF3 and suggest a mechanism by which the NleB effector inhibits type I IFN signaling.

  6. Hypusine modification in eukaryotic initiation factor 5A in rodent cells selected for resistance to growth inhibition by ornithine decarboxylase-inhibiting drugs.

    Science.gov (United States)

    Tome, M E; Gerner, E W

    1996-11-15

    Selection of HTC cells in drugs that inhibit ornithine decarboxylase (ODC) has produced two cell lines, HMOA and DH23A/b, that contain increased amounts of more stable ODC. In addition to alterations in ODC, these cells appear to produce modified eukaryotic initiation factor 5A (eIF-5A) at different rates, a reaction that both requires spermidine and is essential for proliferation. Alterations to the modification of eIF-5A by spermidine cannot be accounted for by changes in eIF-5A protein or modified eIF-5A turnover. Deoxyhypusine synthetase activity is similar in the parental and variant cell lines and is unaltered by growth into plateau phase or by spermidine depletion. The increased rate of eIF-5A modification in DH23A/b cells is due to an increased accumulation of the unmodified eIF-5A precursor. Increased precursor accumulation is not due to increased eIF-5A transcription, but rather it can be attributed to a metabolic accumulation caused by growth under conditions of chronically limiting spermidine. Selection using drugs that inhibit ODC apparently does not cause alterations in the eIF-5A modification pathway. These data support the hypothesis that one of the main effects of spermidine depletion is depletion of the modified eIF-5A pool, and that this is a critical factor in the cytostasis often observed after depletion of cellular polyamines.

  7. Hydrogen Sulfide Inhibits Transforming Growth Factor-β1-Induced EMT via Wnt/Catenin Pathway.

    Directory of Open Access Journals (Sweden)

    Lin Guo

    Full Text Available Hydrogen sulfide (H2S has anti-fibrotic potential in lung, kidney and other organs. The exogenous H2S is released from sodium hydrosulfide (NaHS and can influence the renal fibrosis by blocking the differentiation of quiescent renal fibroblasts to myofibroblasts. But whether H2S affects renal epithelial-to-mesenchymal transition (EMT and the underlying mechanisms remain unknown. Our study is aimed at investigating the in vitro effects of H2S on transforming growth factor-β1 (TGF-β1-induced EMT in renal tubular epithelial cells (HK-2 cells and the associated mechanisms. The induced EMT is assessed by Western blotting analysis on the expressions of α-SMA, E-cadherin and fibronectin. HK-2 cells were treated with NaHS before incubating with TGF-β1 to investigate its effect on EMT and the related molecular mechanism. Results demonstrated that NaHS decreased the expression of α-SMA and fibronectin, and increased the expression of E-cadherin. NaHS reduced the expression of TGF-β receptor type I (TβR I and TGF-β receptor type II (TβR II. In addition, NaHS attenuated TGF-β1-induced increase of β-catenin expression and ERK phosphorylation. Moreover, it inhibited the TGF-β1-induced nuclear translocation of ββ-catenin. These effects of NaHS on fibronectin, E-cadherin and TβR I were abolished by the ERK inhibitor U0126 or β-catenin inhibitor XAV939, or β-catenin siRNA interference. We get the conclusion that NaHS attenuated TGF-β1-induced EMT in HK-2 cells through both ERK-dependent and β-catenin-dependent pathways.

  8. Hydrogen Sulfide Inhibits Transforming Growth Factor-β1-Induced EMT via Wnt/Catenin Pathway

    Science.gov (United States)

    Tao, Jie; Lan, Zhen; Hei, Hongya; Tian, Lulu; Pan, Wanma; Wang, Li; Zhang, Xuemei

    2016-01-01

    Hydrogen sulfide (H2S) has anti-fibrotic potential in lung, kidney and other organs. The exogenous H2S is released from sodium hydrosulfide (NaHS) and can influence the renal fibrosis by blocking the differentiation of quiescent renal fibroblasts to myofibroblasts. But whether H2S affects renal epithelial-to-mesenchymal transition (EMT) and the underlying mechanisms remain unknown. Our study is aimed at investigating the in vitro effects of H2S on transforming growth factor-β1 (TGF-β1)-induced EMT in renal tubular epithelial cells (HK-2 cells) and the associated mechanisms. The induced EMT is assessed by Western blotting analysis on the expressions of α-SMA, E-cadherin and fibronectin. HK-2 cells were treated with NaHS before incubating with TGF-β1 to investigate its effect on EMT and the related molecular mechanism. Results demonstrated that NaHS decreased the expression of α-SMA and fibronectin, and increased the expression of E-cadherin. NaHS reduced the expression of TGF-β receptor type I (TβR I) and TGF-β receptor type II (TβR II). In addition, NaHS attenuated TGF-β1-induced increase of β-catenin expression and ERK phosphorylation. Moreover, it inhibited the TGF-β1-induced nuclear translocation of ββ-catenin. These effects of NaHS on fibronectin, E-cadherin and TβR I were abolished by the ERK inhibitor U0126 or β-catenin inhibitor XAV939, or β-catenin siRNA interference. We get the conclusion that NaHS attenuated TGF-β1-induced EMT in HK-2 cells through both ERK-dependent and β-catenin-dependent pathways. PMID:26760502

  9. Erythropoietin decreases carbon tetrachloride-induced hepatic fibrosis by inhibiting transforming growth factor-beta

    Institute of Scientific and Technical Information of China (English)

    Soo Young Park; Joo Young Lee; Won Young Tak; Young Oh Kweon; Mi Suk Lee

    2012-01-01

    Background In addition to hematopoietic effect,the erythropoietin is known as a multifunctional cytokine with anti-fibrosis and organ-protective activities.The purpose of this study was to evaluate the effect of recombinant human erythropoietin (rhEPO) on hepatic fibrosis and hepatic stellate cells (HSCs).Methods Carbon tetrachloride (CCl4) induced hepatic fibrosis mice models were used for in vivo study and HSCs line for in vitro study.CCl4 and rhEPO (0,200 or 1000 U/kg) was injected intraperitoneally in BALB/c mice three times a week for 4 weeks.Immunohistochemistry and immunoblotting were performed to evaluate expressions of transforming growth factor-β31 (TGF-β1),α-smooth muscle actin (α-SMA),and fibronectin in explanted liver.Immunoblotting of α-SMA,phophorylated Smad-2 and Smad-2/3 was performed in HSCs treated with TGF-β1 and/or rhEPO.Results Expressions of TGF-β1,α-SMA,and fibronectin were increased in CCl4 injected mice livers,but significantly attenuated by co-treatment with CCl4 and rhEPO.Co-treatment of rhEPO markedly suppressed fibrosis in Masson's trichrome compared with treatment of only CCl4.TGF-β1 increased phosphorylated α-SMA,Smad-2 expressions in HSCs,which were decreased by rhEPO co-treatment.Conclusions Treatment of rhEPO effectively suppressed fibrosis in CCl4-induced liver fibrosis mice models.Anti-fibrosis effect of rhEPO could be related to inhibition of TGF-β1 induced activation of HSCs.

  10. Knockdown of lymphoid enhancer factor 1 inhibits colon cancer progression in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Wen-Juan Wang

    Full Text Available Expression of lymphoid enhancer factor 1 (LEF1 is frequently altered in different human cancers. This study aimed to assess LEF1 expression in colon cancer tissues and to explore changed phenotypes, gene expressions, and the possible mechanism after knocked down LEF1 expression in colon cancer cell lines. A total of 106 colon cancer and matched paratumorous normal tissues were used to assess LEF1 expression using immunohistochemistry and qRT-PCR. LEF1 lentivirus was used to knockdown LEF1 expression for the assessment of cell viability, cell cycle distribution, apoptosis, and gene expressions. The nude mouse xenograft assay was performed to detect the effects of LEF1 knockdown in vivo. The data showed that the levels of LEF1 mRNA and protein were significantly increased in human colon cancer tissues compared to the matched paratumorous normal tissues and were associated with infiltration depth, lymph node and distant metastases, advanced TNM (tumor-node-metastasis stages, and shorter overall survival. Furthermore, LEF1 knockdown reduced tumor cell viability, invasion capacity, MMP2 and MMP-9 expression, but induced apoptosis. Nude mouse xenograft assay showed that LEF1 knockdown suppressed tumor formation and growth in vivo. In addition, the expression of Notch pathway-related proteins RBP-jκ and Hes1 was reduced in LEF1 knockdown cells. Taken together, LEF1 protein was overexpressed in colon cancer tissues and knockdown of LEF1 expression inhibited colon cancer growth in vitro and in vivo. These data suggest that targeting of LEF1 expression should be further evaluated for colon cancer prevention and therapy.

  11. Knockdown of lymphoid enhancer factor 1 inhibits colon cancer progression in vitro and in vivo.

    Science.gov (United States)

    Wang, Wen-Juan; Yao, Yu; Jiang, Li-Li; Hu, Ting-Hua; Ma, Jie-Qun; Liao, Zi-Jun; Yao, Jun-Tao; Li, Dong-Fan; Wang, Shu-Hong; Nan, Ke-Jun

    2013-01-01

    Expression of lymphoid enhancer factor 1 (LEF1) is frequently altered in different human cancers. This study aimed to assess LEF1 expression in colon cancer tissues and to explore changed phenotypes, gene expressions, and the possible mechanism after knocked down LEF1 expression in colon cancer cell lines. A total of 106 colon cancer and matched paratumorous normal tissues were used to assess LEF1 expression using immunohistochemistry and qRT-PCR. LEF1 lentivirus was used to knockdown LEF1 expression for the assessment of cell viability, cell cycle distribution, apoptosis, and gene expressions. The nude mouse xenograft assay was performed to detect the effects of LEF1 knockdown in vivo. The data showed that the levels of LEF1 mRNA and protein were significantly increased in human colon cancer tissues compared to the matched paratumorous normal tissues and were associated with infiltration depth, lymph node and distant metastases, advanced TNM (tumor-node-metastasis) stages, and shorter overall survival. Furthermore, LEF1 knockdown reduced tumor cell viability, invasion capacity, MMP2 and MMP-9 expression, but induced apoptosis. Nude mouse xenograft assay showed that LEF1 knockdown suppressed tumor formation and growth in vivo. In addition, the expression of Notch pathway-related proteins RBP-jκ and Hes1 was reduced in LEF1 knockdown cells. Taken together, LEF1 protein was overexpressed in colon cancer tissues and knockdown of LEF1 expression inhibited colon cancer growth in vitro and in vivo. These data suggest that targeting of LEF1 expression should be further evaluated for colon cancer prevention and therapy.

  12. Current status of vascular endothelial growth factor inhibition in age-related macular degeneration.

    Science.gov (United States)

    Mousa, Shaker A; Mousa, Shaymaa S

    2010-06-01

    Angiogenesis, the process by which new vessels are created from pre-existing vasculature, has become the subject of intense research in recent years. Increased rates of angiogenesis are associated with several disease states, including cancer, age-related macular degeneration (AMD), psoriasis, rheumatoid arthritis, and diabetic retinopathy. Vascular endothelial growth factor (VEGF) is an important modulator of angiogenesis, and has been implicated in the pathology of a number of conditions, including AMD, diabetic retinopathy, and cancer. AMD is a progressive disease of the macula and the third major cause of blindness worldwide. If not treated appropriately, AMD can progress to involve both eyes. Until recently, the treatment options for AMD have been limited, with photodynamic therapy (PDT) the mainstay of treatment. Although PDT is effective at slowing disease progression, it rarely results in improved vision. Several therapies have been or are now being developed for neovascular AMD, with the goal of inhibiting VEGF. These VEGF inhibitors include the RNA aptamer pegaptanib, partial and full-length antibodies ranibizumab and bevacizumab, the VEGF receptor decoy aflibercept, small interfering RNA-based therapies bevasiranib and AGN 211745, sirolimus, and tyrosine kinase inhibitors, including vatalanib, pazopanib, TG 100801, TG 101095, AG 013958, and AL 39324. At present, established therapies have met with great success in reducing the vision loss associated with neovascular AMD, whereas those still under investigation offer the potential for further advances. In AMD patients, these therapies slow the rate of vision loss and in some cases increase visual acuity. Although VEGF-inhibitor therapies are a milestone in the treatment of these disease states, several concerns need to be addressed before their impact can be fully realized.

  13. Schwann cells but not olfactory ensheathing cells inhibit CNS myelination via the secretion of connective tissue growth factor.

    Science.gov (United States)

    Lamond, Rebecca; Barnett, Susan C

    2013-11-20

    Cell transplantation is a promising strategy to promote CNS repair and has been studied for several decades with a focus on glial cells. Promising candidates include Schwann cells (SCs) and olfactory ensheathing cells (OECs). Both cell types are thought to be neural crest derived and share many properties in common, although OECs appear to be a better candidate for transplantation by evoking less astrogliosis. Using CNS mixed myelinating rat cultures plated on to a monolayer of astrocytes, we demonstrated that SCs, but not OECs, secrete a heat labile factor(s) that inhibits oligodendrocyte myelination. Comparative qRT-PCR and ELISA showed that SCs expressed higher levels of mRNA and protein for connective tissue growth factor (CTGF) than OECs. Anti-CTGF reversed the SCM-mediated effects on myelination. Both SCM and CTGF inhibited the differentiation of purified rat oligodendrocyte precursor cells (OPCs). Furthermore, pretreatment of astrocyte monolayers with SCM inhibited CNS myelination and led to transcriptional changes in the astrocyte, corresponding to upregulation of bone morphogenic protein 4 mRNA and CTGF mRNA (inhibitors of OPC differentiation) and the downregulation of insulin-like growth factor 2 mRNA (promoter of OPC differentiation). CTGF pretreatment of astrocytes increased their expression of CTGF, suggesting that this inhibitory factor can be positively regulated in astrocytes. These data provide evidence for the advantages of using OECs, and not mature SCs, for transplant-mediated repair and provide more evidence that they are a distinct and unique glial cell type.

  14. Leishmania eukaryotic initiation factor (LeIF inhibits parasite growth in murine macrophages.

    Directory of Open Access Journals (Sweden)

    Olga Koutsoni

    Full Text Available The leishmaniases constitute neglected global public health problems that require adequate control measures, prophylactic clinical vaccines and effective and non-toxic drug treatments. In this study, we explored the potential of Leishmania infantum eukaryotic initiation factor (LieIF, an exosomal protein, as a novel anti-infective therapeutic molecule. More specifically, we assessed the efficacy of recombinant LieIF, in combination with recombinant IFN-γ, in eliminating intracellular L. donovani parasites in an in vitro macrophage model. J774A.1 macrophages were initially treated with LieIF/IFN-γ prior to in vitro infection with L. donovani stationary phase promastigotes (pre-infection treatment, and resistance to infection was observed 72 h after infection. J774A.1 macrophages were also treated with LieIF/IFN-γ after L. donovani infection (post-infection treatment, and resistance to infection was also observed at both time points tested (19 h and 72 h after infection. To elucidate the LieIF/IFN-γ-induced mechanism(s that mediate the reduction of intracellular parasite growth, we examined the generation of potent microbicidal molecules, such as nitric oxide (NO and reactive oxygen species (ROS, within infected macrophages. Furthermore, macrophages pre-treated with LieIF/IFN-γ showed a clear up-regulation in macrophage inflammatory protein 1α (MIP-1α as well as tumor necrosis factor alpha (TNF-α expression. However, significant different protein levels were not detected. In addition, macrophages pre-treated with LieIF/IFN-γ combined with anti-TNF-α monoclonal antibody produced significantly lower amounts of ROS. These data suggest that during the pre-treatment state, LieIF induces intramacrophage parasite growth inhibition through the production of TNF-α, which induces microbicidal activity by stimulating NO and ROS production. The mechanisms of NO and ROS production when macrophages are treated with LieIF after infection are probably

  15. Brain-derived neurotrophic factor inhibits neuromuscular junction maturation in a cAMP-PKA-dependent way.

    Science.gov (United States)

    Song, Wei; Jin, Xiwan Albert

    2015-03-30

    The development of neuromuscular junction (NMJ) is initiated by motor axon's contact with the skeletal muscle cell that is followed by synaptic maturation. Previous studies showed that brain-derived neurotrophic factor (BDNF) enhanced motoneurons' survival and growth but significantly inhibited synaptogenesis. Here, we report that chronic application of BDNF resulted in inhibition in the maturation process both physiologically and morphologically. The response to BDNF was mediated by its cognate receptor TrkB as the effects were abolished by Trk receptor inhibitor K252a. Protein kinase A (PKA) inhibitor reversed the effects of BDNF in inhibiting NMJ maturation. These results indicate that BDNF suppresses NMJ maturation through cAMP-PKA signaling pathway. Together with the previous studies, these results suggest that BDNF suppresses NMJ formation and maturation despite its effects in enhancing neuronal survival and growth.

  16. Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.

    Science.gov (United States)

    Mutoh, Shingo; Sobhany, Mack; Moore, Rick; Perera, Lalith; Pedersen, Lee; Sueyoshi, Tatsuya; Negishi, Masahiko

    2013-05-07

    Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr(52), which then promoted the dephosphorylation of CAR at Thr(38) by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR.

  17. Troglitazone inhibits cell proliferation by attenuation of epidermal growth factor receptor signaling independent of peroxisome proliferator-activated receptor γ

    Institute of Scientific and Technical Information of China (English)

    Xiaoqi Li; Xuanming Yang; Youli Xu; Xuejun Jiang; Xin Li; Fajun Nan; Hong Tang

    2009-01-01

    Peroxisome proliferator-activated receptors (PPAR) belong to the nuclear hormone receptor superfamily of ligand-dependent transcription factors. Recent results have shown that agonists of PPARy, such as troglitazone (TGZ), can inhibit cell proliferation and promote cell differentiation independent of PPARγ. In the present study, we provide evidence that TGZ may bind directly to EGFR and trigger its signaling and internalization independent of PPARγ. Detailed studies revealed that prolonged incubation with TGZ effectively attenuated EGFR signaling by target-ing the receptor to the endo-lysosomal degradation machinery. Although the extracellular signal-regulated kinase-signaling pathway was transiently activated by TGZ in EGFR overexpressing cancer cells, inhibition of EGF-induced Akt phosphorylation most likely accounted for the growth arrest of tumor cells caused by TGZ at pharmacologically achievable concentrations. Therefore, we have provided a new line of evidence indicating that TGZ inhibits cell pro-liferation by promoting EGFR degradation and attenuating Akt phosphorylation.

  18. Nuclear factor-kappa B inhibition can enhance apoptosis of differentiated thyroid cancer cells induced by 131I.

    Directory of Open Access Journals (Sweden)

    Zhaowei Meng

    Full Text Available OBJECTIVE: To evaluate changes of nuclear factor-kappa B (NF-κB during radioiodine 131 ((131I therapy and whether NF-κB inhibition could enhance (131I-induced apoptosis in differentiated thyroid cancer (DTC cells in a synergistic manner. METHODS: Three human DTC cell lines were used. NF-κB inhibition was achieved by using a NF-κB inhibitor (Bay 11-7082 or by p65 siRNA transfection. Methyl-thiazolyl-tetrazolium assay was performed for cell viability assessment. DNA-binding assay, luciferase reporter assay, and Western blot were adopted to determine function and expression changes of NF-κB. Then NF-κB regulated anti-apoptotic factors XIAP, cIAP1, and Bcl-xL were measured. Apoptosis was analyzed by Western blot for caspase 3 and PARP, and by flow cytometry as well. An iodide uptake assay was performed to determine whether NF-κB inhibition could influence radioactive iodide uptake. RESULTS: The methyl-thiazolyl-tetrazolium assay showed significant decrease of viable cells by combination therapy than by mono-therapies. The DNA-binding assay and luciferase reporter assay showed enhanced NF-κB function and reporter gene activities due to (131I, yet significant suppression was achieved by NF-κB inhibition. Western blot proved (131I could increase nuclear NF-κB concentration, while NF-κB inhibition reduced NF-κB concentration. Western blot also demonstrated significant up-regulation of XIAP, cIAP1, and Bcl-xL after (131I therapy. And inhibition of NF-κB could significantly down-regulate these factors. Finally, synergism induced by combined therapy was displayed by significant enhancements of cleaved caspase 3 and PARP from Western blot, and of Annexin V positively staining from flow cytometry. The iodine uptake assay did not show significant changes when NF-κB was inhibited. CONCLUSION: We demonstrated that (131I could induce NF-κB activation, which would attenuate (131I efficacy in DTC cells. NF-κB inhibition by Bay 11-7082 or by p65 si

  19. Borna disease virus nucleoprotein inhibits type I interferon induction through the interferon regulatory factor 7 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Song, Wuqi [The Heilongjiang Key Laboratory of Immunity and Infection, Heilongjiang (China); Department of Microbiology, Harbin Medical University (China); Kao, Wenping [The Key Laboratory of Pathogenic Biology, Heilongjiang Higher Education Institutions (China); Department of Microbiology, Harbin Medical University (China); Zhai, Aixia [The Heilongjiang Key Laboratory of Immunity and Infection, Heilongjiang (China); Qian, Jun; Li, Yujun [The Key Laboratory of Pathogenic Biology, Heilongjiang Higher Education Institutions (China); Zhang, Qingmeng [The Heilongjiang Key Laboratory of Immunity and Infection, Heilongjiang (China); Zhao, Hong; Hu, Yunlong; Li, Hui [Department of Microbiology, Harbin Medical University (China); Zhang, Fengmin, E-mail: fengminzhang@ems.hrbmu.edu.cn [The Heilongjiang Key Laboratory of Immunity and Infection, Heilongjiang (China); The Key Laboratory of Pathogenic Biology, Heilongjiang Higher Education Institutions (China); Department of Microbiology, Harbin Medical University (China)

    2013-09-06

    Highlights: •IRF7 nuclear localisation was inhibited by BDV persistently infected. •BDV N protein resistant to IFN induction both in BDV infected OL cell and N protein plasmid transfected OL cell. •BDV N protein is related to the inhibition of IRF7 nuclear localisation. -- Abstract: The expression of type I interferon (IFN) is one of the most potent innate defences against viral infection in higher vertebrates. Borna disease virus (BDV) establishes persistent, noncytolytic infections in animals and in cultured cells. Early studies have shown that the BDV phosphoprotein can inhibit the activation of type I IFN through the TBK1–IRF3 pathway. The function of the BDV nucleoprotein in the inhibition of IFN activity is not yet clear. In this study, we demonstrated IRF7 activation and increased IFN-α/β expression in a BDV-persistently infected human oligodendroglia cell line following RNA interference-mediated BDV nucleoprotein silencing. Furthermore, we showed that BDV nucleoprotein prevented the nuclear localisation of IRF7 and inhibited endogenous IFN induction by poly(I:C), coxsackie virus B3 and IFN-β. Our findings provide evidence for a previously undescribed mechanism by which the BDV nucleoprotein inhibits type I IFN expression by interfering with the IRF7 pathway.

  20. Motivating and Inhibiting Factors to Oral-Dental Health Behavior in Adolescents: a Cross-Sectional Study

    Directory of Open Access Journals (Sweden)

    Zahra Hosseini

    2016-11-01

    Full Text Available Background Oral-dental diseases, especially tooth decay, are among the most common diseases in the world which usually begin in adolescence. Oral health during this period of life has a huge impact on the reduction of dental problems. This study aimed to determine motivating and inhibiting factors to oral-dental health behavior in adolescents. Materials and Methods This cross-sectional which had a descriptive and analytical design was conducted on 10-12th grade students in Kashan city, Iran. Using multi-stage sampling method and based on sampling size formula, a total of 290 of the students were randomly selected from the schools and were enrolled into the study. Then they received a research-made questionnaire containing questions about the knowledge and motivating and inhibiting factors to oral-dental health behavior. The collected data were analyzed using SPSS V.20 by independent t-test, ANOVA, and Pearson correlation coefficient. Results Of all, 62.8% of students brushed their teeth at least once a day. Moreover, 11.7% used dental floss once a day and 6.6% visited a dentist every six months. Oral-dental health behavior had a significant relationship with gender (P0.05. Conclusion When designing educational plans and interventions for improving oral-dental health behavior in students, it is necessary to adopt measures to enhance motivating factors and eliminate inhibiting factors.

