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Sample records for activated factor ix

  1. Cleavage and activation of human factor IX by serine proteases

    SciTech Connect

    Enfield, D.L.; Thompson, A.R.

    1984-10-01

    Human factor IX circulates as a single-chain glycoprotein. Upon activation in vitro, it is cleaved into disulfide-linked light and heavy chains and an activation peptide. After reduction of activated /sup 125/I-factor IX, the heavy and light chains are readily identified by gel electrophoresis. A direct, immunoradiometric assay for factor IXa was developed to assess activation of factor IX for proteases that cleaved it. The assay utilized radiolabeled antithrombin III with heparin to identify the active site and antibodies to distinguish factor IX. After cleavage of factor IX by factor XIa, factor VIIa-tissue thromboplastin complex, or the factor X-activating enzyme from Russell's viper venom, antithrombin III bound readily to factor IXa. Cleavage of /sup 125/I-factor IX by trypsin, chymotrypsin, and granulocyte elastase in the presence of calcium yielded major polypeptide fragments of the sizes of the factor XIa-generated light and heavy chains. When the immunoradiometric assay was used to assess trypsin-cleaved factor IX, the product bound antithrombin III, but not maximally. After digesting with insolubilized trypsin, clotting activity confirmed activation. In evaluating activation of factor IX, physical evidence of activation cleavages does not necessarily correlate with generation of an active site.

  2. Characterization of the clotting activities of structurally different forms of activated factor IX. Enzymatic properties of normal human factor IXa alpha, factor IXa beta, and activated factor IX Chapel Hill.

    PubMed Central

    Griffith, M J; Breitkreutz, L; Trapp, H; Briet, E; Noyes, C M; Lundblad, R L; Roberts, H R

    1985-01-01

    Two structurally different forms of activated human Factor IX (Factor IXa alpha and IXa beta) have been previously reported to have essentially identical clotting activity in vitro. Although it has been shown that activated Factor IX Chapel Hill, an abnormal Factor IX isolated from the plasma of a patient with mild hemophilia B, and normal Factor IXa alpha are structurally very similar, the clotting activity of activated Factor IX Chapel Hill is much lower (approximately fivefold) than that of normal Factor IXa beta. In the present study we have prepared activated Factor IX by incubating human Factor IX with calcium and Russell's viper venom covalently bound to agarose. Fractionation of the activated Factor IX by high-performance liquid chromatography demonstrated the presence of both Factors IXa alpha and IXa beta. On the basis of active site concentration, determined by titration with antithrombin III, the clotting activities of activated Factor IX Chapel Hill and IXa alpha were similar, but both activities were less than 20% of the clotting activity of Factor IXa beta. Activated Factor IX activity was also measured in the absence of calcium, phospholipid, and Factor VIII, by determination of the rate of Factor X activation in the presence of polylysine. In the presence of polylysine, the rates of Factor X activation by activated Factor IX Chapel Hill, Factor IXa alpha, and Factor IXa beta were essentially identical. We conclude that the clotting activity of activated Factor IX Chapel Hill is reduced when compared with that of Factor IXa beta but essentially normal when compared with that of Factor IXa alpha. PMID:3871202

  3. Evidence for a prevalent dimorphism in the activation peptide of human coagulation factor IX.

    PubMed Central

    McGraw, R A; Davis, L M; Noyes, C M; Lundblad, R L; Roberts, H R; Graham, J B; Stafford, D W

    1985-01-01

    We have independently isolated and characterized cDNA and genomic clones for the human coagulation factor IX. Sequence analysis in both cases indicates that threonine is encoded by the triplet ACT as the third residue of the activation peptide. This is in agreement with some earlier reports but in disagreement with others that show the alanine triplet GCT at this position. The discrepancy can thus be accounted for by natural variation of a single nucleotide in the normal population. Amino acid sequence analyses of activated factor IX from plasma samples of four individuals yielded two cases of alanine and two cases of threonine at the third position of the activation peptide. In factor IX from pooled plasma and in factor IX from a heterozygous individual, however, both alanine and threonine were found. Taken together, the findings show that a prevalent nondeleterious dimorphism exists in the activation peptide of human coagulation factor IX. PMID:3857619

  4. Factor IX Amagasaki: A new mutation in the catalytic domain resulting in the loss of both coagulant and esterase activities

    SciTech Connect

    Miyata, Toshiyuki; Iwanaga, Sadaaki ); Sakai, Toshiyuki; Sugimoto, Mitsuhiko; Naka, Hiroyuki; Yamamoto, Kazukuni; Yoshioka, Akira; Fukui, Hiromu ); Mitsui, Kotoko; Kamiya, Kensyu; Umeyama, Hideaki )

    1991-11-26

    Factor IX Amagasaki (AMG) is a naturally occurring mutant of factor IX having essentially no coagulant activity, even though normal levels of antigen are detected in plasma. Factor IX AMG was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Factor IX AMG was cleaved normally by factor VIIa-tissue factor complex, yielding a two-chain factor IXa. Amino acid composition and sequence analysis of one of the tryptic peptides isolated from factor IX AMG revealed that Gly-311 had been replaced by Glu. The authors identified a one-base substitution of guanine to adenine in exon VIII by amplifying exon VIII using the polymerase chain reaction method and sequencing the product. This base mutation also supported the replacement of Gly-311 by Glu. In the purified system, factor IXa AMG did not activate for factor X in the presence of factor VIII, phospholipids, and Ca{sup 2+}, and no esterase activity toward Z-Arg-p-nitrobenzyl ester was observed. The model building of the serine protease domain of factor IXa suggests that the Gly-311 {yields} Glu exchange would disrupt the specific conformational state in the active site environment, resulting in the substrate binding site not forming properly. This is the first report to show the experimental evidence for importance of a highly conserved Gly-142 (chymotrypsinogen numbering) located in the catalytic site of mammalian serine proteases so far known.

  5. Fixing human factor IX (fIX): correction of a cryptic RNA splice enables the production of biologically active fIX in the mammary gland of transgenic mice.

    PubMed Central

    Yull, F; Harold, G; Wallace, R; Cowper, A; Percy, J; Cottingham, I; Clark, A J

    1995-01-01

    Transgenic mice and sheep secrete only low levels of human factor IX in their milk because of an aberrant splicing of the transgene RNA in the mammary gland. Removal of the cryptic 3' splice site prevents this splicing and leads to the production of relatively high levels of factor IX. The purified protein is fully active showing that the mammary gland is capable of the efficient post-translational modification of this protein and that transgenic animals are a suitable means of its production. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7479906

  6. Polymorphism of normal factor IX detected by mouse monoclonal antibodies.

    PubMed Central

    Wallmark, A; Ljung, R; Nilsson, I M; Holmberg, L; Hedner, U; Lindvall, M; Sjögren, H O

    1985-01-01

    Hemophilia B is an X-chromosomal recessive disease due to deficiency of coagulation factor IX. Three monoclonal antibodies against factor IX were prepared and used to develop immunoradiometric assays (IRMAs) of factor IX antigen (IX-Ag). IX-Ag was measured in 65 normal individuals with one IRMA based on polyclonal anti-IX antibodies and two IRMAs based on three monoclonal anti-IX antibodies. One of the monoclonal antibodies differed in specificity since it neutralized less than 50% of the clotting activity of factor IX (IX-C), whereas the other two monoclonal antibodies neutralized 80-95%. When the former antibody was used as the solid phase in IRMA, two groups of normal individuals were distinguished: group A with measurable IX-Ag, and group B without demonstrable IX-Ag. There were no differences between the groups either in IX-C or in IX-Ag measured with polyclonal antibodies. A subgroup comprising only women could be distinguished in group A, in whom intermediate IX-Ag concentrations were found. Family studies showed the group B variant of normal factor IX to be transmitted according to the pattern of X-linked recessive inheritance. The allelic frequency of group A was 0.66, and that of group B was 0.34. PMID:3873655

  7. Hemophilia as a defect of the tissue factor pathway of blood coagulation: Effect of factors VIII and IX on factor X activation in a continuous-flow reactor

    SciTech Connect

    Repke, D.; Gemmell, C.H.; Guha, A.; Turitto, V.T.; Nemerson, Y. ); Broze, G.J. Jr. )

    1990-10-01

    The effect of factors VIII and IX on the ability of the tissue factor-factor VIIa complex to activate factor X was studied in a continuous-flow tubular enzyme reactor. Tissue factor immobilized in a phospholipid bilayer on the inner surface of the tube was exposed to a perfusate containing factors VIIa, VIII, IX, and X flowing at a wall shear rate of 57, 300, or 1130 sec{sup {minus}1}. The addition of factors VIII and IX at their respective plasma concentrations resulted in a further 2{endash}-to 3{endash}fold increase. The direct activation of factor X by tissue factor-factor VIIa could be virtually eliminated by the lipoprotein-associated coagulation inhibitor. These results suggest that the tissue factor pathway, mediated through factors VIII and IX, produces significant levels of factor Xa even in the presence of an inhibitor of the tissue factor-factor VIIa complex; moreover, the activation is dependent on local shear conditions. These findings are consistent both with a model of blood coagulation in which initiation of the system results from tissue factor and with the bleeding observed in hemophilia.

  8. Influence of factor VIII:C and factor IX activity in plasmas of haemophilic dogs on the activated partial thromboplastin time measured with two commercial reagents.

    PubMed

    Mischke, R

    2000-05-01

    The present study is based on 145 plasma samples with a reduced activity of factor VIII:C (range: 0.009-0.62 IU mL-1) and 28 samples with a reduced factor IX activity (range: 0.035-0.55 IU mL-1). The samples were collected from dogs with haemophilia A (n=22) or haemophilia B (n=3), some of these during substitution therapy. For all samples the activated partial thromboplastin time (APTT) was measured with two commercial reagents containing kaolin as a contact activator. In each case, the deficiency of factor VIII:C or IX was reflected in abnormal results of the APTT. This was true for both reagents. A significant correlation (P < 0.001) was found between factor VIII:C activity and APTT (reagent 1, Pathromtin(R); Spearman's rank correlation coefficient, rS=-0.731, reagent 2, PTT-Reagenz; rS=-0.875) as well as between factor IX activity and APTT (reagent 1, rS=-0.819; reagent 2, rS=-0.955]. In each case, the relationship between coagulation factor activity and APTT could be proven most precisely by geometric regression. The results of this study illustrate the applicability of commercial APTT test kits as a sensitive screening test of factor VIII:C and IX deficiencies in canine plasma. PMID:10792470

  9. Prolonged activity of factor IX as a monomeric Fc fusion protein.

    PubMed

    Peters, Robert T; Low, Susan C; Kamphaus, George D; Dumont, Jennifer A; Amari, John V; Lu, Qi; Zarbis-Papastoitsis, Greg; Reidy, Thomas J; Merricks, Elizabeth P; Nichols, Timothy C; Bitonti, Alan J

    2010-03-11

    Treatment of hemophilia B requires frequent infusions of factor IX (FIX) to prophylax against bleeding episodes. Hemophilia B management would benefit from a FIX protein with an extended half-life. A recombinant fusion protein (rFIXFc) containing a single FIX molecule attached to the Fc region of immunoglobulin G was administered intravenously and found to have an extended half-life, compared with recombinant FIX (rFIX) in normal mice, rats, monkeys, and FIX-deficient mice and dogs. Recombinant FIXFc protein concentration was determined in all species, and rFIXFc activity was measured in FIX-deficient animals. The half-life of rFIXFc was approximately 3- to 4-fold longer than that of rFIX in all species. In contrast, in mice in which the neonatal Fc receptor (FcRn) was deleted, the half-life of rFIXFc was similar to rFIX, confirming the increased circulatory time was due to protection of the rFIXFc via the Fc/FcRn interaction. Whole blood clotting time in FIX-deficient mice was corrected through 144 hours for rFIXFc, compared with 72 hours for rFIX; similar results were observed in FIX-deficient dogs. Taken together, these studies show the enhanced pharmacodynamic and pharmacokinetic properties of the rFIXFc fusion protein and provide the basis for evaluating rFIXFc in patients with hemophilia B. PMID:20056791

  10. Use of factor IX concentrates in active teenagers with hemophilia B.

    PubMed

    Tagariello, Giuseppe

    2004-06-01

    This paper addresses issues specific to the treatment of teenagers with hemophilia B. All teenagers, including those with hemophilia, are at high risk of being exposed to accident-related trauma because of activities inherent within normal adolescent development. To reduce the impact of recurrent joint bleeds, studies have shown that prophylaxis should be started at an early age, with treatment individualized due to variability in patient pharmacokinetics and product recoveries. A key factor in the acceptance and success of prophylaxis in teenagers is the training given at hemophilia treatment centers regarding self-treatment dosing and infusion schedules. Both plasma-derived and recombinant products are appropriate in this cohort, depending on the patient's and family's experiences and preferences. PMID:15322451

  11. Molecular interactions of the intrinsic activation complex of coagulation: binding of native and activated human factors IX and X to defined phospholipid vesicles.

    PubMed

    Burri, B J; Edgington, T S; Fair, D S

    1987-02-20

    The assembly of proteins of the intrinsic activation complex has been partially elucidated. In the present study we examine the association of gamma-carboxylated serine proteinase zymogens factors IX and X, and their proteolytically activated counterparts factors IXa and Xa to unilamellar lipid vesicles of defined composition using three types of physical measurement. Utilizing relative light scatter to estimate the dissociation constants for binding in the presence of calcium ions, it appears that factor IXa (0.93 +/- 0.37 microM) may preferentially associate with phospholipids relative to factor IX (0.35 +/- 0.08 microM). In contrast, factor X (0.34 +/- 0.14 microM), the substrate for factor IXa, appears to bind to phospholipid with a higher affinity than factor Xa (0.58 +/- 0.13 microM). These observations are compatible with the hypothesized dynamics where the forward 'traffic' is facilitated by favoring the association of factor IXa with factor X. The dissociation constants were estimated by molecular exclusion chromatography (1.1 - 2.5 microM) and do not reflect these relative and ordered differences in association with lipid vesicles. Quasi-elastic light scatter analyses indicate that each protein appears to saturate the same vesicle surface, consistent with competition for similar surface lipids, although the molecular shell formed by factor Xa (36 A) is smaller, suggesting that it has a different packing on the phospholipid surface than the other proteins (64-79 A). The pattern of preferential affinities for phospholipid is consistent with a kinetically functional forward traffic through the reaction precursors to products, and suggests that these preferential affinities may assist in the ordering of the four proteins in the intrinsic activation complex. PMID:3493031

  12. Monoclonal antibodies to coagulation factor IX define a high-frequency polymorphism by immunoassays.

    PubMed Central

    Smith, K J

    1985-01-01

    Monoclonal antibodies have been used to demonstrate a polymorphism of human plasma coagulation factor IX antigen in double antibody solid-phase immunoradiometric assays. This polymorphism is detected in an assay where a monoclonal antibody (A-1) adsorbed to microtiter wells is used to bind factor IX from diluted plasma samples. Plasma samples with the factor IX polymorphism have less than 0.2 U/ml of apparent antigen when tested with the A-1 antibody, while assays with other monoclonal antibodies and assays with goat antisera to factor IX show normal amounts of factor IX antigen. Factor IX coagulant activity was normal in samples from donors with the polymorphism. The thin-layer polyacrylamide gel isoelectric focusing pattern of factor IX purified from a donor with the factor IX polymorphism (IXp) was identical to that obtained with factor IX prepared from a donor who did not have the polymorphism (IXn). Purified radiolabeled factor IX prepared from a donor with the polymorphism showed a Ka for the A-1 antibody that was threefold less than that measured for IXn. The gene frequency of IXp in male blood donors is 0.25. This polymorphism may be useful as a marker for the X chromosome in genetic studies on plasma samples. Further studies are necessary to determine the explanation for decreased reaction of IXp with the A-1 monoclonal antibody. Images Fig. 1 PMID:9556657

  13. Identification of the endothelial cell binding site for factor IX.

    PubMed Central

    Cheung, W F; van den Born, J; Kühn, K; Kjellén, L; Hudson, B G; Stafford, D W

    1996-01-01

    We previously demonstrated that the primary region of factor IX and IXa responsible for saturable specific binding to bovine aortic endothelial cells resides in residues 3-11 at the amino terminus of factor IX. We also demonstrated that mutations of lysine to alanine at residue 5, factor IX K5A, or valine to lysine at residue 10, factor IX V10K, resulted in a molecule unable to bind to endothelial cells. Moreover, a mutation with lysine to arginine at residue 5, factor IX K5R, resulted in a factor IX molecule with increased affinity for the endothelial cell binding site. In this paper we report that collagen IV is a strong candidate for the factor IX binding site on endothelial cells. Factor IX and factor IX K5R compete with 125I-labeled factor IX for binding to tetrameric collagen IV immobilized on microtiter plates, while factor X, factor VII, and factor IX K5A or V10K fail to compete. The Kd for wild-type factor IX binding to collagen IV in the presence of heparin was 6.8 +/- 2 nM, and the Kd for factor IX K5R was 1.1 +/- 0.2 nM, which agrees well with our previously published Kd values of 7.4 and 2.4 nM for binding of the same proteins to endothelial cells. Our working assumption is that we have identified the endothelial cell binding site and that it is collagen IV. Its physiological relevance remains to be determined. PMID:8855310

  14. Labeled factor IX kinetics in patients with hemophilia-B

    SciTech Connect

    Smith, K.J.; Thompson, A.R.

    1981-09-01

    Labeled factor IX was infused five time into four patients with hemophilia-B. Ten-minute plasma recovery average 35% (SD +/- 2) and the mean T 1/2 beta-phase elimination was 23 hr (+/- 5). No alteration in the postinfusion 125I-factor-IX could be detected by radioautography of plasma samples run on polyacrylamide gels or on crossed-immunoelectrophoresis. Label was excreted into the urine as free 125I-iodide. Kinetics were similar when the labeled preparation was infused alone or with a commercial concentrate containing unlabeled factor IX. Infusion of factor IX in man is best described by a two-compartment open pharmacokinetic model where factor IX is distributed in a space larger than the plasma volume.

  15. The Conundrum of "Warfarin Hypersensitivity": Prolonged Partial Thromboplastin Time From Factor IX Propeptide Mutation.

    PubMed

    Sekhri, Arunabh; Lisinschi, Adriana; Furqan, Muhammad; Palaniswamy, Chandrasekar; Mukhi, Nikhil; Gupta, Ridhi; Nelson, John C

    2016-01-01

    Carboxylation of glutamic acid residues of vitamin K dependent clotting factors (II, VII, IX, and X) is essential to their biological functioning. Binding of these factors to γ-glutamyl carboxylase enzyme for carboxylation reaction is mediated by wild-type propeptide, a small sequence of amino acids that precede the actual polypeptide. Missense mutations at certain residue severely decrease the affinity of mutated propeptide for the enzyme. Such mutations are reported to occur at codon-10 of factor IX propeptide, a clinically silent metabolic event in normal conditions. However in the presence of warfarin, such mutations and resultant decrease affinity of factor IX propeptide for the enzyme that causes severe selective decrease in factor IX activity. This can potentially leads to life-threatening bleeding complications and known as one of the causes of warfarin hypersensitivity. It is imperative to recognize such cases early on to avoid additional warfarin therapy. Recurrent bleeding episodes, subtherapeutic to therapeutic range international normalized ratio values with relatively prolong partial thromboplastin time should raise the suspicion of underlying factor IX propeptide mutations. PMID:24832385

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

    SciTech Connect

    Shounan Yao; Wilson, J.M.; Nabel, E.G.; Kurachi, Sumiko; Hachiya, H.L.; Kurachi, Kotoku )

    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.

  17. Recombinant Human Factor IX Produced from Transgenic Porcine Milk

    PubMed Central

    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. PMID:24955355

  18. Long-acting recombinant coagulation factor IX albumin fusion protein (rIX-FP) in hemophilia B: results of a phase 3 trial

    PubMed Central

    Martinowitz, Uri; Lissitchkov, Toshko; Pan-Petesch, Brigitte; Hanabusa, Hideji; Oldenburg, Johannes; Boggio, Lisa; Negrier, Claude; Pabinger, Ingrid; von Depka Prondzinski, Mario; Altisent, Carmen; Castaman, Giancarlo; Yamamoto, Koji; Álvarez-Roman, Maria-Teresa; Voigt, Christine; Blackman, Nicole; Jacobs, Iris

    2016-01-01

    A global phase 3 study evaluated the pharmacokinetics, efficacy, and safety of recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in 63 previously treated male patients (12-61 years) with severe hemophilia B (factor IX [FIX] activity ≤2%). The study included 2 groups: group 1 patients received routine prophylaxis once every 7 days for 26 weeks, followed by either 7-, 10-, or 14-day prophylaxis regimen for a mean of 50, 38, or 51 weeks, respectively; group 2 patients received on-demand treatment of bleeding episodes for 26 weeks and then switched to a 7-day prophylaxis regimen for a mean of 45 weeks. The mean terminal half-life of rIX-FP was 102 hours, 4.3-fold longer than previous FIX treatment. Patients maintained a mean trough of 20 and 12 IU/dL FIX activity on prophylaxis with rIX-FP 40 IU/kg weekly and 75 IU/kg every 2 weeks, respectively. There was 100% reduction in median annualized spontaneous bleeding rate (AsBR) and 100% resolution of target joints when subjects switched from on-demand to prophylaxis treatment with rIX-FP (P < .0001). The median AsBR was 0.00 for all prophylaxis regimens. Overall, 98.6% of bleeding episodes were treated successfully, including 93.6% that were treated with a single injection. No patient developed an inhibitor, and no safety concerns were identified. These results indicate rIX-FP is safe and effective for preventing and treating bleeding episodes in patients with hemophilia B at dosing regimens of 40 IU/kg weekly and 75 IU/kg every 2 weeks. This trial was registered at www.clinicaltrials.gov as #NCT0101496274. PMID:26755710

  19. Long-acting recombinant coagulation factor IX albumin fusion protein (rIX-FP) in hemophilia B: results of a phase 3 trial.

    PubMed

    Santagostino, Elena; Martinowitz, Uri; Lissitchkov, Toshko; Pan-Petesch, Brigitte; Hanabusa, Hideji; Oldenburg, Johannes; Boggio, Lisa; Negrier, Claude; Pabinger, Ingrid; von Depka Prondzinski, Mario; Altisent, Carmen; Castaman, Giancarlo; Yamamoto, Koji; Álvarez-Roman, Maria-Teresa; Voigt, Christine; Blackman, Nicole; Jacobs, Iris

    2016-04-01

    A global phase 3 study evaluated the pharmacokinetics, efficacy, and safety of recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in 63 previously treated male patients (12-61 years) with severe hemophilia B (factor IX [FIX] activity ≤2%). The study included 2 groups: group 1 patients received routine prophylaxis once every 7 days for 26 weeks, followed by either 7-, 10-, or 14-day prophylaxis regimen for a mean of 50, 38, or 51 weeks, respectively; group 2 patients received on-demand treatment of bleeding episodes for 26 weeks and then switched to a 7-day prophylaxis regimen for a mean of 45 weeks. The mean terminal half-life of rIX-FP was 102 hours, 4.3-fold longer than previous FIX treatment. Patients maintained a mean trough of 20 and 12 IU/dL FIX activity on prophylaxis with rIX-FP 40 IU/kg weekly and 75 IU/kg every 2 weeks, respectively. There was 100% reduction in median annualized spontaneous bleeding rate (AsBR) and 100% resolution of target joints when subjects switched from on-demand to prophylaxis treatment with rIX-FP (P< .0001). The median AsBR was 0.00 for all prophylaxis regimens. Overall, 98.6% of bleeding episodes were treated successfully, including 93.6% that were treated with a single injection. No patient developed an inhibitor, and no safety concerns were identified. These results indicate rIX-FP is safe and effective for preventing and treating bleeding episodes in patients with hemophilia B at dosing regimens of 40 IU/kg weekly and 75 IU/kg every 2 weeks. This trial was registered atwww.clinicaltrials.govas #NCT0101496274. PMID:26755710

  20. New polymorphic variants of human blood clotting factor IX

    SciTech Connect

    Surin, V.L.; Luk`yanenko, A.V.; Tagiev, A.F.; Smirnova, O.V.; Plutalov, O.V.; Berlin, Yu.A.

    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.

  1. G244E in the canine factor IX gene leads to severe haemophilia B in Rhodesian Ridgebacks.

    PubMed

    Mischke, R; Kühnlein, P; Kehl, A; Langbein-Detsch, I; Steudle, F; Schmid, A; Dandekar, T; Czwalinna, A; Müller, E

    2011-01-01

    Haemophilia B in Rhodesian Ridgebacks is currently the most important canine haemophilia in Germany. The aim of this study was to define the underlying genetic defect. Genetic studies were performed including six phenotypically affected male dogs (factor IX activity: approximately 1%), four suspected carriers (factor IX activity 48-69%, one confirmed by affected offspring), and 12 healthy dogs. Comparison of the entire coding region of the canine factor IX DNA sequences and exon-intron junctions from affected dogs with the wild type canine factor IX DNA revealed a G-A missense mutation in exon 7. This mutation results in a glycine (GGA) to glutamic acid (GAA) exchange in the catalytic domain of the haemophilic factor IX. All affected dogs were hemizygous for the detected mutation and carriers were heterozygous, whereas none of the Rhodesian Ridgebacks with normal factor IX activity showed the mutation. No further alterations in the sequences between affected dogs and the healthy control group could be observed. None of the Rhodesian Ridgebacks with undefined haemophilia B status (n=30) and no individual of three other dog breeds (Doberman Pinscher: n=20; German Wire haired Pointer: n=20; Labrador: n=25) showed the presence of the mutation. Amino acid sequence alignment and protein structural modelling analysis indicate that the detected mutation causes a relevant functional defect. The results of this study suggest that the detected mutation is responsible for a severe form of haemophilia B in Rhodesian Ridgebacks. PMID:20303304

  2. Improved muscle-derived expression of human coagulation factor IX from a skeletal actin/CMV hybrid enhancer/promoter.

    PubMed

    Hagstrom, J N; Couto, L B; Scallan, C; Burton, M; McCleland, M L; Fields, P A; Arruda, V R; Herzog, R W; High, K A

    2000-04-15

    Hemophilia B is caused by the absence of functional coagulation factor IX (F.IX) and represents an important model for treatment of genetic diseases by gene therapy. Recent studies have shown that intramuscular injection of an adeno-associated viral (AAV) vector into mice and hemophilia B dogs results in vector dose-dependent, long-term expression of biologically active F.IX at therapeutic levels. In this study, we demonstrate that levels of expression of approximately 300 ng/mL (6% of normal human F.IX levels) can be reached by intramuscular injection of mice using a 2- to 4-fold lower vector dose (1 x 10(11) vector genomes/mouse, injected into 4 intramuscular sites) than previously described. This was accomplished through the use of an improved expression cassette that uses the cytomegalovirus (CMV) immediate early enhancer/promoter in combination with a 1.2-kilobase portion of human skeletal actin promoter. These results correlated with enhanced levels of F.IX transcript and secreted F.IX protein in transduced murine C2C12 myotubes. Systemic F.IX expression from constructs containing the CMV enhancer/promoter alone was 120 to 200 ng/mL in mice injected with 1 x 10(11) vector genomes. Muscle-specific promoters performed poorly for F.IX transgene expression in vitro and in vivo. However, the incorporation of a sequence from the alpha-skeletal actin promoter containing at least 1 muscle-specific enhancer and 1 enhancer-like element further improved muscle-derived expression of F.IX from a CMV enhancer/promoter-driven expression cassette over previously published results. These findings will allow the design of a clinical protocol for therapeutic levels of F.IX expression with lower vector doses, thus enhancing efficacy and safety of the protocol. (Blood. 2000;95:2536-2542) PMID:10753832

  3. The endothelial cell binding determinant of human factor IX resides in the. gamma. -carboxyglutamic acid domain

    SciTech Connect

    Toomey, J.R.; Roberts, H.R.; Stafford, D.W. ); Smith, K.J. United Blood Services, Albuquerque, NM )

    1992-02-18

    The blood coagulation factor IX(a) binds specifically to a site on endothelial cells with a K{sub d} of 2.0-3.0 nM. A number of previous studies have attempted to define the region(s) of factor IX(a) that mediate this interaction. These studies suggested that there are two regions of factor IX(a), the {gamma}-carboxyglutamic acid (Gla) domain and the epidermal growth factor like (EGF-like) domains, that mediate high-affinity binding to endothelial cells. Recently, however, the participation of the EGF1 domain has been excluded from the interaction. This indicated that if there was an EGF component of factor IX contributing to the binding affinity, then it must be in the second EGF-like domain. In order to further evaluate this relationship, the authors performed competitive binding experiments between {sup 125}I plasma factor IX and a set of six chimeric proteins composed of portions of factor VII and factor IX. The data suggest that the high-affinity interaction between factor IX and the endothelial cell binding site is mediated by the factor IX Gla domain and that the factor IX EGF domains are not involved in binding specificity.

  4. Transforming the treatment for hemophilia B patients: update on the clinical development of recombinant fusion protein linking recombinant coagulation factor IX with recombinant albumin (rIX-FP).

    PubMed

    Santagostino, Elena

    2016-05-01

    Recombinant fusion protein linking recombinant coagulation factor IX with recombinant albumin (rIX-FP; Idelvion®(†)) is an innovative new treatment designed to extend the half-life of factor IX (FIX) and ease the burden of care for hemophilia B patients. The rIX-FP clinical development program - PROLONG-9FP - is in its advanced phases, with pivotal studies in previously treated adults, adolescents, and pediatrics now completed. Across all age groups studied, rIX-FP has demonstrated a markedly improved pharmacokinetic profile compared with plasma-derived and recombinant FIX treatments, with a 30-40% higher incremental recovery, an approximately 5-fold longer half-life, a lower clearance, and a greater area under the curve. rIX-FP has been very well tolerated with an excellent safety profile. In the pivotal studies, there have been no reports of FIX inhibitors or antidrug antibodies, and few treatment-related adverse events have been observed. Prophylactic regimens of rIX-FP administered once weekly to once every 14 days have been highly effective. When used for surgical prophylaxis, a single infusion of rIX-FP has been sufficient to maintain hemostasis, even during major orthopedic surgery. An ongoing study is now enrolling previously untreated patients and evaluating the possibility of extending the dosing interval to every 21 days. There is little doubt that rIX-FP will transform the treatment of hemophilia B. PMID:27288064

  5. Sensitivity of the reaction time of the resonance thrombogram for factor VIII:C and factor IX deficiencies in the blood of dogs with haemophilia A or B.

    PubMed

    Mischke, R

    2000-09-01

    The sensitivity of the reaction time of the resonance thrombogram (RTG-r) was examined for two instruments based on 105 canine samples with a reduced factor VIII:C activity and 26 samples with a reduced factor IX activity. These samples were taken from dogs suffering from haemophilia A (n=18) or B (n=3) before and at different times after the substitution therapy with fresh frozen plasma. The RTG-r sensitivity was low; with reference to the total of all examined samples, it was between 0.39 and 0.65 for the factor VIII:C activity and between 0.46 and 0.62 for the factor IX activity, depending on the instrument and the testing procedure used. Low sensitivity was shown especially by the fact that even some of the samples with a factor VIII:C activity < 5% showed false negative results. Moreover, the correlation found between RTG-r and factor VIII:C activity (Spearman's rank correlation coefficient, r(S)=-0.556 to -0.700) as well as between factor IX activity and RTG-r (r(S)=-0.380 to -0.612) was only moderate. The low sensitivity of the RTG towards a reduced activity of the haemophilia factors VIII:C and IX contradicts the exclusive use of the RTG for monitoring haemophilic dogs as well as a global screening test for the haemostasis of dogs. PMID:11012705

  6. A study of reported factor IX use around the world.

    PubMed

    Stonebraker, J S; Bolton-Maggs, P H B; Brooker, M; Farrugia, A; Srivastava, A

    2011-05-01

    Replacement therapy has significantly improved the life expectancy and lifestyle of people with haemophilia. The objectives of this article were to study the reported factor IX (FIX) use on a country-by-country basis and address the following question: Does the reported FIX use vary by national economies? We obtained data on the reported number of international units (IUs) of FIX used for 90 countries from the Marketing Research Bureau and the World Federation of Hemophilia. Results show that the reported FIX use varies considerably across national economies, even among the wealthiest of countries.Trends suggest that the reported FIX usage increases with increasing economic capacity and has been increasing over time. Trends also suggest that consumption of FIX has been increasing at a greater rate in high income countries. Given these trends, there will likely be an overall increase in the amount of FIX concentrates used in the treatment of haemophilia B. We also found that FIX use both in terms of IUs per capita and IUs per person provide a complete picture of the level of haemophilia care within a country. Such information is critical for planning efforts of national healthcare agencies to determine realistic budget priorities and pharmaceutical manufacturers to determine adequate production levels of FIX concentrates. By improving the data collection and surveillance of FIX use for the treatment of people with haemophilia B, we can identify trends and needs of patients and highlight best treatment practices among countries. PMID:21299742

  7. Factor VIII/factor IX prophylaxis for severe hemophilia.

    PubMed

    Carcao, Manuel; Srivastava, Alok

    2016-01-01

    Experience with clotting factor concentrate (CFC) replacement products over several decades has shown that regular replacement (prophylaxis) is the only way to prevent musculoskeletal damage in hemophilia and impact the natural history of hemophilia. Yet there is a lack of data on the optimal age to start such replacement therapy and the regimens to be used. While very early administration of high doses is certainly more effective in preventing bleeding, cost and compliance are major constraints all over the world. Starting prophylaxis with even lower doses comparable to that used in episodic therapies leads to major reduction in bleeding. Recognition of the clinical heterogeneity of hemophilia even among patients with a label of severe hemophilia in terms of their spontaneous bleeding has led to efforts aimed at individualizing CFC replacement, based on clinical responses or pharmacokinetic data of the CFC. The importance of long-term outcome assessment being combined with CFC replacement therapy cannot be overemphasized. PMID:26805901

  8. Recombinant to modified factor VIII and factor IX - chromogenic and one-stage assays issues.

    PubMed

    Kitchen, S; Kershaw, G; Tiefenbacher, S

    2016-07-01

    The recent development of modified recombinant factor VIII (FVIII) and factor IX (FIX) therapeutic products with extended half-lives will create challenges for the haemostasis laboratory in obtaining recovery estimates of these products in clinical samples using existing assays. The new long-acting therapeutic concentrates contain molecular modifications of Fc fusion, site-specific of polyethylene glycol or albumin fusion. The optimum methods for monitoring each new product will need to be assessed individually and laboratories should select an assay which gives similar results to the assay used to assign potency to the product in question. For some extended half-life FVIII and FIX products some one stage assays are entirely unsuitable for monitoring purposes. For most products and assay reagents studied so far, and reviewed in this manuscript, chromogenic FVIII or FIX assays can be safely used with conventional plasma standards. If one stage assays are used then they should be performed using carefully selected reagents/methods which have been shown to recover activity close to the labelled potency for the specific product being monitored. PMID:27405680

  9. Signaling-mediated cooperativity between glycoprotein Ib-IX and protease-activated receptors in thrombin-induced platelet activation.