  1. Critical Factors Inhibiting Performance of Small- and Medium-Scale Contractors in Sub-Saharan Region: A Case for Malawi

    Directory of Open Access Journals (Sweden)

    Paul John Kulemeka

    2015-01-01

    Full Text Available The construction industry is dominated by small- and medium-scale contractors (SMCs who face an emerging trend of unique challenges in the implementation of projects. The study was aimed at examining inhibiting factors that influence performance of SMCs in terms of “quality of work,” “tender estimation,” “tender preparation,” and “timely completion of construction projects” in Malawi. A survey questionnaire was administered to 370 players in the construction industry which included public sector clients, contractors, consultants, and construction resource trainers in order to elicit data from 118 variables that were identified through a careful literature review. The inhibiting factors were generally dominated by economic issues, which was an emerging trend to what has been previously reported in the sub-Saharan region. The first highest ranked inhibiting factors were high lending interest regimes offered by financial institutions; stringent conditions to access capital; fluctuation of currency; stringent requirements for obtaining bonds; and high taxes. The research lays the foundation for further understanding of inhibitors on performance of SMCs in an evolving world which is being impacted by global factors and punctuated by sudden changes.

  2. Propranolol inhibits angiogenesis via down-regulating the expression of vascular endothelial growth factor in hemangioma derived stem cell

    Science.gov (United States)

    Zhang, Ling; Mai, Hua-Ming; Zheng, Jing; Zheng, Jia-Wei; Wang, Yan-An; Qin, Zhong-Ping; Li, Ke-Lei

    2014-01-01

    Background: Oral propranolol (PRN) has recently been shown to be highly effective for infantile hemangiomas (IHs), and is currently recommended as the first-line treatment of complicated IHs. However, the therapeutic mechanism(s) still remain unclear. Methods: In this study, we tested hemangioma-derived stem cells for expression of vascular endothelial growth factor (VEGF) in vitro and studied the inhibition of VEGF expression. We used PCR, Elisa, Western blotting and immunohistochemistry in vivo and in vitro trial. Results: The study demonstrated that application of PRN at a “normal” concentration equivalent to plasma concentration did not inhibit proliferation or promote apoptosis of hemangioma derived stem cells (HemSCs) isolated from IH patients. PRN suppressed expression of vascular endothelial growth factor (VEGF) and basic Fibroblast Growth Factor (bFGF) in HemSCs in vitro. Morphological, histological and immunohistological improvement were observed in vivo using murine IH model in which HemSCs pre-treated with PRN were implanted into BALB/c-nu mice. In the pre-treated HemSC grafts, mean micro-vessel density (MVD) significantly decreased and protein levels of VEGF markedly decreased, while bFGF was still detectable. Conclusions: The results suggested PRN inhibited angiogenesis via down-regulating the expression of vascular endothelial growth factor in hemangioma derived stem cell. These findings provide critical insight into the potential mechanisms of PRN action on IH. PMID:24427325

  3. The Development of the Ares I-X Flight Test

    Science.gov (United States)

    Ess, Robert H.

    2008-01-01

    The National Aeronautics and Space Administration (NASA) Constellation Program (CxP) has identified a series of tests to provide insight into the design and development of the Ares I Crew Launch Vehicle (CLV) and the Orion Crew Exploration Vehicle (CEV). Ares I-X was created as the first suborbital development flight test to help meet CxP objectives. The Ares I-X flight vehicle is an early operational model of Ares, with specific emphasis on Ares I and ground operation characteristics necessary to meet Ares I-X flight test objectives. Ares I-X will encompass the design and construction of an entire system that includes the Flight Test Vehicle (FTV) and associated operations. The FTV will be a test model based on the Ares I design. Select design features will be incorporated in the FTV design to emulate the operation of the CLV in order to meet the flight test objectives. The operations infrastructure and processes will be customized for Ares I-X, while still providing data to inform the developers of the launch processing system for Ares/Orion. The FTV is comprised of multiple elements and components that will be developed at different locations. The components will be delivered to the launch/assembly site, Kennedy Space Center (KSC), for assembly of the elements and components into an integrated, flight-ready, launch vehicle. The FTV will fly a prescribed trajectory in order to obtain the necessary data to meet the objectives. Ares I-X will not be commanded or controlled from the ground during flight, but the FTV will be equipped with telemetry systems, a data recording capability and a flight termination system (FTS). The in-flight part of the test includes a trajectory to simulate maximum dynamic pressure during flight and perform a stage separation representative of the CLV. The in-flight test also includes separation of the Upper Stage Simulator (USS) from the First Stage and recovery of the First Stage. The data retrieved from the flight test will be analyzed

  4. A novel glucosamine derivative exerts anti-inflammatory actions via inhibition of nuclear factor-kappaB.

    Science.gov (United States)

    Shin, Jin-A; Hwang, Ji-Sun; Kim, Song-Yi; Oh, Sei-Kwan; Nam, Ghilsoo; Han, Inn-Oc

    2013-08-29

    Glucosamine suppresses lipopolysaccharide (LPS)-induced upregulation of pro-inflammatory mediators both in vivo and in culture systems of mouse microglia or macrophage. In the present study, we show that the novel glucosamine derivative, 2-deoxy-2-[(o-methylbenzylidene)]-β-glucopyranoside (NK-4), significantly reduced LPS-induced production of nitric oxide (NO) in BV2 microglia, RAW264.7 macrophage, and primary cultured peritoneal macrophages cells. NK-4 inhibited LPS-induced upregulation of inducible NO synthase (iNOS), cyclooxygenase-2, interleukin-6, tumor necrosis factor-α, and interleukin-1β in RAW264.7 cells in a time- and concentration-dependent manner. Furthermore, administering NK-4 significantly inhibited the induction of inflammatory cytokine mRNAs in the brains of LPS-injected mice. Although NK-4 inhibited LPS-induced nuclear factor-kappaB (NF-κB) activation, IκB-α degradation was not changed. Instead, NK-4 inhibited LPS-induced DNA-binding activity of NF-κB by suppressing p50 and c-Rel binding to NF-κB binding site of the iNOS promoter.

  5. Andrographolide inhibits hypoxia-inducible factor-1 through phosphatidylinositol 3-kinase/AKT pathway and suppresses breast cancer growth

    Science.gov (United States)

    Li, Jie; Zhang, Chao; Jiang, Hongchuan; Cheng, Jiao

    2015-01-01

    Hypoxia-inducible factor-1 (HIF-1) is a master regulator of the transcriptional response to hypoxia. HIF-1α is one of the most compelling anticancer targets. Andrographolide (Andro) was newly identified to inhibit HIF-1 in T47D cells (a half maximal effective concentration [EC50] of 1.03×10−7 mol/L), by a dual-luciferase reporter assay. It suppressed HIF-1α protein and gene accumulation, which was dependent on the inhibition of upstream phosphatidylinositol 3-kinase (PI3K)/AKT pathway. It also abrogated the expression of HIF-1 target vascular endothelial growth factor (VEGF) gene and protein. Further, Andro inhibited T47D and MDA-MB-231 cell proliferation and colony formation. In addition, it exhibited significant in vivo efficacy and antitumor potential against the MDA-MB-231 xenograft in nude mice. In conclusion, these results highlighted the potential effects of Andro, which inhibits HIF-1, and hence may be developed as an antitumor agent for breast cancer therapy in future. PMID:25709476

  6. Arabidopsis Lectin Receptor Kinases LecRK-IX.1 and LecRK-IX.2 Are Functional Analogs in Regulating Phytophthora Resistance and Plant Cell Death.

    Science.gov (United States)

    Wang, Yan; Cordewener, Jan H G; America, Antoine H P; Shan, Weixing; Bouwmeester, Klaas; Govers, Francine

    2015-09-01

    L-type lectin receptor kinases (LecRK) are potential immune receptors. Here, we characterized two closely-related Arabidopsis LecRK, LecRK-IX.1 and LecRK-IX.2, of which T-DNA insertion mutants showed compromised resistance to Phytophthora brassicae and Phytophthora capsici, with double mutants showing additive susceptibility. Overexpression of LecRK-IX.1 or LecRK-IX.2 in Arabidopsis and transient expression in Nicotiana benthamiana increased Phytophthora resistance but also induced cell death. Phytophthora resistance required both the lectin domain and kinase activity, but for cell death, the lectin domain was not needed. Silencing of the two closely related mitogen-activated protein kinase genes NbSIPK and NbNTF4 in N. benthamiana completely abolished LecRK-IX.1-induced cell death but not Phytophthora resistance. Liquid chromatography-mass spectrometry analysis of protein complexes coimmunoprecipitated in planta with LecRK-IX.1 or LecRK-IX.2 as bait, resulted in the identification of the N. benthamiana ABC transporter NbPDR1 as a potential interactor of both LecRK. The closest homolog of NbPDR1 in Arabidopsis is ABCG40, and coimmunoprecipitation experiments showed that ABCG40 associates with LecRK-IX.1 and LecRK-IX.2 in planta. Similar to the LecRK mutants, ABCG40 mutants showed compromised Phytophthora resistance. This study shows that LecRK-IX.1 and LecRK-IX.2 are Phytophthora resistance components that function independent of each other and independent of the cell-death phenotype. They both interact with the same ABC transporter, suggesting that they exploit similar signal transduction pathways.

  7. Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis

    DEFF Research Database (Denmark)

    Lasica, Anna M; Goulas, Theodoros; Mizgalska, Danuta;

    2016-01-01

    Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T...

  8. Na/K ATPase inhibition by digitalis-like factors in neonates

    Energy Technology Data Exchange (ETDEWEB)

    Bottorff, M.B.; Songu-Mize, E.; Hoon, T.J.; Phelps, S.J.; Kamper, C.A.

    1986-03-01

    At the authors institution, 48% of neonates < 1 month of age had false-positive digoxin immunoassay determinations while not receiving digoxin, presumably due to an endogenous digoxin-like immunoreactive substance (DLIS) in the plasma. Plasma from 3 neonates positive for DLIS by fluorescence polarization immunoassay (FPIA) was evaluated for inhibitory activity on human red blood cell (RBC) Na/K ATPase. Neonatal plasma aliquots containing DLIS concentrations (conc) of 0.24, 0.37, 0.43, 0.49 and 0.61 ng/ml (3.07 - 7.81 x 10/sup -10/M) were incubated with human RBC and /sup 86/Rb in order to measure /sup 86/Rb uptake inhibition with respect to DLIS negative neonatal plasma. /sup 86/Rb uptake inhibition by digoxin-spiked human serum (1.07 x 10/sup -10/ - 4.57 x 10/sup -6/M) was also measured. Percent inhibition vs. log molar conc plots for DLIS and digoxin were compared. DLIS inhibited Na/K ATPase in a linear fashion over the range studied. Comparing the linear portions of the conc-inhibition curves for digoxin and DLIS, the molar conc of digoxin producing 40% inhibition of /sup 86/Rb uptake is 333 times greater than the molar conc of DLIS producing similar inhibition. Therefore, DLIS in neonatal serum as measured by FPIA has approximately 300 times greater inhibitory activity than digoxin. The presence of circulating DLIS may reflect an adaptive or maladaptive response to some, as yet unknown, process early in life.

  9. Adipose stem cells can secrete angiogenic factors that inhibit hyaline cartilage regeneration

    National Research Council Canada - National Science Library

    Lee, Christopher Sd; Burnsed, Olivia A; Raghuram, Vineeth; Kalisvaart, Jonathan; Boyan, Barbara D; Schwartz, Zvi

    2012-01-01

    Adipose stem cells (ASCs) secrete many trophic factors that can stimulate tissue repair, including angiogenic factors, but little is known about how ASCs and their secreted factors influence cartilage regeneration...

  10. Overexpression of transcription factor Sp1 leads to gene expression perturbations and cell cycle inhibition.

    Directory of Open Access Journals (Sweden)

    Emmanuelle Deniaud

    Full Text Available BACKGROUND: The ubiquitous transcription factor Sp1 regulates the expression of a vast number of genes involved in many cellular functions ranging from differentiation to proliferation and apoptosis. Sp1 expression levels show a dramatic increase during transformation and this could play a critical role for tumour development or maintenance. Although Sp1 deregulation might be beneficial for tumour cells, its overexpression induces apoptosis of untransformed cells. Here we further characterised the functional and transcriptional responses of untransformed cells following Sp1 overexpression. METHODOLOGY AND PRINCIPAL FINDINGS: We made use of wild-type and DNA-binding-deficient Sp1 to demonstrate that the induction of apoptosis by Sp1 is dependent on its capacity to bind DNA. Genome-wide expression profiling identified genes involved in cancer, cell death and cell cycle as being enriched among differentially expressed genes following Sp1 overexpression. In silico search to determine the presence of Sp1 binding sites in the promoter region of modulated genes was conducted. Genes that contained Sp1 binding sites in their promoters were enriched among down-regulated genes. The endogenous sp1 gene is one of the most down-regulated suggesting a negative feedback loop induced by overexpressed Sp1. In contrast, genes containing Sp1 binding sites in their promoters were not enriched among up-regulated genes. These results suggest that the transcriptional response involves both direct Sp1-driven transcription and indirect mechanisms. Finally, we show that Sp1 overexpression led to a modified expression of G1/S transition regulatory genes such as the down-regulation of cyclin D2 and the up-regulation of cyclin G2 and cdkn2c/p18 expression. The biological significance of these modifications was confirmed by showing that the cells accumulated in the G1 phase of the cell cycle before the onset of apoptosis. CONCLUSION: This study shows that the binding to DNA

  11. A diarylheptanoid from lesser galangal (Alpinia officinarum) inhibits proinflammatory mediators via inhibition of mitogen-activated protein kinase, p44/42, and transcription factor nuclear factor-kappa B.

    Science.gov (United States)

    Yadav, Prem N; Liu, Zhihua; Rafi, Mohamed M

    2003-06-01

    The diarylheptanoid 7-(4'-hydroxy-3'-methoxyphenyl)-1-phenylhept-4-en-3-one (HMP) is a naturally occurring phytochemical found in lesser galangal (Alpinia officinarum). In the present study, we have demonstrated the anti-inflammatory properties of this compound on mouse macrophage cell line (RAW 264.7) and human peripheral blood mononuclear cells (PBMCs) in vitro. Treatment of RAW 264.7 cells with HMP (6.25-25 microM) significantly inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production. This compound also inhibited the release of LPS-induced proinflammatory cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) from human PB-MCs in vitro. In addition, Western blotting and reverse transcription-polymerase chain reaction analysis demonstrated that HMP decreased LPS-induced inducible nitric-oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and mRNA expression in RAW 264.7 cells. Furthermore, HMP treatment also reduced nuclear factor-kappa B (NF-kappa B) DNA binding induced by LPS in RAW 264.7 cells. To elucidate the molecular mechanism for inhibition of proinflammatory mediators by HMP (25 microM), we have studied the effect of HMP on LPS-induced p38 and p44/42 mitogen-activated protein kinase (MAPK). We observed that the phosphorylation of p44/42 MAPK in LPS-stimulated RAW 264.7 cells was markedly inhibited by HMP, whereas activation of p38 MAPK was not affected. These results suggested that HMP from lesser galangal suppressed the LPS-induced production of NO, IL-1 beta, and TNF-alpha and expression of iNOS and COX-2 gene expression by inhibiting NF-kappa B activation and phosphorylation of p44/42 MAPK.

  12. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) induces initiation factor 2 alpha phosphorylation and translation inhibition in PC12 cells.

    Science.gov (United States)

    Muñoz, F; Martín, M E; Salinas, M; Fando, J L

    2001-03-09

    We have investigated the effect of the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) on protein synthesis rate and initiation factor 2 (eIF2) phosphorylation in PC12 cells differentiated with nerve growth factor. FCCP treatment induced a very rapid 2-fold increase in intracellular Ca(2+) concentration that was accompanied by a strong protein synthesis rate inhibition (68%). The translation inhibition correlated with an increased phosphorylation of the alpha subunit of eIF2 (eIF2 alpha) (25% vs. 7%, for FCCP-treated and control cells, respectively) and a 1.7-fold increase in the double-stranded RNA-dependent protein kinase activity. No changes in the PKR endoplasmic reticulum-related kinase or eIF2 alpha phosphatase were found. Translational regulation may play a significant role in the process triggered by mitochondrial calcium mobilization.

  13. Mycalamide A Shows Cytotoxic Properties and Prevents EGF-Induced Neoplastic Transformation through Inhibition of Nuclear Factors

    Science.gov (United States)

    Dyshlovoy, Sergey A.; Fedorov, Sergey N.; Kalinovsky, Anatoly I.; Shubina, Larisa K.; Bokemeyer, Carsten; Stonik, Valentin A.; Honecker, Friedemann

    2012-01-01

    Mycalamide A, a marine natural compound previously isolated from sponges, is known as a protein synthesis inhibitor with potent antitumor activity. However, the ability of this compound to prevent malignant transformation of cells has never been examined before. Here, for the first time, we report the isolation of mycalamide A from ascidian Polysincraton sp. as well as investigation of its cancer preventive properties. In murine JB6 Cl41 P+ cells, mycalamide A inhibited epidermal growth factor (EGF)-induced neoplastic transformation, and induced apoptosis at subnanomolar or nanomolar concentrations. The compound inhibited transcriptional activity of the oncogenic nuclear factors AP-1 and NF-κB, a potential mechanism of its cancer preventive properties. Induction of phosphorylation of the kinases MAPK p38, JNK, and ERK was also observed at high concentrations of mycalamide A. The drug shows promising potential for both cancer-prevention and cytotoxic therapy and should be further developed. PMID:22822368

  14. Star formation rate in Holmberg IX dwarf galaxy

    Directory of Open Access Journals (Sweden)

    Anđelić M.M.

    2011-01-01

    Full Text Available In this paper we use previously determined Hα fluxes for dwarf galaxy Holmberg IX (Arbutina et al. 2009 to calculate star formation rate (SFR in this galaxy. We discuss possible contaminations of Hα flux and, for the first time, we take into account optical emission from supernova remnants (SNRs as a possible source of contamination of Hα flux. Derived SFR for Holmberg IX is 3:4 x 10-4M.yr-1. Our value is lower then in previous studies, due to luminous shock-heated source M&H 9-10, possible hypernova remnant, which we excluded from the total Hα flux in our calculation of SFR.

  15. Ares I-X Best Estimated Trajectory Analysis and Results

    Science.gov (United States)

    Karlgaard, Christopher D.; Beck, Roger E.; Starr, Brett R.; Derry, Stephen D.; Brandon, Jay; Olds, Aaron D.

    2011-01-01

    The Ares I-X trajectory reconstruction produced best estimated trajectories of the flight test vehicle ascent through stage separation, and of the first and upper stage entries after separation. The trajectory reconstruction process combines on-board, ground-based, and atmospheric measurements to produce the trajectory estimates. The Ares I-X vehicle had a number of on-board and ground based sensors that were available, including inertial measurement units, radar, air-data, and weather balloons. However, due to problems with calibrations and/or data, not all of the sensor data were used. The trajectory estimate was generated using an Iterative Extended Kalman Filter algorithm, which is an industry standard processing algorithm for filtering and estimation applications. This paper describes the methodology and results of the trajectory reconstruction process, including flight data preprocessing and input uncertainties, trajectory estimation algorithms, output transformations, and comparisons with preflight predictions.

  16. Bianchi-IX, Darboux-Halphen and Chazy-Ramanujan

    CERN Document Server

    Chanda, Sumanto; Roychowdhury, Raju

    2015-01-01

    Bianchi-IX four metrics are $SU(2)$ invariant solutions of vacuum Einstein equation, for which the connection-wise self-dual case describes the Euler Top, while the curvature-wise self-dual case yields the Ricci flat classical Darboux-Halphen system. It is possible to see such a solution exhibiting Ricci flow. The classical Darboux-Halphen system is a special case of the generalized one that arises from a reduction of the self-dual Yang-Mills equation and the solutions to the related homogeneous quadratic differential equations provide the desired metric. A few integrable and near-integrable dynamical systems related to the Darboux-Halphen system and occur in the study of Bianchi IX gravitational instanton have been listed as well. We explore in details whether self-duality implies integrability.

  17. Inhibition of chemomigration of a human prostatic carcinoma cell (TSU-pr1) line by inhibition of epidermal growth factor receptor function.

    Science.gov (United States)

    Zolfaghari, A; Djakiew, D

    1996-04-01

    Chemoattractants expressed at bony sites and pelvic lymph nodes are thought to promote the preferential metastasis of human prostate tumor cells to these organs. Epidermal growth factor (EGF) is a potent chemoattractant for several human metastatic prostate tumor cell lines, including the TSU-pr1 cell line, and EGF has been localized to the stroma of both bony sites and pelvic lymph nodes in humans. Hence, we investigated whether the TSU-pr1 cell line expresses a functional EGF receptor (EGFR), which when antagonized reduces EGF-mediated chemomigration of this cell line. In this context, the EGFR immunoprecipitated from cell lysates of TSU-pr1 cells comigrated with the EGFR from A431 cells at a molecular weight of 170 kD. Addition of human EGF (hEGF) to the TSU-pr1 cells for 5 min stimulated the dose-dependent biphasic phosphorylation of the EGFR, with maximal stimulation of EGFR phosphorylation occurring at 2 ng/ml hEGF. In addition, treatment of hEGF-stimulated (2 ng/ml) TSU-pr1 cells with 0.5 microgram/ml anti-hEGF monoclonal antibody or 100 nM staurosporine inhibited EGFR phosphorylation. Conversely, as negative controls, treatment of hEGF-stimulated (2 ng/ml) TSU-pr1 cells with K252a or dimethyl sulfoxide (DMSO) vehicle did not inhibit EGFR phosphorylation. TSU-pr1 cells were stimulated to migration in 4 hr across Boyden chambers in response to 10 ng/ml hEGF. Treatment of the TSU-pr1 cells with anti-hEGFR monoclonal antibody inhibited in a dose-dependent manner the chemomigration of the TSU-pr1 cells across Boyden chambers. Similarly, treatment of the TSU-pr1 cells with staurosporine inhibited in a dose-dependent manner the chemomigration of the TSU-pr1 cells across Boyden chambers. These results demonstrate that antagonists of hEGF-mediated hEGFR phosphorylation also antagonize chemomigration of the TSU-pr1 cells across Boyden chambers, suggesting that antagonists of the EGFR in prostate cancer may be useful in the treatment of metastatic disease.

  18. Loop quantum cosmology of Bianchi type IX models

    CERN Document Server

    Wilson-Ewing, Edward

    2010-01-01

    The loop quantum cosmology "improved dynamics" of the Bianchi type IX model are studied. The action of the Hamiltonian constraint operator is obtained via techniques developed for the Bianchi type I and type II models, no new input is required. It is shown that the big bang and big crunch singularities are resolved by quantum gravity effects. We also present the effective equations which provide modifications to the classical equations of motion due to quantum geometry effects.

  19. Loop quantum cosmology of Bianchi type IX models

    Science.gov (United States)

    Wilson-Ewing, Edward

    2010-08-01

    The loop quantum cosmology “improved dynamics” of the Bianchi type IX model are studied. The action of the Hamiltonian constraint operator is obtained via techniques developed for the Bianchi type I and type II models, no new input is required. It is shown that the big bang and big crunch singularities are resolved by quantum gravity effects. We also present effective equations which provide quantum geometry corrections to the classical equations of motion.