    PubMed

    Estevez, Brian; Kim, Kyungho; Delaney, M Keegan; Stojanovic-Terpo, Aleksandra; Shen, Bo; Ruan, Changgeng; Cho, Jaehyung; Ruggeri, Zaverio M; Du, Xiaoping

    2016-02-01

    Thrombin-induced cellular response in platelets not only requires protease-activated receptors (PARs), but also involves another thrombin receptor, the glycoprotein Ib-IX complex (GPIb-IX). It remains controversial how thrombin binding to GPIb-IX stimulates platelet responses. It was proposed that GPIb-IX serves as a dock that facilitates thrombin cleavage of protease-activated receptors, but there are also reports suggesting that thrombin binding to GPIb-IX induces platelet activation independent of PARs. Here we show that GPIb is neither a passive thrombin dock nor a PAR-independent signaling receptor. We demonstrate a novel signaling-mediated cooperativity between PARs and GPIb-IX. Low-dose thrombin-induced PAR-dependent cell responses require the cooperativity of GPIb-IX signaling, and conversely, thrombin-induced GPIb-IX signaling requires cooperativity of PARs. This mutually dependent cooperativity requires a GPIb-IX-specific 14-3-3-Rac1-LIMK1 signaling pathway, and activation of this pathway also requires PAR signaling. The cooperativity between GPIb-IX signaling and PAR signaling thus drives platelet activation at low concentrations of thrombin, which are important for in vivo thrombosis. PMID:26585954

  10. Recombinant Factor IX Fc Fusion Protein Maintains Full Procoagulant Properties and Exhibits Prolonged Efficacy in Hemophilia B Mice

    PubMed Central

    Toby, Garabet G.; Liu, Tongyao; Buyue, Yang; Zhang, Xin; Bitonti, Alan J.; Pierce, Glenn F.; Sommer, Jurg M.; Jiang, Haiyan; Peters, Robert T.

    2016-01-01

    Introduction Hemophilia B is an inherited X chromosome–linked disorder characterized by impaired blood clotting owing to the absence of functional coagulation factor IX. Due to the relatively short half-life of factor IX, patients with hemophilia B require frequent factor IX infusions to maintain prophylaxis. We have developed a recombinant factor IX (rFIX) fused to the Fc region of IgG (rFIXFc) with an extended half-life in animals and humans. Materials and Methods Procoagulant properties of rFIXFc and rFIX (BENEFIX®) were compared to determine the effect of the Fc region on rFIXFc hemostatic function. Specifically, we assessed rFIXFc activation, intermolecular interactions within the Xase complex, inactivation by antithrombin III (AT) and thrombin generation potential compared with rFIX. We also assessed the acute and prophylactic efficacy profiles of rFIXFc and rFIX in vivo in hemophilia B mouse bleeding models. Results and Conclusions The activation by factor XIa or factor VIIa/tissue factor, inhibition by AT, interaction profiles with phospholipids, affinities for factor VIIIa within the context of the Xase complex, and thrombin generation profiles were similar for rFIXFc and rFIX. Xase complexes formed with either molecule exhibited similar kinetic profiles for factor Xa generation. In acute efficacy models, mice infused with rFIXFc or rFIX were equally protected from bleeding. However, in prophylactic efficacy models, protection from bleeding was maintained approximately three times longer in rFIXFc-dosed mice than in those given rFIX; this prolonged efficacy correlates with the previously observed half-life extension. We conclude that rFIXFc retains critical FIX procoagulant attributes and that the extension in rFIXFc half-life translates into prolonged efficacy in hemophilia B mice. PMID:26840952

  11. Concurrent acquired inhibitors to factor VIII and IX, a laboratory artifact: a case report

    PubMed Central

    Doma, Saša Anžej; Hillarp, Andreas; Pajič, Tadej; Andoljšek, Dušan; Černelč, Peter; Preldžnik Zupan, Irena

    2016-01-01

    Acquired inhibitors to coagulation factors other than factor VIII are extremely rare. We describe a case of a 59-year-old woman with abnormal bleeding, diagnosed with concurrent inhibitor antibodies to factor VIII and IX by Bethesda testing. We demonstrate that anti-FVIII antibodies of a very high titre are capable of disturbing the aPTT-based Bethesda assay, resulting in falsely-positive antibodies to factor IX. The case also illustrates the usefulness of the immunological assay (ELISA) in complementing the inhibitor diagnosis. PMID:27346976

  12. Comparison in different species of biliary bilirubin-IX alpha conjugates with the activities of hepatic and renal bilirubin-IX alpha-uridine diphosphate glycosyltransferases.

    PubMed Central

    Fevery, J; Van de Vijver, M; Michiels, R; Heirwegh, K P

    1977-01-01

    The bilrubin-IXalpha conjugates in bile and the activities of bilirubin-IX alpha--UDP-glycosyltransferases in liver and kidney were determined for ten species of mammals and for the chicken. 1. In the mammalian species, bilirubin-IX alpha glucuronide was the predominant bile pigment. Excretion of neutral glycosides was unimportant, except in the cat, the mouse, the rabbit and the dog, where glucose and xylose represented 12--41% of total conjugating groups bound to bilirubin-IX alpha. In chicken bile, glucoside and glucuronide conjugates were of equal importance. They probably represent only a small fraction of the total bile pigment. 2. The transferase activities in liver showed pronounced species variation. This was also apparent with regard to activation by digitonin, pH optimum and relative activities of transferases acting on either UDP-glucuronic acid or neutral UDP-sugars. 3. Man, the dog, the cat and the rat excrete bilirubin-IX alpha largely as diconjugated derivatives. In general, diconjugated bilirubin-IX alpha could also be synthesized in vitro with liver homogenate, bilirubin-IX alpha and UDP-sugar. In contrast, for the other species examined, bilirubin pigments consisted predominantly of monoconjugated bilirubin-IX alpha. Synthesis in vitro with UDP-glucuronic acid, UDP-glucose or UDP-xylose as the sugar donor led exclusively to the formation of monoconjugated bilirubin-IX alpha. 4. The transferase activities in the kidney were restricted to the cortex and were important only for the rat and the dog. No activity at all could be detected for several species, including man. 5. Comparison of the transferase activities in liver with reported values of the maximal rate of excretion in bile suggests a close linkage between conjugation and biliary secretion of bilirubin-IX alpha. PMID:407905

  13. Manic depressive illness not linked to factor IX region in an independent series of pedigrees.

    PubMed

    Gejman, P V; Detera-Wadleigh, S; Martinez, M M; Berrettini, W H; Goldin, L R; Gelernter, J; Hsieh, W T; Gershon, E S

    1990-12-01

    We studied seven informative kindreds segregating for manic depressive illness (MDI), consistent with X-chromosome transmission of the trait (families do not show affective disease in both a father and a son), using markers mapped to the region of Xq27-Xq28. The lod scores were consistently below -2 in the region extending from about 10 cM centromeric from the Factor IX locus (F9) to the colorblindness region. This study does not replicate previous reports of linkage of MDI to Factor IX (Xq27) and colorblindness region (Xq28) chromosomal markers in other kindreds. PMID:1980485

  14. Catalytic activity of human carbonic anhydrase isoform IX is displayed both extra- and intracellularly.

    PubMed

    Klier, Michael; Jamali, Somayeh; Ames, Samantha; Schneider, Hans-Peter; Becker, Holger M; Deitmer, Joachim W

    2016-01-01

    Most carbonic anhydrases catalyse the reversible conversion of carbon dioxide to protons and bicarbonate, either as soluble cytosolic enzymes, in or at intracellular organelles, or at the extracellular face of the cell membrane as membrane-anchored proteins. Carbonic anhydrase isoform IX (CA IX), a membrane-bound enzyme with catalytic activity at the extracellular membrane surface, has come to prominence in recent years because of its association with hypoxic tissue, particularly tumours, often indicating poor prognosis. We have evaluated the catalytic activity of CA IX heterologously expressed in Xenopus laevis oocytes by measuring the amplitude and rate of cytosolic pH changes as well as pH changes at the outer membrane surface (pHs ) during addition and removal of 5% CO2 /25 mm HCO3-, and by mass spectrometry. Our results indicate both extracellular and intracellular catalytic activity of CA IX. Reduced rates of CO2 -dependent intracellular pH changes after knockdown of CA IX confirmed these findings in two breast cancer cell lines: MCF-7 and MDA-MB-231. Our results demonstrate a new function of CA IX that may be important in the search for therapeutic cancer drugs targeting CA IX. PMID:26470855

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

    SciTech Connect

    Thompson, A.R. Puget Sound Blood Center, Seattle, WA ); Darlington, G. ); 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.

  16. Somatic mosaicism and female-to-female transmission in a kindred with hemophilia B (factor IX deficiency)

    SciTech Connect

    Taylor, S.A.M.; Deugau, K.V.; Lillicrap, D.P. )

    1991-01-01

    Studies have shown that hemophilia B (Christmas disease; factor IX deficiency) results from many different mutations in the factor IX gene, of which {gt}95% are single nulceotide substitutions. This study has identified a previously unreported form of hemophilia B in a patient who was a somatic mosaic for a guanine-to-cytosine transversion at nucleotide 31,170 in the factor IX gene. This point mutation changes the codon for residue 350 in the catalytic domain of factor IX from a cysteine to a serine. The authors used differential termination of primer extension to confirm and measure the degree of mosaicism. The study shows that a varying proportion of cells from hepatic, renal, smooth muscle, and hematopoietic populations possessed normal as well as mutant factor IX sequences. These results indicate that the mutation in this patient occurred either as an uncorrected half-chromatid mutation in the female gamete or as a replication or postreplication error in the initial mitotic divisions of the zygote preceding implantation. In addition, this kindred also contains two females in successive generations who have moderately severe factor IX deficiency. The molecular pathogenesis of this latter phenomenon has been studied and seems to relate to the unaccompanied expression of the mutant factor IX gene consequent upon a second, as yet undefined, genetic event that has prevented inactivation of sequences including the mutant factor IX gene on the X chromosome inherited from the affected male.

  17. Improved Induction of Immune Tolerance to Factor IX by Hepatic AAV-8 Gene Transfer

    PubMed Central

    Cooper, Mario; Nayak, Sushrusha; Hoffman, Brad E.; Terhorst, Cox; Cao, Ou

    2009-01-01

    Abstract Gene therapy for hemophilia B has been shown to result in long-term expression and immune tolerance to factor IX (F.IX) after in vivo transduction of hepatocytes with adeno-associated viral (AAV-2) vectors in experimental animals. An optimized protocol was effective in several strains of mice with a factor 9 gene deletion (F9−/−). However, immune responses against F.IX were repeatedly observed in C3H/HeJ F9−/− mice. We sought to establish a gene transfer protocol that results in sustained expression without a requirement for additional manipulation of the immune system. Compared with AAV-2, AAV-8 was more efficient in transgene expression and induction of tolerance to F.IX in three different strains of wild-type mice. At equal vector doses, AAV-8 induced transgene product-specific regulatory CD4+CD25+FoxP3+ T cells at significantly higher frequency. Moreover, sustained correction of hemophilia B in C3H/HeJ F9−/− mice without antibody formation was documented in all animals treated with ≥4 × 1011 vector genomes (VG)/kg and in 80% of mice treated with 8 × 1010 VG/kg. Therefore, it is possible to develop a gene transfer protocol that reliably induces tolerance to F.IX largely independent of genetic factors. A comparison with other studies suggests that additional parameters besides plateau levels of F.IX expression contributed to the improved success rate of tolerance induction. PMID:19309290

  18. Protoporphyrin IX Content Correlates with Activity of Photobleaching Herbicides

    PubMed Central

    Becerril, Jose M.; Duke, Stephen O.

    1989-01-01

    Several laboratories have demonstrated recently that photobleaching herbicides such as acifluorfen and oxadiazon cause accumulation of protoporphyrin IX (PPIX), a photodynamic pigment capable of herbicidal activity. We investigated, in acifluorfen-treated tissues, the in vivo stability of PPIX, the kinetics of accumulation, and the correlation between concentration of PPIX and herbicidal damage. During a 20 hour dark period, PPIX levels rose from barely detectable concentrations to 1 to 2 nanomoles per 50 cucumber (Cucumis sativus L.) cotyledon discs treated with 10 micromolar acifluorfen. When placed in 500 micromoles per square meter per second PAR, PPIX levels decayed logarithmically, with an initial half-life of about 2.5 hours. PPIX levels at each time after exposure to light correlated positively with the cellular damage that occurred during the following 1 hour in both green and yellow (tentoxin-treated) cucumber cotyledon tissues. PPIX levels in discs incubated for 20 hours in darkness correlated positively with the acifluorfen concentration in which they were incubated. In cucumber, the level of herbicidal damage caused by several p-nitrodiphenyl other herbicides, a p-chlorodiphenylether herbicide, and oxadiazon correlated positively with the amount of PPIX induced to accumulate by each of the herbicide treatments. Similar results were obtained with acifluorfen-treated pigweed and velvetleaf primary leaf tissues. In cucumber, PPIX levels increased within 15 and 30 minutes after exposure of discs to 10 micromolar acifluorfen in the dark and light, respectively. These data strengthen the view that PPIX is responsible for all or a major part of the photobleaching activity of acifluorfen and related herbicides. PMID:16666869

  19. Characterization of IXINITY® (Trenonacog Alfa), a Recombinant Factor IX with Primary Sequence Corresponding to the Threonine-148 Polymorph

    PubMed Central

    Monroe, Dougald M.; Jenny, Richard J.; Van Cott, Kevin E.; Saward, Laura L.

    2016-01-01

    The goal of these studies was to extensively characterize the first recombinant FIX therapeutic corresponding to the threonine-148 (Thr-148) polymorph, IXINITY (trenonacog alfa [coagulation factor IX (recombinant)]). Gel electrophoresis, circular dichroism, and gel filtration were used to determine purity and confirm structure. Chromatographic and mass spectrometry techniques were used to identify and quantify posttranslational modifications. Activity was assessed as the ability to activate factor X (FX) both with and without factor VIIIa (FVIIIa) and in a standard clotting assay. All results were consistent across multiple lots. Trenonacog alfa migrated as a single band on Coomassie-stained gels; activity assays were normal and showed <0.002 IU of activated factor IX (FIXa) per IU of FIX. The molecule has >97%  γ-carboxylation and underwent the appropriate structural change upon binding calcium ions. Trenonacog alfa was activated normally with factor XIa (FXIa); once activated it bound to FVIIIa and FXa. When activated to FIXa, it was inhibited efficiently by antithrombin. Glycosylation patterns were similar to plasma-derived FIX with sialic acid content consistent with the literature reports of good pharmacokinetic performance. These studies have shown that trenonacog alfa is a highly pure product with a primary sequence and posttranslational modifications consistent with the common Thr-148 polymorphism of plasma-derived FIX. PMID:26997955

  20. Characterization of IXINITY® (Trenonacog Alfa), a Recombinant Factor IX with Primary Sequence Corresponding to the Threonine-148 Polymorph.

    PubMed

    Monroe, Dougald M; Jenny, Richard J; Van Cott, Kevin E; Buhay, Shelly; Saward, Laura L

    2016-01-01

    The goal of these studies was to extensively characterize the first recombinant FIX therapeutic corresponding to the threonine-148 (Thr-148) polymorph, IXINITY (trenonacog alfa [coagulation factor IX (recombinant)]). Gel electrophoresis, circular dichroism, and gel filtration were used to determine purity and confirm structure. Chromatographic and mass spectrometry techniques were used to identify and quantify posttranslational modifications. Activity was assessed as the ability to activate factor X (FX) both with and without factor VIIIa (FVIIIa) and in a standard clotting assay. All results were consistent across multiple lots. Trenonacog alfa migrated as a single band on Coomassie-stained gels; activity assays were normal and showed <0.002 IU of activated factor IX (FIXa) per IU of FIX. The molecule has >97%  γ-carboxylation and underwent the appropriate structural change upon binding calcium ions. Trenonacog alfa was activated normally with factor XIa (FXIa); once activated it bound to FVIIIa and FXa. When activated to FIXa, it was inhibited efficiently by antithrombin. Glycosylation patterns were similar to plasma-derived FIX with sialic acid content consistent with the literature reports of good pharmacokinetic performance. These studies have shown that trenonacog alfa is a highly pure product with a primary sequence and posttranslational modifications consistent with the common Thr-148 polymorphism of plasma-derived FIX. PMID:26997955

  1. Nonacog gamma, a novel recombinant factor IX with low factor IXa content for treatment and prophylaxis of bleeding episodes.

    PubMed

    Turecek, Peter L; Abbühl, Brigitt; Tangada, Srilatha D; Chapman, Miranda; Gritsch, Herbert; Rottensteiner, Hanspeter; Schrenk, Gerald; Mitterer, Artur; Dietrich, Barbara; Höllriegl, Werner; Schiviz, Alexandra; Horling, Frank; Reipert, Birgit M; Muchitsch, Eva-Maria; Pavlova, Borislava G; Scheiflinger, Friedrich

    2015-03-01

    Nonacog gamma is a new recombinant factor IX to treat factor IX deficiency. It is indicated for control of bleeding episodes, perioperative management and routine prophylaxis to prevent or reduce the frequency of bleeding episodes in adults and children with hemophilia B. Nonacog gamma was first approved in the USA in June 2013 under the trade name RIXUBIS followed by market approvals in Australia and the EU in 2014, and marketing authorization decision is pending in Japan. Nonacog gamma is derived from a recombinant Chinese hamster ovary cell line using a state of the art biotechnological manufacturing process. Recombinant factor IX is produced by Baxter's protein-free fermentation technology, which was first developed for ADVATE. The product is purified and formulated in the absence of any human or animal-derived protein. Nonacog gamma was characterized both in comprehensive in vitro and in vivo non-clinical studies as well as in an extensive clinical trial program. PMID:25660348

  2. The first EGF domain of coagulation factor IX attenuates cell adhesion and induces apoptosis.

    PubMed

    Ishikawa, Tomomi; Kitano, Hisataka; Mamiya, Atsushi; Kokubun, Shinichiro; Hidai, Chiaki

    2016-07-01

    Coagulation factor IX (FIX) is an essential plasma protein for blood coagulation. The first epidermal growth factor (EGF) motif of FIX (EGF-F9) has been reported to attenuate cell adhesion to the extracellular matrix (ECM). The purpose of the present study was to determine the effects of this motif on cell adhesion and apoptosis. Treatment with a recombinant EGF-F9 attenuated cell adhesion to the ECM within 10 min. De-adhesion assays with native FIX recombinant FIX deletion mutant proteins suggested that the de-adhesion activity of EGF-F9 requires the same process of FIX activation as that which occurs for coagulation activity. The recombinant EGF-F9 increased lactate dehydrogenase (LDH) activity release into the medium and increased the number of cells stained with annexin V and activated caspase-3, by 8.8- and 2.7-fold respectively, indicating that EGF-F9 induced apoptosis. Activated caspase-3 increased very rapidly after only 5 min of administration of recombinant EGF-F9. Treatment with EGF-F9 increased the level of phosphorylated p38 mitogen-activated protein kinase (MAPK), but not that of phosphorylated MAPK 44/42 or c-Jun N-terminal kinase (JNK). Inhibitors of caspase-3 suppressed the release of LDH. Caspase-3 inhibitors also suppressed the attenuation of cell adhesion and phosphorylation of p38 MAPK by EGF-F9. Our data indicated that EGF-F9 activated signals for apoptosis and induced de-adhesion in a caspase-3 dependent manner. PMID:27129300

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

    NASA Astrophysics Data System (ADS)

    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.

  4. An unusual complication in a gravida with factor IX deficiency: case report with review of the literature.

    PubMed

    Guy, G P; Baxi, L V; Hurlet-Jensen, A; Chao, C R

    1992-09-01

    Factor IX deficiency (hemophilia B, Christmas disease) is an X-linked recessive coagulation disorder. It occurs in one out of every 25,000-30,000 male births and requires even rarer genetic circumstances for phenotypic expression in females. We report the occurrence of a large, late-trimester subchorionic hematoma in a gravida with factor IX deficiency and with laboratory evidence of consumptive coagulopathy during treatment. The patient was managed conservatively and had a successful outcome at term. The only four reported cases of antepartum management of factor IX deficiency in the English literature are reviewed. PMID:1495722

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

    NASA Technical Reports Server (NTRS)

    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

  6. Inactivation and clearance of viruses during the manufacture of high purity factor IX.

    PubMed

    Johnston, A; Macgregor, A; Borovec, S; Hattarki, M; Stuckly, K; Anderson, D; Goss, N H; Oates, A; Uren, E

    2000-09-01

    Haemophilia is a bleeding disorder characterised by a deficiency in Factor IX. Replacement therapy in the form of a Factor IX concentrate is a widely accepted practice. In this paper we describe a double virus inactivated chromatographic process for producing a high purity Factor IX product, MonoFIX((R))-VF. The process involves separation of the prothrombin complex by cryoprecipitation, fraction I precipitation and DEAE-cellulose adsorption, further ion-exchange chromatography of crude Factor IX, followed by solvent/detergent treatment. Heparin affinity chromatography is then used to further purify Factor IX. Final nanofiltration is sequential through 35 nm then 15 nm membrane filters. The principal virus inactivation/removal steps are solvent/detergent treatment and nanofiltration and the partitioning of relevant and model viruses provides further reduction in virus load through the production process.Solvent/detergent treatment was shown to achieve log reduction factors of 4.5 for HIV-1, 5.1 for Sindbis virus, 6.1 for vesicular stomatitis virus (VSV), 5.1 for bovine viral diarrhoea virus (BVDV) and 5.3 for pseudorabies virus (PRV). BVDV is a model for hepatitis C virus (HCV), and pseudorabies virus (PRV), like hepatitis B virus (HBV) is an enveloped DNA virus. Using scaled down models of the production process, we have also demonstrated the neutralization/partitioning of at least 6 logs of hepatitis A virus (HAV) during cryoprecipitation, Fraction I precipitation, and the DEAE adsorption and elution step, and a further 1.6 log reduction in HAV load as a result of heparin affinity chromatography. The log reduction factors for HAV as a result of the second ion-exchange chromatography step and as a result of enhanced neutralisation associated with solvent/detergent treatment were not significant. Nanofiltration was shown to contribute a further log reduction factor of 6.7 for HAV and 5.8 for BVDV indicating that log reduction factors of this order would be obtained

  7. Computationally designed liver-specific transcriptional modules and hyperactive factor IX improve hepatic gene therapy

    PubMed Central

    Nair, Nisha; Rincon, Melvin Y.; Evens, Hanneke; Sarcar, Shilpita; Dastidar, Sumitava; Samara-Kuko, Emira; Ghandeharian, Omid; Man Viecelli, Hiu; Thöny, Beat; De Bleser, Pieter; Chuah, Marinee K.

    2014-01-01

    The development of the next-generation gene therapy vectors for hemophilia requires using lower and thus potentially safer vector doses and augmenting their therapeutic efficacy. We have identified hepatocyte-specific transcriptional cis-regulatory modules (CRMs) by using a computational strategy that increased factor IX (FIX) levels 11- to 15-fold. Vector efficacy could be enhanced by combining these hepatocyte-specific CRMs with a synthetic codon-optimized hyperfunctional FIX-R338L Padua transgene. This Padua mutation boosted FIX activity up to sevenfold, with no apparent increase in thrombotic risk. We then validated this combination approach using self-complementary adenoassociated virus serotype 9 (scAAV9) vectors in hemophilia B mice. This resulted in sustained supraphysiologic FIX activity (400%), correction of the bleeding diathesis at clinically relevant, low vector doses (5 × 1010 vector genomes [vg]/kg) that are considered safe in patients undergoing gene therapy. Moreover, immune tolerance could be induced that precluded induction of inhibitory antibodies to FIX upon immunization with recombinant FIX protein. PMID:24637359

  8. Why does the human factor IX gene have a G + C content of 40%?

    PubMed Central

    Bottema, C D; Bottema, M J; Ketterling, R P; Yoon, H S; Janco, R L; Phillips, J A; Sommer, S S

    1991-01-01

    The factor IX gene has a G + C content of approximately 40% in all mammalian species examined. In human factor IX, C----T and G----A transitions at the dinucleotide CpG are elevated at least 24-fold relative to other transitions. Can the G + C content be explained solely by this hot spot of mutation? Using our mathematical model, we show that the elevation of mutation at CpG cannot alone lower the G + C content below 45%. To search for other hot spots of mutation that might contribute to the reduction of G + C content, we assessed the relative rates of base substitution in our sample of 160 families with hemophilia B. Seventeen independent single-base substitutions are reported herein for a total of 96 independent point mutations in our sample. The following conclusions emerge from the analysis of our data and, where appropriate, the data of others: (1) Transversions at CpG are elevated an estimated 7.7-fold relative to other transversions. (2) The mutation rates at non-CpG dinucleotides are remarkably uniform; none of the observed rates are either more than twofold above the median for transitions or more than threefold above the median for transversions. (3) The pattern of recent mutation is compatible with the pattern during mammalian evolution that has maintained the G + C content of the factor IX gene at approximately 40%. PMID:1897528

  9. Role of enhanced half-life factor VIII and IX in the treatment of haemophilia.

    PubMed

    Mahdi, Ali J; Obaji, Samya G; Collins, Peter W

    2015-06-01

    Treatment of congenital haemophilia with factor VIII and IX concentrates often requires frequent infusions. This has obvious implications in establishing effective administration strategies and, in turn, adherence. To overcome these issues, three main technologies--polyethylene-glycol, Fc-neonatal IgG1 and albumin fusion products--have emerged into various stages of clinical development. Published data indicates an approximately 1·5- and fivefold increase in half-life of factor VIII and IX, respectively, compared to standard recombinant concentrates. Studies into efficacy and safety are starting to be published. Monitoring and optimal use of these new concentrates remains unknown. Weekly factor IX prophylaxis appears to be a feasible prophylactic regimen in haemophilia B patients. Weekly longer-acting FVIII is unlikely to provide adequate prophylaxis in most patients with haemophilia A but may reduce the frequency of infusions. Ongoing clinical trials and real life experience will help shape how these products can be used in practice and their cost effectiveness. The drive for convenience however should not overshadow the ultimate goal of prophylaxis, namely, preventing bleeding and arthropathy. PMID:25754016

  10. Title IX Resource Guide

    ERIC Educational Resources Information Center

    Office for Civil Rights, US Department of Education, 2015

    2015-01-01

    Title IX of the Education Amendments of 1972 (Title IX) prohibits discrimination based on sex in education programs and activities in federally funded schools at all levels. If any part of a school district or college receives any Federal funds for any purpose, all of the operations of the district or college are covered by Title IX. The essence…

  11. Title IX, Girls' Sports Participation, and Adult Female Physical Activity and Weight

    ERIC Educational Resources Information Center

    Kaestner, Robert; Xu, Xin

    2010-01-01

    Arguably, the most important school-based intervention to increase physical activity was Title IX of the Education Amendments of 1972, which led to a 600% increase in girls' sports participation between 1972 and 1978. We studied the effect of this increase in sports participation and athletic opportunities while young on the physical activity and…

  12. Molecular Basis and Therapeutic Strategies to Rescue Factor IX Variants That Affect Splicing and Protein Function.

    PubMed

    Tajnik, Mojca; Rogalska, Malgorzata Ewa; Bussani, Erica; Barbon, Elena; Balestra, Dario; Pinotti, Mirko; Pagani, Franco

    2016-05-01

    Mutations that result in amino acid changes can affect both pre-mRNA splicing and protein function. Understanding the combined effect is essential for correct diagnosis and for establishing the most appropriate therapeutic strategy at the molecular level. We have identified a series of disease-causing splicing mutations in coagulation factor IX (FIX) exon 5 that are completely recovered by a modified U1snRNP particle, through an SRSF2-dependent enhancement mechanism. We discovered that synonymous mutations and missense substitutions associated to a partial FIX secretion defect represent targets for this therapy as the resulting spliced-corrected proteins maintains normal FIX coagulant specific activity. Thus, splicing and protein alterations contribute to define at the molecular level the disease-causing effect of a number of exonic mutations in coagulation FIX exon 5. In addition, our results have a significant impact in the development of splicing-switching therapies in particular for mutations that affect both splicing and protein function where increasing the amount of a correctly spliced protein can circumvent the basic functional defects. PMID:27227676

  13. Molecular Basis and Therapeutic Strategies to Rescue Factor IX Variants That Affect Splicing and Protein Function

    PubMed Central

    Bussani, Erica; Barbon, Elena; Pinotti, Mirko; Pagani, Franco

    2016-01-01

    Mutations that result in amino acid changes can affect both pre-mRNA splicing and protein function. Understanding the combined effect is essential for correct diagnosis and for establishing the most appropriate therapeutic strategy at the molecular level. We have identified a series of disease-causing splicing mutations in coagulation factor IX (FIX) exon 5 that are completely recovered by a modified U1snRNP particle, through an SRSF2-dependent enhancement mechanism. We discovered that synonymous mutations and missense substitutions associated to a partial FIX secretion defect represent targets for this therapy as the resulting spliced-corrected proteins maintains normal FIX coagulant specific activity. Thus, splicing and protein alterations contribute to define at the molecular level the disease-causing effect of a number of exonic mutations in coagulation FIX exon 5. In addition, our results have a significant impact in the development of splicing-switching therapies in particular for mutations that affect both splicing and protein function where increasing the amount of a correctly spliced protein can circumvent the basic functional defects. PMID:27227676

  14. Potency determination of factor VIII and factor IX for new product labelling and postinfusion testing: challenges for caregivers and regulators.

    PubMed

    Dodt, J; Hubbard, A R; Wicks, S J; Gray, E; Neugebauer, B; Charton, E; Silvester, G

    2015-07-01

    A workshop organized by the European Medicines Agency and the European Directorate for the Quality of Medicines and HealthCare was held in London, UK on November 28-29, 2013, to provide an overview of the current knowledge of the characterization of new factor VIII (FVIII) and factor IX (FIX) concentrates with respect to potency assays and testing of postinfusion material. The objective was to set the basis for regulatory authorities' discussion on the most appropriate potency assay for the individual products, and European Pharmacopoeia (Ph. Eur.) discussion on whether to propose revision of the Ph. Eur. monographs with respect to potency assays in the light of information on new FVIII and FIX concentrates. The workshop showed that for all products valid assays vs. the international concentrate standards were obtained and potency could be expressed in International Units. The Ph. Eur. chromogenic potency assay gave valid assay results which correlate with in vivo functionality of rFVIII products. For some modified rFVIII products and all modified rFIX products, one-stage clotting assay methods result in different potencies depending on the activated partial thromboplastin time reagent. As a consequence, monitoring of patients' postinfusion levels is challenging but it was pointed out that manufacturers are responsible for providing the users with appropriate information for use and laboratory testing of their product. Strategies to avoid misleading determination of patents' plasma levels, e.g. information on suitable assays, laboratory standards or correction factors were discussed. PMID:25623631

  15. Stable and high-level production of recombinant Factor IX in human hepatic cell line.

    PubMed

    de Castilho Fernandes, Andrielle; Fontes, Aparecida; Gonsales, Nathalia; Swiech, Kamilla; Picanco-Castro, Virginia; Faca, Sandra; Covas, Dimas

    2011-01-01

    Hemophilia B is a genetic disease of the coagulation system that affects one in 30,000 males worldwide. Recombinant human Factor IX (rhFIX) has been used for hemophilia B treatment, but the amount of active protein generated by these systems is inefficient, resulting in a high-cost production of rhFIX. In this study, we developed an alternative for rhFIX production. We used a retrovirus system to obtain two recombinant cell lines. We first tested rhFIX production in the human embryonic kidney 293 cells (293). Next, we tested a hepatic cell line (HepG2) because FIX is primarily expressed in the liver. Our results reveal that intracellular rhFIX expression was more efficient in HepG2/rhFIX (46%) than in 293/rhFIX (21%). The activated partial thromboplastin time test showed that HepG2/rhFIX expressed biologically active rhFIX 1.5 times higher than 293/rhFIX (P = 0.016). Recovery of rhFIX from the HepG2 by reversed-phase chromatography was straightforward. We found that rhFIX has a pharmacokinetic profile similar to that of FIX purified from human plasma when tested in hemophilic B model. HepG2/rhFIX cell line produced the highest levels of rhFIX, representing an efficient in vitro expression system. This work opens up the possibility of significantly reducing the costs of rhFIX production, with implications for expanding hemophilia B treatment in developing countries. PMID:21838798

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

    PubMed Central

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

    2016-01-01

    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. PMID:26960037

  17. Functions of AAV-CMV-F.IX And AAV-EF1alpha-F.IX in gene therapy for hemophilia B.

    PubMed

    Wiwanitkit, Viroj

    2007-02-01

    There has been substantial progress in using gene therapy to treat animals with hemophilia. Adeno-associated viral (AAV) gene transfer of coagulation factor IX to skeletal muscle and liver of murine and canine models of hemophilia has resulted in sustained systemic expression and, in several studies, in complete cure of the bleeding disorder. Two AAV vectors widely used at present are AAV-CMV-F.IX and AAV-EF1alpha-F.IX. This work compares the predicted molecular functions of AAV-CMV-F.IX and AAV-EF1alpha -F.IX by sequence docking and gene ontology. It is shown that both AAV-CMV-F.IX and AAV-EF1alpha -F.IX induce coagulation factor IXa activity; however, AAV-CMV-F.IX administration also yields coagulation factor XIa activity and AAV-EF1alpha -F.IX treatment results in coagulation factor Xa activity. Therefore, AAV-CMV-F.IX might be useful for factor XI deficiency. AAV-CMV-F.IX has several additional molecular functions and processes compared with AAV-CMV-F.IX. PMID:17266422

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

    SciTech Connect

    Ketterling, R.P.; Vielhaber, E.L.; Lind, T.J.; Thorland, E.C.; Sommer S.S. )

    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.

  19. Title IX, girls' sports participation, and adult female physical activity and weight.

    PubMed

    Kaestner, Robert; Xin Xu

    2010-02-01

    Arguably, the most important school-based intervention to increase physical activity was Title IX of the Education Amendments of 1972, which led to a 600% increase in girls' sports participation between 1972 and 1978. We studied the effect of this increase in sports participation and athletic opportunities while young on the physical activity and weight of adult women some 20-25 years later. Our results indicate that adult women who were affected by Title IX and had greater opportunity to participate in athletics while young had lower body mass index (BMI) and lower rates of obesity and reported being more physically active than women who were not afforded these opportunities. However, effect sizes were quite modest. PMID:20130236

  20. Effects of Title IX and sports participation on girls' physical activity and weight.

    PubMed

    Kaestner, Robert; Xu, Xin

    2007-01-01

    In this study, we examined the association between girls' participation in high school sports and the physical activity, weight, body mass, and body composition of adolescent females during the 1970s when girls' sports participation was dramatically increasing as a result of Title IX. We found that increases in girls' participation in high school sports, a proxy for expanded athletic opportunities for adolescent females, were associated with an increase in physical activity and an improvement in weight and body mass among girls. In contrast, adolescent boys experienced a decline in physical activity and an increase in weight and body mass during the period when girls' athletic opportunities were expanding. Taken together, these results strongly suggest that Title IX and the increase in athletic opportunities among adolescent females it engendered had a beneficial effect on the health of adolescent girls. PMID:19548549

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

    PubMed Central

    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 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 Galα(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. PMID:18456721

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

    NASA Astrophysics Data System (ADS)

    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.

  3. Inhibitors of propagation of coagulation (factors VIII, IX and XI): a review of current therapeutic practice

    PubMed Central

    Franchini, Massimo; Mannucci, Pier Mannuccio

    2011-01-01

    The management of patients with congenital haemophilia who develop alloantibodies against factors of the propagation phase of blood coagulation, commonly known as inhibitors, is the most important challenge facing haemophilia caregivers at present, as this complication not only compromises the efficacy of replacement therapy but also consumes an enormous amount of economic resources. Development of inhibitors further complicates the clinical course of severe haemophilia, with a prevalence of up to 30% in patients with haemophilia A (factor VIII deficiency) and up to 5% in those with haemophilia B (factor IX deficiency) and haemophilia C (factor XI deficiency). While the short-term goal of treatment of patients who develop alloantibodies is the control of bleeding, the eradication of the inhibitor is the main long-term goal. The management of severe bleeding episodes and the eradication of the autoantibody are also the mainstays of treatment of patients with acquired haemophilia, a rare but life-threatening haemorrhagic condition characterized by the development of inhibitory autoantibodies against coagulation factor VIII. The most recent options available for treating patients with congenital haemophilia complicated by inhibitors and acquired haemophilia because of autoantibodies against factor VIII are summarized in this review article. PMID:21204915

  4. Liver gene therapy by lentiviral vectors reverses anti-factor IX pre-existing immunity in haemophilic mice.

    PubMed

    Annoni, Andrea; Cantore, Alessio; Della Valle, Patrizia; Goudy, Kevin; Akbarpour, Mahzad; Russo, Fabio; Bartolaccini, Sara; D'Angelo, Armando; Roncarolo, Maria Grazia; Naldini, Luigi

    2013-11-01

    A major complication of factor replacement therapy for haemophilia is the development of anti-factor neutralizing antibodies (inhibitors). Here we show that liver gene therapy by lentiviral vectors (LVs) expressing factor IX (FIX) strongly reduces pre-existing anti-FIX antibodies and eradicates FIX inhibitors in haemophilia B mice. Concomitantly, plasma FIX levels and clotting activity rose to 50-100% of normal. The treatment was effective in 75% of treated mice. FIX-specific plasma cells (PCs) and memory B cells were reduced, likely because of memory B-cell depletion in response to constant exposure to high doses of FIX. Regulatory T cells displaying FIX-specific suppressive capacity were induced in gene therapy treated mice and controlled FIX-specific T helper cells. Gene therapy proved safer than a regimen mimicking immune tolerance induction (ITI) by repeated high-dose FIX protein administration, which induced severe anaphylactoid reactions in inhibitors-positive haemophilia B mice. Liver gene therapy can thus reverse pre-existing immunity, induce active tolerance to FIX and establish sustained FIX activity at therapeutic levels. These data position gene therapy as an attractive treatment option for inhibitors-positive haemophilic patients. PMID:24106222

  5. The Ripple Effect of Title IX on Women's Health Issues: Treating an Increasingly Active Population.

    PubMed

    Mees, Patricia D

    2003-04-01

    Perhaps no area in sports medicine has changed as dramatically in the last 30 years as women's health. Title IX of the Education Amendments of 1972 prohibited discrimination on the basis of sex in all curricular and extracurricular activities at educational institutions that receive federal funding. Before 1972, many assumed that women were not interested in sports and that there was no need to provide programs for girls and women, and most primary care physicians had little experience in treating female athletes and other active women. PMID:20086462

  6. Factor IXMadrid 2: a deletion/insertion in factor IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site.

    PubMed Central

    Solera, J; Magallón, M; Martin-Villar, J; Coloma, A

    1992-01-01

    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' end of intron d and the two last coding nucleotides located at the 3' end of exon IV in the normal factor IX gene; this fragment has been replaced by a 47-bp sequence from the normal factor IX gene, although this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends of the deleted DNA fragment. Images Figure 1 PMID:1346483

  7. Effective gene therapy for haemophilic mice with pathogenic factor IX antibodies

    PubMed Central

    Markusic, David M; Hoffman, Brad E; Perrin, George Q; Nayak, Sushrusha; Wang, Xiaomei; LoDuca, Paul A; High, Katherine A; Herzog, Roland W

    2013-01-01

    Formation of pathogenic antibodies is a major problem in replacement therapies for inherited protein deficiencies. For example, antibodies to coagulation factors (‘inhibitors’) seriously complicate treatment of haemophilia. While immune tolerance induction (ITI) protocols have been developed, inhibitors against factor IX (FIX) are difficult to eradicate due to anaphylactic reactions and nephrotic syndrome and thus substantially elevate risks for morbidity and mortality. However, hepatic gene transfer with an adeno-associated virus (AAV) serotype 8 vector expressing FIX (at levels of ≥4% of normal) rapidly reversed pre-existing high-titre inhibitors in haemophilia B mice, eliminated antibody production by B cells, desensitized from anaphylaxis (even if protein therapy was resumed) and provided long-term correction. High levels of FIX protein suppressed memory B cells and increased Treg induction, indicating direct and indirect mechanisms of suppression of inhibitor formation. Persistent presence of Treg was required to prevent relapse of antibodies. Together, these data suggest that hepatic gene transfer-based ITI provides a safe and effective alternative to eradicate inhibitors. This strategy may be broadly applicable to reversal of antibodies in different genetic diseases. PMID:24106230

  8. 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

    SciTech Connect

    Solera, J. ); Magallon, M.; Martin-Villar, J. ); Coloma, A. )

    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.

  9. Missense mutations and evolutionary conservation of amino acids: evidence that many of the amino acids in factor IX function as "spacer" elements.

    PubMed Central

    Bottema, C D; Ketterling, R P; Ii, S; Yoon, H S; Phillips, J A; Sommer, S S

    1991-01-01

    We report 31 point mutations in the factor IX gene and explore the relationship between the level of evolutionary conservation of an amino acid and the probability of a mutation causing hemophilia B. From our total sample of 125 hemophiliacs and from those reported by others, we identify 95 independent missense mutations, 94 of which occur at amino acids that are evolutionarily conserved in the available mammalian factor IX sequences. The likelihood of a missense mutation causing hemophilia B depends on whether the residue is also conserved in the factor IX-related proteases: factor VII, factor X, and protein C. Most of the possible missense mutations in generically conserved residues (i.e., those conserved in factor IX and in all the related proteases) should cause disease. In contrast, missense mutations in factor IX-specific residues (i.e., those conserved in human, cow, dog, and mouse factor IX but not in the related proteases) are sixfold less likely to cause disease. Missense mutations at nonconserved residues are 33-fold less likely to cause disease. At least three models are compatible with these observations. A comparison of sequence alignments from four and nine species of factor IX and an examination of the missense mutations occurring at CpG residues suggest a model in which most residues fall on opposite ends of a spectrum. In about 40% of residues, virtually any missense mutation in a minority of the residues will cause disease, while virtually no missense mutations will cause disease in most of the remaining residues. Thus, many of the residues in factor IX are spacers; that is, the main chains are presumably necessary to keep other amino acid interactions in register, but the nature of the side chain is unimportant. PMID:1680287

  10. Confirmation of warfarin resistance of naturally occurring VKORC1 variants by coexpression with coagulation factor IX and in silico protein modelling

    PubMed Central

    2014-01-01

    Background VKORC1 has been identified some years ago as the gene encoding vitamin K epoxide reductase (VKOR) – the target protein for coumarin derivates like warfarin or phenprocoumon. Resistance against warfarin and other coumarin-type anticoagulants has been frequently reported over the last 50 years in rodents due to problems in pest control as well as in thrombophilic patients showing variable response to anticoagulant treatment. Many different mutations have already been detected in the VKORC1 gene leading to warfarin resistance in rats, mice and in humans. Since the conventional in vitro dithiothreitol (DTT)-driven VKOR enzymatic assay often did not reflect the in vivo status concerning warfarin resistance, we recently developed a cell culture-based method for coexpression of VKORC1 with coagulation factor IX and subsequent measurement of secreted FIX in order to test warfarin inhibition in wild-type and mutated VKORC1. Results In the present study, we coexpressed wild-type factor IX with 12 different VKORC1 variants which were previously detected in warfarin resistant rats and mice. The results show that amino acid substitutions in VKORC1 maintain VKOR activity and are associated with warfarin resistance. When we projected in silico the amino acid substitutions onto the published three-dimensional model of the bacterial VKOR enzyme, the predicted effects matched well the catalytic mechanism proposed for the bacterial enzyme. Conclusions The established cell-based system for coexpression of VKORC1 and factor IX uses FIX activity as an indicator of carboxylation efficiency. This system reflects the warfarin resistance status of VKORC1 mutations from anticoagulant resistant rodents more closely than the traditional DTT-driven enzyme assay. All mutations studied were also predicted to be involved in the reaction mechanism. PMID:24491178

  11. In silico designing of hyper-glycosylated analogs for the human coagulation factor IX.

    PubMed

    Ghasemi, Fahimeh; Zomorodipour, Alireza; Karkhane, Ali Asghar; Khorramizadeh, M Reza

    2016-07-01

    N-glycosylation is a process during which a glycan moiety attaches to the asparagine residue in the N-glycosylation consensus sequence (Asn-Xxx-Ser/Thr), where Xxx can be any amino acid except proline. Introduction of a new N-glycosylation site into a protein backbone leads to its hyper-glycosylation, and may improve the protein properties such as solubility, folding, stability, and secretion. Glyco-engineering is an approach to facilitate the hyper-glycosylation of recombinant proteins by application of the site-directed mutagenesis methods. In this regard, selection of a suitable location on the surface of a protein for introduction of a new N-glycosylation site is a main concern. In this work, a computational approach was conducted to select suitable location(s) for introducing new N-glycosylation sites into the human coagulation factor IX (hFIX). With this aim, the first 45 residues of mature hFIX were explored to find out suitable positions for introducing either Asn or Ser/Thr residues, to create new N-glycosylation site(s). Our exploration lead to detection of five potential positions, for hyper-glycosylation. For each suggested position, an analog was defined and subjected for N-glycosylation efficiency prediction. After generation of three-dimensional structures, by homology-based modeling, the five designed analogs were examined by molecular dynamic (MD) simulations, to predict their stability levels and probable structural distortions caused by amino acid substitutions, relative to the native counterpart. Three out of five suggested analogs, namely; E15T, K22N, and R37N, reached equilibration state with relatively constant Root Mean Square Deviation values. Additional analysis on the data obtained during MD simulations, lead us to conclude that, R37N is the only qualified analog with the most similar structure and dynamic behavior to that of the native counterpart, to be considered for further experimental investigations. PMID:27356208

  12. Activity of Gallium Meso- and Protoporphyrin IX against Biofilms of Multidrug-Resistant Acinetobacter baumannii Isolates

    PubMed Central

    Chang, David; Garcia, Rebecca A.; Akers, Kevin S.; Mende, Katrin; Murray, Clinton K.; Wenke, Joseph C.; Sanchez, Carlos J.

    2016-01-01

    Acinetobacter baumannii is a challenging pathogen due to antimicrobial resistance and biofilm development. The role of iron in bacterial physiology has prompted the evaluation of iron-modulation as an antimicrobial strategy. The non-reducible iron analog gallium(III) nitrate, Ga(NO3)3, has been shown to inhibit A. baumannii planktonic growth; however, utilization of heme-iron by clinical isolates has been associated with development of tolerance. These observations prompted the evaluation of iron-heme sources on planktonic and biofilm growth, as well as antimicrobial activities of gallium meso- and protoporphyrin IX (Ga-MPIX and Ga-PPIX), metal heme derivatives against planktonic and biofilm bacteria of multidrug-resistant (MDR) clinical isolates of A. baumannii in vitro. Ga(NO3)3 was moderately effective at reducing planktonic bacteria (64 to 128 µM) with little activity against biofilms (≥512 µM). In contrast, Ga-MPIX and Ga-PPIX were highly active against planktonic bacteria (0.25 to 8 µM). Cytotoxic effects in human fibroblasts were observed following exposure to concentrations exceeding 128 µM of Ga-MPIX and Ga-PPIX. We observed that the gallium metal heme conjugates were more active against planktonic and biofilm bacteria, possibly due to utilization of heme-iron as demonstrated by the enhanced effects on bacterial growth and biofilm formation. PMID:26999163

  13. Mapping Selective Inhibition of the Cancer-Related Carbonic Anhydrase IX Using Structure-Activity Relationships of Glucosyl-Based Sulfamates.

    PubMed

    Mahon, Brian P; Lomelino, Carrie L; Ladwig, Janina; Rankin, Gregory M; Driscoll, Jenna M; Salguero, Antonieta L; Pinard, Melissa A; Vullo, Daniela; Supuran, Claudiu T; Poulsen, Sally-Ann; McKenna, Robert

    2015-08-27

    Inhibition of human carbonic anhydrase IX (hCA IX) has shown to be therapeutically advantageous for treating many types of highly aggressive cancers. However, designing selective inhibitors for hCA IX has been difficult due to its high structural homology and sequence similarity with off-target hCAs. Recently, the use of glucosyl sulfamate inhibitors has shown promise as selective inhibitors for hCA IX. In this study, we present five X-ray crystal structures, determined to a resolution of 1.7 Å or better, of both hCA II (a ubiquitous CA) and an engineered hCA IX-mimic in complex with selected glucosyl sulfamates and structurally rationalize mechanisms for hCA IX selectivity. Results from this study have allowed us, for the first time, to empirically "map" key interactions of the hCA IX active site in order to establish parameters needed to design novel hCA IX selective inhibitors. PMID:26203869

  14. The propeptide region of clotting factor IX is a signal for a vitamin K dependent carboxylase: evidence from protein engineering of amino acid -4.

    PubMed Central

    Galeffi, P; Brownlee, G G

    1987-01-01

    Homologous "propeptide" regions are present in a family of vitamin K-dependent mammalian proteins, including clotting factors II, VII, IX, X, protein C, protein S and bone "gla" proteins. To test the hypothesis that the propeptide is a signal for the correct gamma-carboxylation of the adjacent gamma-carboxy region, we have mutated amino acid -4 of human factor IX from an arginine to a glutamine residue, by M13-directed site-specific mutagenesis of a cDNA clone. After expression of mutant factor IX in dog kidney cells, we find that it is secreted into the medium in a precursor form containing the propeptide, and is inefficiently gamma-carboxylated compared to the control, wild-type, recombinant factor IX. This result supports the hypothesis that the propeptide region is required for efficient gamma-carboxylation of factor IX in dog kidney cells. Furthermore, it confirms previous results that arginine at amino acid -4 is required for correct propeptide processing. Images PMID:3684602

  15. Results of a phase I/II open-label, safety and efficacy trial of coagulation factor IX (recombinant), albumin fusion protein in haemophilia B patients

    PubMed Central

    Martinowitz, U; Lissitchkov, T; Lubetsky, A; Jotov, G; Barazani-Brutman, T; Voigt, C; Jacobs, I; Wuerfel, T; Santagostino, E

    2015-01-01

    Introduction rIX-FP is a coagulation factor IX (recombinant), albumin fusion protein with more than fivefold half-life prolongation over other standard factor IX (FIX) products available on the market. Aim This prospective phase II, open-label study evaluated the safety and efficacy of rIX-FP for the prevention of bleeding episodes during weekly prophylaxis and assessed the haemostatic efficacy for on-demand treatment of bleeding episodes in previously treated patients with haemophilia B. Methods The study consisted of a 10–14 day evaluation of rIX-FP pharmacokinetics (PK), and an 11 month safety and efficacy evaluation period with subjects receiving weekly prophylaxis treatment. Safety was evaluated by the occurrence of related adverse events, and immunogenic events, including development of inhibitors. Efficacy was evaluated by annualized spontaneous bleeding rate (AsBR), and the number of injections to achieve haemostasis. Results Seventeen subjects participated in the study, 13 received weekly prophylaxis and 4 received episodic treatment only. No inhibitors were detected in any subject. The mean and median AsBR were 1.25, and 1.13 respectively in the weekly prophylaxis arm. All bleeding episodes were treated with 1 or 2 injections of rIX-FP. Three prophylaxis subjects who were treated on demand prior to study entry had >85% reduction in AsBR compared to the bleeding rate prior to study entry. Conclusion This study demonstrated the efficacy for weekly routine prophylaxis of rIX-FP to prevent spontaneous bleeding episodes and for the treatment of bleeding episodes. In addition no safety issues were detected during the study and an improved PK profile was demonstrated. PMID:25990590

  16. Coronary artery bypass grafting in a patient with hemophilia B: continuous recombinant factor IX infusion as per the Japanese guidelines for replacement therapy.

    PubMed

    Suzuki, Tomoyuki; Kawamoto, Shunsuke; Kumagai, Kiichiro; Adachi, Osamu; Kanda, Keisuke; Ishikawa, Masaaki; Okitsu, Yoko; Harigae, Hideo; Kurosawa, Shin; Saiki, Yoshikatsu

    2016-08-01

    We herein report our experience of successfully managing the hemostatic system by controlling serum factor IX levels throughout the perioperative period in a patient with hemophilia B. Coronary artery bypass grafting with cardiopulmonary bypass was planned for a 52-year-old man with moderate severity of hemophilia B. During surgery, recombinant factor IX (rFIX; BeneFIX(®) Pfizer Japan inc., Tokyo, Japan) was administered by bolus infusion followed by continuous infusion as per the guidelines of the Japanese Society on Thrombosis and Hemostasis. The operative course was uneventful without any considerable bleeding or complications. PMID:25523881

  17. Antimicrobial Activity of Gallium Protoporphyrin IX against Acinetobacter baumannii Strains Displaying Different Antibiotic Resistance Phenotypes

    PubMed Central

    Arivett, Brock A.; Fiester, Steven E.; Ohneck, Emily J.; Penwell, William F.; Kaufman, Cynthia M.; Relich, Ryan F.

    2015-01-01

    A paucity of effective, currently available antibiotics and a lull in antibiotic development pose significant challenges for treatment of patients with multidrug-resistant (MDR) Acinetobacter baumannii infections. Thus, novel therapeutic strategies must be evaluated to meet the demands of treatment of these often life-threatening infections. Accordingly, we examined the antibiotic activity of gallium protoporphyrin IX (Ga-PPIX) against a collection of A. baumannii strains, including nonmilitary and military strains and strains representing different clonal lineages and isolates classified as susceptible or MDR. Susceptibility testing demonstrated that Ga-PPIX inhibits the growth of all tested strains when cultured in cation-adjusted Mueller-Hinton broth, with a MIC of 20 μg/ml. This concentration significantly reduced bacterial viability, while 40 μg/ml killed all cells of the A. baumannii ATCC 19606T and ACICU MDR isolate after 24-h incubation. Recovery of ATCC 19606T and ACICU strains from infected A549 human alveolar epithelial monolayers was also decreased when the medium was supplemented with Ga-PPIX, particularly at a 40-μg/ml concentration. Similarly, the coinjection of bacteria with Ga-PPIX increased the survival of Galleria mellonella larvae infected with ATCC 19606T or ACICU. Ga-PPIX was cytotoxic only when monolayers or larvae were exposed to concentrations 16-fold and 1,250-fold higher than those showing antibacterial activity, respectively. These results indicate that Ga-PPIX could be a viable therapeutic option for treatment of recalcitrant A. baumannii infections regardless of the resistance phenotype, clone lineage, time and site of isolation of strains causing these infections and their iron uptake phenotypes or the iron content of the media. PMID:26416873

  18. Alternative pathways of thromboplastin-dependent activation of human factor X in plasma

    SciTech Connect

    Marlar, R.A.; Griffin, J.H.

    1981-01-01

    To determine the interrelationships of the major coagulation pathways, the activation of 3H-labeled factor X in normal and various deficient human plasmas was evaluated when clotting was triggered by dilute rabbit or human thromboplastin. Various dilutions of thromboplastin and calcium were added to plasma samples containing 3H-factor X, and the time course of factor X activation was determined. At a 1/250 dilution of rabbit brain thromboplastin, the rate of factor X activation in plasmas deficient in factor VIII or factor IX was 10% of the activation rate of normal plasma or of factor XI deficient plasma. Reconstitution of the deficient plasmas with factors VIII or IX, respectively, reconstituted normal factor X activation. Similar results were obtained when various dilutions of human thromboplastin replaced the rabbit thromboplastin. From these plasma experiments, it is inferred that the dilute thromboplastin-dependent activation of factor X requires factors VII, IX, and VIII. An alternative extrinsic pathway that involves factors IX and VIII may be the physiologic extrinsic pathway and hence help to explain the consistent clinical observations of bleeding diatheses in patients deficient in factors IX or VIII.

  19. Pharmacokinetics, thrombogenicity and safety of a double viral inactivated factor IX concentrate compared with a prothrombin complex concentrate.

    PubMed

    Ruiz-Sáez, A; Hong, A; Arguello, A; Echenagucia, M; Boadas, A; Fabbrizzi, F; Minichilli, F; Bosch, N B

    2005-11-01

    Therapeutic options for developing countries have to assure an optimum safety and efficacy and low-cost antihaemophilic concentrates. A single blind randomized crossover study was carried out in 12 previously treated HB patients, comparing the pharmacokinetics (PK), thrombogenicity (TG) and safety of two plasma-derived double-inactivated (solvent/detergent heating at 100 degrees C, 30 min) factor IX (FIX) concentrates, UMAN COMPLEX DI (product A) [plasma-derived prothrombin concentrates (PCC)] and a high purity FIX concentrate AIMAFIX DI (product B, HPFIX). In a non-bleeding state, they received one single intravenous dose 50 IU FIX kg(-1) of PCC or HPFIX, and after a wash-out period of 14 days, the other product. We evaluated acute tolerance and determined PK parameters based on FIX levels measured over a 50 h postinfusion period. We studied fibrinogen, platelets, antithrombin, F1 + 2, TAT, D-dimer, over a 360 min postinfusion period. Ten cases remained in on-demand treatment for 6 months, five with PCC and five with HPFIX. PK and anti-FIX inhibitors were repeated at 3 and 6 months. No inhibitors were detected. PK values (PCC vs. HPFIX): clearence (CL; mL h(-1) kg(-1)) 5.2 +/- 1.4 vs. 6.5 +/- 1.4; the volume of distribution at steady state (mL kg(-1)) 154.9 +/- 54.9 vs. 197.5 +/- 72.5; mean residence time (h) 29.7 +/- 8.1 vs. 30.7 +/- 9.2; T(1/2) (h) 22.3 +/- 7 vs. 23.5 +/- 12.3; incremental recovery (IR; U dL(-1) U(-1) kg(-1)) 0.96 +/- 0.17 vs. 0.76 +/- 0.13. HPFIX showed significant lower IR and higher CL. There were no differences in PK at 3 and 6 months. In TG, significant increments in TAT and F1 + 2 at 30 min and 6 h were found with PCC. Product B PK results agrees with reported results for other HPFIX preparations. Use of PCC product A has to consider its thrombogenic activity. PMID:16236107

  20. Synthesis and carbonic anhydrase I, II, IX and XII inhibitory activity of sulfamates incorporating piperazinyl-ureido moieties.

    PubMed

    Congiu, Cenzo; Onnis, Valentina; Deplano, Alessandro; Balboni, Gianfranco; Ceruso, Mariangela; Supuran, Claudiu T

    2015-09-01

    A series of sulfamates were synthesized using as lead compound SLC-0111, a sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor in Phase I clinical trials. The new derivatives incorporated ureido moieties as spacers between the benzene sulfamate fragment which binds the zinc ion from the active site, and the tail of the inhibitor, but the urea moieties were part of a substituted piperazine ring system. The derivatives (and some of their phenol precursors) were tested for the inhibition of the cytosolic, hCA I and II (off target isoforms) and the trans-membrane, tumor-associated hCA IX and XII enzymes (anticancer drug targets). Generally hCA I was not effectively inhibited, whereas many low nanomolar inhibitors were evidenced against hCA II (KIs in the range of 1.0-94.4 nM), IX (KIs in the range of 0.91-36.9 nM), and XII (KIs in the range of 1.0-84.5 nM). The best substitution fragments at the piperazine ring included the following moieties: 3-methylphenyl, 2,3-dimethylphenyl, 4-methoxyphenyl, 6-arylpyrimidine-2-yl. PMID:26233796

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

    PubMed Central

    Cao, Dong-Hua; Liu, Xiao-Li; Mu, Kai; Ma, Xiang-Wei; Sun, Jing-Li; Bai, Xiao-Zhong; Lin, Chang-Kun; Jin, Chun-Lian

    2014-01-01

    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. Materials and 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. PMID:25330515

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

    SciTech Connect

    Satoh, Chiyoko; Takahashi, Norio; Asakawa, Junichi; Hiyama, Keiko; Kodaira, Meiko )

    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.

  3. Low cost industrial production of coagulation factor IX bioencapsulated in lettuce cells for oral tolerance induction in hemophilia B.

    PubMed

    Su, Jin; Zhu, Liqing; Sherman, Alexandra; Wang, Xiaomei; Lin, Shina; Kamesh, Aditya; Norikane, Joey H; Streatfield, Stephen J; Herzog, Roland W; Daniell, Henry

    2015-11-01

    Antibodies (inhibitors) developed by hemophilia B patients against coagulation factor IX (FIX) are challenging to eliminate because of anaphylaxis or nephrotic syndrome after continued infusion. To address this urgent unmet medical need, FIX fused with a transmucosal carrier (CTB) was produced in a commercial lettuce (Simpson Elite) cultivar using species specific chloroplast vectors regulated by endogenous psbA sequences. CTB-FIX (∼1 mg/g) in lyophilized cells was stable with proper folding, disulfide bonds and pentamer assembly when stored ∼2 years at ambient temperature. Feeding lettuce cells to hemophilia B mice delivered CTB-FIX efficiently to the gut immune system, induced LAP(+) regulatory T cells and suppressed inhibitor/IgE formation and anaphylaxis against FIX. Lyophilized cells enabled 10-fold dose escalation studies and successful induction of oral tolerance was observed in all tested doses. Induction of tolerance in such a broad dose range should enable oral delivery to patients of different age groups and diverse genetic background. Using Fraunhofer cGMP hydroponic system, ∼870 kg fresh or 43.5 kg dry weight can be harvested per 1000 ft(2) per annum yielding 24,000-36,000 doses for 20-kg pediatric patients, enabling first commercial development of an oral drug, addressing prohibitively expensive purification, cold storage/transportation and short shelf life of current protein drugs. PMID:26302233

  4. CRISPR/Cas9-mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse.

    PubMed

    Guan, Yuting; Ma, Yanlin; Li, Qi; Sun, Zhenliang; Ma, Lie; Wu, Lijuan; Wang, Liren; Zeng, Li; Shao, Yanjiao; Chen, Yuting; Ma, Ning; Lu, Wenqing; Hu, Kewen; Han, Honghui; Yu, Yanhong; Huang, Yuanhua; Liu, Mingyao; Li, Dali

    2016-01-01

    The X-linked genetic bleeding disorder caused by deficiency of coagulator factor IX, hemophilia B, is a disease ideally suited for gene therapy with genome editing technology. Here, we identify a family with hemophilia B carrying a novel mutation, Y371D, in the human F9 gene. The CRISPR/Cas9 system was used to generate distinct genetically modified mouse models and confirmed that the novel Y371D mutation resulted in a more severe hemophilia B phenotype than the previously identified Y371S mutation. To develop therapeutic strategies targeting this mutation, we subsequently compared naked DNA constructs versus adenoviral vectors to deliver Cas9 components targeting the F9 Y371D mutation in adult mice. After treatment, hemophilia B mice receiving naked DNA constructs exhibited correction of over 0.56% of F9 alleles in hepatocytes, which was sufficient to restore hemostasis. In contrast, the adenoviral delivery system resulted in a higher corrective efficiency but no therapeutic effects due to severe hepatic toxicity. Our studies suggest that CRISPR/Cas-mediated in situ genome editing could be a feasible therapeutic strategy for human hereditary diseases, although an efficient and clinically relevant delivery system is required for further clinical studies. PMID:26964564

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

    PubMed

    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. PMID:24564349

  6. Desensitization and immune tolerance induction in children with severe factor IX deficiency; inhibitors and adverse reactions to replacement therapy: a case-report and literature review.