  20. Inhibition of MHC class I is a virulence factor in herpes simplex virus infection of mice.

    Directory of Open Access Journals (Sweden)

    Mark T Orr

    2005-09-01

    Full Text Available Herpes simplex virus (HSV has a number of genes devoted to immune evasion. One such gene, ICP47, binds to the transporter associated with antigen presentation (TAP 1/2 thereby preventing transport of viral peptides into the endoplasmic reticulum, loading of peptides onto nascent major histocompatibility complex (MHC class I molecules, and presentation of peptides to CD8 T cells. However, ICP47 binds poorly to murine TAP1/2 and so inhibits antigen presentation by MHC class I in mice much less efficiently than in humans, limiting the utility of murine models to address the importance of MHC class I inhibition in HSV immunopathogenesis. To address this limitation, we generated recombinant HSVs that efficiently inhibit antigen presentation by murine MHC class I. These recombinant viruses prevented cytotoxic T lymphocyte killing of infected cells in vitro, replicated to higher titers in the central nervous system, and induced paralysis more frequently than control HSV. This increase in virulence was due to inhibition of antigen presentation to CD8 T cells, since these differences were not evident in MHC class I-deficient mice or in mice in which CD8 T cells were depleted. Inhibition of MHC class I by the recombinant viruses did not impair the induction of the HSV-specific CD8 T-cell response, indicating that cross-presentation is the principal mechanism by which HSV-specific CD8 T cells are induced. This inhibition in turn facilitates greater viral entry, replication, and/or survival in the central nervous system, leading to an increased incidence of paralysis.

  1. Dilazep and dipyridamole inhibit tissue factor expression on monocytes induced by IgG from patients with antiphospholipid syndrome

    Institute of Scientific and Technical Information of China (English)

    Hong ZHON

    2004-01-01

    AIM: To investigate whether antiplatelet agents, dilazep and dipyridamole, inhibit tissue factor (TF) expression on monocytes induced by IgG from patients with antiphospholipid syndrome (APS). METHODS: Freshly isolated peripheral blood monocytes were allowed to adhere on plastic and then cultured in media containing patient or control antibodies and/or other agonists with or without dilazep or dipyridamole. The TF activity on monocytes was investigated by measuring factor VIIa-dependent generation of factor Xa, using a chromogenic substrate and the TF mRNA expression was examined by real-time PCR (TaqMan PCR). RESULTS: The TF activity on monocytes induced by APS IgG (250 mg/L) was inhibited by dilazep (0.15-150 μmol/L) and dipyridamole (0.2-200 μrmol/L) in a dose-dependent fashion. But, the TF mRNA expression induced by APS IgG was not inhibited. Theophylline (500 μmol/L), an adenosine receptor antagonist, could counteract the inhibitory effect of dilazep and dipyridamole on TF activity. CONCLUSION: Antiplatelet agents, dilazep and dipyridamole, block APS IgG-induced monocytes TF expression at a post-transcriptional level, partly by adenosine receptor pathway. Pharmacological agents that block monocytes TF activity, such as dilazep and dipyridamole, are a novel therapeutic approach in APS.

  2. Loop quantum cosmology of Bianchi IX: effective dynamics

    Science.gov (United States)

    Corichi, Alejandro; Montoya, Edison

    2017-03-01

    We study solutions to the effective equations for the Bianchi IX class of spacetimes within loop quantum cosmology (LQC). We consider Bianchi IX models whose matter content is a massless scalar field, by numerically solving the loop quantum cosmology effective equations, with and without inverse triad corrections. The solutions are classified using certain geometrically motivated classical observables. We show that both effective theories—with lapse N  =  V and N  =  1—resolve the big bang singularity and reproduce the classical dynamics far from the bounce. Moreover, due to the positive spatial curvature, there is an infinite number of bounces and recollapses. We study the limit of large field momentum and show that both effective theories reproduce the same dynamics, thus recovering general relativity. We implement a procedure to identify amongst the Bianchi IX solutions, those that behave like k  =  0,1 FLRW as well as Bianchi I, II, and VII0 models. The effective solutions exhibit Bianchi I phases with Bianchi II transitions and also Bianchi VII0 phases, which had not been studied before. We comment on the possible implications of these results for a quantum modification to the classical BKL behaviour.

  3. Loop quantum cosmology of Bianchi IX: Effective dynamics

    CERN Document Server

    Corichi, Alejandro

    2015-01-01

    We study numerically the solutions to the effective equations of Bianchi IX spacetimes within Loop Quantum Cosmology. We consider Bianchi IX models with and without inverse triad corrections whose matter content is a scalar field without mass. The solutions are classified using the classical observables. We show that both effective theories --with lapse N=V and N=1-- solve the big bang singularity and reproduce the classical dynamics far from the bounce. Moreover, due to the spatial compactness, there is an infinity number of bounces and recollapses. We study the limit of large volume and show that both effective theories reproduce the same dynamics, thus recovering general relativity. We implement a procedure to identify amongst the Bianchi IX solutions, those that behave like k=0,1 FLRW as well as Bianchi I, II, and VII_0 models. The effective solutions exhibit Bianchi I phases with Bianchi II transitions and also Bianchi VII_0 phases, which had not been studied before, at the quantum nor effective level. W...

  4. Optimization and characterization of the endogenous production of protoporphyrin IX in a yeast model

    Science.gov (United States)

    Joniová, Jaroslava; Gerelli, Emmanuel; Zellweger, Matthieu; Wagnières, Georges

    2016-12-01

    The availability of reproducible, convenient, and inexpensive model organisms able to generate predictable levels of endogenous porphyrins, including protoporphyrin IX (PpIX), is essential in photomedicine research. Saccharomyces cerevisiae produces endogenous PpIX and was used as a model organism for this study with the aim to maximize endogenous PpIX fluorescence intensity. It was found that PpIX fluorescence was significantly enhanced by administration of 5-aminolevulinic acid (ALA) and 2,2‧-bipyridyl. Fluorescence intensity and spectroscopy of PpIX produced endogenously were measured in diluted yeast solutions under various conditions. The optimal protocol was: 5 μM ALA and 1 mM 2,2‧-bipyridyl administered synchronously at 32°C. After 3 h, PpIX in yeast demonstrated similar steady-state and time-resolved spectroscopy as that of PpIX in DMSO. Moreover, under hypoxic conditions, the reciprocal lifetime of PpIX delayed fluorescence measured in real time was correlated to the partial pressure of oxygen (pO2) measured concomitantly with a commercially available pO2 probe. These data show that yeast can, in optimal conditions, reproducibly generate PpIX. This is of interest in various fields such as photodiagnosis, photodynamic therapy, and photobiomodulation. Use of this model organism focuses on essential mechanisms, without the complexity of a multicellular organism.

  5. Inhibition of HIV-1 gene expression by Ciclopirox and Deferiprone, drugs that prevent hypusination of eukaryotic initiation factor 5A

    Directory of Open Access Journals (Sweden)

    Saxena Deepti

    2009-10-01

    Full Text Available Abstract Background Eukaryotic translation initiation factor eIF5A has been implicated in HIV-1 replication. This protein contains the apparently unique amino acid hypusine that is formed by the post-translational modification of a lysine residue catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase (DOHH. DOHH activity is inhibited by two clinically used drugs, the topical fungicide ciclopirox and the systemic medicinal iron chelator deferiprone. Deferiprone has been reported to inhibit HIV-1 replication in tissue culture. Results Ciclopirox and deferiprone blocked HIV-1 replication in PBMCs. To examine the underlying mechanisms, we investigated the action of the drugs on eIF5A modification and HIV-1 gene expression in model systems. At early times after drug exposure, both drugs inhibited substrate binding to DOHH and prevented the formation of mature eIF5A. Viral gene expression from HIV-1 molecular clones was suppressed at the RNA level independently of all viral genes. The inhibition was specific for the viral promoter and occurred at the level of HIV-1 transcription initiation. Partial knockdown of eIF5A-1 by siRNA led to inhibition of HIV-1 gene expression that was non-additive with drug action. These data support the importance of eIF5A and hypusine formation in HIV-1 gene expression. Conclusion At clinically relevant concentrations, two widely used drugs blocked HIV-1 replication ex vivo. They specifically inhibited expression from the HIV-1 promoter at the level of transcription initiation. Both drugs interfered with the hydroxylation step in the hypusine modification of eIF5A. These results have profound implications for the potential therapeutic use of these drugs as antiretrovirals and for the development of optimized analogs.

  6. Inhibition of HIV-1 gene expression by Ciclopirox and Deferiprone, drugs that prevent hypusination of eukaryotic initiation factor 5A.

    Science.gov (United States)

    Hoque, Mainul; Hanauske-Abel, Hartmut M; Palumbo, Paul; Saxena, Deepti; D'Alliessi Gandolfi, Darlene; Park, Myung Hee; Pe'ery, Tsafi; Mathews, Michael B

    2009-10-13

    Eukaryotic translation initiation factor eIF5A has been implicated in HIV-1 replication. This protein contains the apparently unique amino acid hypusine that is formed by the post-translational modification of a lysine residue catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase (DOHH). DOHH activity is inhibited by two clinically used drugs, the topical fungicide ciclopirox and the systemic medicinal iron chelator deferiprone. Deferiprone has been reported to inhibit HIV-1 replication in tissue culture. Ciclopirox and deferiprone blocked HIV-1 replication in PBMCs. To examine the underlying mechanisms, we investigated the action of the drugs on eIF5A modification and HIV-1 gene expression in model systems. At early times after drug exposure, both drugs inhibited substrate binding to DOHH and prevented the formation of mature eIF5A. Viral gene expression from HIV-1 molecular clones was suppressed at the RNA level independently of all viral genes. The inhibition was specific for the viral promoter and occurred at the level of HIV-1 transcription initiation. Partial knockdown of eIF5A-1 by siRNA led to inhibition of HIV-1 gene expression that was non-additive with drug action. These data support the importance of eIF5A and hypusine formation in HIV-1 gene expression. At clinically relevant concentrations, two widely used drugs blocked HIV-1 replication ex vivo. They specifically inhibited expression from the HIV-1 promoter at the level of transcription initiation. Both drugs interfered with the hydroxylation step in the hypusine modification of eIF5A. These results have profound implications for the potential therapeutic use of these drugs as antiretrovirals and for the development of optimized analogs.

  7. Inhibition of Angiogenic Factor Production from Murine Mast Cells by an Antiallergic Agent (Epinastine Hydrochloride In Vitro

    Directory of Open Access Journals (Sweden)

    K. Asano

    2008-01-01

    Full Text Available Angiogenesis is an important event both in the development of allergic inflammatory responses and in the pathophysiology of tissue remodeling in allergic diseases. In the present study, therefore, we examined the influence of antihistamines on angiogenesis through the choice of epinastine hydrochloride (EP and murine mast cells in vitro. Mast cells (5×105 cells/mL presensitized with murine IgE specific for ovalbumin (OVA were stimulated with 10 ng/mL OVA in the presence of various concentrations of EP for 4 hours. The levels of angiogenesis factors, keratinocyte-derived chemokine (KC, tumor necrosis factor-α (TNF, and vascular endothelial growth factor (VEGF in culture supernatants, were examined by ELISA. We also examined mRNA expression for the angiogenesis factors by RT-PCR. EP significantly inhibited the production of KC, TNF, and VEGF induced by IgE-dependent mechanism at more than 25 ng/mL. Semiquantitative analysis using RT-PCR showed that EP also significantly reduced mRNA expressions for KC, TNF, and VEGF. These results strongly suggest that EP suppresses angiogenesis factor production through the inhibition of mRNA expression in mast cells and results in favorable modification of clinical conditions of allergic diseases.

  8. The relationship between factor inhibiting HIF-1-alpha (HIF1AN( expression and vascular invasion in colon cancer

    Directory of Open Access Journals (Sweden)

    Reza Najafipour

    2016-10-01

    Full Text Available Background: Hypoxia is a common phenomenon in human solid tumors which by increasing in angiogenesis induction cause tumor growth survival and metastasis. Inhibitory factor hypoxia regulatory factor (HIF1AN by binding to transcription co activators CBP/P300(, inhibits hypoxia inducible factor (HIF1α. Objective: The relationship between HIF1AN expression and vascular invasion in colon tumors. Methods: The study included 101 patients with colon adenocarcinoma which were divided to vascular invasion and non-vascular invasion groups. Tumor paraffin blocks were immunohistochemistry stained for HIF1AN and were assessed for intensity and extent of positivity. Statistical relation of marker expression and clinic pathologic findings were assessed. Data were analyzed by SPSS 21 software and logistic regression and chi-square test. Findings: Nuclear immunoreactivity of HIF1AN was different between two groups. Statistical relation between low HIF1AN expression and tumor vascular invasion were seen (P=0.01. No relation was found between tumor differentiation, depth and HIF1AN. Conclusion: Evidence showed that the low expression or incorrect position of HIF1AN in nucleus of tumor cells was effective on HIF1α inhibition failure and factors associated angiogenesis increased. The HIF1AN played an tumor suppressor gene (TSG( role in colon tumors and decreased protein in the nucleus of colon cancer cells increased the expression of angiogenesis factors and vascular invasion.

  9. Artemisia princeps Inhibits Biofilm Formation and Virulence-Factor Expression of Antibiotic-Resistant Bacteria

    Directory of Open Access Journals (Sweden)

    Na-Young Choi

    2015-01-01

    Full Text Available In this study, we used ethanol extract of A. princeps and investigated its antibacterial effects against MRSA. Ethanol extract of A. princeps significantly inhibited MRSA growth and organic acid production during glucose metabolism at concentrations greater than 1 mg/mL (P < 0.05. MRSA biofilm formation was observed using scanning electron microscopy (SEM and safranin staining. A. princeps extract was found to inhibit MRSA biofilm formation at concentrations higher than 2 mg/mL significantly (P < 0.05. Bactericidal effects of the A. princeps were observed using confocal laser microscopy, which showed that A. princeps was bactericidal in a dose-dependent manner. Using real-time PCR, expression of mecA, an antibiotic-resistance gene of MRSA, was observed, along with that of sea, agrA, and sarA. A. princeps significantly inhibited mecA, sea, agrA, and sarA, mRNA expression at the concentrations greater than 1 mg/mL (P < 0.05. The phytochemical analysis of A. princeps showed a relatively high content of organic acids and glycosides. The results of this study suggest that the ethanol extract of A. princeps may inhibit proliferation, acid production, biofilm formation, and virulence gene expressions of MRSA, which may be related to organic acids and glycosides, the major components in the extract.

  10. Dexamethasone Inhibits Tumor Necrosis Factor-α-stimulated Gastric Epithelial Cell Migration

    Directory of Open Access Journals (Sweden)

    Jiing-Chyuan Luo

    2009-10-01

    Conclusion: TNF-α plays a regulatory role in rat gastric epithelial cell migration and dexamethasone inhibited TNF-α-stimulated cell migration, which was associated with a decrease in COX-2 expression and PGE2 formation. [J Chin Med Assoc 2009;72(10:509–514

  11. Transforming growth factor-beta, but not ciliary neurotrophic factor, inhibits DNA synthesis of adrenal medullary cells in vitro

    DEFF Research Database (Denmark)

    Wolf, N; Krohn, K; Bieger, S;

    1999-01-01

    by the neuroendocrine chromaffin cells, which also express the transforming growth factor-beta receptor type II. In contrast to the developmentally related sympathetic neurons, chromaffin cells continue to proliferate throughout postnatal life. Using 5-bromo-2'-deoxyuridine pulse labeling and tyrosine hydroxylase...... regulator of chromaffin cell division.......Transforming growth factor-betas are members of a superfamily of multifunctional cytokines regulating cell growth and differentiation. Their functions in neural and endocrine cells are not well understood. We show here that transforming growth factor-betas are synthesized, stored and released...

  12. (+)-Nootkatone inhibits tumor necrosis factor α/interferon γ-induced production of chemokines in HaCaT cells

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Hyeon-Jae; Lee, Jin-Hwee [College of Pharmacy, Ajou University, Suwon 443-749 (Korea, Republic of); Jung, Yi-Sook, E-mail: yisjung@ajou.ac.kr [College of Pharmacy, Ajou University, Suwon 443-749 (Korea, Republic of); Research Institute of Pharmaceutical Sciences and Technology, Ajou University, Suwon 443-749 (Korea, Republic of)

    2014-05-02

    Highlights: • (+)-Nootkatone inhibits TNF-α/IFN-γ-induced TARC and MDC expression in HaCaT cells. • PKCζ, p38 MAPK, or NF-κB mediate TNF-α/IFN-γ-induced TARC and MDC expression. • (+)-Nootkatone inhibits TNF-α/IFN-γ-induced activation of PKCζ, p38 MAPK, or NF-κB. • (+)-Nootkatone suppresses chemokine expression by inhibiting of PKCζ and p38 pathways. - Abstract: Chemokines are important mediators of cell migration, and thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22) are well-known typical inflammatory chemokines involved in atopic dermatitis (AD). (+)-Nootkatone is the major component of Cyperus rotundus. (+)-Nootkatone has antiallergic, anti-inflammatory, and antiplatelet activities. The purpose of this study was to investigate the effect of (+)-nootkatone on tumor necrosis factor α (TNF-α)/interferon γ (IFN-γ)-induced expression of Th2 chemokines in HaCaT cells. We found that (+)-nootkatone inhibited the TNF-α/IFN-γ-induced expression of TARC/CCL17 and MDC/CCL22 mRNA in HaCaT cells. It also significantly inhibited TNF-α/IFN-γ-induced activation of nuclear factor kappa B (NF-κB), p38 mitogen-activated protein kinase (MAPK), and protein kinase Cζ (PKCζ). Furthermore, we showed that PKCζ and p38 MAPK contributed to the inhibition of TNF-α/IFN-γ-induced TARC/CCL17 and MDC/CCL22 expression by blocking IκBα degradation in HaCaT cells. Taken together, these results suggest that (+)-nootkatone may suppress TNF-α/IFN-γ-induced TARC/CCL17 and MDC/CCL22 expression in HaCaT cells by inhibiting of PKCζ and p38 MAPK signaling pathways that lead to activation of NF-κB. We propose that (+)-nootkatone may be a useful therapeutic candidate for inflammatory skin diseases such as AD.

  13. Potentiation of thrombin generation in hemophilia A plasma by coagulation factor VIII and characterization of antibody-specific inhibition.

    Directory of Open Access Journals (Sweden)

    Bhavya S Doshi

    Full Text Available Development of inhibitory antibodies to coagulation factor VIII (fVIII is the primary obstacle to the treatment of hemophilia A in the developed world. This adverse reaction occurs in 20-30% of persons with severe hemophilia A treated with fVIII-replacement products and is characterized by the development of a humoral and neutralizing immune response to fVIII. Patients with inhibitory anti-fVIII antibodies are treated with bypassing agents including recombinant factor VIIa (rfVIIa. However, some patients display poor hemostatic response to bypass therapy and improved treatment options are needed. Recently, we demonstrated that fVIII inhibitors display widely variable kinetics of inhibition that correlate with their respective target epitopes. Thus, it was hypothesized that for antibodies that display slow rates of inhibition, supplementation of rfVIIa with fVIII would result in improved thrombin generation and be predictive of clinical responses to this novel treatment regimen. In order to test this hypothesis, 10 murine monoclonal antibodies (MAbs with non-overlapping epitopes spanning fVIII, differential inhibition titers, and inhibition kinetics were studied using a thrombin generation assay. Of the 3 MAbs with high inhibitory titers, only the one with fast and complete (classically defined as "type I" kinetics displayed significant inhibition of thrombin generation with no improvement upon supplementation of rfVIIa with fVIII. The other two MAbs that displayed incomplete (classically defined as "type II" inhibition did not suppress the potentiation of thrombin generation by fVIII. All antibodies that did not completely inhibit fVIII activity demonstrated potentiation of thrombin generation by the addition of fVIII as compared to rfVIIa alone. In conclusion, fVIII alone or in combination with rfVIIa corrects the thrombin generation defect produced by the majority of anti-fVIII MAbs better than single agent rfVIIa. Therefore, combined f

  14. [Mechanism of Smad 3 signaling pathway and connective tissue growth factor in the inhibition of form deprivation myopia by pirenzepine].

    Science.gov (United States)

    Ji, Xueying; Zhang, Jinsong; Wang, Yanting; Sun, Hongliang; Jia, Peisheng

    2009-04-01

    To observe the inhibitive effect of pirenzepine on form deprivation myopia in guinea pigs and to explore the mechanism of Smad3 signaling pathway and connective tissue growth factor (CTGF) in the inhibition of myopia by pirenzepine. Forty 1-week-old guinea pigs of either sex were randomly divided into 4 groups: a control group (Group I), a form deprivation group (Group II), a pirenzepine ophthalmic solution group (Group III), and a sodium chloride ophthalmic solution group (Group IV). Translucent blinders were used in the right eyes of Group II, III and IV. The left eyes were not given any treatment as the normal control group. Covered eyes of Group III and IV were given 3% pirenzepine ophthalmic solution and 0.1% azone ophthalmic solution respectively twice every day. Six weeks later, refraction and axial length were measured at the end of the experiment, and immunohistochemistry and Western blot were used to analyze the expression levels of Smad3 and CTGF in the sclera of all 4 groups. There was no significant difference between Group III and I in relative refraction and changes of axial length (P>0.05). The difference of Group II and IV compared with Group I was statistically significant (P0.05), while the difference in Group II, IV and I was significant (P0.05). Pirenzepine ophthalmic solution can inhibit the development of form deprivation myopia. Pirenzepine may affect Smad3 signaling pathway in the sclera by inhibiting the development of form deprivation myopia.

  15. Lactobacillus plantarum inhibits epithelial barrier dysfunction and interleukin-8 secretion induced by tumor necrosis factor

    Institute of Scientific and Technical Information of China (English)

    Jae Sung Ko; Hye Ran Yang; Ju Young Chang; Jeong Kee Seo

    2007-01-01

    AIM: To determine whether Lactobacillus plantarum can modify the deleterious effects of tumor necrosis factor-α(TNF-α) on intestinal epithelial cells.METHODS: Caco-2 cells were incubated with TNF-α alone or in the presence of L. plantarum. Transepithelial electrical resistance was used to measure epithelial barrier function. Interleukin 8 (IL-8) secretion by intestinal epithelial cells was measured using an ELISA.Cellular lysate proteins were immunoblotted using the anti-extracellular regulated kinase (ERK), anti-phospho-ERK and anti-IκB-α.RESULTS: A TNF-α-induced decrease in transepithelial electrical resistance was inhibited by L. plantarum. TNF-α-induced IL-8 secretion was reduced by L. plantarum.L. plantarum inhibited the activation of ERK and the degradation of IκB-α in TNF-α-treated Caco-2 cells.CONCLUSION: Induction of epithelial barrier dysfunction and IL-8 secretion by TNF-α is inhibited by L. plantarum.Probiotics may preserve epithelial barrier function and inhibit the inflammatory response by altering the signal transduction pathway.

  16. Inhibition of proteasome activity, nuclear factor-KappaB translocation and cell survival by the antialcoholism drug disulfiram.