    PubMed

    Bon, Andrea; Morfini, Massimo; Dini, Alessandro; Mori, Francesca; Barni, Simona; Gianluca, Sottilotta; de Martino, Maurizio; Novembre, Elio

    2015-01-01

    Hemophilia B is a rare X-linked recessive disorder with plasma factor IX (FIX) deficiency. 1-3% of patients treated with exogenous FIX-containing products develop inhibitors (i.e. polyclonal high affinity immunoglobulins) that neutralize the procoagulant activity of a specific coagulation factor. Although the incidence of inhibitors in hemophilia B patients is low, most are "high titer" and frequently associated with the development of severe allergic or anaphylactic reactions. Immune tolerance induction as a strategy for inhibitor eradication was first described in 1984. Unfortunately, the overall reported success of immune tolerance induction in FIX deficiency with inhibitors is approximately 25-40%.We report the case of a 2-year-old boy with hemophilia B severe FIX deficiency (<1%), inhibitor antibodies to FIX development, and a history of adverse reactions to FIX infusions, who underwent a successful desensitization and immune tolerance induction with a daily FIX infusion. With this regimen the inhibitor titer decreased with effective bleeding prevention. PMID:25887512

  7. Synthesis of 4-(thiazol-2-ylamino)-benzenesulfonamides with carbonic anhydrase I, II and IX inhibitory activity and cytotoxic effects against breast cancer cell lines.

    PubMed

    Abdel Gawad, Nagwa M; Amin, Noha H; Elsaadi, Mohammed T; Mohamed, Fatma M M; Angeli, Andrea; De Luca, Viviana; Capasso, Clemente; Supuran, Claudiu T

    2016-07-01

    A series of 4-(thiazol-2-ylamino)-benzenesulfonamides was synthesized and screened for their carbonic anhydrase (CA, EC 4.2.1.1) inhibitory and cytotoxic activity on human breast cancer cell line MCF-7. Human (h) CA isoforms I, II and IX were included in the study. The new sulfonamides showed excellent inhibition of all three isoforms, with KIs in the range of 0.84-702nM against hCA I, of 0.41-288nM against hCA II and of 5.6-29.2 against the tumor-associated hCA IX, a validated anti-tumor target, with a sulfonamide (SLC-0111) in Phase I clinical trials for the treatment of hypoxic, metastatic solid tumors overexpressing CA IX. The new compounds showed micromolar inhibition of growth efficacy against breast cancer MCF-7 cell lines. PMID:27234893

  8. Coagulation Factor IX Mediates Serotype-Specific Binding of Species A Adenoviruses to Host Cells ▿ †

    PubMed Central

    Lenman, Annasara; Müller, Steffen; Nygren, Mari I.; Frängsmyr, Lars; Stehle, Thilo; Arnberg, Niklas

    2011-01-01

    Human species A adenoviruses (HAdVs) comprise three serotypes: HAdV-12, -18, and -31. These viruses are common pathogens and cause systemic infections that usually involve the airways and/or intestine. In immunocompromised individuals, species A adenoviruses in general, and HAdV-31 in particular, cause life-threatening infections. By combining binding and infection experiments, we demonstrate that coagulation factor IX (FIX) efficiently enhances binding and infection by HAdV-18 and HAdV-31, but not by HAdV-12, in epithelial cells originating from the airways or intestine. This is markedly different from the mechanism for HAdV-5 and other human adenoviruses, which utilize coagulation factor X (FX) for infection of host cells. Surface plasmon resonance experiments revealed that the affinity of the HAdV-31 hexon-FIX interaction is higher than that of the HAdV-5 hexon-FX interaction and that the half-lives of these interactions are profoundly different. Moreover, both HAdV-31–FIX and HAdV-5–FX complexes bind to heparan sulfate-containing glycosaminoglycans (GAGs) on target cells, but binding studies utilizing cells expressing specific GAGs and GAG-cleaving enzymes revealed differences in GAG dependence and specificity between these two complexes. These findings add to our understanding of the intricate infection pathways used by human adenoviruses, and they may contribute to better design of HAdV-based vectors for gene and cancer therapy. Furthermore, the interaction between the HAdV-31 hexon and FIX may also serve as a target for antiviral treatment. PMID:21976659

  9. A two-component system regulates gene expression of the type IX secretion component proteins via an ECF sigma factor

    PubMed Central

    Kadowaki, Tomoko; Yukitake, Hideharu; Naito, Mariko; Sato, Keiko; Kikuchi, Yuichiro; Kondo, Yoshio; Shoji, Mikio; Nakayama, Koji

    2016-01-01

    The periodontopathogen Porphyromonas gingivalis secretes potent pathogenic proteases, gingipains, via the type IX secretion system (T9SS). This system comprises at least 11 components; however, the regulatory mechanism of their expression has not yet been elucidated. Here, we found that the PorY (PGN_2001)-PorX (PGN_1019)-SigP (PGN_0274) cascade is involved in the regulation of T9SS. Surface plasmon resonance (SPR) analysis revealed a direct interaction between a recombinant PorY (rPorY) and a recombinant PorX (rPorX). rPorY autophosphorylated and transferred a phosphoryl group to rPorX in the presence of Mn2+. These results demonstrate that PorX and PorY act as a response regulator and a histidine kinase, respectively, of a two component system (TCS), although they are separately encoded on the chromosome. T9SS component-encoding genes were down-regulated in a mutant deficient in a putative extracytoplasmic function (ECF) sigma factor, PGN_0274 (SigP), similar to the porX mutant. Electrophoretic gel shift assays showed that rSigP bound to the putative promoter regions of T9SS component-encoding genes. The SigP protein was lacking in the porX mutant. Co-immunoprecipitation and SPR analysis revealed the direct interaction between SigP and PorX. Together, these results indicate that the PorXY TCS regulates T9SS-mediated protein secretion via the SigP ECF sigma factor. PMID:26996145

  10. A two-component system regulates gene expression of the type IX secretion component proteins via an ECF sigma factor.

    PubMed

    Kadowaki, Tomoko; Yukitake, Hideharu; Naito, Mariko; Sato, Keiko; Kikuchi, Yuichiro; Kondo, Yoshio; Shoji, Mikio; Nakayama, Koji

    2016-01-01

    The periodontopathogen Porphyromonas gingivalis secretes potent pathogenic proteases, gingipains, via the type IX secretion system (T9SS). This system comprises at least 11 components; however, the regulatory mechanism of their expression has not yet been elucidated. Here, we found that the PorY (PGN_2001)-PorX (PGN_1019)-SigP (PGN_0274) cascade is involved in the regulation of T9SS. Surface plasmon resonance (SPR) analysis revealed a direct interaction between a recombinant PorY (rPorY) and a recombinant PorX (rPorX). rPorY autophosphorylated and transferred a phosphoryl group to rPorX in the presence of Mn(2+). These results demonstrate that PorX and PorY act as a response regulator and a histidine kinase, respectively, of a two component system (TCS), although they are separately encoded on the chromosome. T9SS component-encoding genes were down-regulated in a mutant deficient in a putative extracytoplasmic function (ECF) sigma factor, PGN_0274 (SigP), similar to the porX mutant. Electrophoretic gel shift assays showed that rSigP bound to the putative promoter regions of T9SS component-encoding genes. The SigP protein was lacking in the porX mutant. Co-immunoprecipitation and SPR analysis revealed the direct interaction between SigP and PorX. Together, these results indicate that the PorXY TCS regulates T9SS-mediated protein secretion via the SigP ECF sigma factor. PMID:26996145

  11. Color formation in nitrite-free dried hams as related to Zn-protoporphyrin IX and Zn-chelatase activity.

    PubMed

    Parolari, Giovanni; Benedini, Riccardo; Toscani, Tania

    2009-08-01

    The development of red pigment Zn-protoporphyrin IX (ZPP) in nitrite-free Parma hams was investigated in 5 leg muscles at several stages of processing and the activity of muscle Zn-chelatase was concurrently assayed for its potential role in ZPP formation. A steady increase of the pigment was observed throughout the manufacturing stages at mild temperatures while no development was observed during the prior cold resting phase. The enzyme was partly inactivated according to a muscle-dependent pattern, resulting in similar ZPP contents, hence color, in finished hams. It is concluded that enzyme-dependent synthesis of ZPP in nitrite-free dried hams contributes to color development, enabling muscles in dried hams to become more similar in redness than in green thighs. Therefore, checking raw meat for the enzyme content may be a means to control color formation in nitrite-free dry-cured meat derivatives. PMID:19723176

  12. Improved expression of recombinant human factor IX by co-expression of GGCX, VKOR and furin.

    PubMed

    Liu, Jianming; Jonebring, Anna; Hagström, Jonas; Nyström, Ann-Christin; Lövgren, Ann

    2014-04-01

    Recombinant human FIX concentrates (rhFIX) are essential in the treatment and prevention of bleeding in the bleeding disorder haemophilia B. However, due to the complex nature of FIX production yields are low which leads to high treatment costs. Here we report the production of rhFIX with substantially higher yield by co-expressing human FIX with GGCX (γ-glutamyl carboxylase), VKOR (vitamin K epoxide reductase) and furin (paired basic amino acid cleaving enzyme) in Chinese hamster ovary (CHO) cells. Our results show that controlled co-expression of GGCX with FIX is critical to obtain high rhFIX titre, and, that co-expression of VKOR further increased the yield of active rhFIX. Furin co-expression improved processing of the leader peptide of rhFIX but had a minor effect on yield of active rhFIX. The optimal expression level of GGCX was surprisingly low and required unusual engineering of expression vector elements. For VKOR and furin the control of expression was less critical and could be achieved by standard vector element. Using our expression vectors an rhFIX-producing clone with an expression level of up to 30 mg/L of active rhFIX was obtained. In addition an efficient single step purification method was developed to obtain pure and active rhFIX with up to 94% yield. PMID:24567122

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

    PubMed

    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 <12 years with severe (FIX level <1%) or moderately severe (FIX level 1-2%) haemophilia B. The aim of this prospective clinical trial was to assess the safety, haemostatic efficacy and pharmacokinetic profile of BAX326 in previously treated paediatric patients. BAX326 was administered as prophylaxis twice a week for a period of 6 months, and on demand for treatment of bleeds. Safety was assessed by the occurrence of related AEs, thrombotic events and immunologic assessments. Efficacy was evaluated by annualized bleeding rate (ABR), and by treatment response rating (excellent, good, fair, none). PK was assessed over 72 h. None of the 23 treated paediatric subjects had treatment-related SAEs or AEs. There were no thrombotic events, inhibitory or specific binding antibodies against FIX, rFurin or CHO protein. Twenty-six bleeds (19 non-joint vs. 7 joint bleeds) occurred (mean ABR 2.7 ± 3.14, median 2.0), of which 23 were injury-related. Twenty subjects (87%) did not experience any bleeds of spontaneous aetiology. Haemostatic efficacy of BAX326 was excellent or good for >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. PMID:25495591

  14. Title IX and Sex Discrimination. Revised.

    ERIC Educational Resources Information Center

    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…

  15. Carnosine inhibits carbonic anhydrase IX-mediated extracellular acidosis and suppresses growth of HeLa tumor xenografts

    PubMed Central

    2014-01-01

    Background Carbonic anhydrase IX (CA IX) is a transmembrane enzyme that is present in many types of solid tumors. Expression of CA IX is driven predominantly by the hypoxia-inducible factor (HIF) pathway and helps to maintain intracellular pH homeostasis under hypoxic conditions, resulting in acidification of the tumor microenvironment. Carnosine (β-alanyl-L-histidine) is an anti-tumorigenic agent that inhibits the proliferation of cancer cells. In this study, we investigated the role of CA IX in carnosine-mediated antitumor activity and whether the underlying mechanism involves transcriptional and translational modulation of HIF-1α and CA IX and/or altered CA IX function. Methods The effect of carnosine was studied using two-dimensional cell monolayers of several cell lines with endogenous CA IX expression as well as Madin Darby canine kidney transfectants, three-dimensional HeLa spheroids, and an in vivo model of HeLa xenografts in nude mice. mRNA and protein expression and protein localization were analyzed by real-time PCR, western blot analysis, and immunofluorescence staining, respectively. Cell viability was measured by a flow cytometric assay. Expression of HIF-1α and CA IX in tumors was assessed by immunohistochemical staining. Real-time measurement of pH was performed using a sensor dish reader. Binding of CA IX to specific antibodies and metabolon partners was investigated by competitive ELISA and proximity ligation assays, respectively. Results Carnosine increased the expression levels of HIF-1α and HIF targets and increased the extracellular pH, suggesting an inhibitory effect on CA IX-mediated acidosis. Moreover, carnosine significantly inhibited the growth of three-dimensional spheroids and tumor xenografts compared with untreated controls. Competitive ELISA showed that carnosine disrupted binding between CA IX and antibodies specific for its catalytic domain. This finding was supported by reduced formation of the functional metabolon of CA IX

  16. Factor IX assay

    MedlinePlus

    ... B. In: Hoffman R, Benz EJ Jr, Silberstein LE, Heslop HE, Weitz JI, Anastasi J, eds. Hematology: ... disorders. In: Hoffman R, Benz EJ Jr, Silberstein LE, Heslop HE, Weitz JI, Anastasi J, eds. Hematology: ...

  17. Factor IX assay

    MedlinePlus

    ... LE, Heslop HE, Weitz JI, Anastasi J, eds. Hematology: Basic Principles and Practice . 6th ed. Philadelphia, PA: ... LE, Heslop HE, Weitz JI, Anastasi J, eds. Hematology: Basic Principles and Practice . 6th ed. Philadelphia, PA: ...

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

    PubMed Central

    Olmedillas López, Susana; Garcia-Arranz, Mariano; Garcia-Olmo, Damian

    2016-01-01

    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. PMID:27114871

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

    NASA Astrophysics Data System (ADS)

    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.

  20. Statistical Evidence and Compliance with Title IX

    ERIC Educational Resources Information Center

    Cheslock, John J.; Eckes, Suzanne E.

    2008-01-01

    The scope of Title IX clearly includes all aspects of education, but the legislation's application to college athletics receives the most attention. Athletics programs, unlike most academic activities, are sex segregated, so the proper interpretation of the intercollegiate athletics provisions of Title IX is less clear-cut. This article examines…

  1. Reconstitution of Platelet Glycoprotein Ib-IX Complex in Phospholipid Bilayer Nanodiscs†

    PubMed Central

    Yan, Rong; Mo, Xi; Paredes, Angel M.; Dai, Kesheng; Lanza, Francois; Cruz, Miguel A.; Li, Renhao

    2012-01-01

    Glycoprotein (GP)Ib-IX complex expressed on platelet plasma membrane is involved in thrombosis and hemostasis by initiating platelet adhesion to von Willebrand factor (VWF) exposed at the injured vessel wall. While most of the knowledge for GPIb-IX is obtained from studies on platelets and transfected mammalian cells expressing GPIb-IX, there is not an in vitro membrane system that allows systematic analysis of this receptor. The phospholipid bilayer Nanodisc composed of a patch of phospholipid surrounded by membrane scaffold protein is an attractive tool for membrane protein study. We show here that GPIb-IX purified from human platelets has been reconstituted into the Nanodisc. Nanodisc-reconstituted GPIb-IX was able to bind various conformation-sensitive monoclonal antibodies. Furthermore, it bound to VWF in the presence of botrocetin with an apparent Kd of 0.73 ± 0.07 nM. The binding to VWF was inhibited by anti-GPIbα antibodies with epitopes overlapping with the VWF-binding site, but not by anti-GPIbβ monoclonal antibody RAM.1. Finally, Nanodisc-reconstituted GPIb-IX exhibited similar ligand-binding activity as the isolated extracellular domain of GPIbα. In conclusion, GPIb-IX in Nanodiscs adopts native-like conformation and possesses the ability to bind its natural ligands, thus making Nanodisc a suitable in vitro platform for further investigation on this hemostatically important receptor complex. PMID:22080766

  2. Title IX: With New Opportunities, Girls' Interest Rises

    ERIC Educational Resources Information Center

    Toporek, Bryan

    2012-01-01

    On June 23, 1972, President Richard M. Nixon signed into law Title IX of the Education Amendments of 1972, which prohibits gender discrimination in any federally financed education program or activity. Title IX is far-reaching, but the law is most often associated with school and college athletics. Title IX allows schools to prove their athletic…

  3. The Restoration of Title IX: Implications for Higher Education.

    ERIC Educational Resources Information Center

    Sandler, Bernice R.

    This booklet helps institutions understand the restoration of Title IX of the Education Amendments of 1972 and changes resulting from the Civil Rights Restoration Act. Title IX prohibits sex discrimination in federally assisted education programs. A 1984 ruling held that Title IX covers only programs or activities funded with federal money. In…

  4. Pharmacokinetic properties of IB1001, an investigational recombinant factor IX, in patients with haemophilia B: repeat pharmacokinetic evaluation and sialylation analysis.

    PubMed

    Martinowitz, U; Shapiro, A; Quon, D V; Escobar, M; Kempton, C; Collins, P W; Chowdary, P; Makris, M; Mannucci, P M; Morfini, M; Valentino, L A; Gomperts, E; Lee, M

    2012-11-01

    IB1001 trenacog alfa is an investigational recombinant factor IX (FIX) for the treatment and prevention of bleeding in individuals with haemophilia B. To compare the pharmacokinetics (PK) of IB1001 with nonacog alfa in individuals with haemophilia B and to assess the relationship between sialylation and PK of IB1001 (NCT00768287). A randomized, double-blind, non-inferiority, cross-over study conducted in participants aged ≥ 12 years weighing ≥ 40 kg, with severe or moderately severe haemophilia B (FIX activity ≤ 2 IU dL (-1) ). PK parameters were derived using observed FIX concentration levels and actual PK sampling times, and repeated in a subset of participants who had received IB1001 prophylaxis for 4-18 months. A retrospective analysis was conducted in subgroups according to the sialylation levels of IB1001 (50.8, 57.8-59.0%, or 71.7%). In the 32 adolescent and adult males evaluated, there were no clinically meaningful differences in PK parameters between those receiving IB1001 75 IU kg(-1) or nonacog alfa. The lower limit of the one-sided 95% confidence interval for the ratio of AUC(0-t) and AUC(0-∞) (IB1001/nonacog alfa) was 0.90, establishing non-inferiority. Terminal phase half-lives were similar (29.7 ± 18.2 h for IB1001 and 33.4 ± 21.2 h for nonacog alfa). The PK results were stable for up to 18 months of IB1001 exposure; the impact of sialylation levels was not clinically meaningful. There were no clinically meaningful PK differences between IB1001 and nonacog alfa. IB1001 was well tolerated and without safety concerns. The non-inferiority of IB1001 to nonacog alfa supports IB1001 becoming a useful alternative recombinant agent for the management of haemophilia B. PMID:22764744

  5. Type IX Ehlers-Danlos syndrome and Menkes syndrome: the decrease in lysyl oxidase activity is associated with a corresponding deficiency in the enzyme protein.

    PubMed Central

    Kuivaniemi, H; Peltonen, L; Kivirikko, K I

    1985-01-01

    Type IX of the Ehlers-Danlos syndrome (E-D IX) and the Menkes syndrome are X-linked recessively inherited disorders characterized by abnormalities in copper metabolism. These abnormalities are associated with a severe reduction in the activity of lysyl oxidase, the extracellular copper enzyme that initiates crosslinking of collagens and elastin. No increase in this deficient enzyme activity was obtained when culture media from fibroblasts of patients with E-D IX or the Menkes syndrome were incubated with copper under various conditions in vitro. A distinct, although small, increase in lysyl oxidase activity was obtained, however, when copper-supplemented media were used during culturing of the fibroblasts, although even under these conditions, the enzyme activity in the media from the affected cells remained markedly below that of the controls. Immunoprecipitation, dot-blotting, and immunoperoxidase staining experiments with antisera to human lysyl oxidase indicated that fibroblasts from patients with E-D IX or the Menkes syndrome do not secrete into their medium, or contain inside the cell, any significant amounts of a copper-deficient, catalytically inactive lysyl oxidase protein. These findings appear to be consistent with the hypothesis that synthesis of the lysyl oxidase protein itself is impaired. The possibility is not excluded, however, that a copper-deficient enzyme protein may be synthesized in normal amounts but become degraded very rapidly inside the cell. The failure to obtain any large increase in the deficient lysyl oxidase activity upon various forms of copper administration suggests that it may not be possible to obtain any significant improvement in the connective tissue manifestations of these disorders by copper therapy. Images Fig. 1 Fig. 2 PMID:9556668

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

    ERIC Educational Resources Information Center

    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…

  7. 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?

    SciTech Connect

    Grouse, L.H.; Ketterling, R.P.; Sommer, S.S.

    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.

  8. Production of transgenic goats expressing human coagulation factor IX in the mammary glands after nuclear transfer using transfected fetal fibroblast cells.

    PubMed

    Amiri Yekta, Amir; Dalman, Azam; Eftekhari-Yazdi, Poopak; Sanati, Mohammad Hossein; Shahverdi, Abdol Hossein; Fakheri, Rahman; Vazirinasab, Hamed; Daneshzadeh, Mohammad Taghi; Vojgani, Mahdi; Zomorodipour, Alireza; Fatemi, Nayeralsadat; Vahabi, Zeinab; Mirshahvaladi, Shahab; Ataei, Fariba; Bahraminejad, Elmira; Masoudi, Najmehsadat; Rezazadeh Valojerdi, Mojtaba; Gourabi, Hamid

    2013-02-01

    There are growing numbers of recombinant proteins that have been expressed in milk. Thus one can consider the placement of any gene of interest under the control of the regulatory elements of a milk protein gene in a dairy farm animal. Among the transgene introducing techniques, only nuclear transfer (NT) allows 100 % efficiency and bypasses the mosaicism associated with counterpart techniques. In this study, in an attempt to produce a transgenic goat carrying the human coagulation factor IX (hFIX) transgene, goat fetal fibroblasts were electroporated with a linearized marker-free construct in which the transgene was juxtaposed to β-casein promoter designed to secret the recombinant protein in goat milk. Two different lines of transfected cells were used as donors for NT to enucleated oocytes. Two transgenic goats were liveborn. DNA sequencing of the corresponding transgene locus confirmed authenticity of the cloning procedure and the complementary experiments on the whey demonstrated expression of human factor IX in the milk of transgenic goats. In conclusion, our study has provided the groundwork for a prosperous and promising approach for large-scale production and therapeutic application of hFIX expressed in transgenic goats. PMID:22869287

  9. Sequence-specific sup 1 H NMR assignments, secondary structure, and location of the calcium binding site in the first epidermal growth factor like domain of blood coagulation factor IX

    SciTech Connect

    Huang, L.H.; Cheng, H.; Sweeney, W.V. ); Pardi, A. ); Tam, J.P. )

    1991-07-30

    Factor IX is a blood clotting protein that contains three regions, including a {gamma}-carboxyglutamic acid (Gla) domain, two tandemly connected epidermal growth factor like (EGF-like) domains, and a serine protease region. The protein exhibits a high-affinity calcium binding site in the first EGF0like domain, in addition to calcium binding in the Gla domain. The first EGF-like domain, factor IX (45-87), has been synthesized. Sequence-specific resonance assignment of the peptide has been made by using 2D NMR techniques, and its secondary structure has been determined. The protein is found to have two antiparallel {beta}-sheets, and preliminary distance geometry calculations indicate that the protein has two domains, separated by Trp{sup 28}, with the overall structure being similar to that of EGF. An NMR investigation of the calcium-bound first EGF-like domain indicates the presence and location of a calcium binding site involving residues on both strands of one of the {beta}-sheets as well as the N-terminal region of the peptide. These results suggest that calcium binding in the first EGF-like domain could induce long-range (possibly interdomain) conformational changes in factor IX, rather than causing structural alterations in the EGF-like domain itself.

  10. Expression and Characterization of Gly-317 Variants of Factor IX Causing Variable Bleeding in Hemophilia B Patients.

    PubMed

    Lu, Qiuya; Yang, Likui; Manithody, Chandrashekhara; Wang, Xuefeng; Rezaie, Alireza R

    2015-06-23

    We recently identified two hemophilia B patients who carried Gly-317 to Arg (FIX-G317R) or Gly-317 to Glu (FIX-G317E) substitutions in their FIX gene. The former mutation caused severe and the latter moderate bleeding in afflicted patients. To understand the molecular basis for the variable clinical manifestation of Gly-317 mutations, we prepared recombinant G317R and G317E derivatives of FIX and compared their kinetic properties to those of recombinant wild-type FIX in appropriate assay systems. Both physiological activators, factor XIa and extrinsic Tenase (factor VIIa-tissue factor), activated both zymogen variants with an ∼1.5-fold elevated K(m); however, extrinsic Tenase activated FIX-G317E with an ∼2-fold improved k(cat). By contrast to zymogen activation, the catalytic activities of both FIXa-G317R and FIXa-G317E enzymes toward the natural substrate, factor X, were dramatically (>4 orders of magnitude) impaired, but their apparent affinity for interaction with factor VIIIa was only slightly (<2-fold) decreased. Further studies revealed that the reactivity of FIXa-G317R and FIXa-G317E with antithrombin has been impaired 10- and 13-fold, respectively, in the absence and 166- and 500-fold, respectively, in the presence of pentasaccharide. As expected, the clotting activities of FIX variants could not be measured by the aPTT assay. These results implicate a critical role for Gly-317 in maintaining normal catalytic function for FIX/FIXa in the clotting cascade. The results further suggest that improved k(cat) of FIX-G317E activation in the extrinsic pathway together with dramatically impaired reactivity of FIXa-G317E with antithrombin may account for the less severe bleeding phenotype of a hemophilia B patient carrying the FIX-G317E mutation. PMID:26023895

  11. The Physical Activity Specialist's Role. Implementing Title IX and Attaining Sex Equity: A Workshop Package for Elementary-Secondary Educators.

    ERIC Educational Resources Information Center

    Landers, Barbara

    This workshop package provides training session outlines and materials for a workshop sequence on Title IX and sex equity in physical education. Three of these sessions are designed to provide general information and experiences relevant to all participants attending the workshop. The other two sessions, called "Application Sessions," are designed…

  12. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 45 Public Welfare 1 2012-10-01 2012-10-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Public Welfare DEPARTMENT OF HEALTH AND... ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Procedures Interim procedures. Pt. 86, Index Subject...

  13. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 45 Public Welfare 1 2013-10-01 2013-10-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Public Welfare DEPARTMENT OF HEALTH AND... ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Procedures Interim procedures. Pt. 86, Index Subject...

  14. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 45 Public Welfare 1 2010-10-01 2010-10-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Public Welfare DEPARTMENT OF HEALTH AND... ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Procedures Interim procedures. Pt. 86, Index Subject...

  15. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 45 Public Welfare 1 2014-10-01 2014-10-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Public Welfare Department of Health and... ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Procedures Interim procedures. Pt. 86, Index Subject...

  16. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 45 Public Welfare 1 2011-10-01 2011-10-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Public Welfare DEPARTMENT OF HEALTH AND... ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Procedures Interim procedures. Pt. 86, Index Subject...

  17. Comparative effectiveness of clinically used light sources for cutaneous protoporphyrin IX-based photodynamic therapy.

    PubMed

    Sayre, Robert M; Dowdy, John C; Gottschalk, Ronald W

    2011-04-01

    This report documents the optical characteristics of a number of photodynamic therapy (PDT) light sources of varied types, measured and indexed relative to estimated effectiveness for activation of the PDT chromaphore protoporphyrin IX (PpIX). PDT sources in use at several clinics, including intense pulsed light (IPL) sources, lasers, and continuous wave (CW) light sources, were spectroradiometrically measured and indexed relative to their overlap to an absorption spectrum of PpIX. The sources were highly disparate, varying in power from irradiance in the mW/cm(2) range for the CW sources up to ∼30 J/cm(2) per flash for the IPL sources. Our PpIX Index ranged by a factor of nearly 100 (0.008-0.630) in estimated PpIX PDT effectiveness following the distinct spectral characteristics of the light sources surveyed. Application of this PpIX Index, tempered with an understanding of the biology of the lesion being treated and effective spectrum of the light source reaching the lesion requiring therapy, provides a rational algorithm to approximate equivalent light doses prior to clinical protocols to establish equivalent patient outcomes employing alternative PDT light sources. PMID:21401379

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

    PubMed

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

    2014-01-01

    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

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

    PubMed Central

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

    2013-01-01

    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

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

    SciTech Connect

    Ketterling, R.P.; Vielhaber, E.; Sommer, S.S. )

    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.

  1. Significant differences in integration sites of Moloney murine leukemia virus/Moloney murine sarcoma virus retroviral vector carrying recombinant coagulation factor IX in two human cell lines.

    PubMed

    Castilho-Fernandes, Andrielle; Fontes, Aparecida Maria; Abraham, Kuruvilla Joseph; de Freitas, Marcela Cristina Corrêa; da Rosa, Nathalia Gonsales; Picanço-Castro, Virginia; de Sousa Russo-Carbolante, Elisa Maria; Covas, Dimas Tadeu

    2015-05-01

    Ligation-mediated-PCR was performed followed by the mapping of 177 and 150 integration sites from HepG2 and Hek293 transduced with chimera vector carrying recombinant human Factor IX (rhFIX) cDNA, respectively. The sequences were analyzed for chromosome preference, CpG, transcription start site (TSS), repetitive elements, fragile sites and target genes. In HepG2, rhFIX was had an increased preference for chromosomes 6 and 17; the median distance to the nearest CpG islands was 15,240 base pairs and 37 % of the integrations occurred in RefSeq genes. In Hek293, rhFIX had an increased preference for chromosome 5; the median distance to the nearest CpG islands was 209,100 base pairs and 74 % of the integrations occurred in RefSeq genes. The integrations in both cell lines were distant from the TSS. The integration patterns associated with this vector are different in each cell line. PMID:25650340

  2. Identification of a point mutation in type IIB von Willebrand disease illustrating the regulation of von Willebrand factor affinity for the platelet membrane glycoprotein Ib-IX receptor

    SciTech Connect

    Ware, J.; Dent, J.A.; Azuma, Hiroyuki; Sugimoto, Mitsuhiko; Kyrle, P.A.; Yoshioka, Akira; Ruggeri, Z.M. )

    1991-04-01

    von Willebrand factor (vWF) supports platelet adhesion on thrombogenic surfaces by binding to platelet membrane glycoprotein (GP) Ib in the GP Ib-IX receptor complex. This interaction is physiologically regulated so that it does not occur between circulating vWF and platelets but, rather, only at a site of vascular injury. The abnormal vWF found in type IIB von Willebrand disease, however, has a characteristically increased affinity for GP Ib and binds to circulating platelets. The authors have analyzed the molecular basis of this abnormality by sequence analysis of a type IIB vWF cDNA and have identified a single amino acid change, Trp{sup 550} to Cys{sup 550}, located in the GP IB-binding domain of the molecule comprising residues 449-728. Bacterial expression of recombinant fragments corresponding to this vWF domain yielded molecules that, whether containing a normal Trp{sup 550} or a mutant Cys{sup 550} residue, bound directly to GP Ib in the absence of modulators and with similar affinity. These results identify a region of vWF that, although not thought to be directly involved in binding to GP Ib, may modulate the interaction through conformational changes.

  3. Factors regulating microglia activation

    PubMed Central

    Kierdorf, Katrin; Prinz, Marco

    2013-01-01

    Microglia are resident macrophages of the central nervous system (CNS) that display high functional similarities to other tissue macrophages. However, it is especially important to create and maintain an intact tissue homeostasis to support the neuronal cells, which are very sensitive even to minor changes in their environment. The transition from the “resting” but surveying microglial phenotype to an activated stage is tightly regulated by several intrinsic (e.g., Runx-1, Irf8, and Pu.1) and extrinsic factors (e.g., CD200, CX3CR1, and TREM2). Under physiological conditions, minor changes of those factors are sufficient to cause fatal dysregulation of microglial cell homeostasis and result in severe CNS pathologies. In this review, we discuss recent achievements that gave new insights into mechanisms that ensure microglia quiescence. PMID:23630462

  4. Coagulation factor VIII, IX and XI levels in north Indian patients with venous thromboembolism: first study from India.