    Science.gov (United States)

    Lövborg, Henrik; Oberg, Fredrik; Rickardson, Linda; Gullbo, Joachim; Nygren, Peter; Larsson, Rolf

    2006-03-15

    The proteasome pathway is an important target for anticancer drug development. Here, we identify the antialcoholism drug disulfiram and its analogue pyrrolidine dithiocarbamate (PDTC) as inhibitors of the 26S proteasome activity in a cell-based screening assay. As expected for proteasome inhibitors, these compounds also inhibited TNF-alpha-induced nuclear factor-KappaB (NF-KappaB) translocation and were cytotoxic. Disulfiram was more cytotoxic against chronic lymphocytic leukemia cells compared to peripheral blood mononuclear cells (PBMC) at clinically achievable concentrations. Proteasome and NF-KappaB inhibition were achieved with a potency in the same range as that of the clinically used proteasome inhibitor bortezomib. Disulfiram was also able to induce accumulation of p27(Kip1) and to prolong the half-life of c-Myc, both targets for proteasome-dependent degradation. It is concluded that the previously observed antitumoral and NF-KappaB inhibiting activity of disulfiram and PDTC could be attributed to their inhibition of the 26S proteasome.

  17. Inhibition of cyclo-oxygenase 2 reduces tumor metastasis and inflammatory signaling during blockade of vascular endothelial growth factor

    Directory of Open Access Journals (Sweden)

    Fisher Jason C

    2011-10-01

    Full Text Available Abstract Vascular endothelial growth factor (VEGF blockade is an effective therapy for human cancer, yet virtually all neoplasms resume primary tumor growth or metastasize during therapy. Mechanisms of progression have been proposed to include genes that control vascular remodeling and are elicited by hypoperfusion, such as the inducible enzyme cyclooxygenase-2 (COX-2. We have previously shown that COX-2 inhibition by the celecoxib analog SC236 attenuates perivascular stromal cell recruitment and tumor growth. We therefore examined the effect of combined SC236 and VEGF blockade, using the metastasizing orthotopic SKNEP1 model of pediatric cancer. Combined treatment perturbed tumor vessel remodeling and macrophage recruitment, but did not further limit primary tumor growth as compared to VEGF blockade alone. However, combining SC236 and VEGF inhibition significantly reduced the incidence of lung metastasis, suggesting a distinct effect on prometastatic mechanisms. We found that SC236 limited tumor cell viability and migration in vitro, with effects enhanced by hypoxia, but did not change tumor proliferation or matrix metalloproteinase expression in vivo. Gene set expression analysis (GSEA indicated that the addition of SC236 to VEGF inhibition significantly reduced expression of gene sets linked to macrophage mobilization. Perivascular recruitment of macrophages induced by VEGF blockade was disrupted in tumors treated with combined VEGF- and COX-2-inhibition. Collectively, these findings suggest that during VEGF blockade COX-2 may restrict metastasis by limiting both prometastatic behaviors in individual tumor cells and mobilization of macrophages to the tumor vasculature.

  18. Up-regulation of insulin-like growth factor 2 by ketamine requires glycogen synthase kinase-3 inhibition.

    Science.gov (United States)

    Grieco, Steven F; Cheng, Yuyan; Eldar-Finkelman, Hagit; Jope, Richard S; Beurel, Eléonore

    2017-01-04

    An antidepressant dose of the rapidly-acting ketamine inhibits glycogen synthase kinase-3 (GSK3) in mouse hippocampus, and this inhibition is required for the antidepressant effect of ketamine in learned helplessness depression-like behavior. Here we report that treatment with an antidepressant dose of ketamine (10mg/kg) increased expression of insulin-like growth factor 2 (IGF2) in mouse hippocampus, an effect that required ketamine-induced inhibition of GSK3. Ketamine also inhibited hippocampal GSK3 and increased expression of hippocampal IGF2 in mice when administered after the induction of learned helplessness. Treatment with the specific GSK3 inhibitor L803-mts was sufficient to up-regulate hippocampal IGF2 expression. Administration of IGF2 siRNA reduced ketamine's antidepressant effect in the learned helplessness paradigm. Mice subjected to the learned helplessness paradigm were separated into two groups, those that were resilient (non-depressed) and those that were susceptible (depressed). Non-depressed resilient mice displayed higher expression of IGF2 than susceptible mice. These results indicate that IGF2 contributes to ketamine's antidepressant effect and that IGF2 may confer resilience to depression-like behavior. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Noradrenergic inhibition of canine gallbladder contraction and murine pancreatic secretion during stress by corticotropin-releasing factor.

    Science.gov (United States)

    Lenz, H J; Messmer, B; Zimmerman, F G

    1992-02-01

    Gastrointestinal secretory and motor responses are profoundly altered during stress; but the effects of stress and its mediator(s) on the two major gut functions, exocrine pancreatic secretion and gallbladder motility, are unknown. We therefore developed two animal models that allowed us to examine the effects of acoustic stress on canine gallbladder contraction and restraint stress on rat exocrine pancreatic secretion. Acoustic stress inhibited cholecystokinin-8 (CCK)- and meal-induced gallbladder contraction, and restraint stress inhibited basal and CCK/secretin-stimulated pancreatic secretion. These inhibitory responses were mimicked by cerebral injection of corticotropin-releasing factor (CRF) and abolished by the CRF antagonist, alpha-helical CRF-(9-41). The effects of stress and exogenous CRF were simulated by intravenous infusion of norepinephrine but prevented by ganglionic, noradrenergic, and alpha-adrenergic but not beta-adrenergic receptor blockade. Vagotomy, adrenalectomy, and--in rats--hypophysectomy did not alter the effects produced by stress and CRF. These results indicate that endogenous CRF released in response to different stressors in distinct species inhibits canine gallbladder contraction and murine exocrine pancreatic secretion via activation of sympathetic efferents. Release of norepinephrine appears to be the final common pathway producing inhibition of biliary and pancreatic digestive function during stress mediated by cerebral CRF.

  20. Carotenoid derivatives inhibit nuclear factor kappa B activity in bone and cancer cells by targeting key thiol groups.

    Science.gov (United States)

    Linnewiel-Hermoni, Karin; Motro, Yair; Miller, Yifat; Levy, Joseph; Sharoni, Yoav

    2014-10-01

    Aberrant activation of the nuclear factor kappa B (NFkB) transcription system contributes to cancer progression, and has a harmful effect on bone health. Several major components of the NFkB pathway such as IkB Kinase (IKK) and the NFkB subunits contain cysteine residues that are critical for their activity. The interaction of electrophiles with these cysteine residues results in NFkB inhibition. Carotenoids, hydrophobic plant pigments, are devoid of electrophilic groups, and we have previously demonstrated that carotenoid derivatives, but not the native compounds activate the Nrf2 transcription system. The aim of the current study was to examine whether carotenoid derivatives inhibit NFkB, and, if so, to determine the molecular mechanism underpinning the inhibitory action. We report in the present study that a mixture of oxidized derivatives, prepared by ethanol extraction from partially oxidized lycopene preparation, inhibited NFkB reporter gene activity. In contrast, the intact carotenoid was inactive. A series of synthetic dialdehyde carotenoid derivatives inhibited reporter activity as well as several stages of the NFkB pathway in both cancer and bone cells. The activity of the carotenoid derivatives depended on the reactivity of the electrophilic groups in reactions such as Michael addition to sulfhydryl groups of proteins. Specifically, carotenoid derivatives directly interacted with two key proteins of the NFkB pathway: the IKKβ and the p65 subunit. Direct interaction with IKKβ was found in an in vitro kinase assay with a recombinant enzyme. The inhibition by carotenoid derivatives of p65 transcriptional activity was observed in a reporter gene assay performed in the presence of excess p65. This inhibition action resulted, at least in part, from direct interaction of the carotenoid derivative with p65 leading to reduced binding of the protein to DNA as evidenced by electrophoretic mobility shift assay (EMSA) experiments. Importantly, we found by using

  1. Infiltration anesthetic lidocaine inhibits cancer cell invasion by modulating ectodomain shedding of heparin-binding epidermal growth factor-like growth factor (HB-EGF).

    Science.gov (United States)

    Mammoto, Tadanori; Higashiyama, Shigeki; Mukai, Mutsuko; Mammoto, Akiko; Ayaki, Masako; Mashimo, Takashi; Hayashi, Yukio; Kishi, Yoshihiko; Nakamura, Hiroyuki; Akedo, Hitoshi

    2002-09-01

    Although the mechanism is unknown, infiltration anesthetics are believed to have membrane-stabilizing action. We report here that such a most commonly used anesthetic, lidocaine, effectively inhibited the invasive ability of human cancer (HT1080, HOS, and RPMI-7951) cells at concentrations used in surgical operations (5-20 mM). Ectodomain shedding of heparin-binding epidermal growth factor-like growth factor (HB-EGF) from the cell surface plays an important role in invasion by HT1080 cells. Lidocaine reduced the invasion ability of these cells by partly inhibiting the shedding of HB-EGF from the cell surface and modulation of intracellular Ca2+ concentration contributed to this action. The anesthetic action of lidocaine (sodium channel blocking ability) did not contribute to this anti-invasive action. In addition, lidocaine (5-30 mM), infiltrated around the inoculation site, inhibited pulmonary metastases of murine osteosarcoma (LM 8) cells in vivo. These data point to previously unrecognized beneficial actions of lidocaine and suggest that lidocaine might be an ideal infiltration anesthetic for surgical cancer operations.

  2. Inhibition of corneal neovascularization with new Tyrosine Kinase Inhibitors targeting vascular endothelial growth factor receptors: Sunitinib malate and Sorafenib

    Directory of Open Access Journals (Sweden)

    Delnia Arshadi

    2007-06-01

    Full Text Available Corneal neovascularization (NV is a significant, sight-threatening, complication of many ocular surface disorders. Presence of new vessels in cornea can compromise clarity and thus vision. The data supporting a causal role for vascular endothelial growth factor (VEGF in corneal NV are extensive. Inhibition of VEGF remains as a main strategy for treating corneal NV. There is a growing body of evidence that corneal NV can be reduced by using anti-VEGF agents. Sunitinib malate and Sorafenib are new orally bio-available anti-angiogenic agents undergoing tests of efficacy in the treatment of various types of cancers. The main mechanism of these drugs is inhibiting angiogenesis by diminishing signaling through VEGF receptor1 (VEGFR1, VEGFR2, and platelet-derived growth factor receptors. Since VEGF exerts its angiogenic effects through tyrosine kinase receptors in cornea, any mechanisms which reduce VEGF signaling may inhibit corneal NV or at least attenuate it. Based on this fact we herein hypothesize that Sunitinib malate and Sorafenib can be prepared in topical form and be used in corneal neovascularization states. These approaches offer new hope for the successful treatment of corneal NV. Further investigations in animal models are needed to place these two drugs alongside corneal NV therapeutics.

  3. Salvianic acid A inhibits induction of inflammatory mediators by blocking Nuclear Factor-kB activation in macrophages

    Institute of Scientific and Technical Information of China (English)

    YUAN Jun; YAO Ji-hong; ZHOU Qin

    2008-01-01

    Objective To investigate the anti-inflammation effect and possible mechanism of Salvianic acid A (SAA) in mouse peritoneal macrophages. Methods Peritoneal macrophages were obtained from BALB/c mice. LPS induced nitric oxide (NO), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in supernatant, protein expression of inducible nitric oxide synthase (iNOS), matrix metalloproteinase-9 (MMP-9) and activation of nuclear factor-kappa B (NF-kB) in the extract were measured. Results SAA strongly inhibited the excessive production of NO, TNF-α and IL-6 in LPS-induced peritoneal macrophages in a concentration-dependent manner and blocked the expression of iNOS and MMP-9. Treatment with LPS alone increased the translocation of NF-kB (1065) from cytosol to the nucleus, but the SAA inhibited the translocation of NF-kB (p65). Conclusions The results showed that SAA had strong anti-inflammatory effects in LPS-stimulated peritoneal macrophages. The important mechanism is due to its inhibition of NF-kB activation.

  4. Evaluation of the ability of carprofen and flunixin meglumine to inhibit activation of nuclear factor kappa B.

    Science.gov (United States)

    Bryant, Clare E; Farnfield, Belinda A; Janicke, Heidi J

    2003-02-01

    To determine whether the nonsteroidal anti-inflammatory drugs (NSAIDs) carprofen, flunixin meglumine, and phenylbutazone have cyclooxygenase (COX)-independent effects that specifically inhibit activation of the proinflammatory transcription factor nuclear factor kappa B (NfkappaB). Purified ovine COX-1 and -2 and cultures of RAW 264.7 murine macrophages. The COX-1 and -2 inhibitory effects of the NSAIDs were tested in assays that used purified ovine COX-1 and -2. Prostaglandin production was analyzed by use of a radioimmunoassay. Inhibitory effects of these drugs on lipopolysaccharide (LPS)-induction of inducible nitric oxide synthase (iNOS) and LPS-stimulated translocation of NficB were determined by use of RAW 264.7 murine macrophages. Flunixin meglumine and phenylbutazone were selective inhibitors of COX-1. Carprofen and flunixin meglumine, but not phenylbutazone, inhibited LPS-induction of iNOS. Carprofen and, to a lesser degree, flunixin meglumine had inhibitory effects on NFkappaB activation. The ability of drugs such as carprofen and flunixin meglumine to inhibit activation of NfkappaB-dependent genes such as iNOS, in addition to their effects on COX, suggests an additional mechanism for their anti-inflammatory effects and may explain the ability of flunixin meglumine to be an effective inhibitor of the effects of endotoxin in horses with endotoxemia.

  5. Mechanism of Hepatocyte Growth Factor Inhibition of Angiotensin II-induced Apoptosis in Primary Lung Cells

    Science.gov (United States)

    2010-02-19

    et al., Inhibition of amiodarone-induced lung fibrosis but not alveolitis by angiotensin system antagonists. Pharmacol Toxicol, 2003. 92(2): p. 81...670-5. 46. Hubbard, R., et al., Occupational exposure to metal or wood dust and aetiology of cryptogenic fibrosing alveolitis . Lancet, 1996. 347(8997...1): p. 242-8. 48. Hubbard, R., et al., Exposure to commonly prescribed drugs and the etiology of cryptogenic fibrosing alveolitis : a case-control

  6. Identification of factors in human urine that inhibit the binding of Escherichia coli adhesins.

    OpenAIRE

    1988-01-01

    Earlier studies on the binding of Escherichia coli adhesins to the human urinary tract have indicated that the ability to recognize binding sites on the urinary tract epithelial cells is not a characteristic for P fimbriae only, but is also shared by some other adhesins that are not associated with pyelonephritis, especially S fimbriae. In the present study we have investigated whether human urine contains inhibitors of the binding of E. coli adhesins. Normal human urine was found to inhibit ...

  7. Agents described in the Molecular Imaging and Contrast Agent Database for imaging carbonic anhydrase IX expression.

    Science.gov (United States)

    Sneddon, Deborah; Poulsen, Sally-Ann

    2014-10-01

    Carbonic anhydrase IX (CA IX) is selectively expressed in a range of hypoxic tumours and is a validated endogenous hypoxia marker with prognostic significance; hence, CA IX is of great interest as a molecular imaging target in oncology. In this review, we present an overview of the different imaging agents and imaging modalities that have been applied for the in vivo detection of CA IX. The imaging agents reviewed are all entries in the Molecular Imaging and Contrast Agent Database (MICAD) and comprise antibody, antibody fragments and small molecule imaging agents. The effectiveness of these agents for imaging CA IX in vivo gave variable performance; however, a number of agents proved very capable. As molecular imaging has become indispensable in current medical practice we anticipate that the clinical significance of CA IX will see continued development and improvements in imaging agents for targeting this enzyme.

  8. Adenovirus protein IX sequesters host-cell promyelocytic leukaemia protein and contributes to efficient viral proliferation.

    Science.gov (United States)

    Rosa-Calatrava, Manuel; Puvion-Dutilleul, Francine; Lutz, Pierre; Dreyer, Dominique; de Thé, Hugues; Chatton, Bruno; Kedinger, Claude

    2003-10-01

    The product of adenovirus type 5 (Ad5) gene IX, protein IX (pIX), is a multifunctional protein that stabilizes the viral capsid and has transcriptional activity. We show that pIX also contributes to the Ad5-induced reorganization of the host-cell nuclear ultrastructure: pIX induces the formation of specific and dynamic nuclear inclusions, and the host promyelocytic leukaemia (PML) protein, which is the main structural organizer of PML bodies, is stably relocated and confined within the pIX-induced inclusions late in infection. Our results suggest that Ad5 has evolved a unique strategy that leads to the sustained neutralization of PML bodies throughout infection, thereby ensuring optimal viral proliferation.

  9. Generation and characterization of small single domain antibodies inhibiting human tumor necrosis factor receptor 1.

    Science.gov (United States)

    Steeland, Sophie; Puimège, Leen; Vandenbroucke, Roosmarijn E; Van Hauwermeiren, Filip; Haustraete, Jurgen; Devoogdt, Nick; Hulpiau, Paco; Leroux-Roels, Geert; Laukens, Debby; Meuleman, Philip; De Vos, Martine; Libert, Claude

    2015-02-13

    The cytokine TNF is a well known drug target for several inflammatory diseases such as Crohn disease. Despite the great success of TNF blockers, therapy could be improved because of high costs and side effects. Selective inhibition of TNF receptor (TNFR) 1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, thereby preserving the advantageous immunomodulatory signals mediated by TNFR2. We generated a selective human TNFR1 inhibitor based on Nanobody (Nb) technology. Two anti-human TNFR1 Nbs were linked with an anti-albumin Nb to generate Nb Alb-70-96 named "TNF Receptor-One Silencer" (TROS). TROS selectively binds and inhibits TNF/TNFR1 and lymphotoxin-α/TNFR1 signaling with good affinity and IC50 values, both of which are in the nanomolar range. Surface plasmon resonance analysis reveals that TROS competes with TNF for binding to human TNFR1. In HEK293T cells, TROS strongly reduces TNF-induced gene expression, like IL8 and TNF, in a dose-dependent manner; and in ex vivo cultured colon biopsies of CD patients, TROS inhibits inflammation. Finally, in liver chimeric humanized mice, TROS antagonizes inflammation in a model of acute TNF-induced liver inflammation, reflected in reduced human IL8 expression in liver and reduced IL6 levels in serum. These results demonstrate the considerable potential of TROS and justify the evaluation of TROS in relevant disease animal models of both acute and chronic inflammation and eventually in patients.

  10. Quantitative PET Imaging of Tissue Factor Expression Using 18F-labled Active Site Inhibited Factor VII

    DEFF Research Database (Denmark)

    Nielsen, Carsten H; Erlandsson, Maria; Jeppesen, Troels E

    2016-01-01

    after the last imaging session, and tumor tissue was preserved for molecular analysis. A blocking experiment was performed in a second set of mice. The expression pattern of TF in the tumors was visualized by immunohistochemistry and the amount of TF in tumor homogenates was measured by ELISA......Tissue factor (TF) is up regulated in many solid tumors and its expression is linked to tumor angiogenesis, invasion, metastasis and prognosis. A non-invasive assessment of tumor TF expression status is therefore of obvious clinical relevance. Factor VII (FVII) is the natural ligand to TF. Here we...

  11. Endothelins Inhibit Osmotic Swelling of Rat Retinal Glial and Bipolar Cells by Activation of Growth Factor Signaling.

    Science.gov (United States)

    Vogler, Stefanie; Grosche, Antje; Pannicke, Thomas; Wiedemann, Peter; Reichenbach, Andreas; Bringmann, Andreas

    2016-10-01

    Water accumulation in retinal glial (Müller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Here, we show that endothelin-1 (ET-1) dose-dependently inhibits the hypoosmotic swelling of Müller cells in freshly isolated retinal slices of control and diabetic rats, with a maximal inhibition at 100 nM. Osmotic Müller cell swelling was also inhibited by ET-2. The effect of ET-1 was mediated by activation of ETA and ETB receptors resulting in transactivation of metabotropic glutamate receptors, purinergic P2Y1, and adenosine A1 receptors. ET-1 (but not ET-2) also inhibited the osmotic swelling of bipolar cells in retinal slices, but failed to inhibit the swelling of freshly isolated bipolar cells. The inhibitory effect of ET-1 on the bipolar cell swelling in retinal slices was abrogated by inhibitors of the FGF receptor kinase (PD173074) and of TGF-β1 superfamily activin receptor-like kinase receptors (SB431542), respectively. Both Müller and bipolar cells displayed immunoreactivities of ETA and ETB receptor proteins. The data may suggest that neuroprotective effects of ETs in the retina are in part mediated by prevention of the cytotoxic swelling of retinal glial and bipolar cells. ET-1 acts directly on Müller cells, while the inhibitory effect of ET-1 on bipolar cell swelling is indirectly mediated, via stimulation of the release of growth factors like bFGF and TGF-β1 from Müller cells.

  12. Inhibition of iodine-125-labeled human follitropin binding to testicular receptor by epidermal growth factor and synthetic peptides

    Energy Technology Data Exchange (ETDEWEB)

    Sluss, P.M.; Krystek, S.R. Jr.; Andersen, T.T.; Melson, B.E.; Huston, J.S.; Ridge, R.; Reichert, L.E. Jr.

    1986-05-06

    Two tetrapeptide sequence homologies between mouse epidermal growth factor precursor (mEGFP) and human follitropin (FSH) were revealed by a computer program that identifies identical residues among polypeptide sequences. The two tetrapeptides, Lys-Thr-Cys-Thr (KTCT) and Thr-Arg-Asp-Leu (TRDL), are present in the hormone-specific beta subunit of FSH from all species studied. These tetrapeptides are not present in the alpha subunit, which is common to all pituitary glycoprotein hormones. Both tetrapeptides are also found in mEGFP, and one tetrapeptide, TRDL, is located within the 53-residue form of mEGF purified from mouse submaxillary glands. Computer-generated hydropathy profiles predicted that both tetrapeptides are located in hydrophilic portions of the FSH beta subunit and that TRDL is in a hydrophilic portion of commercially available mEGF. Therefore, the tetrapeptides might be accessible to receptor binding sites for FSH. We report that mEGF inhibits binding of /sup 125/I-labeled human FSH to receptors in testis by 50% (I50) at a concentration of 1.8 X 10(-5) M. No binding inhibition was observed by GnRH or arginine-vasopressin at 10(-4) M, neither of which contain the tetrapeptide sequences. FSH beta subunit, which contains both tetrapeptides, also inhibited binding (I50 = 9 X 10(-8) M) of /sup 125/I-labeled human FSH to testis receptor. Thus, it appears that FSH beta subunit and mEGF are capable of inhibiting binding of FSH to testicular FSH receptors, presumably through interactions that include the homologous tetrapeptides. This presumption was supported by the observation that the synthetic tetrapeptides (KTCT or TRDL) were also active in inhibiting binding of /sup 125/I-labeled human FSH to testis receptor.

  13. Amphypterygium adstringens anacardic acid mixture inhibits quorum sensing-controlled virulence factors of Chromobacterium violaceum and Pseudomonas aeruginosa.

    Science.gov (United States)

    Castillo-Juárez, Israel; García-Contreras, Rodolfo; Velázquez-Guadarrama, Norma; Soto-Hernández, Marcos; Martínez-Vázquez, Mariano

    2013-10-01

    Quorum sensing (QS) is a process of bacterial cell-cell communication that controls a large number of systems affecting pathogenicity. Interrupting this communication system can provide nonvirulent pathogenic bacteria. The aim of this study was to evaluate the anti-quorum sensing (anti-QS) potential of an anacardic acids mixture isolated from Amphipterygium adstringens, a medicinal plant known as "cuachalalate", to prevent the onset of bacterial infections as an alternate to antibiotics. Initially we investigated the anti-QS activity of A. adstringens hexane extract (HE) by the inhibition of violacein production in Chromobacterium violaceum. From the active HE, an anacardic acid mixture (AAM) was obtained. The anti-quorum sensing activity of AAM was investigated by the rhamnolipid and pyocyanin production constraint as well as decrease of elastase activity, all being quorum sensing-controlled virulence factors expressed in the pathogenic bacteria Pseudomonas aeruginosa. HE induced a 91.6% of inhibition of the violecin production at 55 μg/mL concentration, whereas AAM showed 94% of inhibition at 166 μg/mL. In both cases, inhibition of violacein production did not affect the viability of the bacterium. AAM inhibited pyocyanin (86% at 200 μg/mL) and rhamnolipid (91% at 500 μg/mL) production in a dose/response form and decrease the elastase (75% at 500 μg/mL) activity in P. aeruginosa without affecting its development. Because an anacardic acids mixture isolated from A. adstringens demonstrated anti-QS, it could be further exploited for novel molecules to treat the emerging infections of antibiotic-resistant bacterial pathogens. Copyright © 2013 IMSS. Published by Elsevier Inc. All rights reserved.

  14. The Tiam1 Guanine Nucleotide Exchange Factor is Auto-inhibited by its Pleckstrin Homology Coiled-Coil Extension Domain.

    Science.gov (United States)

    Xu, Zhen; Gakhar, Lokesh; Bain, Fletcher E; Spies, Maria; Fuentes, Ernesto J

    2017-09-07

    T-cell lymphoma invasion and metastasis 1 (Tiam1) is a Dbl-family guanine nucleotide exchange factor (GEF) that specifically activates the Rho-family GTPase Rac1 in response to upstream signals, thereby regulating cellular processes including cell adhesion and migration. Tiam1 contains multiple domains, including an N-terminal Pleckstrin homology coiled-coiled extension (PHn-CC-Ex) and catalytic Dbl-homology and C-terminal Pleckstrin homology (DH-PHc) domain. Previous studies indicate that larger fragments of Tiam1, such the region encompassing the N-terminal to C-terminal Pleckstrin homology domains (PHn-PHc) are auto-inhibited. However, the domains in this region responsible for inhibition remain unknown. Here, we show that the PHn-CC-Ex domain inhibits Tiam1 GEF activity by directly interacting with the catalytic DH-PHc domain, preventing Rac1 binding and activation. Enzyme kinetics experiments suggested that Tiam1 is auto-inhibited through occlusion of the catalytic site, rather than by allostery. Small angle X-ray scattering and ensemble modeling yielded models of the PHn-PHc fragment that indicate it is in equilibrium between open and closed conformational states. Finally, single-molecule experiments support a model in which conformational sampling between the open and closed states of Tiam1 contributes to Rac1 dissociation. Our results highlight the role of the PHn-CC-Ex domain in Tiam1 GEF regulation, and suggest a combinatorial model for GEF inhibition and activation of the Rac1 signaling pathway. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  15. Thiopental inhibits global protein synthesis by repression of eukaryotic elongation factor 2 and protects from hypoxic neuronal cell death.

    Directory of Open Access Journals (Sweden)

    Christian I Schwer

    Full Text Available Ischemic and traumatic brain injury is associated with increased risk for death and disability. The inhibition of penumbral tissue damage has been recognized as a target for therapeutic intervention, because cellular injury evolves progressively upon ATP-depletion and loss of ion homeostasis. In patients, thiopental is used to treat refractory intracranial hypertension by reducing intracranial pressure and cerebral metabolic demands; however, therapeutic benefits of thiopental-treatment are controversially discussed. In the present study we identified fundamental neuroprotective molecular mechanisms mediated by thiopental. Here we show that thiopental inhibits global protein synthesis, which preserves the intracellular energy metabolite content in oxygen-deprived human neuronal SK-N-SH cells or primary mouse cortical neurons and thus ameliorates hypoxic cell damage. Sensitivity to hypoxic damage was restored by pharmacologic repression of eukaryotic elongation factor 2 kinase. Translational inhibition was mediated by calcium influx, activation of the AMP-activated protein kinase, and inhibitory phosphorylation of eukaryotic elongation factor 2. Our results explain the reduction of cerebral metabolic demands during thiopental treatment. Cycloheximide also protected neurons from hypoxic cell death, indicating that translational inhibitors may generally reduce secondary brain injury. In conclusion our study demonstrates that therapeutic inhibition of global protein synthesis protects neurons from hypoxic damage by preserving energy balance in oxygen-deprived cells. Molecular evidence for thiopental-mediated neuroprotection favours a positive clinical evaluation of barbiturate treatment. The chemical structure of thiopental could represent a pharmacologically relevant scaffold for the development of new organ-protective compounds to ameliorate tissue damage when oxygen availability is limited.

  16. Resveratrol attenuates renal injury and fibrosis by inhibiting transforming growth factor-β pathway on matrix metalloproteinase 7.

    Science.gov (United States)

    Xiao, Zhou; Chen, Chen; Meng, Ting; Zhang, Wenzheng; Zhou, Qiaoling

    2016-01-01

    Renal injury has a strong relationship to the subsequent development of renal fibrosis. In developing renal fibrosis, tubular epithelial cells in the kidney underwent epithelial-mesenchymal transition (EMT). Matrix metalloproteinase 7 (MMP7) was reported to reduce E-cadherin and induce EMT by up-regulation of β-catenin/lymphoid enhancer-binding factor 1 (LEF1) signaling. In this research, we tried to evaluate the role of resveratrol (RSV) on EMT process in renal injury and fibrosis. Human tubular epithelial cell HK-2 cells were treated with aristolochic acid (AAs) and transforming growth factor-β(TGF-β) to induce EMT with or without the administration of RSV. The inhibitory role of RSV on EMT in renal injury and fibrosis was determined by Western blotting, real-time PCR, and immunofluorescence staining. The EMT repressing role of RSV was also evaluated in vivo by renal ischemia-reperfusion (I/R) injury and unilateral ureteral obstruction (UUO) models. The underlying mechanism was investigated by shRNA interfering MMP7 and sirtuin 1 (SIRT1) expression. The results indicated that RSV reversed human kidney 2 (HK-2) cell EMT, renal I/R injury, and renal fibrosis. MMP7 inhibition was responsible for RSV-induced EMT repression. SIRT1 was up-regulated by RSV inhibited TGF-β pathway on MMP7 via deacetylating Smad4. In conclusion, RSV attenuated renal injury and fibrosis by inhibiting EMT process which was attributed to the fact that the up-regulated SIRT1 by RSV deacetylated Smad4 and inhibited MMP7 expression.

  17. Identification of small peptides inhibiting the integrase-LEDGF/p75 interaction through targeting the cellular co-factor.

    Science.gov (United States)

    Cavalluzzo, Claudia; Christ, Frauke; Voet, Arnout; Sharma, Ajendra; Singh, Brajendra Kumar; Zhang, Kam Y J; Lescrinier, Eveline; De Maeyer, Marc; Debyser, Zeger; Van der Eycken, Erik

    2013-10-01

    The integration of the viral DNA into the host genome is one of the essential steps in the HIV replication cycle. This process is mediated by the viral enzyme integrase (IN) and lens epithelium-derived growth factor (LEDGF/p75). LEDGF/p75 has been identified as a crucial cellular co-factor of integration that acts by tethering IN to the cellular chromatin. Recently, circular peptides were identified that bind to the C-terminal domain of IN and disrupt the interaction with LEDGF/p75. Starting from the circular peptides, we identified a short peptidic sequence able to inhibit the LEDGF/p75-IN interaction at low μM concentration through its binding to the IN binding site of LEDGF/p75. This discovery can lead to the synthesis of peptidomimetics with high anti-HIV activity targeting the cellular co-factor LEDGF/p75 and not the viral protein IN.

  18. PET Imaging of Tissue Factor in Pancreatic Cancer Using 64Cu-Labeled Active Site-Inhibited Factor VII

    DEFF Research Database (Denmark)

    Nielsen, Carsten H; Jeppesen, Troels E; Kristensen, Lotte K

    2016-01-01

    with advanced stage, increased microvessel density, metastasis, and poor overall survival. Imaging of TF expression is of clinical relevance as a prognostic biomarker and as a companion diagnostic for TF-directed therapies currently under clinical development. Factor VII (FVII) is the natural ligand to TF...

  19. Distinct domains of M-T2, the myxoma virus tumor necrosis factor (TNF) receptor homolog, mediate extracellular TNF binding and intracellular apoptosis inhibition.

    OpenAIRE

    Schreiber, M; Sedger, L; McFadden, G

    1997-01-01

    The myxoma virus tumor necrosis factor (TNF) receptor homolog, M-T2, is expressed both as a secreted glycoprotein that inhibits the cytolytic activity of rabbit TNF-alpha and as an endoglycosidase H-sensitive intracellular species that prevents myxoma virus-infected CD4+ T lymphocytes from undergoing apoptosis. To compare the domains of M-T2 mediating extracellular TNF inhibition and intracellular apoptosis inhibition, recombinant myxoma viruses expressing nested C-terminal truncations of M-T...

  20. The NC2 domain of type IX collagen determines the chain register of the triple helix.

    Science.gov (United States)

    Boudko, Sergei P; Bächinger, Hans Peter

    2012-12-28

    Precise mapping and unraveling the mechanism of interaction or degradation of a certain type of collagen triple helix requires the generation of short and stable collagenous fragments. This is a great challenge especially for hetero-trimeric collagens, where chain composition and register (stagger) are important factors. No system has been reported that can be efficiently used to generate a natural collagenous fragment with exact chain composition and desired chain register. The NC2 domain (only 35-50 residues) of FACIT collagens is a potent trimerization domain. In the case of type IX collagen it provides the efficient selection and hetero-trimerization of three distinct chains. The ability of the NC2 domain to determine the chain register of the triple helix is studied. We generated three possible sequence combinations (α1α1α2, α1α2α1, α2α1α1) of a type I collagen fragment (the binding region for the von Willebrand factor A3 domain) attached to the NC2 domain. In addition, two control combinations were produced that constitute homo-trimers of (α1)(3) or (α2)(3). For the hetero-trimeric constructs, α1α1α2 demonstrated a higher melting temperature than the other two. Binding experiments with the von Willebrand factor A3 domain revealed the homo-trimer of (α1)(3) as the strongest binding construct, whereas the homo-trimer of (α2)(3) showed no binding. For hetero-trimers, α1α1α2 was found to be the strongest binding construct. Differences in thermal stability and binding to the A3 domain unambiguously demonstrate that the NC2 domain of type IX collagen determines not only the chain composition but also the chain register of the adjacent triple helix.

  1. Evaluation of PpIX formation in Cervical Intraepithelial Neoplasia I (CIN) using widefield fluorescence images

    Science.gov (United States)

    Carbinatto, Fernanda M.; Inada, Natalia M.; Fortunato, Thereza C.; Lombardi, Welington; da Silva, Eduardo V.; Vollet Filho, José D.; Kurachi, Cristina; Pratavieira, Sebastião.; Bagnato, Vanderlei S.

    2016-03-01

    Optical techniques has been described as auxiliary technology for screening of neoplasia because shows the potential for tissues differentiation in real-time and it is a noninvasive detection and safe. However, only endogenous fluorophores presents the lesion may be insufficient and needed of the administration of the fluorophores synthesized, such as, precursor molecule of protoporphyrin IX (PpIX) induced by 5- aminolevulinic acid and your derivatives. Topical application of methylaminolevulinate (MAL), induces formation of the endogenous photosensitizer, PpIX in tissues where carcinogenesis has begun. The PpIX tend to accumulate in premalignant and malignant tissues and the illumination with light with appropriate wavelength beginning to excitation of PpIX fluorescence, which helps to localize PpIX-rich areas and identify potentially malignant tissues. The aim of the study is to evaluate the production of PpIX in the cervix with CIN I through of the fluorescence images captured after 1 hour of cream application. It was possible to visualize PpIX fluorescence in cervix and it was possible to observe the selectivity in fluorescence in squamous-columnar junction, which a pre-cancerous condition (CIN) and usually is localized. Through the image processing it was possible to quantify the increase of red fluorescence. For the CIN I the increase of red fluorescence was approximately of 4 times indicating a good PpIX formation.

  2. Enhanced cellular uptake of protoporphyrine IX/linolenic acid-conjugated spherical nanohybrids for photodynamic therapy.

    Science.gov (United States)

    Lee, Hye-In; Kim, Young-Jin

    2016-06-01

    Protoporphyrin IX (PpIX) has wide applications in photodynamic diagnosis and photodynamic therapy (PDT) in many human diseases. However, poor water solubility and cancer cell localization limit its direct application for PDT. We improved the water-solubility and cellular internalization of PpIX to enhance PDT efficacy by developing biocompatible PpIX/linolenic acid-conjugated polyhedral oligomeric silsesquioxane (PPLA) nanohybrids. The resulting PPLA nanohybrids exhibited a quasi-spherical shape with a size of gastric cancer cells. These results imply that the PPLA nanohybrid system may be applicable in PDT.

  3. Interleukin-10 inhibits the expression of nuclear factor kappa B in rats with cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Zhihua Wu; Nan Liu; Houwei Du; Ronghua Chen; An Zheng; Huapin Huang

    2006-01-01

    BACKGROUND: Plenty of studies have demonstrated that inflammatory reaction is involved in ischemic cerebral damage, and the expression of inflammatory cytokines can be observed at the initial sites of cerebral damage at early period, including interleukin-6, interleukin-8, etc., which are all the target gene products of nuclear factor kappa B (NF-кB). The process of ischemic damage can be affected by adjusting and controlling NF-кB activity via multi-links.OBJECTIVE: To investigate the inhibitory effect of interleukin-10 on the expression of NF-кB in the ischemic sites of rats with focal cerebral ischemia in rats and its molecular mechanisms.DESIGN: A randomized and controlled animal trial.SETTING: Department of Neurology, the Affiliated Union Hospital of Fujian Medical University.MATERIALS: Thirty-two adult male Sprague-Dawley rats weighing (250±30) g were used. NF-кB p65 (RelA)rabbit anti-rat monoclonal primary antibody was the product of Neomarkers Company; Immunohistochemical kit of the SP two-step method was purchased from Beijing Zhongshan Biotechnology Co., Ltd.METHODS: The experiment was carried out in the Affiliated Union Hospital of Fujian Medical University from August 2005 to April 2006. The rats were randomly assigned into sham-operated group, middle cerebral artery occlusion (MCAO) group, vehicle-treated group and interleukin-10 treated group, 8 rats in each group. Focal cerebral ischemia was induced by occlusion of the middle cerebral artery as previously described. Rats in the MCAO group were anesthetized intraperitoneally, thyroid was bluntly dissected. Right common, external and internal carotid arteries were isolated, the trunk of external carotid artery was ligated and freed, an artery clamp was placed at the internal carotid artery, then a "V" shape incision was made at the free section of external carotid artery, filament was inserted for a depth of (18.5±0.5) mm. The rats in the sham-operated group were given the same treatments with

  4. A RNA antagonist of hypoxia-inducible factor-1alpha, EZN-2968, inhibits tumor cell growth

    DEFF Research Database (Denmark)

    Greenberger, Lee M; Horak, Ivan D; Filpula, David

    2008-01-01

    Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays a critical role in angiogenesis, survival, metastasis, drug resistance, and glucose metabolism. Elevated expression of the alpha-subunit of HIF-1 (HIF-1alpha), which occurs in response to hypoxia or activation of growth facto...

  5. Lignans from Saururus chinensis inhibiting the transcription factor NF-kappaB.

    Science.gov (United States)

    Hwang, Bang Yeon; Lee, Jeong Hyung; Nam, Jeong Bum; Hong, Young Soo; Lee, Jung Joon

    2003-10-01

    The sesquineolignans, saucerneol D and saucerneol E were isolated from the roots of Saururus chinensis together with four known lignans, manassantin A, manassantin B, (-)-saucerneol methyl ether, and (+)-saucernetin. Structure elucidation was based on the analysis of spectroscopic data and anti-inflammatory activity was studied using HeLa cells transfected with NF-kappaB reporter construct. All compounds except for (+)-saucernetin inhibited NF-kappaB dependent reporter gene expression with IC50 values of 2.5-16.9 microM.

  6. Ghrelin inhibits the development of acute pancreatitis and nuclear factor kappaB activation in pancreas and liver.

    Science.gov (United States)

    Zhou, Xiaolei; Xue, Chengrui

    2009-10-01

    To investigate the influence of ghrelin on the development of severe acute pancreatitis (SAP) and the expression of nuclear factor kappaB (NF-kappaB) p65 in the pancreas and liver. Severe acute pancreatitis was induced in rat by sodium taurocholate injection in the pancreaticobiliary duct. Ghrelin was administrated twice at the dose 10 or 20 nmol/kg per injection, respectively. Then, serum amylase activity; serum tumor necrosis factor alpha, interleukin 1beta, and interleukin 6 concentrations; and morphological signs of pancreatitis and hepatic damage were measured. Meanwhile, determination of pancreatic and hepatic NF-kappaB p65 expression was performed by Western blotting and immunohistochemistry. The serumal parameters increased, and morphological damages were observed in the pancreas and liver in SAP rats. Nuclear factor kappaB p65 expression was significantly higher in the pancreas and liver than sham-operated rats (P pancreas and liver. Ghrelin inhibits the development of acute pancreatitis induced by sodium taurocholate. It exerts the therapeutic effects through inhibiting NF-kappaB expression, thereby blocks the inflammatory signal transduction pathway and reduces the release of inflammatory media and cytokines.

  7. Yersinia virulence factor YopJ acts as a deubiquitinase to inhibit NF-κB activation

    Science.gov (United States)

    Zhou, Honglin; Monack, Denise M.; Kayagaki, Nobuhiko; Wertz, Ingrid; Yin, Jianpin; Wolf, Beni; Dixit, Vishva M.

    2005-01-01

    The bacterial pathogens of the genus Yersinia, the causative agents of plague, septicemia, and gastrointestinal syndromes, use a type III secretion system to inject virulence factors into host target cells. One virulence factor, YopJ, is essential for the death of infected macrophages and can block host proinflammatory responses by inhibiting both the nuclear factor κB (NF-κB) and mitogen-activated protein kinase pathways, which might be important for evasion of the host immune response and aid in establishing a systemic infection. Here, we show that YopJ is a promiscuous deubiquitinating enzyme that negatively regulates signaling by removing ubiquitin moieties from critical proteins, such as TRAF2, TRAF6, and IκBα. In contrast to the cylindromatosis tumor suppressor CYLD, which attenuates NF-κB signaling by selectively removing K63-linked polyubiquitin chains that activate IκB kinase, YopJ also cleaves K48-linked chains and thereby inhibits proteasomal degradation of IκBα. YopJ, but not a catalytically inactive YopJ mutant, promoted deubiquitination of cellular proteins and cleaved both K48- and K63-linked polyubiquitin. Moreover, an in vitro assay was established to demonstrate directly the deubiquitinating activity of purified YopJ. PMID:16301742

  8. The enteropathogenic E. coli (EPEC Tir effector inhibits NF-κB activity by targeting TNFα receptor-associated factors.

    Directory of Open Access Journals (Sweden)

    Marie-Hélène Ruchaud-Sparagano

    2011-12-01

    Full Text Available Enteropathogenic Escherichia coli (EPEC disease depends on the transfer of effector proteins into epithelia lining the human small intestine. EPEC E2348/69 has at least 20 effector genes of which six are located with the effector-delivery system genes on the Locus of Enterocyte Effacement (LEE Pathogenicity Island. Our previous work implied that non-LEE-encoded (Nle effectors possess functions that inhibit epithelial anti-microbial and inflammation-inducing responses by blocking NF-κB transcription factor activity. Indeed, screens by us and others have identified novel inhibitory mechanisms for NleC and NleH, with key co-operative functions for NleB1 and NleE1. Here, we demonstrate that the LEE-encoded Translocated-intimin receptor (Tir effector has a potent and specific ability to inhibit NF-κB activation. Indeed, biochemical, imaging and immunoprecipitation studies reveal a novel inhibitory mechanism whereby Tir interaction with cytoplasm-located TNFα receptor-associated factor (TRAF adaptor proteins induces their proteasomal-independent degradation. Infection studies support this Tir-TRAF relationship but reveal that Tir, like NleC and NleH, has a non-essential contribution in EPEC's NF-κB inhibitory capacity linked to Tir's activity being suppressed by undefined EPEC factors. Infections in a disease-relevant intestinal model confirm key NF-κB inhibitory roles for the NleB1/NleE1 effectors, with other studies providing insights on host targets. The work not only reveals a second Intimin-independent property for Tir and a novel EPEC effector-mediated NF-κB inhibitory mechanism but also lends itself to speculations on the evolution of EPEC's capacity to inhibit NF-κB function.

  9. Pharmacological inhibition of microsomal prostaglandin E synthase-1 suppresses epidermal growth factor receptor-mediated tumor growth and angiogenesis.

    Directory of Open Access Journals (Sweden)

    Federica Finetti

    Full Text Available BACKGROUND: Blockade of Prostaglandin (PG E(2 production via deletion of microsomal Prostaglandin E synthase-1 (mPGES-1 gene reduces tumor cell proliferation in vitro and in vivo on xenograft tumors. So far the therapeutic potential of the pharmacological inhibition of mPGES-1 has not been elucidated. PGE(2 promotes epithelial tumor progression via multiple signaling pathways including the epidermal growth factor receptor (EGFR signaling pathway. METHODOLOGY/PRINCIPAL FINDINGS: Here we evaluated the antitumor activity of AF3485, a compound of a novel family of human mPGES-1 inhibitors, in vitro and in vivo, in mice bearing human A431 xenografts overexpressing EGFR. Treatment of the human cell line A431 with interleukin-1beta (IL-1β increased mPGES-1 expression, PGE(2 production and induced EGFR phosphorylation, and vascular endothelial growth factor (VEGF and fibroblast growth factor-2 (FGF-2 expression. AF3485 reduced PGE(2 production, both in quiescent and in cells stimulated by IL-1β. AF3485 abolished IL-1β-induced activation of the EGFR, decreasing VEGF and FGF-2 expression, and tumor-mediated endothelial tube formation. In vivo, in A431 xenograft, AF3485, administered sub-chronically, decreased tumor growth, an effect related to inhibition of EGFR signalling, and to tumor microvessel rarefaction. In fact, we observed a decrease of EGFR phosphorylation, and VEGF and FGF-2 expression in tumours explanted from treated mice. CONCLUSION: Our work demonstrates that the pharmacological inhibition of mPGES-1 reduces squamous carcinoma growth by suppressing PGE(2 mediated-EGFR signalling and by impairing tumor associated angiogenesis. These results underscore the potential of mPGES-1 inhibitors as agents capable of controlling tumor growth.

  10. Using recombinant CD74 protein to inhibit the activity of macrophage migration inhibitory factor (MIF) in vitro

    Institute of Scientific and Technical Information of China (English)

    Zhi-xinSHAN; Xi-yongYU; Qiu-xiongLIN; Yong-hengFU

    2005-01-01

    AIM Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine involved in the pathogenesis of a variety of autoimmune and inflammatory diseases, including arthritis, glomerulonephritis, Gram-positive and Gram-negative sepsis, and atherogenesis. Recent studies showed that CD74(antigen-associated invariant chain Ⅱ) is a high-affinity membrane-binding protein for MIF. The purpose of the present study was to express the recombinant human CD74 in E. coli and inhibit the activity of MIF by using recombinant CD74 in vitro.

  11. Azadirachtin interacts with the tumor necrosis factor (TNF) binding domain of its receptors and inhibits TNF-induced biological responses.

    OpenAIRE

    Thoh, Maikho; Kumar, Pankaj; Nagarajaram, Hampathalu A.; Manna, Sunil K.

    2013-01-01

    The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor κB (NF-κB) and also expression of NF-κB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-κB (IκBα) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IκBα kinase (IKK...