    PubMed

    Chougule, Abhijit; Rajpal, Sweta; Ahluwalia, Jasmina; Bose, Sunil Kumar; Masih, Joseph; Das, Reena; Kumar, Narender; Malhotra, Pankaj; Suri, Vikas

    2016-01-01

    Studies have shown elevated levels of certain coagulation factors as risk factors for venous thromboembolism (VTE). In this study, we investigated the levels of coagulation factor VIII (FVIII), FIX and FXI in north Indian patients with VTE. A total of 123 patients with VTE were screened prospectively for FVIII, FIX and FXI levels and the conventional risk factors - deficiencies of protein C, S and antithrombin, positivity for antiphospholipid antibodies and the factor V Leiden mutation. Age-matched and sex-matched controls were included. VTE was secondary to known circumstantial and thrombophilic risk factors in 66 (53.7%) patients. In 46.3% (idiopathic VTE) patients, no cause was identified. The mean FVIII levels in idiopathic (187 IU/dl) and secondary VTE patients (185.4 IU/dl) were significantly higher compared with controls (129.6 IU/dl; P < 0.001). However, there was no statistically significant difference in the levels of FIX and FXI between patients and controls (P = 0.214 and 0.198, respectively). Patients with elevated FVIII levels had increased risk of VTE compared with controls (odds ratio: 9.4, 95% confidence interval: 4.7-18.79). On logistic regression analysis after adjusting for surgery and presence of antiphospholipid antibodies, this risk remained unchanged (odds ratio: 9.54, 95% confidence interval: 4.68-19.44). A dose-response relationship was observed with progressive increase in FVIII levels. Elevated FVIII levels constitute an independent risk factor for VTE in the north Indian population. Elevated levels of FIX and FXI were not associated with increased risk of VTE. PMID:26340461

  5. Title IX Lawsuits.

    ERIC Educational Resources Information Center

    Tungate, David E.; Orie, Daniel P.

    1998-01-01

    Since Brown University lost its four-year court battle over athletic program equality issues, most colleges and secondary schools have learned to settle when sued under Title IX. Virginia Tech, University of Kansas, and Howard University are illustrative cases. Since nearly all high schools and colleges are vulnerable, it is wise to prepare for…

  6. Absence of in vitro Procoagulant Activity in Immunoglobulin Preparations due to Activated Coagulation Factors

    PubMed Central

    Oviedo, Adriana E.; Bernardi, María E.; Guglielmone, Hugo A.; Vitali, María S.

    2015-01-01

    Summary Background Immunoglobulin (IG) products, including intravenous (IVIG) or subcutaneous (SCIG) immunoglobulins are considered safe and effective for medical therapy; however, a sudden and unexpected increase in thromboembolic events (TE) after administration of certain batches of IVIG products has been attributed to the presence of activated coagulation factors, mainly factor XIa. Our aims were to examine the presence of enduring procoagulant activity during the manufacturing process of IGs, with special focus on monitoring factor XIa, and to evaluate the presence of in vitro procoagulant activity attributed to coagulation factors in different lots of IVIG and SCIG. Methods Samples of different steps of IG purification, 19 lots of IVIG and 9 of SCIG were analyzed and compared with 1 commercial preparation of IVIG and 2 of SCIG, respectively. Factors II, VII, IX, XI and XIa and non-activated partial thromboplastin time (NAPTT) were assayed. Results The levels of factors II, VII, IX, X and XI were non-quantifiable once fraction II had been re-dissolved and in all analyzed lots of IVIG and SCIG. The level of factor XIa at that point was under the detection limits of the assay, and NAPTT yielded values greater than the control during the purification process. In SCIG, we detected higher concentrations of factor XIa in the commercial products, which reached values up to 5 times higher than the average amounts found in the 9 batches produced by UNC-Hemoderivados. Factor XIa in commercial IVIG reached levels slightly higher than those of the 19 batches produced by UNC-Hemoderivados. Conclusion IVIG and SCIG manufactured by UNC-Hemoderivados showed a lack of thrombogenic potential, as demonstrated not only by the laboratory data obtained in this study but also by the absence of any reports of TE registered by the post marketing pharmacovigilance department. PMID:26733772

  7. A role for factor XIIa–mediated factor XI activation in thrombus formation in vivo

    PubMed Central

    Cheng, Qiufang; Tucker, Erik I.; Pine, Meghann S.; Sisler, India; Matafonov, Anton; Sun, Mao-fu; White-Adams, Tara C.; Smith, Stephanie A.; Hanson, Stephen R.; McCarty, Owen J. T.; Renné, Thomas; Gruber, András

    2010-01-01

    Mice lacking factor XII (fXII) or factor XI (fXI) are resistant to experimentally–induced thrombosis, suggesting fXIIa activation of fXI contributes to thrombus formation in vivo. It is not clear whether this reaction has relevance for thrombosis in pri mates. In 2 carotid artery injury models (FeCl3 and Rose Bengal/laser), fXII-deficient mice are more resistant to thrombosis than fXI- or factor IX (fIX)–deficient mice, raising the possibility that fXII and fXI function in distinct pathways. Antibody 14E11 binds fXI from a variety of mammals and interferes with fXI activation by fXIIa in vitro. In mice, 14E11 prevented arterial occlusion induced by FeCl3 to a similar degree to total fXI deficiency. 14E11 also had a modest beneficial effect in a tissue factor–induced pulmonary embolism model, indicating fXI and fXII contribute to thrombus formation even when factor VIIa/tissue factor initiates thrombosis. In baboons, 14E11 reduced platelet-rich thrombus growth in collagen-coated grafts inserted into an arteriovenous shunt. These data support the hypothesis that fXIIa-mediated fXI activation contributes to thrombus formation in rodents and primates. Since fXII deficiency does not impair hemostasis, targeted inhibition of fXI activation by fXIIa may be a useful antithrombotic strategy associated with a low risk of bleeding complications. PMID:20634381

  8. Apoptosis of THP-1 macrophages induced by protoporphyrin IX-mediated sonodynamic therapy

    PubMed Central

    Guo, Shuyuan; Sun, Xin; Cheng, Jiali; Xu, Haobo; Dan, Juhua; Shen, Jing; Zhou, Qi; Zhang, Yun; Meng, Lingli; Cao, Wenwu; Tian, Ye

    2013-01-01

    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. Production of intracellular singlet oxygen and secondary disruption of the cytoskeleton were also observed after SDT with PpIX. Conclusion PpIX-mediated SDT had apoptotic effects on THP-1 macrophages via generation of intracellular singlet oxygen and disruption of the cytoskeleton. PpIX-mediated SDT may be a potential treatment to attenuate progression of atherosclerotic plaque. PMID:23818780

  9. Identification of a point mutation in type IIB von Willebrand disease illustrating the regulation of von Willebrand factor affinity for the platelet membrane glycoprotein Ib-IX receptor.

    PubMed Central

    Ware, J; Dent, J A; Azuma, H; Sugimoto, M; Kyrle, P A; Yoshioka, A; Ruggeri, Z M

    1991-01-01

    von Willebrand factor (vWF) supports platelet adhesion on thrombogenic surfaces by binding to platelet membrane glycoprotein (GP) Ib in the GP Ib-IX receptor complex. This interaction is physiologically regulated so that it does not occur between circulating vWF and platelets but, rather, only at a site of vascular injury. The abnormal vWF found in type IIB von Willebrand disease, however, has a characteristically increased affinity for GP Ib and binds to circulating platelets. We have analyzed the molecular basis of this abnormality by sequence analysis of a type IIB vWF cDNA and have identified a single amino acid change, Trp550 to Cys550, located in the GP Ib-binding domain of the molecule comprising residues 449-728. Bacterial expression of recombinant fragments corresponding to this vWF domain yielded molecules that, whether containing a normal Trp550 or a mutant Cys550 residue, bound directly to GP Ib in the absence of modulators and with similar affinity. In contrast, mammalian cell expression of the same segment of sequence yielded molecules that, when containing the normal Trp550, did not bind to GP Ib directly but, like native vWF, bound in the presence of ristocetin. However, molecules containing the point mutation (Cys550) behaved like type IIB vWF--namely, bound to GP Ib even without ristocetin modulation and, in the presence of ristocetin, had 10-fold higher affinity than molecules with normal sequence. These results identify a region of vWF that, although not thought to be directly involved in binding to GP Ib, may modulate the interaction through conformational changes. Images PMID:2011604

  10. Genetic modification of bone-marrow mesenchymal stem cells and hematopoietic cells with human coagulation factor IX-expressing plasmids.

    PubMed

    Sam, Mohammad Reza; Azadbakhsh, Azadeh Sadat; Farokhi, Farrah; Rezazadeh, Kobra; Sam, Sohrab; Zomorodipour, Alireza; Haddad-Mashadrizeh, Aliakbar; Delirezh, Nowruz; Mokarizadeh, Aram

    2016-05-01

    Ex-vivo gene therapy of hemophilias requires suitable bioreactors for secretion of hFIX into the circulation and stem cells hold great potentials in this regard. Viral vectors are widely manipulated and used to transfer hFIX gene into stem cells. However, little attention has been paid to the manipulation of hFIX transgene itself. Concurrently, the efficacy of such a therapeutic approach depends on determination of which vectors give maximal transgene expression. With this in mind, TF-1 (primary hematopoietic lineage) and rat-bone marrow mesenchymal stem cells (BMSCs) were transfected with five hFIX-expressing plasmids containing different combinations of two human β-globin (hBG) introns inside the hFIX-cDNA and Kozak element and hFIX expression was evaluated by different methods. In BMSCs and TF-1 cells, the highest hFIX level was obtained from the intron-less and hBG intron-I,II containing plasmids respectively. The highest hFIX activity was obtained from the cells that carrying the hBG intron-I,II containing plasmids. BMSCs were able to produce higher hFIX by 1.4 to 4.7-fold increase with activity by 2.4 to 4.4-fold increase compared to TF-1 cells transfected with the same constructs. BMSCs and TF-1 cells could be effectively bioengineered without the use of viral vectors and hFIX minigene containing hBG introns could represent a particular interest in stem cell-based gene therapy of hemophilias. PMID:26928674

  11. Coagulant Activity of Leukocytes. TISSUE FACTOR ACTIVITY

    PubMed Central

    Niemetz, J.

    1972-01-01

    Peritoneal leukocytes harvested from rabbits which have received two spaced doses of endotoxin have significantly greater (10-fold) coagulant activity than leukocytes from control rabbits. The coagulant activity accelerates the clotting of normal plasma and activates factor X in the presence of factor VII and calcium and is therefore regarded as tissue factor. A total of 40-80 mg tissue factor activity was obtained from the peritoneal cavity of single endotoxin-treated rabbits. In leukocyte subcellular fractions, separated by centrifugation, the specific tissue factor activity sedimented mainly at 14,500 g and above. The procoagulant activity was destroyed after heating for 10 min at 65°C but was preserved at lower temperatures. Polymyxin B, when given with the first dose of endotoxin, reduced both the number of peritoneal leukocytes and their tissue factor activity by two-thirds. When given immediately before the second dose of endotoxin, polymyxin B had no inhibitory effect. PMID:4333021

  12. Preparation of factor VII concentrate using CNBr-activated Sepharose 4B immunoaffinity chromatography

    PubMed Central

    Mousavi Hosseini, Kamran; Nasiri, Saleh

    2015-01-01

    Background: Factor VII concentrates are used in patients with congenital or acquired factor VII deficiency or treatment of hemophilia patients with inhibitors. In this research, immunoaffinity chromatography was used to purify factor VII from prothrombin complex (Prothrombin- Proconvertin-Stuart Factor-Antihemophilic Factor B or PPSB) which contains coagulation factors II, VII, IX and X. The aim of this study was to improve purity, safety and tolerability as a highly purified factor VII concentrate. Methods: PPSB was prepared using DEAE-Sephadex and was used as the starting material for purification of coagulation factor VII. Prothrombin complex was treated by solvent/detergent at 24°C for 6 h with constant stirring. The mixture of PPSB in the PBS buffer was filtered and then chromatographed using CNBr-activated Sepharose 4B coupled with specific antibody. Factors II, IX, VII, X and VIIa were assayed on the fractions. Fractions of 48-50 were pooled and lyophilized as a factor VII concentrate. Agarose gel electrophoresis was performed and Tween 80 was measured in the factor VII concentrate. Results: Specific activity of factor VII concentrate increased from 0.16 to 55.6 with a purificationfold of 347.5 and the amount of activated factor VII (FVIIa) was found higher than PPSB (4.4-fold). Results of electrophoresis on agarose gel indicated higher purity of Factor VII compared to PPSB; these finding revealed that factor VII migrated as alpha-2 proteins. In order to improve viral safety, solvent-detergent treatment was applied prior to further purification and nearly complete elimination of tween 80 (2 μg/ml). Conclusion: It was concluded that immuonoaffinity chromatography using CNBr-activated Sepharose 4B can be a suitable choice for large-scale production of factor VII concentrate with higher purity, safety and activated factor VII. PMID:26034723

  13. Title IX: Boom or Bust?

    ERIC Educational Resources Information Center

    Mather, Marilyn J.

    2003-01-01

    Athletics has been significantly impacted by Title IX through an increase the number of female athletes, the number of teams available, and indirectly, the development of women's professional leagues. However, women in leadership positions in athletics have declined significantly since Title IX was signed into law. A concern about the…

  14. Characterization of carbonic anhydrase IX (CA IX) as an endogenous marker of chronic hypoxia in live human tumor cells

    SciTech Connect

    Vordermark, Dirk . E-mail: vordermark_d@klinik.uni-wuerzburg.de; Kaffer, Anja; Riedl, Susanne; Katzer, Astrid; Flentje, Michael

    2005-03-15

    Purpose: Published clinical studies provide conflicting data regarding the prognostic significance of carbonic anhydrase IX (CA IX) overexpression as an endogenous marker of tumor hypoxia and its comparability with other methods of hypoxia detection. We performed a systematic analysis of CA IX protein levels under various in vitro conditions of tumor hypoxia in HT 1080 human fibrosarcoma and FaDu human pharyngeal carcinoma cells. Because sorting of live CA IX positive cells from tumors provides a tool to study the radiosensitivity of chronically hypoxic cells, we modified and tested a CA IX flow cytometry protocol on mixed hypoxic/aerobic suspensions of HT 1080 and FaDu cells. Methods and materials: HT 1080 and FaDu cells were treated with up to 24 h of in vitro hypoxia and up to 96 h of reoxygenation. To test the effect of nonhypoxic stimuli, glucose and serum availability, pH and cell density were modified. CA IX protein was quantified in Western blots of whole-cell lysates. Mixed suspensions with known percentages of hypoxic cells were prepared for CA IX flow cytometry. The same mixtures were assayed for clonogenic survival after 10 Gy. Results: Hypoxia-induced CA IX protein expression was seen after >6 h at {<=}5% O{sub 2}, and protein was stable over 96 h of reoxygenation in both cell lines. Glucose deprivation abolished the hypoxic CA IX response, and high cell density caused CA IX induction under aerobic conditions. Measured percentages of CA IX-positive cells in mixtures closely reflected known percentages of hypoxic cells in HT 1080 and were associated with radioresistance of mixtures after 10 Gy. Conclusion: CA IX is a stable marker of current or previous chronic hypoxia but influenced by nonhypoxic stimuli. Except the time course of accumulation, all properties of this marker resembled our previous findings for hypoxia-inducible factor-1{alpha}. A modified flow cytometry protocol provided good separability of CA IX-negative and -positive cells in vitro

  15. ALA-induced PpIX fluorescence in epileptogenic tissue

    NASA Astrophysics Data System (ADS)

    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.

  16. Structural Insights into Carbonic Anhydrase IX Isoform Specificity of Carbohydrate-Based Sulfamates

    PubMed Central

    2015-01-01

    Carbonic anhydrase IX (CA IX) is an extracellular transmembrane homodimeric zinc metalloenzyme that has been validated as a prognostic marker and therapeutic target for several types of aggressive cancers. CA IX shares a close homology with other CA isoforms, making the design of CA IX isoform selective inhibitors challenging. In this paper, we describe the development of a new class of CA IX inhibitors that comprise a sulfamate as the zinc binding group, a variable linker, and a carbohydrate “tail” moiety. Seven compounds inhibited CA IX with low nM Ki values of 1–2 nM and also exhibited permeability profiles to preferentially target the binding of extracellular CA IX over cytosolic CAs. The crystal structures of two of these compounds in complex with a CA IX-mimic (a variant of CA II, with active site residues that mimic CA IX) and one compound in complex with CA II have been determined to 1.7 Å resolution or better and demonstrate a selective mechanism of binding between the hydrophilic and hydrophobic pockets of CA IX versus CA II. These compounds present promising candidates for anti-CA IX drugs and the treatment for several aggressive cancer types. PMID:25254302

  17. Phosphorylation of carbonic anhydrase IX controls its ability to mediate extracellular acidification in hypoxic tumors.

    PubMed

    Ditte, Peter; Dequiedt, Franck; Svastova, Eliska; Hulikova, Alzbeta; Ohradanova-Repic, Anna; Zatovicova, Miriam; Csaderova, Lucia; Kopacek, Juraj; Supuran, Claudiu T; Pastorekova, Silvia; Pastorek, Jaromir

    2011-12-15

    In the hypoxic regions of a tumor, carbonic anhydrase IX (CA IX) is an important transmembrane component of the pH regulatory machinery that participates in bicarbonate transport. Because tumor pH has implications for growth, invasion, and therapy, determining the basis for the contributions of CA IX to the hypoxic tumor microenvironment could lead to new fundamental and practical insights. Here, we report that Thr443 phosphorylation at the intracellular domain of CA IX by protein kinase A (PKA) is critical for its activation in hypoxic cells, with the fullest activity of CA IX also requiring dephosphorylation of Ser448. PKA is activated by cAMP, which is elevated by hypoxia, and we found that attenuating PKA in cells disrupted CA IX-mediated extracellular acidification. Moreover, following hypoxia induction, CA IX colocalized with the sodium-bicarbonate cotransporter and other PKA substrates in the leading edge membranes of migrating tumor cells, in support of the concept that bicarbonate metabolism is spatially regulated at cell surface sites with high local ion transport and pH control. Using chimeric CA IX proteins containing heterologous catalytic domains derived from related CA enzymes, we showed that CA IX activity was modulated chiefly by the intracellular domain where Thr443 is located. Our findings indicate that CA IX is a pivotal mediator of the hypoxia-cAMP-PKA axis, which regulates pH in the hypoxic tumor microenvironment. PMID:22037869

  18. The effects of CA IX catalysis products within tumor microenvironment.

    PubMed

    Santi, Alice; Caselli, Anna; Paoli, Paolo; Corti, Denise; Camici, Guido; Pieraccini, Giuseppe; Taddei, Maria Letizia; Serni, Sergio; Chiarugi, Paola; Cirri, Paolo

    2013-01-01

    Solid tumors are composed of both cancer cells and various types of accessory cells, mainly fibroblasts, that collectively compose the so called tumor-microenvironment. Cancer-associated fibroblasts have been described to actively participate in cancer progression by establishing a cytokine-mediated as well as metabolic crosstalk with cancer cells. In the present paper we show that activated human fibroblasts are able to boost tumor cells proliferation and that this effect is greatly dependent on stromal carbonic anhydrase IX (CA IX) activity. In fact fibroblasts show a strong upregulation of CA IX expression upon activation by cancer cells, while CA IX products, protons and bicarbonate, exert differential effects on cancer cells proliferation. While acidification of extracellular pH, a typical condition of rapidly growing solid tumors, is detrimental for tumor cells proliferation, bicarbonate, through its organication, supplies cancer cells with intermediates useful to sustain their high proliferation rate. Here we propose a new kind of fibroblasts/tumor cells crosstalk within tumor microenvironment, mediated by stromal CA IX products, aimed to favor cancer cells growth, opening new perspectives on CA IX role in tumor microenvironment. PMID:24168032

  19. Learning about Body Systems. Superific Science Book IX. A Good Apple Science Activity Book for Grades 5-8+.

    ERIC Educational Resources Information Center

    Conway, Lorraine

    This document offers an explanation and drawings of each of the major systems of the human body and of the five senses. It provides teachers with classroom activities, demonstrations, and experiments which are intended to involve students in the acquisition of knowledge concerning the structure and function of their bodies. The drawings of the…

  20. Design and synthesis of novel 4-(4-oxo-2-arylthiazolidin-3-yl)benzenesulfonamides as selective inhibitors of carbonic anhydrase IX over I and II with potential anticancer activity.

    PubMed

    Suthar, Sharad Kumar; Bansal, Sumit; Lohan, Sandeep; Modak, Vikarm; Chaudhary, Anil; Tiwari, Amit

    2013-08-01

    The novel 4-(4-oxo-2-arylthiazolidin-3-yl)benzenesulfonamide derivatives were designed and synthesized for selective carbonic anhydrase IX (CA IX) inhibitory activity with anticancer potential. In the CA inhibition assay, 3f was found to be the most potent and selective inhibitor of CA IX with inhibitory constant (K(I)) value of 2.2 nM. Among the synthesized compounds, 3f showed IC₅₀ values of 5.03 μg/ml (cisplatin: 6.56 μg/ml), 5.81 μg/ml (cisplatin: 5.85 μg/ml), and 23.93 μg/ml (cisplatin: 2.75 μg/ml) against COLO-205, MDA-MB-231, and DU-145 cell lines, respectively. At IC₅₀, 3f caused cell shrinkage, nuclear condensation, and nuclear fragmentation events characteristic to apoptosis in the Hoechst 33258 and acridine orange-ethidium bromide staining studies of COLO-205 cells. In the Dalton's lymphoma ascites (DLA) solid tumor model 3f decreased tumor volume by 64.83% (cisplatin: 71.62%), while increase in mean body weight was found to be only 4.09% (cisplatin: 3.47%). PMID:23827177

  1. Employing a Gain-of-Function Factor IX Variant R338L to Advance the Efficacy and Safety of Hemophilia B Human Gene Therapy: Preclinical Evaluation Supporting an Ongoing Adeno-Associated Virus Clinical Trial

    PubMed Central

    Sun, Junjiang; Gui, Tong; Hu, Genlin; Hannah, William B.; Wichlan, David G.; Wu, Zhijian; Grieger, Joshua C.; Li, Chengwen; Suwanmanee, Thipparat; Stafford, Darrel W.; Booth, Carmen J.; Samulski, Jade J.; Kafri, Tal; McPhee, Scott W.J.

    2015-01-01

    Abstract Vector capsid dose-dependent inflammation of transduced liver has limited the ability of adeno-associated virus (AAV) factor IX (FIX) gene therapy vectors to reliably convert severe to mild hemophilia B in human clinical trials. These trials also identified the need to understand AAV neutralizing antibodies and empty AAV capsids regarding their impact on clinical success. To address these safety concerns, we have used a scalable manufacturing process to produce GMP-grade AAV8 expressing the FIXR338L gain-of-function variant with minimal (<10%) empty capsid and have performed comprehensive dose–response, biodistribution, and safety evaluations in clinically relevant hemophilia models. The scAAV8.FIXR338L vector produced greater than 6-fold increased FIX specific activity compared with wild-type FIX and demonstrated linear dose responses from doses that produced 2–500% FIX activity, associated with dose-dependent hemostasis in a tail transection bleeding challenge. More importantly, using a bleeding model that closely mimics the clinical morbidity of hemophilic arthropathy, mice that received the scAAV8.FIXR338L vector developed minimal histopathological findings of synovitis after hemarthrosis, when compared with mice that received identical doses of wild-type FIX vector. Hemostatically normal mice (n=20) and hemophilic mice (n=88) developed no FIX antibodies after peripheral intravenous vector delivery. No CD8+ T cell liver infiltrates were observed, despite the marked tropism of scAAV8.FIXR338L for the liver in a comprehensive biodistribution evaluation (n=60 animals). With respect to the role of empty capsids, we demonstrated that in vivo FIXR338L expression was not influenced by the presence of empty AAV particles, either in the presence or absence of various titers of AAV8-neutralizing antibodies. Necropsy of FIX–/– mice 8–10 months after vector delivery revealed no microvascular or macrovascular thrombosis in mice expressing FIXR338L (plasma

  2. Employing a gain-of-function factor IX variant R338L to advance the efficacy and safety of hemophilia B human gene therapy: preclinical evaluation supporting an ongoing adeno-associated virus clinical trial.

    PubMed

    Monahan, Paul E; Sun, Junjiang; Gui, Tong; Hu, Genlin; Hannah, William B; Wichlan, David G; Wu, Zhijian; Grieger, Joshua C; Li, Chengwen; Suwanmanee, Thipparat; Stafford, Darrel W; Booth, Carmen J; Samulski, Jade J; Kafri, Tal; McPhee, Scott W J; Samulski, R Jude

    2015-02-01

    Vector capsid dose-dependent inflammation of transduced liver has limited the ability of adeno-associated virus (AAV) factor IX (FIX) gene therapy vectors to reliably convert severe to mild hemophilia B in human clinical trials. These trials also identified the need to understand AAV neutralizing antibodies and empty AAV capsids regarding their impact on clinical success. To address these safety concerns, we have used a scalable manufacturing process to produce GMP-grade AAV8 expressing the FIXR338L gain-of-function variant with minimal (<10%) empty capsid and have performed comprehensive dose-response, biodistribution, and safety evaluations in clinically relevant hemophilia models. The scAAV8.FIXR338L vector produced greater than 6-fold increased FIX specific activity compared with wild-type FIX and demonstrated linear dose responses from doses that produced 2-500% FIX activity, associated with dose-dependent hemostasis in a tail transection bleeding challenge. More importantly, using a bleeding model that closely mimics the clinical morbidity of hemophilic arthropathy, mice that received the scAAV8.FIXR338L vector developed minimal histopathological findings of synovitis after hemarthrosis, when compared with mice that received identical doses of wild-type FIX vector. Hemostatically normal mice (n=20) and hemophilic mice (n=88) developed no FIX antibodies after peripheral intravenous vector delivery. No CD8(+) T cell liver infiltrates were observed, despite the marked tropism of scAAV8.FIXR338L for the liver in a comprehensive biodistribution evaluation (n=60 animals). With respect to the role of empty capsids, we demonstrated that in vivo FIXR338L expression was not influenced by the presence of empty AAV particles, either in the presence or absence of various titers of AAV8-neutralizing antibodies. Necropsy of FIX(-/-) mice 8-10 months after vector delivery revealed no microvascular or macrovascular thrombosis in mice expressing FIXR338L (plasma FIX activity

  3. A sensitive, accurate assay for extrinsic pathway inhibitor (EPI) activity in rabbit plasma: Paradoxical effect of excess exogenous factor X

    SciTech Connect

    Warr, T.A.; Rao, L.V.; Rapaport, S.I. )

    1990-08-15

    A sensitive assay is described for the measurement of rabbit plasma EPI activity in experimental studies of induced hypercoagulable states in this species. It is based upon the ability of a dilution of rabbit plasma to inhibit human factor VIIa/rabbit brain tissue factor (TF) catalyzed activation of human factor IX (tritiated activation peptide release assay). Addition of {sup 3}H-factor IX to the reaction mixture is delayed for 45 minutes to allow full inhibition by EPI/factor Xa complex before the residual catalytic activity of factor VIIa/TF is measured. Although the diluted rabbit plasma test sample contains both EPI and factor X, supplemental factor X is added to the reaction mixture to assure that only EPI content of the test sample affects the assay result. However, the final concentration of factor X in the reaction mixture is critical. Too high a concentration of factor X diminishes the sensitivity of the assay. The reason for this phenomenon, which was observed with both human and rabbit factor X preparations, is unknown.

  4. Experimental Mg IX photorecombination rate coefficient

    NASA Astrophysics Data System (ADS)

    Schippers, S.; Schnell, M.; Brandau, C.; Kieslich, S.; Müller, A.; Wolf, A.

    2004-07-01

    The rate coefficient for radiative and dielectronic recombination of beryllium-like magnesium ions was measured with high resolution at the Heidelberg heavy-ion storage ring TSR. In the electron-ion collision energy range 0-207 eV resonances due to 2s -> 2p (Δ N = 0) and 2s -> 3l (Δ N=1) core excitations were detected. At low energies below 0.15 eV the recombination rate coefficient is dominated by strong 1s2 (2s 2p 3P) 7l resonances with the strongest one occuring at an energy of only 21 meV. These resonances decisively influence the Mg IX recombination rate coefficient in a low temperature plasma. The experimentally derived Mg IX dielectronic recombination rate coefficient (±15% systematical uncertainty) is compared with the recommendation by Mazzotta et al. (1998, A&AS, 133, 403) and the recent calculations by Gu (2003, ApJ, 590, 1131) and by Colgan et al. (2003, A&A, 412, 597). These results deviate from the experimental rate coefficient by 130%, 82% and 25%, respectively, at the temperature where the fractional abundance of Mg IX is expected to peak in a photoionized plasma. At this temperature a theoretical uncertainty in the 1s2 (2s 2p 3P) 7l resonance positions of only 100 meV would translate into an uncertainty of the plasma rate coefficient of almost a factor 3. This finding emphasizes that an accurate theoretical calculation of the Mg IX recombination rate coefficient from first principles is challenging.

  5. Effect of 5-aminolevulinic acid on kinetics of protoporphyrin IX production in CHO cells.

    PubMed

    Wołuń-Cholewa, M; Warchoł, W

    2004-01-01

    5-aminolevulinic acid (ALA) is utilized in a photodynamic therapy as a compound capable of augmenting intracellular pool of protoporphyrin IX (PpIX), which exhibits properties of a photosensitizer. The studies were aimed at monitoring accumulation of endogenous protoporphyrin IX in CHO cells under effect of various concentrations of ALA in culture medium and following removal of the compound from the culture medium. Cell content of PpIX was determined following incubation of the cells for 72 h in a culture medium containing different concentration of ALA. Moreover, the cells were preincubated for 2 h in ALA at various concentrations and separated from the compound by medium change and their PpIX content was monitored following incubation. PpIX content was defined by a fluorescent technique under the confocal microscope. In the course of continuous incubation of cells with ALA, biphasic alterations were noted in cellular PpIX concentration. Removal of ALA from the incubation medium resulted at first in a decrease in PpIX content in cells, which was followed by an evidently augmented accumulation of the compound in the cells. The results suggested that in the case of CHO cells, exogenous ALA was not an exclusive source of PpIX synthesis and that alterations in enzyme activities were responsible for production of PpIX. PMID:15253138

  6. Inhibition of carbonic anhydrase IX as a novel anticancer mechanism

    PubMed Central

    Supuran, Claudiu T

    2012-01-01

    Carbonic anhydrases (CA, EC 4.2.1.1) catalyze the interconversion bewteen carbon dioxide and bicarbonate with generation of protons. The carbonic anhydrase isozyme IX (CA IX) is highly overexpresed in hypoxic tumors and shows very restricted expression in normal tissues. CA IX is a dimeric protein possessing very high catalytic activity for the hydration of carbon dioxide to protons and bicarbonate. Its quaternary structure is unique among members of this family of enzymes, allowing for structure-based drug design campaigns of selective inhibitors. Inhibition of CA IX with sulfonamide and/or coumarin inhibitors was recently shown to lead to a potent retardation for the growth of both primary tumors and metastases. Some fluorescent sulfonamides were shown to accumulate only in hypoxic tumor cells overexpressing CA IX, and might be used as diagnostic tools for imaging of hypoxic cancers. Sulfonamide inhibitors were also more effective in inhibiting the growth of the primary tumors when associated with irrdiation. CA IX is thus both a diagnostic and therapeutic validated target for the management of hypoxic tumors normally non-responsive to classical chemio- and radiotherapy. PMID:22787577

  7. Fibrinogen blocks the autoactivation and thrombin-mediated activation of factor XI on dextran sulfate.

    PubMed Central

    Scott, C F; Colman, R W

    1992-01-01

    The intrinsic pathway of blood coagulation is activated when factor XIa, one of the three contact-system enzymes, is generated and then activates factor IX. Factor XI has been shown to be efficiently activated in vitro by surface-bound factor XIIa after factor XI is transported to the surface by its cofactor, high molecular weight kininogen (HK). However, individuals lacking any of the three contact-system proteins--namely, factor XII, prekallikrein, and HK--do not suffer from bleeding abnormalities. This mystery has led several investigators to search for an "alternate" activation pathway for factor XI. Recently, factor XI has been reported to be autoactivated on the soluble "surface" dextran sulfate, and thrombin was shown to accelerate the autoactivation. However, it was also reported that HK, the cofactor for factor XIIa-mediated activation of factor XI, actually diminishes the thrombin-catalyzed activation rate of factor XI. Nonetheless, it was suggested that thrombin was a more efficient activator than factor XIIa. In this report we investigated the effect of fibrinogen, the major coagulation protein in plasma, on the activation rate of factor XI. Fibrinogen, the preferred substrate for thrombin in plasma, virtually prevented autoactivation of factor XI as well as the thrombin-mediated activation of factor XI, while having no effect on factor XIIa-catalyzed activation. HK dramatically curtailed the autoactivation of factor XI in addition to the thrombin-mediated activation. These data indicate that factor XI would not be autoactivated in a plasma environment, and thrombin would, therefore, be unlikely to potentiate the activation. We believe that the "missing pathway" for factor XI activation remains an enigma that warrants further investigation. PMID:1454798

  8. Phytoestrogen Suppresses Efflux of the Diagnostic Marker Protoporphyrin IX in Lung Carcinoma.

    PubMed

    Fujita, Hirofumi; Nagakawa, Keisuke; Kobuchi, Hirotsugu; Ogino, Tetsuya; Kondo, Yoichi; Inoue, Keiji; Shuin, Taro; Utsumi, Toshihiko; Utsumi, Kozo; Sasaki, Junzo; Ohuchi, Hideyo

    2016-04-01

    One promising method to visualize cancer cells is based on the detection of the fluorescent photosensitizer protoporphyrin IX (PpIX) synthesized from 5-aminolevulinic acid (ALA), but this method cannot be used in cancers that exhibit poor PpIX accumulation. PpIX appears to be pumped out of cancer cells by the ABC transporter G2 (ABCG2), which is associated with multidrug resistance. Genistein is a phytoestrogen that appears to competitively inhibit ABCG2 activity. Therefore, we investigated whether genistein can promote PpIX accumulation in human lung carcinoma cells. Here we report that treatment of A549 lung carcinoma cells with genistein or a specific ABCG2 inhibitor promoted ALA-mediated accumulation of PpIX by approximately 2-fold. ABCG2 depletion and overexpression studies further revealed that genistein promoted PpIX accumulation via functional repression of ABCG2. After an extended period of genistein treatment, a significant increase in PpIX accumulation was observed in A549 cells (3.7-fold) and in other cell lines. Systemic preconditioning with genistein in a mouse xenograft model of lung carcinoma resulted in a 1.8-fold increase in accumulated PpIX. Long-term genistein treatment stimulated the expression of genes encoding enzymes involved in PpIX synthesis, such as porphobilinogen deaminase, uroporphyrinogen decarboxylase, and protoporphyrinogen oxidase. Accordingly, the rate of PpIX synthesis was also accelerated by genistein pretreatment. Thus, our results suggest that genistein treatment effectively enhances ALA-induced PpIX accumulation by preventing the ABCG2-mediated efflux of PpIX from lung cancer cells and may represent a promising strategy to improve ALA-based diagnostic approaches in a broader set of malignancies. Cancer Res; 76(7); 1837-46. ©2016 AACR. PMID:26837765

  9. Temporal variations in plasma vitamin K and lipid concentrations and clotting factor activity in humans.

    PubMed

    Kamali, F; Edwards, C; Wood, P; Wynne, H A; Kesteven, P

    2001-11-01

    There is no information available on temporal variability in plasma vitamin K concentrations and its relationship to coagulation processes. We investigated the possible existence of temporal changes in plasma vitamin K and lipid concentrations and activity of clotting factors II, VII, IX, and X and relationships between these variables. Plasma vitamin K and lipid concentrations and clotting factor activity were measured at four-hour intervals for 28 hours in a group of healthy volunteers. Temporal variations existed in plasma vitamin K concentrations, with a mean maximum at 22:00 hr and a mean minimum (32% of the maximum) at 10:00 hr. Plasma triglycerol concentrations mirrored the changes in vitamin K concentrations. Mean factor VII activity was positively correlated with mean total plasma cholesterol concentrations (r = 0.714; P < 0.0001) and with mean plasma low density lipoprotein (LDL) cholesterol concentrations (r = 0.461; P < 0.0001). No distinct correlations were found between plasma vitamin K concentrations and either high density lipoprotein (HDL) or LDL cholesterol concentrations, or between triglycerol, HDL, or LDL cholesterol concentrations and functional activity of factors II, IX, and X. Plasma vitamin K concentrations did not correlate with the functional activity of any of the clotting factors. The presence of a correlation between plasma cholesterol concentrations and factor VII activity for blood samples collected at four-hour intervals suggests that plasma cholesterol concentrations may have a more acute effect on factor VII activity. Temporal variations in plasma vitamin K concentrations indicate that a single time point measurement may be an inappropriate method of establishing vitamin K status in an individual. PMID:11754396

  10. In-vivo fluorescence dosimetry of aminolevulinate-based protoporphyrin IX (PpIX) accumulation in human nonmelanoma skin cancers and precancers

    NASA Astrophysics Data System (ADS)

    Warren, Christine B.; Lohser, Sara; Chang, Sung; Bailin, Philip A.; Maytin, Edward V.