  12. Responses to the Economic Crisis among Immigrants in the Czech Republic: Impeding and Inhibiting Factors for Staying

    Directory of Open Access Journals (Sweden)

    Marketa Rulikova

    2012-10-01

    This paper is based on research conducted among participants of the government-assisted Voluntary Return Programme and a follow-up ethnographic study in the Vietnamese, Ukrainian and Mongolian communities in Prague. It can be shown that most immigrants decided to stay despite extreme declines in their living conditions. While the motivations of immigrants to leave or stay are multifaceted, this paper offers an alternative to the “pull-push” model that takes into consideration economic as well as cultural factors, which both impede and inhibit migrants from returning “home”.

  13. A novel monoclonal antibody of human stem cell factor inhibits umbilical cord blood stem cell ex vivo expansion

    Directory of Open Access Journals (Sweden)

    Fan Jie

    2012-12-01

    Full Text Available Abstract Stem cell factor (SCF activates hematopoietic stem cell (HSC self-renewal and is being used to stimulate the ex vivo expansion of HSCs. The mechanism by which SCF supports expansion of HSCs remains poorly understood. In cord blood ex vivo expansion assays, a newly produced anti-SCF monoclonal antibody (clone 23C8 was found to significantly inhibit the expansion of CD34+ cells. This antibody appears to bind directly to a part of SCF that is critical for biological activity toward expansion of CD34+ cells, which is located in the first 104 amino acids from the NH2-terminus.

  14. Aspirin inhibits colon cancer cell and tumor growth and downregulates specificity protein (Sp transcription factors.

    Directory of Open Access Journals (Sweden)

    Satya Pathi

    Full Text Available Acetylsalicylic acid (aspirin is highly effective for treating colon cancer patients postdiagnosis; however, the mechanisms of action of aspirin in colon cancer are not well defined. Aspirin and its major metabolite sodium salicylate induced apoptosis and decreased colon cancer cell growth and the sodium salt of aspirin also inhibited tumor growth in an athymic nude mouse xenograft model. Colon cancer cell growth inhibition was accompanied by downregulation of Sp1, Sp3 and Sp4 proteins and decreased expression of Sp-regulated gene products including bcl-2, survivin, VEGF, VEGFR1, cyclin D1, c-MET and p65 (NFκB. Moreover, we also showed by RNA interference that β-catenin, an important target of aspirin in some studies, is an Sp-regulated gene. Aspirin induced nuclear caspase-dependent cleavage of Sp1, Sp3 and Sp4 proteins and this response was related to sequestration of zinc ions since addition of zinc sulfate blocked aspirin-mediated apoptosis and repression of Sp proteins. The results demonstrate an important underlying mechanism of action of aspirin as an anticancer agent and, based on the rapid metabolism of aspirin to salicylate in humans and the high salicylate/aspirin ratios in serum, it is likely that the anticancer activity of aspirin is also due to the salicylate metabolite.

  15. Adaptation in Response of Excitation and Inhibition Factors of Angiogenesis after 4 Weeks of Progressive Resistant Training in Sedentary Men

    Directory of Open Access Journals (Sweden)

    s. Karami

    2016-09-01

    Full Text Available Aims: The sport activity is an important factor affecting the capillary density and angiogenesis. Nitric oxide (NO and vascular endothelial growth factor (VEGF are the most important stimulative regulators in the angiogenesis. In addition, endostatin is one of the inhibitors of angiogenesis. The aim of this study was to investigate the adaptation in the responses of the angiogenesis inhibition and stimulating factors after 4-week increasing resistive exercises in the sedentary men. Materials & Methods: In the semi-experimental study, 20 healthy and inactive male students, aged between 20 and 25 years, who were residents of Tehran University Dormitory, were studied in the first semester of the academic year 2015-16. The subjects, selected via available sampling method, were divided into two groups including experimental and control groups (n=10 per group. 4-week resistive exercises were done three sessions per week. Blood-sampling was done before and 48 hours after the last exercise session. VEGF, NO, and endostatin were then measured. Data was analyzed by SPSS 18 software using independent and dependent T tests, as well as Pearson correlation coefficient test. Findings: In experimental group, VEGF and No significantly increased at the posttest stage than the pretest (p=0.001. Nevertheless, no significant difference was observed in control group (p>0.05. In both experimental and control groups, endostatin level did not significantly increase at the posttest stage than the pretest (p>0.05. In addition, VEGF and NO were the only variables that were significantly correlated (p=0.016; r=0.82. Conclusion: 4-week increasing resistive exercises in the sedentary men significantly affect the angiogenes stimulating factors, i. e. VEGF and NO, while such exercises do not significantly affect the angiogenesis inhibition factor, i. e. endostatin.

  16. Symbolic Dynamics, Modular Curves, and Bianchi IX Cosmologies

    OpenAIRE

    Manin, Yuri I.; Marcolli, Matilde

    2015-01-01

    It is well known that the so called Bianchi IX spacetimes with SO(3)-symmetry in a neighbourhood of the Big Bang exhibit a chaotic behaviour of typical trajectories in the backward movement of time. This behaviour (Mixmaster Model of the Universe) can be encoded by the shift of two-sided continued fractions. Exactly the same shift encodes the sequences of intersections of hyperbolic geodesics with purely imaginary axis in the upper complex half-plane, that is geodesic flow on an appropriate m...

  17. Aportes de "una empresa docente" a la IX CIAM

    OpenAIRE

    Gómez, Pedro; Carulla, Cristina; De Castro, Mauricio; Fernández, Felipe; Gómez, Cristina; Mesa, Vilma María; Perry, Patricia; Valero, Paola

    1995-01-01

    Este volumen contiene los aportes del grupo de investigadores de "una empresa docente" a la IX Conferencia Interamericana de Educación Matemática, realizada en Santiago de Chile en agosto de 1995. Los artículos que se incluyen representan parcialmente los intereses y las realizaciones de este centro de investigación de la Universidad de los Andes durante los últimos años. Los primeros dos capítulos, La potenciación del sistema de educación matemática en Colombia y La interdisciplinareidad ...

  18. Althaea rosea Cavanil and Plantago major L. suppress neoplastic cell transformation through the inhibition of epidermal growth factor receptor kinase.

    Science.gov (United States)

    Choi, Eun-Sun; Cho, Sung-Dae; Shin, Ji-Ae; Kwon, Ki Han; Cho, Nam-Pyo; Shim, Jung-Hyun

    2012-10-01

    For thousands of years in Asia, Althaea rosea Cavanil (ARC) and Plantago major L. (PML) have been used as powerful non-toxic therapeutic agents that inhibit inflammation. However, the anticancer mechanisms and molecular targets of ARC and PML are poorly understood, particularly in epidermal growth factor (EGF)-induced neoplastic cell transformation. The aim of this study was to evaluate the chemopreventive effects and mechanisms of the methanol extracts from ARC (MARC) and PML (MPML) in EGF-induced neoplastic cell transformation of JB6 P+ mouse epidermal cells using an MTS assay, anchorage-independent cell transformation assay and western blotting. Our results showed that MARC and MPML significantly suppressed neoplastic cell transformation by inhibiting the kinase activity of the EGF receptor (EGFR). The activation of EGFR by EGF was suppressed by MARC and MPML treatment in EGFR(+/+) cells, but not in EGFR(-/-) cells. In addition, MARC and MPML inhibited EGF-induced cell proliferation in EGFR-expressing murine embryonic fibroblasts (EGFR(+/+)). These results strongly indicate that EGFR targeting by MARC and MPML may be a good strategy for chemopreventive or chemotherapeutic applications.

  19. Endosulfan inhibiting the meiosis process via depressing expressions of regulatory factors and causing cell cycle arrest in spermatogenic cells.

    Science.gov (United States)

    Guo, Fang-Zi; Zhang, Lian-Shuang; Wei, Jia-Liu; Ren, Li-Hua; Zhang, Jin; Jing, Li; Yang, Man; Wang, Ji; Sun, Zhi-Wei; Zhou, Xian-Qing

    2016-10-01

    Endosulfan is a persistent organic pollutant and widely used in agriculture as a pesticide. It is present in air, water, and soil worldwide; therefore, it is a health risk affecting especially the reproductive system. The aim of this study was to evaluate the toxicity of endosulfan in the reproductive system. To investigate the effect of endosulfan on meiosis process, 32 rats were divided into four groups, treated with 0, 1, 5, and 10 mg/kg/day endosulfan, respectively, and sacrificed after the 21 days of treatments. Results show that endosulfan caused the reductions in sperm concentration and motility rate, which resulted into an increased in sperm abnormality rate; further, endosulfan induced downregulation of spermatogenesis- and oogenesis-specific basic helix-loop-helix transcription factor (Sohlh1) which controls the switch on meiosis in mammals, as well cyclin A1, cyclin-dependent kinases 1 (CDK1), and cyclin-dependent kinases 2 (CDK2). In vitro, endosulfan induced G2/M phase arrest in the spermatogenic cell cycle and caused proliferation inhibition. Moreover, endosulfan induced oxidative stress and DNA damage in vivo and vitro. The results suggested that endosulfan could inhibit the start of meiosis by downregulating the expression of Sohlh1 and induce G2/M phase arrest of cell cycle by decreasing the expression of cyclin A1, CDK1, and CDK2 via oxidative damage, which inhibits the meiosis process, and therefore decrease the amount of sperm.

  20. A functional connection between pRB and transforming growth factor beta in growth inhibition and mammary gland development.

    Science.gov (United States)

    Francis, Sarah M; Bergsied, Jacqueline; Isaac, Christian E; Coschi, Courtney H; Martens, Alison L; Hojilla, Carlo V; Chakrabarti, Subrata; Dimattia, Gabriel E; Khoka, Rama; Wang, Jean Y J; Dick, Frederick A

    2009-08-01

    Transforming growth factor beta (TGF-beta) is a crucial mediator of breast development, and loss of TGF-beta-induced growth arrest is a hallmark of breast cancer. TGF-beta has been shown to inhibit cyclin-dependent kinase (CDK) activity, which leads to the accumulation of hypophosphorylated pRB. However, unlike other components of TGF-beta cytostatic signaling, pRB is thought to be dispensable for mammary development. Using gene-targeted mice carrying subtle missense changes in pRB (Rb1(DeltaL) and Rb1(NF)), we have discovered that pRB plays a critical role in mammary gland development. In particular, Rb1 mutant female mice have hyperplastic mammary epithelium and defects in nursing due to insensitivity to TGF-beta growth inhibition. In contrast with previous studies that highlighted the inhibition of cyclin/CDK activity by TGF-beta signaling, our experiments revealed that active transcriptional repression of E2F target genes by pRB downstream of CDKs is also a key component of TGF-beta cytostatic signaling. Taken together, our work demonstrates a unique functional connection between pRB and TGF-beta in growth control and mammary gland development.

  1. Total saponins from Albizia julibrissin inhibit vascular endothelial growth factor-mediated angiogenesis in vitro and in vivo.

    Science.gov (United States)

    Cai, Weiwei; Li, Yue; Yi, Qingqing; Xie, Fengshan; Du, Bin; Feng, Lei; Qiu, Liying

    2015-05-01

    Dried stem bark from Albizia julibrissin (AJ) is a highly valued Traditional Chinese Medicine, which has been shown to suppress tumor growth and angiogenesis. Total saponins from AJ (TSAJ) are one of the most bioactive components of AJ extract. The present study evaluated the anti‑tumor and anti‑angiogenic effects of TSAJ in vitro and in vivo. The anti‑angiogenic activity of TSAJ was investigated by measuring the effects on vascular endothelial growth factor (VEGF)‑induced proliferation, migration and tube formation of Ea.hy926 endothelial cells in vitro. The expression levels of proteins associated with VEGF‑induced angiogenesis were determined by western blotting. Furthermore, in vivo Matrigel™ plug and H22 hepatoma tumor models were used to verify the anti‑angiogenic effects of TSAJ. The present study demonstrated that TSAJ significantly inhibited VEGF‑mediated endothelial cell proliferation, migration and tube formation of Ea.hy926 cells in vitro. The anti‑angiogenic effects of TSAJ were modulated by suppression of phosphorylated‑(p‑) focal adhesion kinase, p‑Akt, and p‑extracellular signal‑regulated kinase in the VEGF/VEGF receptor 2 (R2) signaling pathway. Furthermore, oral administration of TSAJ significantly inhibited tumor growth and tumor‑induced angiogenesis, as well as the formation of functional vessels, in the Matrigel™ plug model. These results suggest that TSAJ may be a potential anti‑angiogenic agent that targets the VEGF/VEGFR2 signaling pathway, and inhibits tumor‑induced angiogenesis.

  2. Resveratrol Inhibits the Secretion of Vascular Endothelial Growth Factor and Subsequent Proliferation in Human Leukemia U937 Cells

    Institute of Scientific and Technical Information of China (English)

    TANG Zehai; LIU Xinyue; ZOU Ping

    2007-01-01

    This study examined the effect of resveratrol on the secretion of vascular endothelial growth factor (VEGF) and subsequent proliferation of human leukemia U937 cells, and explored the mechanisms involved. Human leukemia U937 cells were treated with resveratrol of different concen- trations (12.5-200 μmol/L) for different time lengths (12-48 h). The proliferation of the U937 leu- kemic cells was determined by MTT assay. Apoptosis was observed by Annexin-Ⅴ-FIFC/PI double staining and flow cytometry (FCM). Cells cycle was analyzed by PI staining and FCM. The content of VEGF was determined by ELISA. Human umbibical vein endothelial cells were examined for vasoformation in vitro after exposures to resveratrol of various concetrations. The results showed that resveratrol inhibited the proliferation of U937 leukemia cells in a dose- and time-dependent manner. Resveratrol induced apoptosis and S-phase cell cycle arrest in human leukemic U937 cells. Resvera-trol inhibited the secretion of VEGF in U937 cells. Resveratrol inhibited the vasoformation of human vein endothelial cells in a dose-dependent manner. It was concluded that resveratrol could down-regulate the secretion of VEGE induce apoptosis and suppress the proliferation of U937 cells.

  3. Widdrol, a sesquiterpene isolated from Juniperus chinensis, inhibits angiogenesis by targeting vascular endothelial growth factor receptor 2 signaling.

    Science.gov (United States)

    Jin, Soojung; Yun, Hee Jung; Jeong, Hyun Young; Oh, You Na; Park, Hyun-Jin; Yun, Seung-Geun; Kim, Byung Woo; Kwon, Hyun Ju

    2015-09-01

    Widdrol is an odorous compound derived from Juniperus chinensis that is widely used in traditional medicine to treat fever, inflammation and cancer. It was previously reported that widdrol has antitumor activity by apoptosis induction in cancer cells in vitro. However, its anti-angiogenic activity remains elusive. In the present study, we investigated the anti‑angiogenic activity of widdrol and the molecular mechanisms involved. Widdrol inhibited cell proliferation via G1 phase arrest induction in human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Additionally, it was associated with a decreased expression of cyclin-dependent kinase 2 (CDK2) and an increased expression of p21, a CDK inhibitor. Widdrol significantly inhibited the cell migration and tube formation of HUVECs using an in vitro angiogenesis assay. The results showed that widdrol suppressed phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and its downstream proteins, such as AKT, focal adhesion kinase (FAK) and endothelial nitric oxide synthase (eNOS). Moreover, widdrol effectively reduced tumor growth and blood vessel formation in colon tumor xenograft mice. Collectively, these results suggested that widdrol may act as a potential anti-angiogenic agent by inhibiting vessel sprouting and growth, which may have implications for angioprevention.

  4. ENDOCANNABINOIDS INHIBIT RELEASE OF NERVE GROWTH FACTOR BY INFLAMMATION-ACTIVATED MAST CELLS

    OpenAIRE

    2011-01-01

    Abstract Nerve growth factor (NGF) is a pleiotropic member of the neurotrophin family. Beside its neuronal effects, NGF plays a role in various processes, including angiogenesis. Mast cells release NGF and are among elements contributing to angiogenesis, a process regulated by arrays of factors, including the inhibitory cannabinoids. The possible inhibitory role of cannabinoids on mast cell-related NGF mitogenic effect on endothelial cells was then investigated. Human mastocytic ce...

  5. Sulindac sulfide inhibits colon cancer cell growth and downregulates specificity protein transcription factors

    OpenAIRE

    Li, Xi; Pathi, Satya S.; Safe, Stephen

    2015-01-01

    Background Specificity protein (Sp) transcription factors play pivotal roles in maintaining the phenotypes of many cancers. We hypothesized that the antineoplastic effects of sulindac and its metabolites were due, in part, to targeting downregulation of Sp transcription factors. Methods The functional effects of sulindac, sulindac sulfone and sulindac sulfide on colon cancer cell proliferation were determined by cell counting. Effects of these compounds on expression of Sp1, Sp3, Sp4 and pro-...

  6. Intermedin 1-53 Inhibits Myocardial Fibrosis in Rats by Down-Regulating Transforming Growth Factor

    Science.gov (United States)

    Fang, Jian; Luan, Jiangwei; Zhu, Gaohong; Qi, Chang; Yang, Zhiyong; Zhao, Sheng; Li, Bin; Zhang, Xinzhong; Guo, Naipeng; Li, Xiaodong; Wang, Dandan

    2017-01-01

    Background Myocardial fibrosis is the result of persistent anoxia and ischemic myocardial fibers caused by coronary atherosclerotic stenosis, which lead to heart failure, threatening the patient’s life. This study aimed to explore the regulatory role of intermedin 1-53 (IMD1-53) in cardiac fibrosis using neonatal rat cardiac fibroblasts and a myocardial infarction (MI) rat model both in vitro and in vivo. Material/Methods The Western blot method was used to detect the protein expression of collagen I and collagen III in myocardial fibroblasts. The SYBR Green I real-time quantitative polymerase chain reaction (PCR) assay was used to detect the mRNA expression of collagen type I and III, IMD1-53 calcitonin receptor-like receptor (CRLR), transforming growth factor-β (TGF-β), and matrix metalloproteinase-2 (MMP-2). Masson staining was used to detect the area changes of myocardial fibrosis in MI rats. Results Results in vivo showed that IMD1-53 reduced the scar area on the heart of MI rats and inhibited the expression of collagen type I and III both in mRNA and protein. Results of an in vitro study showed that IMD1-53 inhibited the transformation of cardiomyocytes into myofibroblasts caused by angiotensin II (Ang II). The further mechanism study showed that IMD1-53 inhibited the expression of TGF-β and the phosphorylation of smad3, which further up-regulated the expression of MMP-2. Conclusions IMD1-53 is an effective anti-fibrosis hormone that inhibits cardiac fibrosis formation after MI by down-regulating the expression of TGF-β and the phosphorylation of smad3, blocking fibrous signal pathways, and up-regulating the expression of MMP-2, thereby demonstrating its role in regression of myocardial fibrosis. PMID:28065931

  7. Chamaecyparis obtusa Essential Oil Inhibits Methicillin-Resistant Staphylococcus aureus Biofilm Formation and Expression of Virulence Factors.

    Science.gov (United States)

    Kim, Eun-Sook; Kang, Sun-Young; Kim, Young-Hoi; Lee, Young-Eun; Choi, Na-Young; You, Yong-Ouk; Kim, Kang-Ju

    2015-07-01

    The emergence of antibiotic-resistant bacteria has caused difficulty in treating infectious diseases. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most commonly recognized antibiotic-resistant bacteria. Novel antibiotics are urgently required to treat these bacteria. Raw materials derived from natural sources can be used for the development of novel antibiotics, such as Chamaecyparis obtusa (C. obtusa), which has been traditionally used in treating asthmatic disease. In this study, the antibacterial activity of the essential oil (EO) extracted from C. obtusa leaves against MRSA was investigated. MRSA growth and acid production from glucose metabolism were inhibited at concentrations greater than 0.1 mg/mL C. obtusa EO. MRSA biofilm formation was observed using scanning electron microscopy and safranin staining. C. obtusa EO inhibited MRSA biofilm formation at concentrations greater than 0.1 mg/mL. Using real-time polymerase chain reaction, mRNA expression of virulence factor genes, sea, agrA, and sarA, was observed. agrA expression was inhibited with C. obtusa EO concentrations greater than 0.2 mg/mL, whereas inhibition of sea and sarA expression was also observed at a concentration of 0.3 mg/mL. C. obtusa EO was analyzed by gas chromatography (GC) and GC coupled for mass spectrometry, which identified 59 constituents, accounting to 98.99% of the total EO. These findings suggest that C. obtusa EO has antibacterial effects against MRSA, which might be associated with the major components of C. obtusa EO, such as sabinene (19.06%), α-terpinyl acetate (16.99%), bornyl acetate (10.48%), limonene (8.54%), elemol (7.47%), myrcene (5.86%), γ-terpinene (4.04%), and hibaene (3.01%).

  8. Inhibition of vascular endothelial growth factor signaling facilitates liver repair from acute ethanol-induced injury in zebrafish

    Directory of Open Access Journals (Sweden)

    Changwen Zhang

    2016-11-01

    Full Text Available Alcoholic liver disease (ALD results from alcohol overconsumption and is among the leading causes of liver-related morbidity and mortality worldwide. Elevated expression of vascular endothelial growth factor (VEGF and its receptors has been observed in ALD, but how it contributes to ALD pathophysiology is unclear. Here, we investigated the impact of VEGF signaling inhibition on an established zebrafish model of acute alcoholic liver injury. Kdrl activity was blocked by chemical inhibitor treatment or by genetic mutation. Exposing 4-day-old zebrafish larvae to 2% ethanol for 24 h induced hepatic steatosis, angiogenesis and fibrogenesis. The liver started self-repair once ethanol was removed. Although inhibiting Kdrl did not block the initial activation of hepatic stellate cells during ethanol treatment, it suppressed their proliferation, extracellular matrix protein deposition and fibrogenic gene expression after ethanol exposure, thus enhancing the liver repair. It also ameliorated hepatic steatosis and attenuated hepatic angiogenesis that accelerated after the ethanol treatment. qPCR showed that hepatic stellate cells are the first liver cell type to increase the expression of VEGF ligand and receptor genes in response to ethanol exposure. Both hepatic stellate cells and endothelial cells, but not hepatic parenchymal cells, expressed kdrl upon ethanol exposure and were likely the direct targets of Kdrl inhibition. Ethanol-induced steatosis and fibrogenesis still occurred in cloche mutants that have hepatic stellate cells but lack hepatic endothelial cells, and Kdrl inhibition suppressed both phenotypes in the mutants. These results suggest that VEGF signaling mediates interactions between activated hepatic stellate cells and hepatocytes that lead to steatosis. Our study demonstrates the involvement of VEGF signaling in regulating sustained liver injuries after acute alcohol exposure. It also provides a proof of principle of using the

  9. Is copper an inhibiting factor for primary production in the upwelling waters of Cabo Frio?

    Directory of Open Access Journals (Sweden)

    Diniz Antônia G.

    2003-01-01

    Full Text Available The goal of the present work was to test the hypothesis that inorganic species of copper may inhibit primary production in upwelling waters from the Cabo Frio region. Water samples were collected from four different depths up to 50 m at three stations of Cabo Frio and nutrient concentration, pH, dissolved oxygen, alkalinity, seston, chlorophyll a, primary production, NPP, copper speciation, Cu(II and Cu(I and complexing capacity were determined. The results indicate that the rate of photosynthesis in nutrient rich waters with higher copper content and lower complexing capacity is significantly reduced. Cu(I made up 2-15% of the total copper, however, there was no evidence to suggest that the photochemical production of Cu(I affects NPP.

  10. Topical glycerol monooleate/propylene glycol formulations enhance 5-aminolevulinic acid in vitro skin delivery and in vivo protophorphyrin IX accumulation in hairless mouse skin.