    2009-06-01

    PDT is clinically useful for precancers (actinic keratoses; AK) of the skin, but the optimal duration for 5-ALA application is still controversial. For basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), cure rates remain inferior to surgical excision. Lack of knowledge about regional levels of PpIX levels within target tissues clearly contribute to these suboptimal results. To investigate PpIX levels achievable in human skin neoplasias in-vivo, a clinical study to monitor PpIX accumulation in vivo was performed. PpIX-fluorescence in patients undergoing ALA-PDT for facial AK was monitored via real-time in-vivo fluorescence dosimetry, with measurements q20 min following application of 5-ALA (Levulan Kerastick). PpIX accumulation followed linear kinetics in nearly all cases. The slopes varied widely, and did not correlate with clinical outcome in all patients. Some patients with a low accumulation of PpIX fluorescence had a good response to therapy, whereas others with high PpIX accumulation required repeat treatment (although not necessarily of the same lesion). PpIX accumulation rates did correlate to a certain degree with the overall amount of erythema. We conclude that unknown factors besides PpIX levels must be critical for the response to treatment. To assess the relationship between PpIX levels in various skin cancers, patients undergoing routine Mohs surgery for BCC or SCC were measured by in-vivo dosimetry at 2 h after 5-ALA application. Overall, a progressive increase in PpIX signal during malignant progression was observed, in the following rank order: Normal skin < AK < SCC ~ BCC.

  11. Cell cycle dependence of protophorphyrin IX generation in 9L rat gliosarcoma

    NASA Astrophysics Data System (ADS)

    Luo, Shiming; Da, Xing; Chen, Qun

    2006-09-01

    Photodynamic therapy (PDT) is a cancer therapy that utilizes optical energy to activate a photosensitizer drug in a target tissue. Always, the curative effect is dependent on the light fluence, the concentration of the photosensitizer and the concentration of the oxygen. To date, Protophorphyrin IX (PpIX) as the only one endogenous photosensitizer is widely used in PDT of brain tumors. Since PpIX is synthesized in intracellular structure, and is likely dependent on the phase of the cell cycle. The cell cycle dependence of PpIX production is thus investigated in the current work in 9L gliosarcoma cells.

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

    PubMed

    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

    2016-08-23

    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 properties that allow for low pH stability and activity. Furthermore, the unfolding process of hCA IX-c appears to be reversible, and its catalytic efficiency is thought to be correlated directly with its stability between pH 3.0 and 8.0 but not above pH 8.0. To rationalize this, we determined the X-ray crystal structure of hCA IX-c to 1.6 Å resolution. Insights from this study suggest an understanding of hCA IX-c stability and activity in low-pH tumor microenvironments and may be applicable to determining pH-related effects on enzymes. PMID:27439028

  13. Reconsidering the Status of Title IX.

    ERIC Educational Resources Information Center

    Hammer, Ben

    2003-01-01

    Discusses the controversy over Title IX and women's participation in college athletics. Critics say the mandate shortchanges men's teams, while proponents say that women's sports programs remain underfunded in spite of Title IX. Describes some proposed modifications to Title IX and their potential effects. (SLD)

  14. Triangulated loop quantum cosmology: Bianchi IX universe and inhomogeneous perturbations

    SciTech Connect

    Battisti, Marco Valerio; Marciano, Antonino; Rovelli, Carlo

    2010-03-15

    We develop the triangulated version of loop quantum cosmology, recently introduced in the literature. We focus on the dipole cosmology, where space is a three-sphere and the triangulation is formed by two tetrahedra. We show that the discrete fiducial connection has a simple and appealing geometrical interpretation and we correct the ansatz on the relation between the model variables and the Friedmann-Robertson-Walker scale factor. The modified ansatz leads to the convergence of the Hamiltonian constraint to the continuum one. We then ask which degrees of freedom are captured by this model. We show that the model is rich enough to describe the (anisotropic) Bianchi IX universe, and give the explicit relation between the Bianchi IX variables and the variables of the model. We discuss the possibility of using this path in order to define the quantization of the Bianchi IX universe. The model contains more degrees of freedom than Bianchi IX, and therefore captures some inhomogeneous degrees of freedom as well. Inhomogeneous degrees of freedom can be expanded in representations of the SU(2) Bianchi IX isometry group, and the dipole model captures the lowest integer representation of these, connected to hyperspherical harmonic of angular momentum j=1.

  15. Critical Issue Bibliography (CRIB) Sheet: Title IX of the Educational Amendments of 1972.

    ERIC Educational Resources Information Center

    ERIC Clearinghouse on Higher Education, Washington, DC.

    This CRitical Issue Bibliography (CRIB) Sheet cites resources that give an overview of Title IX legislation and offer information about compliance, litigation, and related issues. Title IX of the Education Amendments of 1972 protects individuals from gender-based discrimination in education programs or activities that receive federal financial…

  16. Activation of human factor V by factor Xa and thrombin

    SciTech Connect

    Monkovic, D.D.; Tracy, P.B. )

    1990-02-06

    The activation of human factor V by factor Xa and thrombin was studied by functional assessment of cofactor activity and sodium dodecyl sulfate-polycarylamide gel electrophoresis followed by either autoradiography of {sup 125}I-labeled factor V activation products or Western blot analyses of unlabeled factor V activation products. Cofactor activity was measured by the ability of the factor V/Va peptides to support the activation of prothrombin. The factor Xa catalyzed cleavage of factor V was observed to be time, phospholipid, and calcium ion dependent, yielding a cofactor with activity equal to that of thrombin-activated factor V (factor Va). The cleavage pattern differed markedly from the one observed in the bovine system. The factor Xa activated factor V subunits expressing cofactor activity were isolated and found to consist of peptides of M{sub r} 220,000 and 105,000. Although thrombin cleaved the M{sub r} 220,000 peptide to yield peptides previously shown to be products of thrombin activation, cofactor activity did not increase. N-Terminal sequence analysis confirmed that both factor Xa and thrombin cleave factor V at the same bond to generate the M{sub r} 220,000 peptide. The factor Xa dependent functional assessment of {sup 125}I-labeled factor V coupled with densitometric analyses of the cleavage products indicated that the cofactor activity of factor Xa activated factor V closely paralleled the appearance of the M{sub r} 220,000 peptide. The data indicate that factor Xa is as efficient an enzyme toward factor V as thrombin.

  17. Ongoing advances in quantitative PpIX fluorescence guided intracranial tumor resection (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Olson, Jonathan D.; Kanick, Stephen C.; Bravo, Jaime J.; Roberts, David W.; Paulsen, Keith D.

    2016-03-01

    Aminolevulinc-acid induced protoporphyrin IX (ALA-PpIX) is being investigated as a biomarker to guide neurosurgical resection of brain tumors. ALA-PpIX fluorescence can be observed visually in the surgical field; however, raw fluorescence emissions can be distorted by factors other than the fluorophore concentration. Specifically, fluorescence emissions are mixed with autofluorescence and attenuated by background absorption and scattering properties of the tissue. Recent work at Dartmouth has developed advanced fluorescence detection approaches that return quantitative assessments of PpIX concentration, which are independent of background optical properties. The quantitative fluorescence imaging (qFI) approach has increased sensitivity to residual disease within the resection cavity at the end of surgery that was not visible to the naked eye through the operating microscope. This presentation outlines clinical observations made during an ongoing investigation of ALA-PpIX based guidance of tumor resection. PpIX fluorescence measurements made in a wide-field hyperspectral imaging approach are co-registered with point-assessment using a fiber optic probe. Data show variations in the measured PpIX accumulation among different clinical tumor grades (i.e. high grade glioma, low grade glioma), types (i.e. primary tumors. metastases) and normal structures of interest (e.g. normal cortex, hippocampus). These results highlight the contrast enhancement and underscore the potential clinical benefit offered from quantitative measurements of PpIX concentration during resection of intracranial tumors.

  18. The novel CA IX inhibition antibody chKM4927 shows anti-tumor efficacy in vivo.

    PubMed

    Yamaguchi, Ayami; Usami, Katsuaki; Shimabe, Munetake; Hasegawa, Kazumasa; Asada, Masao; Motoki, Kazuhiro; Tahara, Tomoyuki; Masuda, Kazuhiro

    2015-04-01

    Carbonic anhydrase IX (CA IX) is an attractive target for cancer therapy. Many anti-CA IX antibodies have been reported but few have been shown to possess inhibition activity. Furthermore, effective use of CA IX-inhibition antibodies for cancer immunotherapy has not been well-validated since data are mainly limited to in vitro assays. In this study, we established that chKM4927, an anti-CA IX chimeric antibody, recognizes CA IX and has CA IX-specific inhibition activity. ChKM4927 also retains antibody-dependent cellular cytotoxicity (ADCC) activity against CA IX-expressing cancer cells. Compared to controls, chKM4927 treatment (10 mg/kg) showed anti-tumor activity in the VMRC-RCW xenograft model in vivo. ChKM4927-attenuated ADCC activity showed equally effective anti-tumor activity. These results suggest that the CA IX-inhibition antibody chKM4927 has an anti-tumor effect in the VMRC-RCW xenograft model via an ADCC-independent mechanism. PMID:25862852

  19. Discovery and characterization of novel selective inhibitors of carbonic anhydrase IX.

    PubMed

    Dudutienė, Virginija; Matulienė, Jurgita; Smirnov, Alexey; Timm, David D; Zubrienė, Asta; Baranauskienė, Lina; Morkūnaite, Vaida; Smirnovienė, Joana; Michailovienė, Vilma; Juozapaitienė, Vaida; Mickevičiūtė, Aurelija; Kazokaitė, Justina; Bakšytė, Sandra; Kasiliauskaitė, Aistė; Jachno, Jelena; Revuckienė, Jurgita; Kišonaitė, Miglė; Pilipuitytė, Vilma; Ivanauskaitė, Eglė; Milinavičiūtė, Goda; Smirnovas, Vytautas; Petrikaitė, Vilma; Kairys, Visvaldas; Petrauskas, Vytautas; Norvaišas, Povilas; Lingė, Darius; Gibieža, Paulius; Capkauskaitė, Edita; Zakšauskas, Audrius; Kazlauskas, Egidijus; Manakova, Elena; Gražulis, Saulius; Ladbury, John E; Matulis, Daumantas

    2014-11-26

    Human carbonic anhydrase IX (CA IX) is highly expressed in tumor tissues, and its selective inhibition provides a potential target for the treatment of numerous cancers. Development of potent, highly selective inhibitors against this target remains an unmet need in anticancer therapeutics. A series of fluorinated benzenesulfonamides with substituents on the benzene ring was designed and synthesized. Several of these exhibited a highly potent and selective inhibition profile against CA IX. Three fluorine atoms significantly increased the affinity by withdrawing electrons and lowering the pKa of the benzenesulfonamide group. The bulky ortho substituents, such as cyclooctyl or even cyclododecyl groups, fit into the hydrophobic pocket in the active site of CA IX but not CA II, as shown by the compound's co-crystal structure with chimeric CA IX. The strongest inhibitor of recombinant human CA IX's catalytic domain in human cells achieved an affinity of 50 pM. However, the high affinity diminished the selectivity. The most selective compound for CA IX exhibited 10 nM affinity. The compound that showed the best balance between affinity and selectivity bound with 1 nM affinity. The inhibitors described in this work provide the basis for novel anticancer therapeutics targeting CA IX. PMID:25358084

  20. The Transmembrane Domains of β and IX Subunits Mediate the Localization of the Platelet Glycoprotein Ib-IX Complex to the Glycosphingolipid-enriched Membrane Domain.

    PubMed

    Xu, Guofeng; Shang, Dan; Zhang, Zuping; Shaw, Tanner S; Ran, Yali; López, José A; Peng, Yuandong

    2015-09-01

    We have previously reported that the structural elements of the GP Ib-IX complex required for its localization to glycosphingolipid-enriched membranes (GEMs) reside in the Ibβ and IX subunits. To identify them, we generated a series of cell lines expressing mutant GP Ibβ and GP IX where 1) the cytoplasmic tails (CTs) of either or both GP Ibβ and IX are truncated, and 2) the transmembrane domains (TMDs) of GP Ibβ and GP IX were swapped with the TMD of a non-GEMs associating molecule, human transferrin receptor. Sucrose density fractionation analysis showed that the removal of either or both of the CTs from GP Ibβ and GP IX does not alter GP Ibα-GEMs association when compared with the wild type. In contrast, swapping of the TMDs of either GP Ibβ or GP IX with that of transferrin receptor results in a significant loss (∼ 50%) of GP Ibα from the low density GEMs fractions, with the largest effect seen in the dual TMD-replaced cells (> 80% loss) when compared with the wild type cells (100% of GP Ibα present in the GEMs fractions). Under high shear flow, the TMD-swapped cells adhere poorly to a von Willebrand factor-immobilized surface to a much lesser extent than the previously reported disulfide linkage dysfunctional GP Ibα-expressing cells. Thus, our data demonstrate that the bundle of GP Ibβ and GP IX TMDs instead of their individual CTs is the structural element that mediates the β/IX complex localization to the membrane GEMs, which through the α/β disulfide linkage brings GP Ibα into the GEMs. PMID:26203189

  1. 34 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

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    Code of Federal Regulations, 2010 CFR

    1999-10-01

    ... 45 Public Welfare 1 1999-10-01 1999-10-01 false Subject Index to Title IX Preamble and Regulation \\1\\ Index Subject Index to Title IX Preamble and Regulation \\1\\ Public Welfare DEPARTMENT OF.... Pt. 86, Index Subject Index to Title IX Preamble and Regulation \\1\\ 1 Preamble paragraph numbers...

  17. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation \\1\\

    Code of Federal Regulations, 2010 CFR

    1998-10-01

    ... 45 Public Welfare 1 1998-10-01 1998-10-01 false Subject Index to Title IX Preamble and Regulation \\1\\ Index Subject Index to Title IX Preamble and Regulation \\1\\ GENERAL ADMINISTRATION... FINANCIAL ASSISTANCE Procedures Interim procedures. Pt. 86, Index Subject Index to Title IX Preamble...

  18. 34 CFR Subject Index to Title IX... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2015-07-01

    ... 34 Education 1 2015-07-01 2015-07-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Education Regulations of the Offices of the...] Procedures. Subject Index Subject Index to Title IX Preamble and Regulation 1 1 Preamble paragraph...

  19. 34 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2004-07-01

    ... 34 Education 1 2004-07-01 2004-07-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Education Regulations of the Offices of the.... Subject Index Subject Index to Title IX Preamble and Regulation 1 1 Preamble paragraph numbers are...

  20. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2001-10-01

    ... 45 Public Welfare 1 2001-10-01 2001-10-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Public Welfare DEPARTMENT OF HEALTHAND..., Index Subject Index to Title IX Preamble and Regulation 1 1 Preamble paragraph numbers are in...

  1. 34 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2006-07-01

    ... 34 Education 1 2006-07-01 2006-07-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Education Regulations of the Offices of the.... Subject Index Subject Index to Title IX Preamble and Regulation 1 1 Preamble paragraph numbers are...

  2. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation \\1\\

    Code of Federal Regulations, 2010 CFR

    1997-10-01

    ... 45 Public Welfare 1 1997-10-01 1997-10-01 false Subject Index to Title IX Preamble and Regulation \\1\\ Index Subject Index to Title IX Preamble and Regulation \\1\\ GENERAL ADMINISTRATION... FINANCIAL ASSISTANCE Procedures Interim procedures. Pt. 86, Index Subject Index to Title IX Preamble...

  3. 34 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2005-07-01

    ... 34 Education 1 2005-07-01 2005-07-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Education Regulations of the Offices of the.... Subject Index Subject Index to Title IX Preamble and Regulation 1 1 Preamble paragraph numbers are...

  4. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2004-10-01

    ... 45 Public Welfare 1 2004-10-01 2004-10-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Public Welfare DEPARTMENT OF HEALTHAND..., Index Subject Index to Title IX Preamble and Regulation 1 1 Preamble paragraph numbers are in...

  5. Title IX: A Brief History. 25 Years of Title IX. WEEA Digest.

    ERIC Educational Resources Information Center

    Valentin, Iram

    This brief history of Title IX points out that the role of women and girls in education and the work force began to change significantly with the passage of Title IX as part of the Education Amendments to the Civil Rights Act of 1964. Title IX ensures legal protection against discrimination for students and employees. This article discusses the…

  6. 34 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Education Regulations of the Offices of the.... Subject Index Subject Index to Title IX Preamble and Regulation 1 1 Preamble paragraph numbers are...

  7. 34 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 34 Education 1 2012-07-01 2012-07-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Education Regulations of the Offices of the.... Subject Index Subject Index to Title IX Preamble and Regulation 1 1 Preamble paragraph numbers are...

  8. 34 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 34 Education 1 2014-07-01 2014-07-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Education Regulations of the Offices of the.... Subject Index Subject Index to Title IX Preamble and Regulation 1 1 Preamble paragraph numbers are...

  9. 34 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 34 Education 1 2011-07-01 2011-07-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Education Regulations of the Offices of the.... Subject Index Subject Index to Title IX Preamble and Regulation 1 1 Preamble paragraph numbers are...

  10. 34 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 34 Education 1 2013-07-01 2013-07-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Education Regulations of the Offices of the.... Subject Index Subject Index to Title IX Preamble and Regulation 1 1 Preamble paragraph numbers are...

  11. Identification of Ectonucleotide Pyrophosphatase/Phosphodiesterase 3 (ENPP3) as a Regulator of N-Acetylglucosaminyltransferase GnT-IX (GnT-Vb)*

    PubMed Central

    Korekane, Hiroaki; Park, Jong Yi; Matsumoto, Akio; Nakajima, Kazuki; Takamatsu, Shinji; Ohtsubo, Kazuaki; Miyamoto, Yasuhide; Hanashima, Shinya; Kanekiyo, Kenji; Kitazume, Shinobu; Yamaguchi, Yoshiki; Matsuo, Ichiro; Taniguchi, Naoyuki

    2013-01-01

    Our previous studies on a β1,6-N-acetylglucosaminyltransferase, GnT-IX (GnT-Vb), a homolog of GnT-V, indicated that the enzyme has a broad GlcNAc transfer activity toward N-linked and O-mannosyl glycan core structures and that its brain-specific gene expression is regulated by epigenetic histone modifications. In this study, we demonstrate the existence of an endogenous inhibitory factor for GnT-IX that functions as a key regulator for GnT-IX enzymatic activity in Neuro2a (N2a) cells. We purified this factor from N2a cells and found that it is identical to ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3), as evidenced by mass spectrometry and by the knockdown and overexpression of ENPP3 in cultured cells. Kinetic analyses revealed that the mechanism responsible for the inhibition of GnT-IX caused by ENPP3 is the ENPP3-mediated hydrolysis of the nucleotide sugar donor substrate, UDP-GlcNAc, with the resulting generation of UMP, a potent and competitive inhibitor of GnT-IX. Indeed, ENPP3 knockdown cells had significantly increased levels of intracellular nucleotide sugars and displayed changes in the total cellular glycosylation profile. In addition to chaperones or other known regulators of glycosyltransferases, the ENPP3-mediated hydrolysis of nucleotide sugars would have widespread and significant impacts on glycosyltransferase activities and would be responsible for altering the total cellular glycosylation profile and modulating cellular functions. PMID:23960081

  12. A frequent human coagulation Factor VII mutation (A294V, c152) in loop 140s affects the interaction with activators, tissue factor and substrates.

    PubMed Central

    Toso, Raffaella; Pinotti, Mirko; High, Katherine A; Pollak, Eleanor S; Bernardi, Francesco

    2002-01-01

    Activated Factor VII (FVIIa) is a vitamin-K-dependent serine protease that initiates blood clotting after interacting with its cofactor tissue factor (TF). The complex FVIIa-TF is responsible for the activation of Factor IX (FIX) and Factor X (FX), leading ultimately to the formation of a stable fibrin clot. Activated FX (FXa), a product of FVIIa enzymic activity, is also the most efficient activator of zymogen FVII. Interactions of FVII/FVIIa with its activators, cofactor and substrates have been investigated extensively to define contact regions and residues involved in the formation of the complexes. Site-directed mutagenesis and inhibition assays led to the identification of sites removed from the FVIIa active site that influence binding specificity and affinity of the enzyme. In this study we report the characterization of a frequent naturally occurring human FVII mutant, A294V (residue 152 in the chymotrypsin numbering system), located in loop 140s. This region undergoes major rearrangements after FVII activation and is relevant to the development of substrate specificity. FVII A294V shows delayed activation by FXa as well as reduced activity towards peptidyl and macromolecular substrates without impairing the catalytic efficiency of the triad. Also, the interaction of this FVII variant with TF was altered, suggesting that this residue, and more likely loop 140s, plays a pivotal role not only in the recognition of FX by the FVIIa-TF complex, but also in the interaction of FVII with both its activators and cofactor TF. PMID:11931672

  13. Sonodynamic therapy induces apoptosis of human leukemia HL-60 cells in the presence of protoporphyrin IX.

    PubMed

    Su, Xiaomin; Wang, Xiaobing; Zhang, Kun; Yang, Shuang; Liu, Quanhong; Leung, Albert W; Xu, Chuanshan; Wang, Pan

    2016-04-01

    Sonodynamic therapy (SDT) is expected to be a novel therapeutic strategy for tumor. The protoporphyrin IX disodium salt (PpIX), a photosensitizer, can be activated by ultrasound. The present study aims to investigate apoptosis of HL-60 cells induced by PpIX-mediated SDT. 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was adopted to examine cell toxicity. Apoptosis was detected using Annexin V-PE/7-amino-actinomycin D (7-AAD) double staining. Detection of apoptotic bodies was examined by Hoechst33342 (HO) staining. Western blotting was used to analyze the protein of caspase-3 and poly ADP-ribose polymerase (PARP). Intracellular reactive oxygen species (ROS) was detected by a flow cytometer after exposures. Compared with PpIX alone and ultrasound alone groups, the synergistic cytotoxicity of PpIX plus ultrasound were significantly boosted. In addition, as determined by Annexin V-PE/7-AAD staining, SDT significantly induced HL-60 cell apoptosis, the obvious nuclear condensation was also found with HO staining at 4 hours post-SDT treatment. Furthermore, Western blotting showed visible enhancement of caspase-3 and PARP cleavage in this process. Besides, intracellular ROS production was significantly enhanced after SDT. Our findings demonstrate that PpIX-mediated SDT could induce apoptosis on HL-60 cells, suggesting that apoptosis is an important mechanism of cell death induced by PpIX-mediated SDT. PMID:26891272

  14. Topical glycerol monooleate/propylene glycol formulations enhance 5-aminolevulinic acid in vitro skin delivery and in vivo protophorphyrin IX accumulation in hairless mouse skin.

    PubMed

    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. PMID:15996585

  15. A galactopoiesis accordant yield of functional recombinant human factor IX from homozygous transgenic pigs requires a large amount of vitamin K supplementation.

    PubMed

    Yen, Chon-Ho; Yang, Tien-Shuh; Lin, Yin-Shen; Lee, Meng-Hwan; Yu, Kuo-Cheng; Huang, Chung-Lin; Hsieh, How-Hong; Tu, Ching-Fu

    2016-08-01

    Transgenic pigs failed to accord milk yield curve to lactate rhFIX-a vitamin K (VK) dependent protein even fed with VK enriched to 8 times higher than nutritional requirement. A further higher VK supplementation may be required. Homozygous transgenic sows (n = 4, 200 kg) at their 3rd nursing were divided into control and treatment groups and respectively received VK enriched and further menadione (soluble VK) supplemented diet (220 mg/kg VK enriched diet) for 33 days. At next lactation, control sows than received treatment and previous treated were fed on control diet. Results revealed that menadione treatment increased milk bioactivity of rhFIX from the 7th day of 73 to the 21st day of 153 IU/mL; it gradually decreased to 96 IU/mL on 35th day of lactation. Under control feeding, bioactivity remained relatively unchanged. However, milk rhFIX concentration and ratio of activated rhFIX responded little to the treatment. The menadione-induced bioactivity curve agrees with the known lactation pattern of sow means rhFIX secretion is still galactopoietic but requires high VK intake to show. The ineffectual VK spend on lactational carboxylation might be common in other mammary VK dependent expression system but can be effectively overcome by a high supplementation of menadione with a 5-folds improvement in quality. PMID:27160182

  16. Zn protoporphyrin IX is formed not from heme but from protoporphyrin IX.

    PubMed

    Wakamatsu, Jun-Ichi; Okui, Jun; Hayashi, Nobutaka; Nishimura, Takanori; Hattori, Akihito

    2007-12-01

    We examined the effects of exogenous myoglobin, a bivalent chelator, and nitrite on Zn protoporphyrin IX (ZPP) formation by using model systems. ZPP was formed in a model solution without addition of exogenous myoglobin. After incubation, the amount of ZPP in a model solution was increased but that of heme was not decreased compared with the amounts before incubation. Protoporphyrin IX (PPIX) instead of ZPP also accumulated in a model solution with addition of EDTA, but the amount of heme was not reduced. These results suggested that ZPP was not formed by the Fe-Zn substitution in heme but was formed by the insertion of Zn into PPIX, which was formed independently. The fact that the effects of various factors in model systems with/without addition of a bivalent chelator were similar suggested that ZPP formation was strongly affected by PPIX formation. Inhibition of PPIX formation by nitrite might be the reason for the low levels of ZPP in cured meats. PMID:22061944

  17. Activity factors of the Korean exposure factors handbook.

    PubMed

    Jang, Jae-Yeon; Jo, Soo-Nam; Kim, So-Yeon; Lee, Kyung-Eun; Choi, Kyung-Ho; Kim, Young-Hee

    2014-01-01

    Exposure factors based on the Korean population are required for making appropriate risk assessment. It is expected that handbooks for exposure factors will be applied in many fields, as well as by health department risk assessors. The present article describes the development of an exposure factors handbook that specifically focuses on human activities in situations involving the possible risk of exposure to environmental contaminants. We define majour exposure factors that represent behavioral patterns for risk assessment, including time spent on routine activities, in different places, on using transportation, and engaged in activities related to water contact including swimming, bathing and washing. Duration of residence and employment are also defined. National survey data were used to identify recommended levels of exposure factors in terms of time spent on routine activities and period of residence and employment. An online survey was conducted with 2073 subjects who were selected using a stratified random sampling method in order to develop a list of exposure factors for the time spent in different places and in performing water-related activities. We provide the statistical distribution of the variables, and report reference levels of average exposure based on the reliable data in our exposure factors handbook. PMID:24570804

  18. An activation factor of liver phosphofructokinase.

    PubMed Central

    Furuya, E; Uyeda, K

    1980-01-01

    Pure phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from liver is strongly inhibited by ATP, whereas crude phosphofructokinase is only slightly inhibited by ATP. A factor that is removed from the enzyme during purification and can prevent the inhibition of phosphofructokinase by ATP has been isolated. The factor can be resolved into three components that differ in molecular weights, as shown by gel filtration on Sephadex G-25. These factors overcome the ATP inhibition but have no effect on the catalytic activity under the optimum assay conditions. Furthermore, AMP acts syngeristically with the activation factor in reversing ATP inhibition. It is proposed that the activation of phosphofructokinase by the activation factor and AMP is sufficient to account for the glycolytic flux in the liver. PMID:6449699

  19. Activation of factor X by rat hepatocytes

    SciTech Connect

    Willingham, A.K.; Matschiner, J.T.

    1986-05-01

    Synthesis and secretion of blood coagulation factor X was studied in hepatocytes prepared by perfusion of rat livers with collagenase. Hepatocytes were incubated in the presence of vitamin K and /sup 3/H-leucine for up to 4h at 37/sup 0/C. Factor X was isolated from the incubation medium by immunochemical techniques and analyzed by SDS-PAGE. The recovered /sup 3/H-labeled proteins migrated, after reduction of disulfides, as two polypeptide chains with apparent molecular weights (M/sub r/) of approximately 42,000 and 22,000 representing the heavy and light chains of factor X respectively. The apparent M/sub r/ of the heavy chain was about 10,000 daltons lighter than seen with the heavy chain of factor X isolated from rat plasma and was more characteristic of the heavy chain of factor Xa. When the levels of factor X secreted by hepatocytes were determined by clotting assays, activity was present as factor Xa. Also, when purified plasma factor X was added to incubations of hepatocytes (>95% parenchymal cells) the added factor X was rapidly converted to factor Xa. Plasma membranes prepared from isolated hepatocytes or from liver homogenates contained an enzyme that converted factor X to factor Xa in a calcium dependent reaction. The physiological significance of a factor X activating enzyme on hepatocyte plasma membranes is not clear.

  20. A sucrose-binding site provides a lead towards an isoform-specific inhibitor of the cancer-associated enzyme carbonic anhydrase IX.

    PubMed

    Pinard, Melissa A; Aggarwal, Mayank; Mahon, Brian P; Tu, Chingkuang; McKenna, Robert

    2015-10-01

    Human carbonic anhydrase (CA; EC 4.2.1.1) isoform IX (CA IX) is an extracellular zinc metalloenzyme that catalyzes the reversible hydration of CO2 to HCO3(-), thereby playing a role in pH regulation. The majority of normal functioning cells exhibit low-level expression of CA IX. However, in cancer cells CA IX is upregulated as a consequence of a metabolic transition known as the Warburg effect. The upregulation of CA IX for cancer progression has drawn interest in it being a potential therapeutic target. CA IX is a transmembrane protein, and its purification, yield and crystallization have proven challenging to structure-based drug design, whereas the closely related cytosolic soluble isoform CA II can be expressed and crystallized with ease. Therefore, we have utilized structural alignments and site-directed mutagenesis to engineer a CA II that mimics the active site of CA IX. In this paper, the X-ray crystal structure of this CA IX mimic in complex with sucrose is presented and has been refined to a resolution of 1.5 Å, an Rcryst of 18.0% and an Rfree of 21.2%. The binding of sucrose at the entrance to the active site of the CA IX mimic, and not CA II, in a non-inhibitory mechanism provides a novel carbohydrate moiety binding site that could be further exploited to design isoform-specific inhibitors of CA IX. PMID:26457530

  1. Hantavirus Prevalence in the IX Region of Chile

    PubMed Central

    Vial, Pablo C.; Castillo, Constanza H.; Godoy, Paula M.; Hjelle, Brian; Ferrés, Marcela G.

    2003-01-01

    An epidemiologic and seroprevalence survey was conducted (n=830) to assess proportion of persons exposed to hantavirus in IX Region Chile, which accounts for 25% of reported cases of hantavirus cardiopulmonary syndrome. This region has three geographic areas with different disease incidences and a high proportion of aboriginals. Serum samples were tested for immunoglobulin (Ig) G antibodies by enzyme-linked immunosorbent assay against Sin Nombre virus N antigen by strip immunoblot assay against Sin Nombre, Puumala, Río Mamoré, and Seoul N antigens. Samples from six patients were positive for IgG antibodies reactive with Andes virus; all patients lived in the Andes Mountains. Foresting was also associated with seropositivity; but not sex, age, race, rodent exposure, or farming activities. Exposure to hantavirus varies in different communities of IX Region. Absence of history of pneumonia or hospital admission in persons with specific IgG antibodies suggests that infection is clinically inapparent. PMID:12890323

  2. Identification of a juxtamembrane mechanosensitive domain in the platelet mechanosensor glycoprotein Ib-IX complex.

    PubMed

    Zhang, Wei; Deng, Wei; Zhou, Liang; Xu, Yan; Yang, Wenjun; Liang, Xin; Wang, Yizhen; Kulman, John D; Zhang, X Frank; Li, Renhao

    2015-01-15

    How glycoprotein (GP)Ib-IX complex on the platelet surface senses the blood flow through its binding to the plasma protein von Willebrand factor (VWF) and transmits a signal into the platelet remains unclear. Here we show that optical tweezer-controlled pulling of the A1 domain of VWF (VWF-A1) on GPIb-IX captured by its cytoplasmic domain induced unfolding of a hitherto unidentified structural domain before the dissociation of VWF-A1 from GPIb-IX. Additional studies using recombinant proteins and mutant complexes confirmed its existence in GPIb-IX and enabled localization of this quasi-stable mechanosensitive domain of ∼60 residues between the macroglycopeptide region and the transmembrane helix of the GPIbα subunit. These results suggest that VWF-mediated pulling under fluid shear induces unfolding of the mechanosensitive domain in GPIb-IX, which may possibly contribute to platelet mechanosensing and/or shear resistance of VWF-platelet interaction. The identification of the mechanosensitive domain in GPIb-IX has significant implications for the pathogenesis and treatment of related blood diseases. PMID:25359992

  3. Spectral action for Bianchi type-IX cosmological models

    NASA Astrophysics Data System (ADS)

    Fan, Wentao; Fathizadeh, Farzad; Marcolli, Matilde

    2015-10-01

    A rationality result previously proved for Robertson-Walker metrics is extended to a homogeneous anisotropic cosmological model, namely the Bianchi type-IX minisuperspace. It is shown that the Seeley-de Witt coefficients appearing in the expansion of the spectral action for the Bianchi type-IX geometry are expressed in terms of polynomials with rational coefficients in the cosmic evolution factors w 1( t) , w 2( t) , w 3( t) , and their higher derivates with respect to time. We begin with the computation of the Dirac operator of this geometry and calculate the coefficients a 0 ,a 2 ,a 4 of the spectral action by using heat kernel methods and parametric pseudodifferential calculus. An efficient method is devised for computing the Seeley-de Witt coefficients of a geometry by making use of Wodzicki's noncommutative residue, and it is confirmed that the method checks out for the cosmological model studied in this article. The advantages of the new method are discussed, which combined with symmetries of the Bianchi type-IX metric, yield an elegant proof of the rationality result.

  4. Nuclear transcription factors: a new approach to enhancing cellular responses to ALA-mediated photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Maytin, Edward V.; Anand, Sanjay; Sato, Nobuyuki; Moore, Brian; Mack, Judith; Gasbarre, Christopher; Keevey, Samantha; Ortel, Bernhard; Sinha, Alok; Khachemoune, Amor

    2006-02-01

    Photodynamic therapy (PDT) using aminolevulinic acid (ALA) relies upon the uptake of ALA into cancer cells, where it is converted into a porphyrin intermediate, protoporphyrin IX (PpIX) that is highly photosensitizing. For large or resistant tumors, however, ALA/PDT is often not completely effective due to inadequate PpIX levels. Therefore, new approaches to enhance the intracellular production of PpIX are sought. Here, we describe a general approach to improve intracellular PpIX accumulation via manipulations that increase the expression of an enzyme, coproporphyrinogen oxidase (CPO), that is rate-determining for PpIX production. We show that nuclear hormones that promote terminal differentiation, e.g. vitamin D or androgens, can also increase the accumulation of PpIX and the amount of killing of the target cells upon exposure to light. These hormones bind to intracellular hormone receptors that translocate to the nucleus, where they act as transcription factors to increase the expression of target genes. We have found that several other transcription factors associated with terminal differentiation, including members of the CCAAT enhancer binding (C/EBP) family, and a homeobox protein named Hoxb13, are also capable of enhancing PpIX accumulation. These latter transcription factors appear to interact directly with the CPO gene promoter, resulting in enhanced CPO transcriptional activity. Our data in several different cell systems, including epithelial cells of the skin and prostate cancer cells, indicate that enhancement of CPO expression and PpIX accumulation represents a viable new approach toward improving the efficacy of ALA/PDT.

  5. Analysis of cell line variation in biochemical production of protoporphyrin IX

    NASA Astrophysics Data System (ADS)

    Gibbs, Summer L.; Chen, Bin; O'Hara, Julia A.; Hoopes, P. Jack; Hasan, Tayyaba; Pogue, Brian W.

    2006-02-01

    Protoporphyrin IX (PpIX) is produced via the heme synthesis pathway by the cell following administration of aminolevulinic acid (ALA). ALA synthase, the enzyme that produces ALA in the cell from glycine and succinyl-coenzyme A, is inhibited in a feedback mechanism by heme and thus is the rate limiting enzyme in the heme synthesis pathway. Since ALA is administered systemically, the rate limiting step that naturally exists in the cells is bypassed, however it is currently unclear why cells have different rate limiting steps in the ALA-PpIX synthesis pathway, and more specifically which types of cancer cells are most productive. It has been determined that when the same amount of ALA is administered to a wide panel of cancer cells in vitro that vastly differing amounts of PpIX are produced. The steps for the ALA-PpIX pathway occur in and around the mitochondria of the cell, but interestingly no correlation is seen between PpIX production and mitochondrial content of the cell, following ALA administration. However, total cell area shows positive correlation with PpIX production. Administration of the iron chelator, 1,2-dimethyl-3-hydroxy-4-pyridone (L1) in combination with ALA allows the final step in the heme synthesis pathway, conversion of PpIX to heme, to be delayed and thus increases the detectable amount of PpIX in each cell line. The cell lines that have the lowest PpIX production following administration of ALA alone show the largest increase in production following the combined administration of ALA and L1. PpIX fluorescence is thought to be a measure of cellular activity and the goal of the current study was to determine which cell lines would be the most promising targets for fluorescence detection or monitoring response to therapy. The results indicate that the cells with larger size and larger numbers of mitochondria may be good potential targets for this therapy. While this conclusion may appear obvious, it is not universally true, and cellular specific

  6. A novel mouse PKC{delta} splice variant, PKC{delta}IX, inhibits etoposide-induced apoptosis

    SciTech Connect

    Kim, Jung D.; Seo, Kwang W.; Lee, Eun A.; Quang, Nguyen N.; Cho, Hong R.; Kwon, Byungsuk

    2011-07-01

    Highlights: {yields} A novel PKC{delta} isoform, named PKC{delta}IX, that lacks the C1 domain and the ATP-binding site is ubiquitously expressed. {yields} PKC{delta}IX inhibits etoposide-induced apoptosis. {yields} PKC{delta}IX may function as an endogenous dominant negative isoform for PKC{delta}. -- Abstract: Protein kinase C (PKC) {delta} plays an important role in cellular proliferation and apoptosis. The catalytic fragment of PKC{delta} generated by caspase-dependent cleavage is essential for the initiation of etoposide-induced apoptosis. In this study, we identified a novel mouse PKC{delta} isoform named PKC{delta}IX (Genebank Accession No. (HQ840432)). PKC{delta}IX is generated by alternative splicing and is ubiquitously expressed, as seen in its full-length PKC{delta}. PKC{delta}IX lacks the C1 domain, the caspase 3 cleavage site, and the ATP binding site but preserves an almost intact c-terminal catalytic domain and a nuclear localization signal (NLS). The structural characteristics of PKC{delta}IX provided a possibility that this PKC{delta} isozyme functions as a novel dominant-negative form for PKC{delta} due to its lack of the ATP-binding domain that is required for the kinase activity of PKC{delta}. Indeed, overexpression of PKC{delta}IX significantly inhibited etoposide-induced apoptosis in NIH3T3 cells. In addition, an in vitro kinase assay showed that recombinant PKC{delta}IX protein could competitively inhibit the kinase activity of PKC{delta}. We conclude that PKC{delta}IX can function as a natural dominant-negative inhibitor of PKC{delta}in vivo.

  7. New selective carbonic anhydrase IX inhibitors: synthesis and pharmacological evaluation of diarylpyrazole-benzenesulfonamides.

    PubMed

    Rogez-Florent, Tiphaine; Meignan, Samuel; Foulon, Catherine; Six, Perrine; Gros, Abigaëlle; Bal-Mahieu, Christine; Supuran, Claudiu T; Scozzafava, Andrea; Frédérick, Raphaël; Masereel, Bernard; Depreux, Patrick; Lansiaux, Amélie; Goossens, Jean-François; Gluszok, Sébastien; Goossens, Laurence

    2013-03-15

    Carbonic anhydrase (CA) IX expression is increased upon hypoxia and has been proposed as a therapeutic target since it has been associated with poor prognosis, tumor progression and pH regulation. We report the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors, 4-(5-aryl-2-hydroxymethyl-pyrazol-1-yl)-benzenesulfonamides. A molecular modeling study was conducted in order to simulate the binding mode of this new family of enzyme inhibitors within the active site of hCA IX. Pharmacological studies revealed high hCA IX inhibitory potency in the parameters nanomolar range. This study showed that the position of sulfonamide group in meta of the 1-phenylpyrazole increase a selectivity hCA IX versus hCA II of our compounds. An in vitro antiproliferative screening has been performed on the breast cancer MDA-MB-231 cell using doxorubicin as cytotoxic agent and in presence of selected CA IX inhibitor. The results shown that the cytotoxic efficiency of doxorubicin in an hypoxic environment, expressed in IC50 value, is restored at 20% level with 1μM CA IX inhibitor. PMID:23168081

  8. Technical Progress on the Ares I-X Flight Test

    NASA Technical Reports Server (NTRS)

    Davis, S.R.; Robinson, K.F.; Flynn, K.C.

    2008-01-01

    Ares I-X will be NASA's first test flight for a new human-rated launch vehicle since 1981, and the team is well on its way toward completing the vehicle's design and hardware fabrication for an April 2009 launch. This uncrewed suborbital development test flight gives NASA its first opportunities to: gather critical data about the flight dynamics of the integrated launch vehicle; understand how to control its roll during flight; better characterize the stage separation environments during future flight; and demonstrate the first stage recovery system. The Ares I-X Flight Test Vehicle (FTV) incorporates a mix of flight and mockup hardware. It is powered by a four-segment solid rocket booster, and will be modified to include a fifth, spacer segment; the upper stage, Orion crew exploration vehicle, and launch abort system are simulator hardware to make the FTV aerodynamically similar to the same size, shape, and weight of Ares I. The Ares IX first stage includes an existing Shuttle solid rocket motor and thrust vector control system controlled by an Ascent Thrust Vector Controller (ATVC) designed and built by Honeywell International. The avionics system will be tested in a dedicated System Integration Laboratory located at Lockheed Martin Space Systems (LMSS) in Denver, Colorado. The Upper Stage Simulator (USS) is made up of cylindrical segments that will be stacked and integrated at Kennedy Space Center (KSC) for launch. Glenn Research Center is already building these segments, along with their internal access structures. The active Roll Control System (RoCS) includes two thruster units harvested from Peacekeeper missiles. Duty cycle testing for RoCS was conducted, and fuel tanking and detanking tests will occur at KSC in early 2008. This important flight will provide valuable experience for the ground operations team in integrating, stacking, and launching Ares I. Data from Ares I-X will ensure the safety and reliability of America's newest launch vehicle.

  9. Ares I-X: On the Threshold of Exploration

    NASA Technical Reports Server (NTRS)

    Davis, Stephan R.; Askins, Bruce

    2009-01-01

    Ares I-X, the first flight of the Ares I crew launch vehicle, is less than a year from launch. Ares I-X will test the flight characteristics of Ares I from liftoff to first stage separation and recovery. The flight also will demonstrate the computer hardware and software (avionics) needed to control the vehicle; deploy the parachutes that allow the first stage booster to land in the ocean safely; measure and control how much the rocket rolls during flight; test and measure the effects of first stage separation; and develop and try out new ground handling and rocket stacking procedures in the Vehicle Assembly Building (VAB) and first stage recovery procedures at Kennedy Space Center (KSC) in Florida. All Ares I-X major elements have completed their critical design reviews, and are nearing final fabrication. The first stage--four-segment solid rocket booster from the Space Shuttle inventory--incorporates new simulated forward structures to match the Ares I five-segment booster. The upper stage, Orion crew module, and launch abort system will comprise simulator hardware that incorporates developmental flight instrumentation for essential data collection during the mission. The upper stage simulator consists of smaller cylindrical segments, which were transported to KSC in fall 2008. The crew module and launch abort system simulator were shipped in December 2008. The first stage hardware, active roll control system (RoCS), and avionics components will be delivered to KSC in 2009. This paper will provide detailed statuses of the Ares I-X hardware elements as NASA's Constellation Program prepares for this first flight of a new exploration era in the summer of 2009.

  10. The role of polymorphisms of genes encoding collagen IX and XI in lumbar disc disease.

    PubMed

    Janeczko, Łukasz; Janeczko, Magdalena; Chrzanowski, Robert; Zieliński, Grzegorz

    2014-01-01

    The intervertebral disc disease (IDD) is one of the most common musculoskeletal disorders. A number of environment and anthropometric risk factors may contribute to it. The recent reports have suggested the importance of genetic factors, especially these which encode collagen types IX and XI. The allelic variants in the collagen IX genes - COL9A2 (Trp2) and COL9A3 (Trp3) have been identified as genetic risk factors for IDD, because they interfere the cross-linking between collagen types II, IX and XI and result in decreased stability of intervertebral discs. Type XI collagen is a minor component of cartilage collagen fibrils, but it is present in the annulus fibrosus and nucleus pulposus of intervertebral discs. Some studies have shown the association between gene COL11A1 polymorphism c.4603C>T and IDD. The frequency of 4603T allele was significantly higher in the patients with IDD than in the healthy controls. PMID:24636772

  11. PECAM-1 negatively regulates GPIb/V/IX signaling in murine platelets.

    PubMed

    Rathore, Vipul; Stapleton, Michelle A; Hillery, Cheryl A; Montgomery, Robert R; Nichols, Timothy C; Merricks, Elizabeth P; Newman, Debra K; Newman, Peter J

    2003-11-15

    Platelet adhesion at sites of vascular injury is mediated, in part, by interaction of the platelet plasma membrane glycoprotein (GP) Ib/V/IX complex with von Willebrand Factor (VWF) presented on collagen-exposed surfaces. Recent studies indicate that GPIb/V/IX may be functionally coupled with the Fc receptor gamma (FcR gamma)-chain, which, by virtue of its cytoplasmic immunoreceptor tyrosine-based activation motif, sends activation signals into the cell. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is an inhibitory receptor that has previously been shown to negatively regulate platelet responses to collagen, which transduces activation signals via the GPVI/FcR gamma-chain complex. To determine whether PECAM-1 might similarly regulate signals emanating from GPIb/FcR gamma, we compared activation and aggregation responses to VWF of PECAM-1-positive and PECAM-1-deficient murine platelets. PECAM-1 and the FcR gamma-chain became rapidly tyrosine phosphorylated in platelets following botrocetin-induced VWF binding, but FcR gamma-chain tyrosine phosphorylation was delayed in PECAM-1-positive, versus PECAM-1-deficient, platelets. PECAM-1-deficient platelets were hyperaggregable to VWF, exhibited enhanced spreading and, under conditions of arterial flow, formed markedly larger thrombi on immobilized VWF than did wild-type platelets. Taken together, these data support the notion that engagement of the GPIb complex, in addition to sending activation signals, also initiates a negative feedback loop involving PECAM-1 that controls the rate and extent of platelet activation. PMID:12893757

  12. A central role of GPIb-IX in the procoagulant function of platelets that is independent of the 45-kDa GPIbalpha N-terminal extracellular domain.

    PubMed

    Ravanat, Catherine; Strassel, Catherine; Hechler, Béatrice; Schuhler, Simone; Chicanne, Gaëtan; Payrastre, Bernard; Gachet, Christian; Lanza, François

    2010-08-19

    Activated platelets become procoagulant and efficiently promote the conversion of prothrombin to thrombin. A role of the GPIb-V-IX complex has long been postulated in view of the decreased prothrombin consumption in Bernard-Soulier patients. We evaluated the impact of GPIb-V-IX deficiency and the requirement for the GPIbalpha extracellular domain. In GPIbbeta(-/-) mice, thrombin generation was profoundly decreased in tissue factor- or collagen-related peptide (CRP)-activated platelet-rich plasma and in washed platelets supplemented with normal plasma or with FVa, FXa, and prothrombin. Phosphatidylserine (PS) exposure was similarly decreased in response to thrombin, CRP, or CRP + PAR4 peptide despite a normal platelet phospholipid composition. The hypothesis that these defects originate from lack of the GPIbalpha N-terminal domain was evaluated after its removal from normal mouse and human platelets with Nk protease or O-sialoglycoprotein endopeptidase. Unexpectedly, the treated platelets exhibited normal thrombin generation and PS exposure, indicating that GPIb-V-IX regulates procoagulant activity independently of its GPIbalpha-binding region. These results suggested a more general structuring role through intracellular cytoskeleton-anchoring portions regulating responses leading to PS exposure. This hypothesis was supported by the decreased calcium mobilization observed in GPIbbeta(-/-) platelets in response to several agonists, some acting independently of GPIb, in contrast to the normal calcium responses in Nk protease-treated platelets. PMID:20457869

  13. Quantum supersymmetric Bianchi IX cosmology

    NASA Astrophysics Data System (ADS)

    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

  14. Complement activation induced by rabbit rheumatoid factor.

    PubMed Central

    Meyer, R R; Brown, J C

    1980-01-01

    Rabbit rheumatoid factor produced in animals by hyperimmunized with group C streptococcal vaccine activated guinea pig complement. Anti-streptococcal serum was fractionated by Sephacryl S-200 chromatography into excluded (19S) and included (7S) material and examined for hemolytic activity in a sensitive homologous hemolytic assay system. In the presence of complement, both 19S and 7S antistreptococcal serum fractions induced lysis of bovine (ox) erythrocytes coated with mildly reduced and carboxymethylated rabbit anti-erythrocyte immunoglobulin G. That rabbit rheumatoid factor was responsible for the observed hemolytic activity was substantiated by hemolytic inhibition assays. Significant inhibition of hemolysis was effected when antistreptococcal serum fractions were incubated in the presence of human immunoglobulin G, rabbit immunoglobulin G, and Fc, whereas, no inhibition was detected when the same fractions were tested in the presence of rabbit Fab or F(ab')2 fragments. Deaggregation of inhibitor preparations revealed a preferential reactivity of rheumatoid factor for rabbit immunoglobulin G. In addition to the rheumatoid factor-dependent hemolytic activity observed in humoral preparations, immunoglobulin G-specific antibody-forming cells in spleen and peripheral blood lymphocyte isolates were enumerated by plaque-forming cell assay. PMID:7399707

  15. Factors Associated with Evaluating Public Relations Activities.

    ERIC Educational Resources Information Center

    McElreath, Mark P.

    More than 150 public relations practitioners responded to a survey designed to identify and clarify factors associated with evaluative research in public relations. Responses indicated that (1) no more than half the practitioners formally evaluate their public relations activities on a regular basis; (2) the majority of evaluation is done…

  16. White light-informed optical properties improve ultrasound-guided fluorescence tomography of photoactive protoporphyrin IX

    NASA Astrophysics Data System (ADS)

    Flynn, Brendan P.; DSouza, Alisha V.; Kanick, Stephen C.; Davis, Scott C.; Pogue, Brian W.

    2013-04-01

    Subsurface fluorescence imaging is desirable for medical applications, including protoporphyrin-IX (PpIX)-based skin tumor diagnosis, surgical guidance, and dosimetry in photodynamic therapy. While tissue optical properties and heterogeneities make true subsurface fluorescence mapping an ill-posed problem, ultrasound-guided fluorescence-tomography (USFT) provides regional fluorescence mapping. Here USFT is implemented with spectroscopic decoupling of fluorescence signals (auto-fluorescence, PpIX, photoproducts), and white light spectroscopy-determined bulk optical properties. Segmented US images provide a priori spatial information for fluorescence reconstruction using region-based, diffuse FT. The method was tested in simulations, tissue homogeneous and inclusion phantoms, and an injected-inclusion animal model. Reconstructed fluorescence yield was linear with PpIX concentration, including the lowest concentration used, 0.025 μg/ml. White light spectroscopy informed optical properties, which improved fluorescence reconstruction accuracy compared to the use of fixed, literature-based optical properties, reduced reconstruction error and reconstructed fluorescence standard deviation by factors of 8.9 and 2.0, respectively. Recovered contrast-to-background error was 25% and 74% for inclusion phantoms without and with a 2-mm skin-like layer, respectively. Preliminary mouse-model imaging demonstrated system feasibility for subsurface fluorescence measurement in vivo. These data suggest that this implementation of USFT is capable of regional PpIX mapping in human skin tumors during photodynamic therapy, to be used in dosimetric evaluations.

  17. Exactin: A specific inhibitor of Factor X activation by extrinsic tenase complex from the venom of Hemachatus haemachatus

    PubMed Central

    Girish, Vallerinteavide Mavelli; Kini, R. Manjunatha

    2016-01-01

    Unwanted clots lead to heart attack and stroke that result in a large number of deaths. Currently available anticoagulants have some drawbacks including their non-specific actions. Therefore novel anticoagulants that target specific steps in the coagulation pathway are being sought. Here we describe the identification and characterization of a novel anticoagulant protein from the venom of Hemachatus haemachatus (African Ringhals cobra) that specifically inhibits factor X (FX) activation by the extrinsic tenase complex (ETC) and thus named as exactin. Exactin belongs to the three-finger toxin (3FTx) family, with high sequence identity to neurotoxins and low identity to the well-characterized 3FTx anticoagulants-hemextin and naniproin. It is a mixed-type inhibitor of ETC with the kinetic constants, Ki’ and Ki determined as 30.62 ± 7.73 nM and 153.75 ± 17.96 nM, respectively. Exactin does not bind to the active site of factor VIIa and factor Xa based on its weak inhibition (IC50 ≫ 300 μM) to the amidolytic activities of these proteases. Exactin shows exquisite macromolecular specificity to FX activation as compared to factor IX activation by ETC. Exactin thus displays a distinct mechanism when compared to other anticoagulants targeting ETC, with its selective preference to ETC-FX [ES] complex. PMID:27558950

  18. Exactin: A specific inhibitor of Factor X activation by extrinsic tenase complex from the venom of Hemachatus haemachatus.

    PubMed

    Girish, Vallerinteavide Mavelli; Kini, R Manjunatha

    2016-01-01

    Unwanted clots lead to heart attack and stroke that result in a large number of deaths. Currently available anticoagulants have some drawbacks including their non-specific actions. Therefore novel anticoagulants that target specific steps in the coagulation pathway are being sought. Here we describe the identification and characterization of a novel anticoagulant protein from the venom of Hemachatus haemachatus (African Ringhals cobra) that specifically inhibits factor X (FX) activation by the extrinsic tenase complex (ETC) and thus named as exactin. Exactin belongs to the three-finger toxin (3FTx) family, with high sequence identity to neurotoxins and low identity to the well-characterized 3FTx anticoagulants-hemextin and naniproin. It is a mixed-type inhibitor of ETC with the kinetic constants, Ki' and Ki determined as 30.62 ± 7.73 nM and 153.75 ± 17.96 nM, respectively. Exactin does not bind to the active site of factor VIIa and factor Xa based on its weak inhibition (IC50 ≫ 300 μM) to the amidolytic activities of these proteases. Exactin shows exquisite macromolecular specificity to FX activation as compared to factor IX activation by ETC. Exactin thus displays a distinct mechanism when compared to other anticoagulants targeting ETC, with its selective preference to ETC-FX [ES] complex. PMID:27558950

  19. Ares I-X Vibroacoustic Environments

    NASA Technical Reports Server (NTRS)

    Larsen, Curtis E.; Schuster, David M.; Kaufman, Daniel S.

    2009-01-01

    This paper provides a summary of the NASA Engineering and Safety Center (NESC) team recommendations and observations following participation with the Ares I-X Vibroacoustic (VA) Environments Panel in meetings at the Kennedy Space Center (KSC) and the Marshall Space Flight Center (MSFC) in March and April 2008, respectively.

  20. Open to All: Title IX at 30.

    ERIC Educational Resources Information Center

    Department of Education, Washington, DC. Office of the Secretary.

    This document is a report to the secretary of education on the findings of the Secretary's Commission on Opportunities in Athletics. The Commission was charged with examining Title IX. Starting in June 2002, the 15-member commission collected information, analyzed issues, and obtained broad public input directed at improving the application of…

  1. Calculation of Electronic Transitions in S IX

    NASA Astrophysics Data System (ADS)

    Bogdanovich, P.; Karpuškienė, R.; Rudzikas, Z.

    Wavelengths and oscillator strengths of electric dipole transitions from the 2p33l configurations of S IX are calculated. Relativistic and correlation effects are accounted for in Hartree-Fock-Pauli approximation and in the basis of transformed radial orbitals. Fairly high accuracy of results is achieved.

  2. Title IX Enforcement Called 'Deeply Troubling'

    ERIC Educational Resources Information Center

    Lipka, Sara; Wolverton, Brad

    2007-01-01

    The government agency responsible for enforcing gender equity in college sports is falling down on the job, according to a report released by the National Women's Law Center. Over the past five years, the U.S. Education Department's Office for Civil Rights -- the administrative guardian of Title IX of the Education Amendments of 1972, the law that…

  3. Feminizing Science: The Alchemy of Title IX

    ERIC Educational Resources Information Center

    Hausman, Patricia

    2008-01-01

    The author scrutinizes the National Academy of Sciences report "Beyond Bias and Barriers: Fulfilling the Potential of Women in Academic Science and Engineering" and its dangerous call to place the sciences under the sledgehammer of Title IX. Her findings: A one-sided, inaccurate, and internally contradictory report prepared by a committee lacking…

  4. Title IX Indian Education Toolkit. A Step-by-Step Guide to the Title IX Indian Education Formula Grant.

    ERIC Educational Resources Information Center

    Beller, Floyd

    Title IX is a federal Indian Education Formula Grant Program approved in 1972 and reauthorized five times, most recently in 1994. Title IX formula grants assist groups in providing sound educational programs and opportunities for American Indian and Alaska Native students. A 1997-98 survey of Title IX grantees revealed their need for help in…

  5. Enhanced cellular uptake of protoporphyrine IX/linolenic acid-conjugated spherical nanohybrids for photodynamic therapy.

    PubMed

    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 <200nm. (1)H NMR analysis confirmed the synthesis of PPLA. The singlet oxygen formation of PPLA nanohybrids on laser irradiation was detected by photoluminescence emission. Fluorescence-activated cell sorting (FACS) analysis displayed higher cellular internalization of PPLA compared with free PpIX. In addition, PPLA nanohybrids exhibited significantly reduced dark-toxicity and a high phototoxicity mostly because of apoptotic cell death against human gastric cancer cells. These results imply that the PPLA nanohybrid system may be applicable in PDT. PMID:26954084

  6. Recombinant activated factor VII in patients at high risk of bleeding.

    PubMed

    Kubisz, Peter; Stasko, Ján

    2004-01-01

    Currently, recombinant activated factor VII (rFVIIa) (NovoSeven) is indicated for the treatment of spontaneous and surgical bleeding in congenital haemophilia A and B patients with inhibitors to factors VIII (FVIII) and IX (FIX) >5 Bethesda units (BU) worldwide, and in patients with acquired haemophilia, congenital FVII deficiency and Glanzmann's thrombasthenia in Europe. Until April 2003, almost three-quarters of a milion doses of rFVIIa have been administered proving its efficacy and excellent safety record. According to results from initial clinical trials and a large number of case reports, the rFVIIa may be effective not only in treating haemophilia patients but also in treatment of bleeding in patients on oral anticoagulation or heparin, patients with liver diseases, von Willebrand disease (vWD), thrombocytopenia, various platelet defects, congenital or acquired deficiency of FVII, and in subjects without any pre-existing coagulopathy with diffuse life-threatening bleeding triggered by surgery or trauma. This review will briefly summarize rFVIIa mode of action in haemostasis, the current clinical experience with rFVIIa and focus on the alternative use of rFVIIa in patients at the high risk of bleeding in both spontaneous cases and clinical trials reports. PMID:15763970

  7. Activating transcription factor 2 in mesenchymal tumors.

    PubMed

    Endo, Makoto; Su, Le; Nielsen, Torsten O

    2014-02-01

    Activating transcription factor 2 (ATF2) is a member of activator protein 1 superfamily, which can heterodimerize with other transcription factors regulating cell differentiation and survival. ATF2 assembles into a complex with the synovial sarcoma translocation, chromosome 18 (SS18)-synovial sarcoma, X breakpoint (SSX) fusion oncoprotein, and the transducin-like enhancer of split 1 (TLE1) corepressor, driving oncogenesis in synovial sarcoma. The fusion oncoproteins in many other translocation-associated sarcomas incorporate transcription factors from the ATF/cAMP response element binding or E26 families, which potentially form heterodimers with ATF2 to regulate transcription. ATF2 may therefore play an important role in the oncogenesis of many mesenchymal tumors, but as yet, little is known about its protein expression in patient specimens. Herein we perform immunohistochemical analyses using a validated specific antibody for ATF2 expression and intracellular localization on a cohort of 594 malignant and 207 benign mesenchymal tumors representing 47 diagnostic entities. Melanoma served as a positive control for nuclear and cytoplasmic staining. High nuclear ATF2 expression was mainly observed in translocation-associated and/or spindle cell sarcomas including synovial sarcoma, desmoplastic small round cell tumor, endometrial stromal sarcoma, gastrointestinal stromal tumor, malignant peripheral nerve sheath tumor, and solitary fibrous tumor. Cytoplasmic ATF2 expression was less frequently seen than nuclear expression in malignant mesenchymal tumors. Benign mesenchymal tumors mostly showed much lower nuclear and cytoplasmic ATF2 expression. PMID:24289970

  8. Platelet activating factor activity in the phospholipids of bovine spermatozoa

    SciTech Connect

    Parks, J.E.; Hough, S.; Elrod, C. )

    1990-11-01

    Platelet activating factor (PAF) has been detected in sperm from several mammalian species and can affect sperm motility and fertilization. Because bovine sperm contain a high percentage of ether-linked phospholipid precursors required for PAF synthesis, a study was undertaken to determine the PAF activity of bovine sperm phospholipids. Total lipids of washed, ejaculated bull sperm were extracted, and phospholipids were fractionated by thin-layer chromatography. Individual phospholipid fractions were assayed for PAF activity on the basis of (3H)serotonin release from equine platelets. PAF activity was detected in the PAF fraction (1.84 pmol/mumol total phospholipid) and in serine/inositol (PS/PI), choline (CP), and ethanolamine phosphoglyceride (EP) and cardiolipin (CA) fractions. Activity was highest in the CP fraction (8.05 pmol/mumol total phospholipid). Incomplete resolution of PAF and neutral lipids may have contributed to the activity in the PS/PI and CA fractions, respectively. Phospholipids from nonsperm sources did not stimulate serotonin release. Platelet activation by purified PAF and by sperm phospholipid fractions was inhibited by the receptor antagonist SRI 63-675. These results indicate that bovine sperm contain PAF and that other sperm phospholipids, especially CP and EP, which are high in glycerylether components, are capable of receptor-mediated platelet activation.