    Science.gov (United States)

    Steluti, Regilene; De Rosa, Fernanda Scarmato; Collett, John; Tedesco, Antônio Cláudio; Bentley, Maria Vitória Lopes Badra

    2005-08-01

    Photodynamic therapy (PDT), a potential therapy for cancer treatment, utilizes exogenously applied or endogenously formed photosensitizers, further activated by light in an appropriate wavelength and dose to induce cell death through free radical formation. 5-Aminolevulinic acid (5-ALA) is a pro-drug which can be converted to the effective photosensitizer, protoporphyrin IX (PpIX). However, the use of 5-ALA in PDT is limited by the low penetration capacity of this highly hydrophilic molecule into appropriate skin layers. In the present study, we propose to increase 5-ALA penetration by using formulations containing glycerol monooleate (GMO), an interesting and useful component of pharmaceutical formulations. Propylene glycol solutions containing different concentrations of GMO significantly increased the in vitro skin permeation/retention of 5-ALA in comparison to control solutions. In vivo studies also showed increased PpIX accumulation in mouse hairless skin, after the use of topical 5-ALA formulations containing GMO in a concentration-dependent manner. The results show that skin 5-ALA penetration and PpIX accumulation, important factors for the success of topical 5-ALA-PDT in skin cancer, are optimized by GMO/propylene glycol formulations.

  11. Effect of IX dosing on polypropylene and PVDF membrane fouling control

    KAUST Repository

    Myat, Darli Theint

    2013-07-01

    The performance of ion exchange (IX) resin for organics removal from wastewater was assessed using advanced characterisation techniques for varying doses of IX. Organic characterisation using liquid chromatography with a photodiode array (PDA) and fluorescence spectroscopy (Method A), and UV254, organic carbon and organic nitrogen detectors (Method B), was undertaken on wastewater before and after magnetic IX treatment. Results showed partial removal of the biopolymer fraction at high IX doses. With increasing concentration of IX, evidence for nitrogen-containing compounds such as proteins and amino acids disappeared from the LC-OND chromatogram, complementary to the fluorescence response. A greater fluorescence response of tryptophan-like proteins (278nm/343nm) for low IX concentrations was consistent with aggregation of tryptophan-like compounds into larger aggregates, either by self-aggregation or with polysaccharides. Recycling of IX resin through multiple adsorption steps without regeneration maintained the high level of humics removal but there was no continued removal of biopolymer. Subsequent membrane filtration of the IX treated waters resulted in complex fouling trends. Filtration tests with either polypropylene (PP) or polyvinylidene fluoride (PVDF) membranes showed higher rates of initial fouling following treatment with high IX doses (10mL/L) compared to filtration of untreated water, while treatment with lower IX doses resulted in decreased fouling rates relative to the untreated water. However, at longer filtration times the rate of fouling of IX treated waters was lower than untreated water and the relative fouling rates corresponded to the amount of biopolymer material in the feed. It was proposed that the mode of fouling changed from pore constriction during the initial filtration period to filter cake build up at longer filtration times. The organic composition strongly influenced the rate of fouling during the initial filtration period due to

  12. Determination of factor Xa inhibition doses of low-molecular heparin, nadroparin and reviparin in urological patients

    Directory of Open Access Journals (Sweden)

    Pavlović Svetlana

    2007-01-01

    Full Text Available Background/Aim. The inhibition of factor Xa (FX by the use of low-molecular heparin (LMH is important clinical procedure in patients with moderate and high risk for the developament of venous thromboembolism (VTE and pulmonary embolism (PE. The aim of this study was to determine the level of inhibition of FXa by the use of prophylactic doses of LMH nadroparin-calcium and reviparine-sodium which were applied in urological patients with moderate risk for VTE and PE. Methods. The examination included 80 urological patients divided into 4 groups after urological, uroradiological and anesthesiological preoperative preparation and categorization of anesthesiological risk according to the ASA III classification. The first two groups, of 20 patients each, received the recommended doses of LMH in accordance with the preoperative risk, and an inhibition of FXa 48 hours after the surgical operation and four hours after the administration of LMH was determined. Heptest and homogenous anti-Xa test were used for monitoring of FXa inhibition. Since the obtained anti-Xa values were not satisfactory, two more groups were formed and given double the recommended doses. In these new groups, inhibition of FXa was in recommended range. Standard descriptive statistical parameters were used for describing the charateristics of the people from the formed groups. Results. All the patients examined were clinically estimated as patients of moderate risk, for VTE and PE. There were no statistically singificant difference in body weight of the patients who received nadroparin-calcium 0.3 ml and reviparine-sodium 0.25 ml and those who received their double doses, respectively. The level of FXa inhibition in the group in which the dose of nadroparin-calcium of 0.6 ml was applied was statistically significantly higher than in the group which received the dose of 0.3 ml (Mann-Whitney U test: Z = 5.416; p < 0.0001. The level of FXa in the group given reviparine-sodium 0.5 ml was

  13. Nerve Growth Factor Inhibits Gd3+-sensitive Calcium Influx and Reduces Chemical Anoxic Neuronal Death

    Institute of Scientific and Technical Information of China (English)

    Hui JIANG; Shunlian TIAN; Yan ZENG; Jing SHI

    2008-01-01

    To investigate whether glutamate and voltage-gated calcium channels-independent calcium influx exists during acute anoxic neuronal damage and its possible relationship to neuronal protective function of NGF. In in vitro model of acute anoxia, hippocampal cultures from newborn rats were exposed to 3 mmol/L KCN. Changes of intracellular Ca2+ concentration ([Ca2+]i) were monitored by con-focal imaging and cell viability was assayed by PI and cFDA staining. The results showed that after treatment with primary hippocampal cultures with 3 mmol/L KCN for 15 min,[Ca2+]i was significantly increased 6.27-fold compared to pre-anoxia level and 73.3% of the cells died.When combination of 20 μmol/L MK-801 (glutamate receptor antagonist), 40 μmol/L CNQX (AMPA receptor antagonist) and 5 μmol/L nimodipine (voltage-gated calcium channel antagonist) (hereafter denoted as MCN) were administrated to hippocampal cultures, levels of [Ca2+]i and cell death rate induced by KCN were partially reduced by 35.9% and 47.5% respectively. However, Gd3+ (10μmol/L) almost completely blocked KCN-mediated [Ca2+]i elevation by 81.9% and reduced neuronal death by 88.8% in the presence of MCN. It is noteworthy that NGF, used in combination with MCN,inhibited KCN-induced [Ca2+]i increase by 77.4% and reduced cell death by 87.1%. Only PLC inhibitor U73122 (10 μmol/L) abolished NGF effects. It is concluded that Gd3+-sensitive calcium influx,which is NMDA (glutamate receptor) and voltage-gated calcium channels-independent, is responsible for acute anoxic neuronal death. NGF can inhibit Gd3+-sensitive calcium influx and reduce anoxic neuronal death through activating PLC pathway.

  14. Axon guidance factor SLIT2 inhibits neural invasion and metastasis in pancreatic cancer.

    Science.gov (United States)

    Göhrig, Andreas; Detjen, Katharina M; Hilfenhaus, Georg; Körner, Jan L; Welzel, Martina; Arsenic, Ruza; Schmuck, Rosa; Bahra, Marcus; Wu, Jane Y; Wiedenmann, Bertram; Fischer, Christian

    2014-03-01

    Pancreatic ductal adenocarcinoma (PDAC) metastasizes by neural, vascular, and local invasion routes, which limit patient survival. In nerves and vessels, SLIT2 and its ROBO receptors constitute repellent guidance cues that also direct epithelial branching. Thus, the SLIT2-ROBO system may represent a key pinch point to regulate PDAC spread. In this study, we examined the hypothesis that escaping from repellent SLIT2-ROBO signaling is essential to enable PDAC cells to appropriate their local stromal infrastructure for dissemination. Through immunohistochemical analysis, we detected SLIT2 receptors ROBO1 and ROBO4 on epithelia, nerves, and vessels in healthy pancreas and PDAC specimens, respectively. SLIT2 mRNA expression was reduced in PDAC compared with nontransformed pancreatic tissues and cell lines, suggesting a reduction in SLIT2-ROBO pathway activity in PDAC. In support of this interpretation, restoring the SLIT2 expression in SLIT2-deficient PDAC cells inhibited their bidirectional chemoattraction with neural cells, and more specifically, impaired unidirectional PDAC cell navigation along outgrowing neurites in models of neural invasion. Restoring autocrine/paracrine SLIT2 signaling was also sufficient to inhibit the directed motility of PDAC cells, but not their random movement. Conversely, RNA interference-mediated silencing of ROBO1 stimulated the motility of SLIT2-competent PDAC cells. Furthermore, culture supernatants from SLIT2-competent PDAC cells impaired migration of endothelial cells (human umbilical vein endothelial cells), whereas an N-terminal SLIT2 cleavage fragment stimulated such migration. In vivo investigations of pancreatic tumors with restored SLIT2 expression demonstrated reduced invasion, metastasis, and vascularization, with opposing effects produced by ROBO1 silencing in tumor cells or sequestration of endogenous SLIT2. Analysis of clinical specimens of PDAC showed that those with low SLIT2 mRNA expression exhibited a higher incidence

  15. Synergistic Induction of Cyclooxygenase-2 by Transforming Growth Factor-β1 and Epidermal Growth Factor Inhibits Apoptosis in Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Debabrata Saha

    1999-12-01

    Full Text Available Increased expression of cyclooxygenase-2 (COX-2 expression has been observed in several human tumor types and in selected animal and cell culture models of carcinogenesis, including lung cancer. Increased expression of COX-2 and production of prostaglandins appear to provide a survival advantage to transformed cells through the inhibition of apoptosis, increased attachment to extracellular matrix, increased invasiveness, the stimulation of angiogenesis. In the present studies, we found that transforming growth factor β1 (TGF-β1 and epidermal growth factor (EGF synergistically induced the expression of COX-2 and prostaglandin E2 (PGE2 production in mink lung epithelial (Mvi Lu cells. EGF, but not PDGF or IGF-1, was able to inhibit TGF-β1-induced apoptosis in Mvi Lu cells and this effect was blocked by NS-398, a selective inhibitor of COX-2 activity, suggesting a possible role for COX-2 in the anti-apoptosic effect of EGF receptor ligands. The combination of TGF-β1 and EGF also significantly induced COX-2 expression in rat intestinal epithelial (RIE-1 cells and completely prevented sodium butyrate (NaBu-induced apoptosis. The synergistic induction of COX-2 by TGF-β1 and EGF was not observed in R1B-L17 cells, a line derived from Mvi Lu cells that lacks the TGF-β type-I receptor. AG1478, a selective inhibitor of EGF receptor tyrosine kinase activity, completely suppressed the induction of COX-2 expression by either EGF or TGF-β1+EGF. Also, PD98059, a specific inhibitor of MEK/ERK pathway, SB203580, a specific inhibitor of p38 MAPK activity, significantly inhibited the induction of COX-2 in response to combined EGF and TGF-β1. These results suggest an important collaborative interaction of TGF-β1 and EGF signaling in the induction of COX-2 and prostaglandin production in Mv1Lu cells.

  16. Epidermal growth factor receptor inhibition reduces angiogenesis via hypoxia-inducible factor-1α and Notch1 in head neck squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Wei-Ming Wang

    Full Text Available Angiogenesis, a marker of cancer development, affects response to radiotherapy sensibility. This preclinical study aims to understand the receptor tyrosine kinase-mediated angiogenesis in head neck squamous cell carcinoma (HNSCC. The receptor tyrosine kinase activity in a transgenic mouse model of HNSCC was assessed. The anti-tumorigenetic and anti-angiogenetic effects of cetuximab-induced epidermal growth factor receptor (EGFR inhibition were investigated in xenograft and transgenic mouse models of HNSCC. The signaling transduction of Notch1 and hypoxia-inducible factor-1α (HIF-1α was also analyzed. EGFR was overexpressed and activated in the Tgfbr1/Pten deletion (2cKO mouse model of HNSCC. Cetuximab significantly delayed tumor onset by reducing tumor angiogenesis. This drug exerted similar effects on heterotopic xenograft tumors. In the human HNSCC tissue array, increased EGFR expression correlated with increased HIF-1α and micro vessel density. Cetuximab inhibited tumor-induced angiogenesis in vitro and in vivo by significantly downregulating HIF-1α and Notch1. EGFR is involved in the tumor angiogenesis of HNSCC via the HIF-1α and Notch1 pathways. Therefore, targeting EGFR by suppressing hypoxia- and Notch-induced angiogenesis may benefit HNSCC therapy.

  17. Transforming growth factor-beta1 inhibits tissue engineering cartilage absorption via inducing the generation of regulatory T cells.

    Science.gov (United States)

    Li, Chichi; Bi, Wei; Gong, Yiming; Ding, Xiaojun; Guo, Xuehua; Sun, Jian; Cui, Lei; Yu, Youcheng

    2016-02-01

    The objective of the present study was to explore the mechanisms of transforming growth factor (TGF)-β1 inhibiting the absorption of tissue engineering cartilage. We transfected TGF-β1 gene into bone marrow mesenchymal stem cells (BMMSCs) and co-cultured with interferon (IFN)-γ and tumour necrosis factor (TNF)-α and CD4(+) CD25(-) T lymphocytes. We then characterized the morphological changes, apoptosis and characterization of chondrogenic-committed cells from TGF-β1(+) BMMSCs and explored their mechanisms. Results showed that BMMSCs apoptosis and tissue engineering cartilage absorption in the group with added IFN-γ and TNF-α were greater than in the control group. In contrast, there was little BMMSC apoptosis and absorption by tissue engineering cartilage in the group with added CD4(+) CD25(-) T lymphocytes; Foxp3(+) T cells and CD25(+) CD39(+) T cells were found. In contrast, no type II collagen or Foxp3(+) T cells or CD25(+) CD39(+) T cells was found in the TGF-β1(-) BMMSC group. The data suggest that IFN-γ and TNF-α induced BMMSCs apoptosis and absorption of tissue engineering cartilage, but the newborn regulatory T (Treg) cells inhibited the function of IFN-γ and TNF-α and protected BMMSCs and tissue engineering cartilage. TGF-β1not only played a cartilage inductive role, but also inhibited the absorption of tissue engineering cartilage. The pathway proposed in our study may simulate the actual reaction procedure after implantation of BMMSCs and tissue engineering cartilage in vivo. Copyright © 2013 John Wiley & Sons, Ltd.

  18. Nimbolide Inhibits Nuclear Factor-КB Pathway in Intestinal Epithelial Cells and Macrophages and Alleviates Experimental Colitis in Mice.

    Science.gov (United States)

    Seo, Ji Yeon; Lee, Changhyun; Hwang, Sung Wook; Chun, Jaeyoung; Im, Jong Pil; Kim, Joo Sung

    2016-10-01

    Nimbolide is a limonoid extracted from neem tree (Azadirachta indica) that has antiinflammatory properties. The effect of nimbolide on the nuclear factor-kappa B (NF-κB) pathway in intestinal epithelial cells (IECs), macrophages and in murine colitis models was investigated. The IEC COLO 205, the murine macrophage cell line RAW 264.7, and peritoneal macrophages from interleukin-10-deficient (IL-10(-/-) ) mice were preconditioned with nimbolide and then stimulated with tumor necrosis factor-α (TNF-α) or lipopolysaccharide. Dextran sulfate sodium-induced acute colitis model and chronic colitis model in IL-10(-/-) mice were used for in vivo experiments. Nimbolide significantly suppressed the expression of inflammatory cytokines (IL-6, IL-8, IL-12, and TNF-α) and inhibited the phosphorylation of IκBα and the DNA-binding affinity of NF-κB in IECs and macrophages. Nimbolide ameliorated weight loss, colon shortening, disease activity index score, and histologic scores in dextran sulfate sodium colitis. It also improved histopathologic scores in the chronic colitis of IL-10(-/-) mice. Staining for phosphorylated IκBα was significantly decreased in the colon tissue after treatment with nimbolide in both models. Nimbolide inhibits NF-κB signaling in IECs and macrophages and ameliorates experimental colitis in mice. These results suggest nimbolide could be a potentially new treatment for inflammatory bowel disease. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  19. Colchicine inhibits epidermal growth factor degradation in 3T3 cells.

    OpenAIRE

    Brown, K. D.; Friedkin, M; Rozengurt, E

    1980-01-01

    Colchicine (2 microM) did not affect the initial rate of association of 125I-labeled epidermal growth factor (125I-EGF) to Swiss 3T3 cells but continued incubation (up to 24 hr) led to an increase in cell-associated radioactivity. The effect is also produced by Colcemid, vinblastine, and podophyllotoxin but not by lumicolchicine. Disruption of microtubules with colchicine does not alter the rate of "down regulation" of EGF receptors, suggesting the binding and internalization of the factor pr...

  20. The interferon-gamma-induced GTPase, mGBP-2, inhibits tumor necrosis factor alpha (TNF-alpha) induction of matrix metalloproteinase-9 (MMP-9) by inhibiting NF-kappaB and Rac protein.

    Science.gov (United States)

    Balasubramanian, Sujata; Fan, Meiyun; Messmer-Blust, Angela F; Yang, Chuan H; Trendel, Jill A; Jeyaratnam, Jonathan A; Pfeffer, Lawrence M; Vestal, Deborah J

    2011-06-03

    Matrix metalloproteinase-9 (MMP-9) is important in numerous normal and pathological processes, including the angiogenic switch during tumor development and tumor metastasis. Whereas TNF-α and other cytokines up-regulate MMP-9 expression, interferons (IFNs) inhibit MMP-9 expression. We found that IFN-γ treatment or forced expression of the IFN-induced GTPase, mGBP-2, inhibit TNF-α-induced MMP-9 expression in NIH 3T3 fibroblasts, by inhibiting MMP-9 transcription. The NF-κB transcription factor is required for full induction of MMP-9 by TNF-α. Both IFN-γ and mGBP-2 inhibit the transcription of a NF-κB-dependent reporter construct, suggesting that mGBP-2 inhibits MMP-9 induction via inhibition of NF-κB-mediated transcription. Interestingly, mGBP-2 does not inhibit TNF-α-induced degradation of IκBα or p65/RelA translocation into the nucleus. However, mGBP-2 inhibits p65 binding to a κB oligonucleotide probe in gel shift assays and to the MMP-9 promoter in chromatin immunoprecipitation assays. In addition, TNF-α activation of NF-κB in NIH 3T3 cells is dependent on Rac activation, as evidenced by the inhibition of TNF-α induction of NF-κB-mediated transcription by a dominant inhibitory form of Rac1. A role for Rac in the inhibitory action of mGBP-2 on NF-κB is further shown by the findings that mGBP-2 inhibits TNF-α activation of endogenous Rac and constitutively activate Rac can restore NF-κB transcription in the presence of mGBP-2. This is a novel mechanism by which IFNs can inhibit the cytokine induction of MMP-9 expression.

  1. IX Draconis - a curious ER UMa-type dwarf nova

    CERN Document Server

    Otulakowska-Hypka, M; de Miguel, E; Rutkowski, A; Koff, R; B\\kakowska, K

    2012-01-01

    We report results of an extensive world-wide observing campaign devoted to a very active dwarf nova star - IX Draconis. We investigated photometric behaviour of the system to derive its basic outburst properties and understand peculiarities of IX Dra as well as other active cataclysmic variables, in particular dwarf novae of the ER Uma-type. In order to measure fundamental parameters of the system, we carried out analyses of the light curve, O-C diagram, and power spectra. During over two months of observations we detected two superoutbursts and several normal outbursts. The V magnitude of the star varied in the range 14.6 - 18.2 mag. Superoutbursts occur regularly with the supercycle length of 58.5+/-0.5 d. When analysing data over the past 20 years, we found that the supercycle length is increasing at a rate of P_dot = 1.8 * 10^{-3}. Normal outbursts appear to be irregular, with typical occurrence times in the range 3.1 - 4.1 d. We detected a double-peaked structure of superhumps during superoutburst, with ...

  2. Ares I-X Upper Stage Simulator Residual Stress Analysis

    Science.gov (United States)

    Raju, Ivatury S.; Brust, Frederick W.; Phillips, Dawn R.; Cheston, Derrick

    2008-01-01

    The structural analyses described in the present report were performed in support of the NASA Engineering and Safety Center (NESC) Critical Initial Flaw Size (CIFS) assessment for the Ares I-X Upper Stage Simulator (USS) common shell segment. An independent assessment was conducted to determine the critical initial flaw size (CIFS) for the flange-to-skin weld in the Ares I-X Upper Stage Simulator (USS). The Ares system of space launch vehicles is the US National Aeronautics and Space Administration s plan for replacement of the aging space shuttle. The new Ares space launch system is somewhat of a combination of the space shuttle system and the Saturn launch vehicles used prior to the shuttle. Here, a series of weld analyses are performed to determine the residual stresses in a critical region of the USS. Weld residual stresses both increase constraint and mean stress thereby having an important effect on fatigue and fracture life. The results of this effort served as one of the critical load inputs required to perform a CIFS assessment of the same segment.

  3. Symbolic Dynamics, Modular Curves, and Bianchi IX Cosmologies

    CERN Document Server

    Manin, Yuri

    2015-01-01

    It is well known that the so called Bianchi IX spacetimes with SO(3)-symmetry in a neighbourhood of the Big Bang exhibit a chaotic behaviour of typical trajectories in the backward movement of time. This behaviour (Mixmaster Model of the Universe) can be encoded by the shift of two-sided continued fractions. Exactly the same shift encodes the sequences of intersections of hyperbolic geodesics with purely imaginary axis in the upper complex half-plane, that is geodesic flow on an appropriate modular surface. A physical interpretation of this coincidence was suggested in arXiv:1402.2158: namely, that Mixmaster chaos is an approximate description of the passage from a hot quantum Universe at the Big Bang moment to the cooling classical Universe. Here we discuss and elaborate this suggestion, looking at the Mixmaster Model from the perspective of the second class of Bianchi IX spacetimes: those with SU(2)-symmetry (self-dual Einstein metrics). We also extend it to the more general context related to Painleve' VI ...

  4. Atomic Data and Spectral Line Intensities for Ca IX

    Science.gov (United States)

    Landi, E.; Bhatia, A. K.

    2012-01-01

    Electron impact collision strengths, energy levels, oscillator strengths and spontaneous radiative decay rates are calculated for Ca IX. We include in the calculations the 33 lowest configurations in the n = 3, 4, 5 complexes, corresponding to 283 fine structure levels in the 3l3l ', 3l4l'' and 3l4l''' configurations, where l,l' = s, p, d, l '' = s, p, d, f and l''' = s, p, d, f, g. Collision strengths are calculated at five incident energies for all transitions: 5.8, 13.6, 24.2, 38.6 and 57.9 Ry above the threshold of each transition. An additional energy, very close to the transition threshold, has been added, whose value is between 0.0055 Ry and 0.23 Ry depending on the levels involved. Calculations have been carried out using the Flexible Atomic Code and the distorted wave approximation. Excitation rate coefficients are calculated as a function of electron temperature by assuming a Maxwellian electron velocity distribution. Using the excitation rate coefficients and the radiative transition rates calculated in the present work, statistical equilibrium equations for level populations are solved at electron densities covering the 10(exp 8)-10(exp 14)/cubic cm range and at an electron temperature of log T(sub e)(K)=5.8, corresponding to the maximum abundance of Ca IX. Spectral line intensities are calculated, and their diagnostic relevance is discussed.

  5. Localization of type II collagen, long form alpha 1(IX) collagen, and short form alpha 1(IX) collagen transcripts in the developing chick notochord and axial skeleton.

    Science.gov (United States)

    Swiderski, R E; Solursh, M

    1992-06-01

    In this study we compare, by in situ hybridization, the spatial and temporal expression patterns of transcripts of avian type II collagen and the long and short forms of the (alpha 1) chain of type IX collagen during the development of the notochord and axial skeleton. We observed type II collagen and short form type IX collagen transcripts in the developing (stage 25-28) nonchondrogenic notochord. Conversely, long form type IX transcripts were not detectable in the notochord or perinotochordal sheath. Interestingly, all three transcripts colocalized in the developing chondrogenic vertebrae of the axial skeleton as well as in the chondrocranium and Meckel's cartilage. The expression of the short form of type IX collagen in these regions was more restricted than that of the long form. This report provides additional support for a complex regulatory pathway of cartilage marker gene expression in chondrogenic vs. nonchondrogenic tissues during avian embryogenesis.

  6. Coagulation Factor Xa inhibits cancer cell migration via Protease-activated receptor-1 activation

    NARCIS (Netherlands)

    Borensztajn, Keren; Bijlsma, Maarten F.; Reitsma, Pieter H.; Peppelenbosch, Maikel R.; Spek, C. Arnold

    2009-01-01

    Cell migration is critically important in (patho) physiological processes. The metastatic potential of cancer cells partly depends on activation of the coagulation cascade. The aim of the present study was to determine whether coagulation factor X (FXa) can regulate the migration and invasion of can

  7. Factors that Motivate and Inhibit Educators to Apply for the Principalship

    Science.gov (United States)

    Morton, William

    2011-01-01

    The national principal shortage caused by the competing factors of an aging workforce, along with recent changes to the role expectations of the principalship making the position less desirable to potential candidates have been well documented in recent years. Effective schools research shows that principals are second only to classroom teachers…

  8. Through thick and thin: A circulating growth factor inhibits age-related cardiac hypertrophy

    OpenAIRE

    McPherron, Alexandra C.

    2013-01-01

    In an intriguing new study, Loffredo et al., report that joining the circulation of old mice with that of young mice reduces age-related cardiac hypertrophy. They also found that the growth factor GDF11 is a circulating negative regulator of cardiac hypertrophy which suggests that raising GDF11 levels may be useful to treat cardiac hypertrophy associated with aging.

  9. A Place on the Team: The Triumph and Tragedy of Title IX

    Science.gov (United States)

    Suggs, Welch

    2006-01-01

    "A Place on the Team" is the inside story of how Title IX revolutionized American sports. The federal law guaranteeing women's rights in education, Title IX opened gymnasiums and playing fields to millions of young women previously locked out. Journalist Welch Suggs chronicles both the law's successes and failures-the exciting…

  10. Modelling topical photodynamic therapy treatment including the continuous production of Protoporphyrin IX

    Science.gov (United States)

    Campbell, C. L.; Brown, C. T. A.; Wood, K.; Moseley, H.

    2016-11-01

    Most existing theoretical models of photodynamic therapy (PDT) assume a uniform initial distribution of the photosensitive molecule, Protoporphyrin IX (PpIX). This is an adequate assumption when the prodrug is systematically administered; however for topical PDT this is no longer a valid assumption. Topical application and subsequent diffusion of the prodrug results in an inhomogeneous distribution of PpIX, especially after short incubation times, prior to light illumination. In this work a theoretical simulation of PDT where the PpIX distribution depends on the incubation time and the treatment modality is described. Three steps of the PpIX production are considered. The first is the distribution of the topically applied prodrug, the second in the conversion from the prodrug to PpIX and the third is the light distribution which affects the PpIX distribution through photobleaching. The light distribution is modelled using a Monte Carlo radiation transfer model and indicates treatment depths of around 2 mm during daylight PDT and approximately 3 mm during conventional PDT. The results suggest that treatment depths are not only limited by the light penetration but also by the PpIX distribution.

  11. 75 FR 18245 - Public Federal Regulatory Enforcement Fairness Hearing Region IX Regulatory Fairness Board

    Science.gov (United States)

    2010-04-09

    ... ADMINISTRATION Public Federal Regulatory Enforcement Fairness Hearing Region IX Regulatory Fairness Board.... Small Business Administration (SBA) Region IX Regulatory Fairness Board and the SBA Office of the National Ombudsman will hold a National Regulatory Fairness Hearing on Monday, April 26, 2010, at 1:30 p.m...

  12. Not Second-Class: Title IX, Equity, and Girls' High School Sports

    Science.gov (United States)

    Stader, David L.; Surface, Jeanne L.

    2014-01-01

    Title IX is designed to protect students from discrimination based on sex in any educational institution that receives financial assistance. This article focuses on Title IX as it applies to high school athletic programs by considering the trial of a high school district in California. A federal court found considerable inequalities between boys…

  13. "What Do I Think about Title IX?" Voices from a University Community

    Science.gov (United States)

    Paule-Koba, Amanda L.; Harris, Othello; Freysinger, Valeria J.

    2013-01-01

    Despite the apparent benefits of Title IX, the implementation of the law remains controversial, and there are divergent beliefs regarding its impact on collegiate sport. The purpose of this study was to examine how members of a university community, whose intercollegiate sport programs have changed, perceive and make sense of Title IX and the…

  14. Inhibition of elongation factor-2 kinase augments the antitumor activity of Temozolomide against glioma.

    Directory of Open Access Journals (Sweden)

    Xiao-Yuan Liu

    Full Text Available BACKGROUND: Glioblastoma multiforme (GBM, the most common form of brain cancer with an average survival of less than 12 months, is a highly aggressive and fatal disease characterized by survival of glioma cells following initial treatment, invasion through the brain parenchyma and destruction of normal brain tissues, and ultimately resistance to current treatments. Temozolomide (TMZ is commonly used chemotherapy for treatment of primary and recurrent high-grade gliomas. Nevertheless, the therapeutic outcome of TMZ is often unsatisfactory. In this study, we sought to determine whether eEF-2 kinase affected the sensitivity of glioma cells to treatment with TMZ. METHODOLOGY/PRINCIPAL FINDINGS: Using RNA interference approach, a small molecule inhibitor of eEF-2 kinase, and in vitro and in vivo glioma models, we observed that inhibition of eEF-2 kinase could enhance sensitivity of glioma cells to TMZ, and that this sensitizing effect was associated with blockade of autophagy and augmentation of apoptosis caused by TMZ. CONCLUSIONS/SIGNIFICANCE: These findings demonstrated that targeting eEF-2 kinase can enhance the anti-glioma activity of TMZ, and inhibitors of this kinase may be exploited as chemo-sensitizers for TMZ in treatment of malignant glioma.

  15. Cinnamaldehyde Inhibits Staphylococcus aureus Virulence Factors and Protects against Infection in a Galleria mellonella Model

    Science.gov (United States)

    Ferro, Thiago A. F.; Araújo, Jéssica M. M.; dos Santos Pinto, Bruna L.; dos Santos, Jéssica S.; Souza, Eliene B.; da Silva, Bruna L. R.; Colares, Valderlane L. P.; Novais, Tânia M. G.; Filho, Clovis M. B.; Struve, Carsten; Calixto, João B.; Monteiro-Neto, Valério; da Silva, Luís C. N.; Fernandes, Elizabeth S.

    2016-01-01

    Bacterial resistance to the available marketed drugs has prompted the search of novel therapies; especially in regards of anti-virulence strategies that aim to make bacteria less pathogenic and/or decrease their probability to become resistant to therapy. Cinnamaldehyde is widely known for its antibacterial properties through mechanisms that include the interaction of this compound with bacterial cell walls. However, only a handful of studies have addressed its effects on bacterial virulence, especially when tested at sub-inhibitory concentrations. Herein, we show for the first time that cinnamaldehyde is bactericidal against Staphylococcus aureus and Enterococcus faecalis multidrug resistant strains and does not promote bacterial tolerance. Cinnamaldehyde actions were stronger on S. aureus as it was able to inhibit its hemolytic activity on human erythrocytes and reduce its adherence to latex. Furthermore, cinnamaldehyde enhanced the serum-dependent lysis of S. aureus. In vivo testing of cinnamaldehyde in Galleria mellonella larvae infected with S. aureus, showed this compound improves larvae survival whilst diminishing bacterial load in their hemolymph. We suggest that cinnamaldehyde may represent an alternative therapy to control S. aureus-induced bacterial infections as it presents the ability to reduce bacterial virulence/survival without promoting an adaptive phenotype. PMID:28066373

  16. Lichen-forming fungus Caloplaca flavoruscens inhibits transcription factors and chromatin remodeling system in fungi.

    Science.gov (United States)

    Kwon, Youngho; Cha, Jaeyul; Chiang, Jennifer; Tran, Grant; Nislow, Corey; Hur, Jae-Seoun; Kwak, Youn-Sig

    2016-06-01

    Lichen-forming fungi and extracts derived from them have been used as alternative medicine sources for millennia and recently there has been a renewed interest in their known bioactive properties for anticancer agents, cosmetics and antibiotics. Although lichen-forming fungus-derived compounds are biologically and commercially valuable, few studies have been performed to determine their modes of action. This study used chemical-genetic and chemogenomic high-throughput analyses to gain insight into the modes of action of Caloplaca flavoruscens extracts. High-throughput screening of 575 lichen extracts was performed and 39 extracts were identified which inhibited yeast growth. A C. flavoruscens extract was selected as a promising antifungal and was subjected to genome-wide haploinsufficiency profiling and homozygous profiling assays. These screens revealed that yeast deletion strains lacking Rsc8, Pro1 and Toa2 were sensitive to three concentrations (IC25.5, IC25 and IC50, respectively) of C. flavoruscens extract. Gene-enrichment analysis of the data showed that C. flavoruscens extracts appear to perturb transcription and chromatin remodeling.

  17. Response of malignant scalp dermatofibrosarcoma to presurgical targeted growth factor inhibition.

    Science.gov (United States)

    Mattox, Austin K; Mehta, Ankit I; Grossi, Peter M; Cummings, Thomas J; Adamson, D Cory

    2010-05-01

    Dermatofibrosarcoma protuberans (DFSP) is an uncommon, locally aggressive, malignant cutaneous tumor that sparingly presents on the scalp. Dermatofibrosarcomas often result from the formation of a fusion oncogene on translocated or supernumerary ring chromosomes 17 and 22, causing the overexpression of PDGFRbeta driven by the COL1A1 promoter. Because of uncertainty surrounding appropriate treatment of aggressive scalp DFSP, the authors performed an extensive review of the available data from a MEDLINE (Ovid) search to describe the clinical presentation and treatment options for this rare tumor. Their search identified 39 different cases, including the illustrative case presented in this study. Adjuvant therapy for this malignant lesion is not universally established in the literature. In the present case, the authors successfully treated a locally invasive scalp DFSP with presurgical therapy that specifically inhibited the PDGFbeta receptor. Imatinib significantly shrank the DFSP tumor mass, reduced hypervascularity, reduced metabolic activity on PET scanning, and permitted a safe gross-total resection. Although wide excision and Mohs micrographic surgery remain the standard surgical treatments for DFSP, the authors illustrate that presurgical chemotherapeutic treatment by imatinib provides a critical adjunct to traditional therapy.

  18. Specific inhibiting effects of Ilexonin A on von Willebrand factor-dependent platelet aggregation under high shear rate

    Institute of Scientific and Technical Information of China (English)

    李敏; 吴伟康; 刘良; 廖福龙; 篠原幸人; 半田俊之介; 後藤信哉

    2004-01-01

    Background Ilexonin A (IA), purified from the Chinese herbal medicine Maodongqing (Ilex pubescens Hook, et Am) has been commonly used in south China to treat thrombotic disorders. In this study, we aimed to study the inhibiting effects and mechanism of lA on von Willebrand factor (vWF)-dependent high shear-induced platelet aggregation. Methods vWF-dependent high shear (10 800 s-1) induced aggregation of platelets obtained from normal donors in the presence or absence of lA was measured by a modified cone-plate viscometer and shear-induced vWF binding was measured by quantitative flowcytometry with monoclonal antibody known to bind exclusively to the C-terminal domain of vWF (LJ-C3) directly labeled with fluorescein isothiocyanate (FITC). P-selectin surface expression was also measured by a similar method with FITC conjugated anti-P-selectin monoclonal antibody (WGA1).Results Shear-induced platelet aggregation was inhibited by IA in a dose-dependent manner. The extent of aggregation decreased from (78.6±4.6)% in the absence of lA to (36.5±2.1 )% in the presence of lA (3.3 mmol/L) (P<0.0001, n=9) with a high shear rate of 10800 s-1. vWF binding and P-selectin expression were also inhibited by lA in a dose dependent manner. The number of binding FITC-LJ-C3 molecules increased after exposure of platelet-rich plasma to a high shear rate of 10 800 s-1 for 6 minutes, but this shear-induced increased binding platelet surface vWF molecules and P-selectin expression can be decreased in the presence of IA.Conclusion vWF binding and vWF mediated platelet activation, aggregation occurring under high shear rate were inhibited by IA. lA may be a unique antithrombotic drug inhibiting the vWF-GP Ib α interaction, and may thus facilitate drug design targeting arterial thrombosis.

  19. Occurrence and functionality of cycle inhibiting factor, cytotoxic necrotising factors and cytolethal distending toxins in Escherichia coli isolated from calves and dogs in Italy.

    Science.gov (United States)

    Salvarani, S; Tramuta, C; Nebbia, P; Robino, P

    2012-06-01

    Escherichia coli isolated from animals up to three months of age, with diarrhea (255 calves and 29 dogs (pups)), without diarrhea (21 calves and 11 pups, used as controls), and 58 adult dogs with cystitis were tested to investigate the occurrence and functional expression of cyclomodulins cycle inhibiting factor (CIF), cytotoxic necrotizing factors (CNFs) and cytolethal distending toxins (CDTs). In cyclomodulin-positive isolates the association was assessed with other virulence genotypes and phylogenetic groups. Of 374 E. coli isolates, 80 (21.4%) were positive for at least one cyclomodulin and 14 of the latter (3.7%) showed different combinations of more than one. cif-positive isolates showed a low number of additional virulence factors, and were commonly associated with phylogroup B1, while cnf- and cdt-positive isolates, harboring many extraintestinal virulence factors, belonged to phylogroups B2 and D. Almost all isolates showed an irreversible cytopathic effect (CPE), displaying functionality of cyclomodulins. Five isolates that presented a mutation of cif were CPE-negative.

  20. Inhibition of B-NHEJ in Plateau-Phase Cells Is Not a Direct Consequence of Suppressed Growth Factor Signaling

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Satyendra K.; Bednar, Theresa; Zhang Lihua [Institute of Medical Radiation Biology, University Duisburg-Essen Medical School, Essen (Germany); Wu, Wenqi [First Affiliated Hospital of Guangzhou, Urology Department, Minimally Invasive Center, Medical College, 510230 Guangzhou (China); Mladenov, Emil [Institute of Medical Radiation Biology, University Duisburg-Essen Medical School, Essen (Germany); Iliakis, George, E-mail: Georg.Iliakis@uk-essen.de [Institute of Medical Radiation Biology, University Duisburg-Essen Medical School, Essen (Germany)

    2012-10-01

    Purpose: It has long been known that the proliferation status of a cell is a determinant of radiation response, and the available evidence implicates repair of DNA double-strand breaks (DSBs) in the underlying mechanism. Recent results have shown that a novel, highly error-prone pathway of nonhomologous end joining (NHEJ) operating as backup (B-NHEJ) processes DSBs in irradiated cells when the canonical, DNA-PK (DNA-dependent protein kinase)-dependent pathway of NHEJ (D-NHEJ) is compromised. Notably, B-NHEJ shows marked reduction in efficiency when D-NHEJ-deficient cells cease to grow and enter a plateau phase. This phenomenon is widespread and observed in cells of different species with defects in core components of D-NHEJ, with the notable exception of DNA-PKcs (DNA-dependent protein kinase, catalytic subunit). Using new, standardized serum-deprivation protocols, we re-examine the growth requirements of B-NHEJ and test the role of epidermal growth factor receptor (EGFR) signaling in its regulation. Methods and Materials: DSB repair was measured by pulsed-field gel electrophoresis in cells maintained under different conditions of growth. Results: Serum deprivation in D-NHEJ-deficient cells causes a rapid reduction in B-NHEJ similar to that measured in normally growing cells that enter the plateau phase of growth. Upon serum deprivation, reduction in B-NHEJ activity is evident at 4 h and reaches a plateau reflecting maximum inhibition at 12-16 h. The inhibition is reversible, and B-NHEJ quickly recovers to the levels of actively growing cells upon supply of serum to serum-deprived cells. Chemical inhibition of EGFR in proliferating cells inhibits only marginally B-NHEJ and addition of EGFR in serum-deprived cells increases only a marginally B-NHEJ. Conclusions: The results document a rapid and fully reversible adaptation of B-NHEJ to growth activity and point to factors beyond EGFR in its regulation. They show notable differences in the regulation of error

  1. Apoptosis of THP-1 macrophages induced by protoporphyrin IX-mediated sonodynamic therapy

    Directory of Open Access Journals (Sweden)

    Guo S

    2013-06-01

    Full Text Available Shuyuan Guo,1* Xin Sun,1,2* Jiali Cheng,1 Haobo Xu,1 Juhua Dan,2 Jing Shen,3 Qi Zhou,4 Yun Zhang,1 Lingli Meng,1 Wenwu Cao,4,5 Ye Tian1,2 1Division of Cardiology, the First Affiliated Hospital, Cardiovascular Institute, Harbin Medical University, Harbin, People's Republic of China; 2Division of Pathophysiology, the State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education, Harbin, People's Republic of China; 3Division of Oncology, the Third Affiliated Hospital, Harbin Medical University, Harbin, People's Republic of China; 4Laboratory of Photo- and Sono-theranostic Technologies and Condensed Matter Science and Technology Institute, Harbin Institute of Technology, Harbin, People's Republic of China; 5Department of Mathematics and Materials Research Institute, Pennsylvania State University, University Park, PA, USA *These authors contributed equally to this work Background: Sonodynamic therapy (SDT was developed as a localized ultrasound-activated cytotoxic therapy for cancer. The ability of SDT to destroy target tissues selectively is especially appealing for atherosclerotic plaque, in which selective accumulation of the sonosensitizer, protoporphyrin IX (PpIX, had been demonstrated. Here we investigate the effects of PpIX-mediated SDT on macrophages, which are the main culprit in progression of atherosclerosis. Methods and results: Cultured THP-1 derived macrophages were incubated with PpIX. Fluorescence microscopy showed that the intracellular PpIX concentration increased with the concentration of PpIX in the incubation medium. MTT assay demonstrated that SDT with PpIX significantly decreased cell viability, and this effect increased with duration of ultrasound exposure and PpIX concentration. PpIX-mediated SDT induced both apoptosis and necrosis, and the maximum apoptosis to necrosis ratio was obtained after SDT with 20 µg/mL PpIX and five minutes of sonication

  2. Mactosylceramide Prevents Glial Cell Overgrowth by Inhibiting Insulin and Fibroblast Growth Factor Receptor Signaling

    DEFF Research Database (Denmark)

    Gerdøe-Kristensen, Stine; Lund, Viktor K; Wandall, Hans H

    2017-01-01

    , in which the mannosyltransferase Egghead controls conversion of glucosylceramide (GlcCer) to mactosylceramide (MacCer). Lack of elongated GSL in egghead (egh) mutants causes overgrowth of subperineurial glia (SPG), largely due to aberrant activation of phosphatidylinositol 3-kinase (PI3K). However, to what...... extent this effect involves changes in upstream signaling events is unresolved. We show here that glial overgrowth in egh is strongly linked to increased activation of Insulin and Fibroblast Growth Factor receptors (FGFR). Glial hypertrophy is phenocopied when overexpressing gain-of-function mutants...... hyperactivation is caused by absence of MacCer and not by GlcCer accumulation. We conclude that an early product in GSL biosynthesis, MacCer, prevents inappropriate activation of Insulin and Fibroblast Growth Factor Receptors in Drosophila glia. This article is protected by copyright. All rights reserved....

  3. Synthesis and Biological Evaluation of Manassantin Analogues for Hypoxia-Inducible FactorInhibition

    OpenAIRE

    Kwon, Do-Yeon; Lee, Hye Eun; Weitzel, Douglas H.; PARK, KYUNGHYE; Lee, Sun Hee; Lee, Chen-Ting; Stephenson, Tesia N.; Park, Hyeri; Fitzgerald, Michael C.; Chi, Jen-Tsan; Mook, Robert A.; Dewhirst, Mark W.; Lee, You Mie; Hong, Jiyong

    2015-01-01

    To cope with hypoxia, tumor cells have developed a number of adaptive mechanisms mediated by hypoxia-inducible factor 1 (HIF-1) to promote angiogenesis and cell survival. Due to significant roles of HIF-1 in the initiation, progression, metastasis, and resistance to treatment of most solid tumors, a considerable amount of effort has been made to identify HIF-1 inhibitors for treatment of cancer. Isolated from Saururus cernuus, manassantins A (1) and B (2) are potent inhibitors of HIF-1 activi...

  4. Electrical inhibition of lens epithelial cell proliferation: an additional factor in secondary cataract?

    OpenAIRE

    Wang, Entong; Reid, Brian; Lois, Noemi; Forrester, John V.; McCaig, Colin D.; Zhao, Min

    2005-01-01

    Cataract is the most common cause of blindness but is at least curable by surgery. Unfortunately, many patients gradually develop the complication of posterior capsule opacification (PCO) or secondary cataract. This arises from stimulated cell growth within the lens capsule and can greatly impair vision. It is not fully understood why residual lens epithelial cell growth occurs after surgery. We propose and show that cataract surgery might remove an important inhibitory factor for lens cell g...

  5. The Maf factor Traffic jam both enables and inhibits collective cell migration in Drosophila oogenesis.

    Science.gov (United States)

    Gunawan, Felix; Arandjelovic, Mimi; Godt, Dorothea

    2013-07-01

    Border cell cluster (BCC) migration in the Drosophila ovary is an excellent system to study the gene regulatory network that enables collective cell migration. Here, we identify the large Maf transcription factor Traffic jam (Tj) as an important regulator of BCC migration. Tj has a multifaceted impact on the known core cascade that enables BCC motility, consisting of the Jak/Stat signaling pathway, the C/EBP factor Slow border cells (Slbo), and the downstream effector DE-cadherin (DEcad). The initiation of BCC migration coincides with a Slbo-dependent decrease in Tj expression. This reduction of Tj is required for normal BCC motility, as high Tj expression strongly impedes migration. At high concentration, Tj has a tripartite negative effect on the core pathway: a decrease in Slbo, an increase in the Jak/Stat inhibitor Socs36E, and a Slbo-independent reduction of DEcad. However, maintenance of a low expression level of Tj in the BCC during migration is equally important, as loss of tj function also results in a significant delay in migration concomitant with a reduction of Slbo and consequently of DEcad. Taken together, we conclude that the regulatory feedback loop between Tj and Slbo is necessary for achieving the correct activity levels of migration-regulating factors to ensure proper BCC motility.

  6. Berberine inhibits cyclin D1 expression via suppressed binding of AP-1 transcription factors to CCND1 AP-1 motif

    Institute of Scientific and Technical Information of China (English)

    Ye LUO; Yu HAO; Tai-ping SHI; Wei-wei DENG; Na LI

    2008-01-01

    Aim: To verify the suppressive effect of berberine on the proliferation of the human pulmonary giant cell carcinoma cell line PG and to demonstrate the mecha-nisms behind the antitumoral effects of berberine. Methods: The proliferative effects of PG cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetry. The cell cycle was examined by flow cytometry. The expression level of cyclin D1 was detected by RT-PCR. The activities of the activating protein-1 (AP-1) and NF-κB signaling pathways related to cyclin D1 were examined by luciferase assay. The cytoplasmic level of c-Jun was detected by Western blot analysis. An electrophoretic mobility shift assay was used to examiae the binding of transcription factors to the cyclin D1 gene (CCNDl) AP-1 motif. Results: The results showed that the proliferation of PG cells treated with different concentrations (10, 20, and 40 μg/mL) of berberine for 24 and 48 h was suppressed