  9. Particle emission factors during cooking activities

    NASA Astrophysics Data System (ADS)

    Buonanno, G.; Morawska, L.; Stabile, L.

    Exposure to particles emitted by cooking activities may be responsible for a variety of respiratory health effects. However, the relationship between these exposures and their subsequent effects on health cannot be evaluated without understanding the properties of the emitted aerosol or the main parameters that influence particle emissions during cooking. Whilst traffic-related emissions, stack emissions and concentrations of ultrafine particles (UFPs, diameter < 100 nm) in urban ambient air have been widely investigated for many years, indoor exposure to UFPs is a relatively new field and in order to evaluate indoor UFP emissions accurately, it is vital to improve scientific understanding of the main parameters that influence particle number, surface area and mass emissions. The main purpose of this study was to characterise the particle emissions produced during grilling and frying as a function of the food, source, cooking temperature and type of oil. Emission factors, along with particle number concentrations and size distributions were determined in the size range 0.006-20 μm using a Scanning Mobility Particle Sizer (SMPS) and an Aerodynamic Particle Sizer (APS). An infrared camera was used to measure the temperature field. Overall, increased emission factors were observed to be a function of increased cooking temperatures. Cooking fatty foods also produced higher particle emission factors than vegetables, mainly in terms of mass concentration, and particle emission factors also varied significantly according to the type of oil used.

  10. Mechanisms of Specificity for Hox Factor Activity

    PubMed Central

    Zandvakili, Arya; Gebelein, Brian

    2016-01-01

    Metazoans encode clusters of paralogous Hox genes that are critical for proper development of the body plan. However, there are a number of unresolved issues regarding how paralogous Hox factors achieve specificity to control distinct cell fates. First, how do Hox paralogs, which have very similar DNA binding preferences in vitro, drive different transcriptional programs in vivo? Second, the number of potential Hox binding sites within the genome is vast compared to the number of sites bound. Hence, what determines where in the genome Hox factors bind? Third, what determines whether a Hox factor will activate or repress a specific target gene? Here, we review the current evidence that is beginning to shed light onto these questions. In particular, we highlight how cooperative interactions with other transcription factors (especially PBC and HMP proteins) and the sequences of cis-regulatory modules provide a basis for the mechanisms of Hox specificity. We conclude by integrating a number of the concepts described throughout the review in a case study of a highly interrogated Drosophila cis-regulatory module named “The Distal-less Conserved Regulatory Element” (DCRE). PMID:27583210

  11. eEF1A1 binds and enriches protoporphyrin IX in cancer cells in 5-aminolevulinic acid based photodynamic therapy

    PubMed Central

    Fan, Zhichao; Cui, Xiaojun; Wei, Dan; Liu, Wei; Li, Buhong; He, Hao; Ye, Huamao; Zhu, Naishuo; Wei, Xunbin

    2016-01-01

    Photodynamic therapy (PDT) with protoporphyrin IX (PpIX), which is endogenously derived from 5-aminolevulinic acid (5-ALA) or its derivatives, is a promising modality for the treatment of both pre-malignant and malignant lesions. However, the mechanisms of how ALA-induced PpIX selectively accumulated in the tumors are not fully elucidated. Here we discovered that eukaryotic translation elongation factor 1 alpha 1 (eEF1A1) interacted with PpIX (with an affinity constant of 2.96 × 106 M−1). Microscopy imaging showed that ALA-induced PpIX was co-localized with eEF1A1 in cancer cells. eEF1A1 was found to enrich ALA-induced PpIX in cells by competitively blocking the downstream bioavailability of PpIX. Taken together, our study discovered eEF1A1 as a novel photosensitizer binding protein, which may play an essential role in the enrichment of ALA-induced PpIX in cancer cells during PDT. These suggested eEF1A1 as a molecular marker to predict the selectivity and efficiency of 5-ALA based PDT in cancer therapy. PMID:27150264

  12. eEF1A1 binds and enriches protoporphyrin IX in cancer cells in 5-aminolevulinic acid based photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Fan, Zhichao; Cui, Xiaojun; Wei, Dan; Liu, Wei; Li, Buhong; He, Hao; Ye, Huamao; Zhu, Naishuo; Wei, Xunbin

    2016-05-01

    Photodynamic therapy (PDT) with protoporphyrin IX (PpIX), which is endogenously derived from 5-aminolevulinic acid (5-ALA) or its derivatives, is a promising modality for the treatment of both pre-malignant and malignant lesions. However, the mechanisms of how ALA-induced PpIX selectively accumulated in the tumors are not fully elucidated. Here we discovered that eukaryotic translation elongation factor 1 alpha 1 (eEF1A1) interacted with PpIX (with an affinity constant of 2.96 × 106 M‑1). Microscopy imaging showed that ALA-induced PpIX was co-localized with eEF1A1 in cancer cells. eEF1A1 was found to enrich ALA-induced PpIX in cells by competitively blocking the downstream bioavailability of PpIX. Taken together, our study discovered eEF1A1 as a novel photosensitizer binding protein, which may play an essential role in the enrichment of ALA-induced PpIX in cancer cells during PDT. These suggested eEF1A1 as a molecular marker to predict the selectivity and efficiency of 5-ALA based PDT in cancer therapy.

  13. eEF1A1 binds and enriches protoporphyrin IX in cancer cells in 5-aminolevulinic acid based photodynamic therapy.

    PubMed

    Fan, Zhichao; Cui, Xiaojun; Wei, Dan; Liu, Wei; Li, Buhong; He, Hao; Ye, Huamao; Zhu, Naishuo; Wei, Xunbin

    2016-01-01

    Photodynamic therapy (PDT) with protoporphyrin IX (PpIX), which is endogenously derived from 5-aminolevulinic acid (5-ALA) or its derivatives, is a promising modality for the treatment of both pre-malignant and malignant lesions. However, the mechanisms of how ALA-induced PpIX selectively accumulated in the tumors are not fully elucidated. Here we discovered that eukaryotic translation elongation factor 1 alpha 1 (eEF1A1) interacted with PpIX (with an affinity constant of 2.96 × 10(6) M(-1)). Microscopy imaging showed that ALA-induced PpIX was co-localized with eEF1A1 in cancer cells. eEF1A1 was found to enrich ALA-induced PpIX in cells by competitively blocking the downstream bioavailability of PpIX. Taken together, our study discovered eEF1A1 as a novel photosensitizer binding protein, which may play an essential role in the enrichment of ALA-induced PpIX in cancer cells during PDT. These suggested eEF1A1 as a molecular marker to predict the selectivity and efficiency of 5-ALA based PDT in cancer therapy. PMID:27150264

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

    PubMed

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

    2016-08-01

    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. PMID:27508443

  15. Construction and radiolabeling of adenovirus variants that incorporate human metallothionein into protein IX for analysis of biodistribution.

    PubMed

    Liu, Lei; Rogers, Buck E; Aladyshkina, Natalia; Cheng, Bing; Lokitz, Stephen J; Curiel, David T; Mathis, J Michael

    2014-01-01

    Using adenovirus (Ad)-based vectors is a promising strategy for novel cancer treatments; however, current tracking approaches in vivo are limited. The C-terminus of the Ad minor capsid protein IX (pIX) can incorporate heterologous reporters to monitor biodistribution. We incorporated metallothionein (MT), a low-molecular-weight metal-binding protein, as a fusion to pIX. We previously demonstrated 99mTc binding in vitro to a pIX-MT fusion on the Ad capsid. We investigated different fusions of MT within pIX to optimize functional display. We identified a dimeric MT construct fused to pIX that showed significantly increased radiolabeling capacity. After Ad radiolabeling, we characterized metal binding in vitro. We explored biodistribution in vivo in control mice, mice pretreated with warfarin, mice preimmunized with wild-type Ad, and mice that received both warfarin pretreatment and Ad preimmunization. Localization of activity to liver and bladder was seen, with activity detected in spleen, intestine, and kidneys. Afterwards, the mice were euthanized and selected organs were dissected for further analysis. Similar to the imaging results, most of the radioactivity was found in the liver, spleen, kidneys, and bladder, with significant differences between the groups observed in the liver. These results demonstrate this platform application for following Ad dissemination in vivo. PMID:25060486

  16. Electron Impact Collision Strength in Si IX

    NASA Astrophysics Data System (ADS)

    Noman, Hala; Gokce, Y.; Nahar, Sultana; Pradhan, Anil

    2016-05-01

    Results from work in progress under Iron Project on the electron impact excitation collision strengths and rate coefficients for transitions between the fine-structure levels of the 2s2 2p2 , 2 s 2p3 , 2p4 , 2s2 2 p 3 s , 2s2 2 p 3 p , and 2s2 2 p 3 d configurations in Si IX will be presented. The fine structure collision strength has been calculated at very fine energy mesh using relativistic effects in Breit-Pauli R-matrix method. Maxwellian averaged collision strengths have been tabulated for all possible transitions among all 46 enrgy levels. We made comparisions of our results with the previously reported results in the literature and found significant differences in low the temperature range (Te < 106 K) for few of the transitions. The correction to the previous reported values results due to more extensive expansion for Si IX target states.

  17. Disorders of Cranial Nerves IX and X

    PubMed Central

    Erman, Audrey B.; Kejner, Alexandra E.; Hogikyan, Norman D.; Feldman, Eva L.

    2014-01-01

    The glossopharyngeal and vagus nerves mediate the complex interplay between the many functions of the upper aerodigestive tract. Defects may occur anywhere from the brainstem to the peripheral nerve and can result in significant impairment in speech, swallowing, and breathing. Multiple etiologies can produce symptoms. This review will broadly examine the normal functions, clinical examination, and various pathologies of cranial nerves IX and X. PMID:19214937

  18. Ares I-X 30 Day Report

    NASA Technical Reports Server (NTRS)

    Ess, Bob; Smith, Marshall

    2009-01-01

    This slide presentation represents the 30 day report on the Ares I-X test flight. Included in the review is information on the following areas: (1) Ground Systems, (2) Guidance, Navigation and Control, (3) Roll Response, (4) Vehicle Response, (5) Control System Performance, (6) Structural Damping, (7) Thrust Oscillation, (8) Stage Separation, (9) Connector Assessment, (10) USS Splashdown, (11) Data Recorder and (12) FS Hardware Assessment.

  19. [Ultrasmall nanoparticles for radiotherapy: AGuIX].

    PubMed

    Lux, F; Detappe, A; Dufort, S; Sancey, L; Louis, C; Carme, S; Tillement, O

    2015-10-01

    Since twenty years, many nanoparticles based on high atomic number elements have been developed as radiosensitizers. The design of these nanoparticles is limited by the classical rules associated with the development of nanoparticles for oncology and by the specific ones associated to radiosensitizers, which aim to increase the effect of the dose in the tumor area and to spare the health tissues. For this application, systemic administration of nanodrugs is possible. This paper will discuss the development of AGuIX nanoparticles and will emphasize on this example the critical points for the development of a nanodrug for this application. AGuIX nanoparticles display hydrodynamic diameters of a few nanometers and are composed of polysiloxane and gadolinium chelates. This particle has been used in many preclinical studies and is evaluated for a further phase I clinical trial. Finally, in addition to its high radiosensitizing potential, AGuIX display MRI functionality and can be used as theranostic nanodrug for personalized medicine. PMID:26343033

  20. Complement activating factor(s) of Trypanosoma lewisi: some physiochemical characteristics of the active components.

    PubMed Central

    Nielsen, K; Sheppard, J; Tizard, I; Holmes, W

    1978-01-01

    Of the complement activating factors present in Trypanosoma lewisi, the major component, a carbohydrate containing substance was further investigated. This component was found to have a lag time of complete activation of 2 CH50 units of bovine complement of approximately 15 minutes while 1% trypsin (a known activator of complement, used as a control system) was capable of instant consumption of a similar quantity of complement. In addition, the complement activating factor of trypanosomes was observed to be stable at 100 degrees C for 15 minutes and over a pH range of 3.0 to 11.0. Thin layer chromatography studies suggested that at least part of the active component contained lipid, perhaps indicating that it may be glycolipid in nature. PMID:25701

  1. Zinc protoporphyrin IX enhances chemotherapeutic response of hepatoma cells to cisplatin

    PubMed Central

    Liu, Yang-Sui; Li, Huan-Song; Qi, Dun-Feng; Zhang, Jun; Jiang, Xin-Chun; Shi, Kui; Zhang, Xiao-Jun; Zhang, Xin-Hui

    2014-01-01

    AIM: To investigate the effect of zinc protoporphyrin IX on the response of hepatoma cells to cisplatin and the possible mechanism involved. METHODS: Cytotoxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was determined by a flow cytometric assay. Western blotting was used to measure protein expression. Heme oxygenase (HO)-1 activity was measured by determining the level of bilirubin generated in isolated microsomes. Reactive oxygen species (ROS) production was monitored by flow cytometry. Caspase-3 activity was measured with a colorimetric assay kit. Mice were inoculated with 1 × 107 tumor cells subcutaneously into the right flanks. All mice were sacrificed 6 wk after the first treatment and tumors were weighed and measured. RESULTS: Overexpression of HO-1 in HepG2 cell line was associated with increased chemoresistance to cis-diaminedichloroplatinum (cisplatin; CDDP) compared to other cell lines in vitro. Inhibition of HO-1 expression or activity by zinc protoporphyrin IX (ZnPP IX) markedly augmented CDDP-mediated cytotoxicity towards all liver cancer cell lines in vitro and in vivo. In contrast, induction of HO-1 with hemin increased resistance of tumor cells to CDDP-mediated cytotoxicity in vitro and in vivo. Furthermore, cells treated with ZnPP IX plus CDDP exhibited marked production of intracellular ROS and caspase-3 activity, which paralleled the incidence of cell apoptosis, whereas hemin decreased cellular ROS and caspase-3 activity induced by CDDP. CONCLUSION: ZnPP IX increases cellular sensitivity and susceptibility of liver cancer cell lines to CDDP and this may represent a mechanism of increasing ROS. PMID:25024611

  2. Photodynamic action of protoporphyrin IX derivatives on Trichophyton rubrum*

    PubMed Central

    Ramos, Rogério Rodrigo; Kozusny-Andreani, Dora Inês; Fernandes, Adjaci Uchôa; Baptista, Mauricio da Silva

    2016-01-01

    BACKGROUND Dermatophytes are filamentous keratinophilic fungi. Trichophyton rubrum is a prevalent infectious agent in tineas and other skin diseases. Drug therapy is considered to be limited in the treatment of such infections, mainly due to low accessibility of the drug to the tissue attacked and development of antifungal resistance in these microorganisms. In this context, Photodynamic Therapy is presented as an alternative. OBJECTIVE Evaluate, in vitro, the photodynamic activity of four derivatives of Protoporphyrin IX by irradiation with LED 400 nm in T. rubrum. METHOD Assays were subjected to irradiation by twelve cycles of ten minutes at five minute intervals. RESULT Photodynamic action appeared as effective with total elimination of UFCs from the second irradiation cycle. CONCLUSION Studies show that the photodynamic activity on Trichophyton rubrum relates to a suitable embodiment of the photosensitizer, which can be maximized by functionalization of peripheral groups of the porphyrinic ring. PMID:27192510

  3. Structural requirements within protoporphyrin IX in the inhibition of heat shock protein 90.

    PubMed

    Lee, Woo Hyung; Lee, Jung Min; Lim, Chaemin; Kim, Sanghee; Kim, Sang Geon

    2013-06-25

    Porphyrins are used for photodynamic therapy for their light-absorbing properties, and some of them were approved for the treatment of certain types of cancers. Porphyrins prevent activation of hypoxia inducible factor-1α (HIF-1α) by inhibiting heat shock protein 90 (HSP90). This study investigated the structural requirements within protoporphyrin IX (PPIX) for the inhibition of HSP90 activity. In HCT116, HT29 and DLD-1 cells, PPIX treatment directly hindered the binding between HSP90 and HIF-1α; PPIX treatment inhibited the chaperone activity of HSP90, accelerating protein degradation of HIF-1α. In addition, PPIX treatment suppressed cancer cell migration, endothelial cell tube formation, and aortic ring sprouting, being consistent with its anti-tumor and anti-angiogenic activities. In silico analysis, molecular docking model indicated that a tetrapyrrole macrocycle and two propionate chains within PPIX are necessary for the binding to the adenosine triphosphate (ATP)-binding pocket of HSP90. The predicted structural requirement was verified by the differential inhibitory effects of PPIX analogs, or the precursor of PPIX, on HIF-1α; compounds lacking either the tetrapyrrole macrocycle or the propionate chains were inactive. Our results show that a tetrapyrrole macrocycle and two attached propionate chains in PPIX coordinately interact with the ATP-binding pocket of HSP90, offering structural information on the inhibitory effect of porphyrins on angiogenesis. PMID:23624237

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

    PubMed

    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. PMID:26408817

  5. Recent Developments in Targeting Carbonic Anhydrase IX for Cancer Therapeutics

    PubMed Central

    McDonald, Paul C.; Winum, Jean-Yves; Supuran, Claudiu T.; Dedhar, Shoukat

    2012-01-01

    Carbonic anhydrase IX (CAIX) is a hypoxia-inducible enzyme that is overexpressed by cancer cells from many tumor types, and is a component of the pH regulatory system invoked by these cells to combat the deleterious effects of a high rate of glycolytic metabolism. CAIX functions to help produce and maintain an intracellular pH (pHi) favorable for tumor cell growth and survival, while at the same time participating in the generation of an increasingly acidic extracellular space, facilitating tumor cell invasiveness. Pharmacologic interference of CAIX catalytic activity using monoclonal antibodies or CAIX-specific small molecule inhibitors, consequently disrupting pH regulation by cancer cells, has been shown recently to impair primary tumor growth and metastasis. Many of these agents are in preclinical or clinical development and constitute a novel, targeted strategy for cancer therapy. PMID:22289741

  6. Latex carrier for improving protoporphyrin IX for photodynamic therapy.

    PubMed

    Bui, Brian; Liu, Li; Chen, Wei

    2016-06-01

    Attachment of Protoporphyrin IX (PPIX) to poly (styrene-co-4-vinylpyridine) (PS4VP) nanobeads was carried out to improve its properties in aqueous solutions. After using an oil-in-water heated emulsion polymerization technique to synthesize PS4VP, PPIX was bonded to the particles via the carboxylic acid of PPIX hydrogen-bonding to the nitrogen at the surface of PS4VP, thereby preventing self-reactions between the carboxyl groups and the porphyrin core. Refraining the two parts from interacting while attached to the nanobeads prevented PPIX from aggregating, which then increased water solubility, enhanced luminescence and singlet oxygen production. Attachment also improved cell uptake and cell destruction by photodynamic activity. This shows that PS4VP-PPIX may help improve aspects of photodynamic therapy for the treatment of cancer. PMID:27020668

  7. A Model Community College Grievance Procedure for Title IX.

    ERIC Educational Resources Information Center

    Noonan, Roberta L.

    Through a review of the literature, analysis of eleven Title IX grievance plans, and interviews with four compliance officers, twelve criteria essential to an effective grievance procedure for use by students were identified and incorporated into a model Title IX grievance procedure for Moraine Valley Community College (Illinois). The twelve…

  8. Rape on College Campuses: Reform through Title IX.

    ERIC Educational Resources Information Center

    Steinberg, Terry Nicole

    1991-01-01

    This article first, analyzes the growing problem of campus rape; second, evaluates some college rape reduction programs; third, uses case law to demonstrate that rape should be considered sex discrimination under Title IX; and, fourth, suggests an amendment to Title IX, defining rape as sex discrimination. Appropriate implementation measures by…

  9. Tilting the Playing Field: Schools, Sports, Sex and Title IX.

    ERIC Educational Resources Information Center

    Gavora, Jessica

    This book suggests that Title IX of the Education Amendments is not creating more female athletes but instead eliminating some of the most prestigious men's sports programs in the name of gender equity. It shows how Title IX has affected every aspect of education, from kindergarten through graduate school, making profound changes in areas as…

  10. School Environment and Academic Achievement of Standard IX Students

    ERIC Educational Resources Information Center

    Lawrence, A. S. Arul; Vimala, A.

    2012-01-01

    The present study School Environment and Academic Achievement of standard IX students was probed to find the relationship between School Environment and Academic Achievement of standard IX students. Data for the study were collected using self-made School Environment Scale (SES). The investigator used stratified random sampling technique for…

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

    NASA Technical Reports Server (NTRS)

    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).

  12. A License for Bias: Sex Discrimination, Schools, and Title IX.

    ERIC Educational Resources Information Center

    Morse, Susan Ed.

    This report discusses non-sports-related Title IX complaints filed with the Department of Education's Office for Civil Rights (OCR) from 1993-1997. Its purpose is to dispel the popular belief that Title IX is a sports-equity law and to determine the effectiveness of the legislation. The document examines the kinds of complaints filed, the status…

  13. 77 FR 64401 - Order of Succession for HUD Region IX

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-19

    ... URBAN DEVELOPMENT Order of Succession for HUD Region IX AGENCY: Office of Field Policy and Management, HUD. ACTION: Notice of Order of Succession. SUMMARY: In this notice, the Assistant Deputy Secretary... Succession for the San Francisco Regional Office and its Field Offices (Region IX). This Order of...

  14. "North Haven" and "Dougherty": Narrowing the Scope of Title IX.

    ERIC Educational Resources Information Center

    Salomone, Rosemary C.

    1981-01-01

    Compares the recent opinions of the Second and Fifth Circuit Courts concerning the legislative intent of Title IX with earlier opinions of the First, Sixth, and Eighth Circuits, which declared the Title IX employment regulations invalid. A middle approach to interpretation of the law is proposed. (Author/MLF)

  15. Lessons Learned in Launching Ares I-X

    NASA Technical Reports Server (NTRS)

    Davis, Stephan R.; Askins, Bruce R.

    2010-01-01

    The Ares I-X flight test, scheduled for 2009, is the first opportunity for the Constellation Program and Ares Projects to obtain important data on the in-flight loads, first stage recovery, and ground-handling characteristics of the Ares I crew launch vehicle. The flight test vehicle will incorporate a mix of flight and mockup hardware, reflecting an acceptable representation of the mass and outer mold line characteristics of the operational Ares I vehicle. It will be powered by a four-segment solid rocket motor from the Space Shuttle inventory and will include a fifth, inert spacer segment and new forward structures to make it the same shape as the Ares I first stage. The vehicle also includes mass simulators for the upper stage, Orion crew module, and launch abort system to match the outer mold line of Ares I; an active roll control system; and avionics derived from Shuttle and Atlas hardware. This suborbital mission will take the flight test vehicle from stacking in the Vehicle Assembly Building (VAB) at Kennedy Space Center (KSC) through liftoff, maximum dynamic pressure, first stage separation, and recovery. Ares I-X presented NASA with unique project management challenges. This presentation will address those unique challenges, including managing a virtual nationwide team under a constrained timeline; designing and building the launch vehicle and ground systems; working concurrently with Space Shuttle activities; and integrating technical and management functions. It also will identify key data collected and lessons learned from the flight that will be applied to future exploration missions.

  16. Ares I-X Ground Diagnostic Prototype

    NASA Technical Reports Server (NTRS)

    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.

  17. Enhancing protoporphyrin IX-induced PDT

    NASA Astrophysics Data System (ADS)

    Curnow, Alison; Pye, Andrew; Campbell, Sandra

    2009-06-01

    Photodynamic therapy (PDT) using porphyrin precursors is commonly used in dermatology. Evidence indicates that good clinical outcomes (associated with excellent cosmesis) can be achieved in superficial precancers and basal cell carcinoma (BCC), however, efficacy appears less favorable for thicker nodular BCC (nBCC) unless multiple PDT treatment cycles are performed. Enhancement is therefore required if nBCC lesions are to be treated effectively with a single PDT treatment. The most common technique currently being routinely employed clinically is the use of aminolevulinic acid (ALA) esters (usually methyl (MAL) or hexyl (HAL)). Standard dermatological PDT employing these porphyrin precursors already manipulates the normal heme biosynthesis pathway resulting in a temporary accumulation of the natural photosensitizer, protoporphyrin IX (PpIX). Further manipulation using iron chelating agents is possible however. In normal and malignant human cells in vitro, the novel iron chelating agent CP94 produced greater PPIX fluorescence when administered with ALA or MAL than either congener produced alone. CP94 was also significantly more effective than the clinically established iron chelating agent desferrioxamine (DFO). Topical application of ALA+CP94 to clinical nBCC lesions was a simple and safe treatment modification which produced a significant increase in clinical clearance when CP94 was included in the cream.

  18. Cranial Nerves IX, X, XI, and XII

    PubMed Central

    Sanders, Richard D.

    2010-01-01

    This article concludes the series on cranial nerves, with review of the final four (IX–XII). To summarize briefly, the most important and common syndrome caused by a disorder of the glossopharyngeal nerve (craniel nerve IX) is glossopharyngeal neuralgia. Also, swallowing function occasionally is compromised in a rare but disabling form of tardive dyskinesia called tardive dystonia, because the upper motor portion of the glossopharyngel nerve projects to the basal ganglia and can be affected by lesions in the basal ganglia. Vagus nerve funtion (craniel nerve X) can be compromised in schizophrenia, bulimia, obesity, and major depression. A cervical lesion to the nerve roots of the spinal accessory nerve (craniel nerve XI) can cause a cervical dystonia, which sometimes is misdiagnosed as a dyskinesia related to neuroleptic use. Finally, unilateral hypoglossal (craniel nerve XII) nerve palsy is one of the most common mononeuropathies caused by brain metastases. Supranuclear lesions of cranial nerve XII are involved in pseudobulbar palsy and ALS, and lower motor neuron lesions of cranial nerve XII can also be present in bulbar palsy and in ALS patients who also have lower motor neuron involvement. This article reviews these and other syndromes related to cranial nerves IX through XII that might be seen by psychiatry. PMID:20532157

  19. Ares I-X Ascent Base Environments

    NASA Technical Reports Server (NTRS)

    Mobley, B. L.; Bender, R. L.; Canabal, F.; Smith, Sheldon D.

    2011-01-01

    Plume induced base heating environments were measured during the flight of the NASA Constellation Ares I-X developmental launch vehicle, successfully flown on October 28, 2009. The Ares IX first stage is a four segment Space Shuttle derived booster with base consisting of a flared aft skirt, deceleration and tumble motors, and a thermal curtain surrounding the first stage 7.2 area ratio nozzle. Developmental Flight Instrumentation (DFI) consisted of radiometers, calorimeters, pressure transducers and gas temperature probes installed on the aft skirt and nozzle to measure the base environments. In addition, thermocouples were also installed between the layers of the flexible thermal curtain to provide insight into the curtain response to the base environments and to assist in understanding curtain failure during reentry. Plume radiation environment predictions were generated by the Reverse Monte Carlo (RMC) code and the convective base heating predictions utilized heritage MSFC empirical methods. These predictions were compared to the DFI data and results from the flight videography. Radiation predictions agreed with the flight measured data early in flight but gauge failures prevented high altitude comparisons. The convective environment comparisons demonstrated the need to improve the prediction methodology; particularly for low altitude, local plume recirculation. The convective comparisons showed relatively good agreement at altitudes greater than 50,000 feet.

  20. Electron Impact Exciation of Fe IX

    NASA Astrophysics Data System (ADS)

    Tayal, Swaraj; Zatsarinny, Oleg

    2015-05-01

    Transition probabilities and electron impact excitation collision strengths and rates for astrophysically important extreme ultraviolet lines of Fe IX are calculated. The 322 fine-structure levels of the 3s2 3p6 , 3s2 3p5 3 d , 3 s 3p6 3 d , 3s2 3p5 4 s , and 3s2 3p4 3d2 configurations are included in our calculations. The collision strengths have been calculated using the B-spline Breit-Pauli R-matrix method for all fine-structure transitions among the 322 levels. The mass, Darwin, and spin-orbit relativistic effects are included in the Breit-Pauli Hamiltonian in the scattering calculation. The one-body and two-body relativistic operators are included in the multi-configuration Hartree-Fock calculations of transition probabilities. Several sets of non-orthogonal spectroscopic and correlation radial orbitals are used to obtain accurate description of Fe IX levels and to represent the scattering functions. The calculated excitation energies are in very good agreement with experiment and represents an improvement over the previous calculations. The present collision strengths show reasonable agreement with the previously available R-matrix and distorted-wave calculations. This research is supported by NASA grant from the Solar and Heliophysics Program.

  1. Ares I-X Ground Diagnostic Prototype

    NASA Technical Reports Server (NTRS)

    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

  2. Core-shell AgSiO2-protoporphyrin IX nanoparticle: Effect of the Ag core on reactive oxygen species generation

    NASA Astrophysics Data System (ADS)

    Lismont, M.; Pá; ez-Martinez, C.; Dreesen, L.

    2015-03-01

    Photodynamic therapy (PDT) for cancer is based on the use of a light sensitive molecule to produce, under specific irradiation, toxic reactive oxygen species (ROS). A way to improve the therapy efficiency is to increase the amount of produced ROS near cancer cells. This aim can be achieved by using a metal enhanced process arising when an optically active molecule is located near a metallic nanoparticle (NP). Here, the coupling effect between silver (Ag) NPs and protoporphyrin IX (PpIX) molecules, a clinically approved photosensitizer, is studied compared first, to PpIX fluorescence yield and second, to ROS production efficiency. By applying a modified Stöber process, PpIX was encapsulated into a silica (SiO2) shell, surrounding a 60 nm sized Ag core. We showed that, compared to SiO2-PpIX NPs, Ag coated SiO2-PpIX NPs dramatically decreased PpIX fluorescence together with singlet oxygen production efficiency. However, after incubation time in the dark, the amount of superoxide anions generated by the Ag doped sample was higher than the control sample one.

  3. Factors Influencing Cypriot Children's Physical Activity Levels

    ERIC Educational Resources Information Center

    Loucaides, Constantinos A.; Chedzoy, Sue M.

    2005-01-01

    The purpose of this paper is to present selected findings from a larger study, which set out to examine the physical activity levels of Cypriot primary school children and determinants of their activity. Twenty parents of children who obtained high and low activity scores based on pedometer counts and self-reports scores were interviewed. By…

  4. Quantitative determination of Zn protoporphyrin IX, heme and protoporphyrin IX in Parma ham by HPLC.

    PubMed

    Wakamatsu, Jun-Ichi; Odagiri, Hiroko; Nishimura, Takanori; Hattori, Akihito

    2009-05-01

    We measured the contents of Zn protoporphyrin IX (ZPP), heme and protoporphyrin IX (PPIX) in Parma ham by simultaneous analysis using high-performance liquid chromatography (HPLC). Extraction with ethyl acetate-acetic acid (4:1) was suitable for the quantitative analysis of ZPP. The contents of heme, ZPP and PPIX in Parma ham were 15.0-29.9, 27.7-47.0 and 0.4-1.1μg/g, respectively, and total content of porphyrin was 43.7-76.6μg/g. The amount of ZPP in Parma ham was larger than that of heme, and ZPP accounted for 60-70% of all porphyrins. PMID:20416611

  5. Nitric oxide inhibits the formation of zinc protoporphyrin IX and protoporphyrin IX.

    PubMed

    Wakamatsu, Jun-ichi; Hayashi, Nobutaka; Nishimura, Takanori; Hattori, Akihito

    2010-01-01

    The aim of this study was to elucidate the mechanism by which curing agents, especially nitrite, inhibit the formation of zinc protoporphyrin IX (ZPP) in dry-cured hams such as Parma ham. The oxidation-reduction potential of model solutions was increased by the addition of nitrite, but it was not clear whether the formation of ZPP is inhibited by the oxidizing property of nitrite. The effect of nitric oxide (NO) produced from nitrite on the formation of ZPP was examined. The amount of ZPP formed was decreased by the addition of NO donors. The amount of protoporphyrin IX (PPIX), which is the precursor of ZPP, was also decreased by the addition of NO donors. It is concluded that NO produced from nitrite inhibited the formation of PPIX and ZPP was therefore not formed in cured meat products with the addition of nitrite or nitrate. PMID:20374763

  6. The plant S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase is located in both envelope and thylakoid chloroplast membranes.

    PubMed

    Block, Maryse A; Tewari, Arun Kumar; Albrieux, Catherine; Maréchal, Eric; Joyard, Jacques

    2002-01-01

    Chlorophyll biosynthesis requires a metabolic dialog between the chloroplast envelope and thylakoids where biosynthetic activities are localized. Here, we report the first plant S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase (MgP(IX)MT) sequence identified in the Arabidopsis genome owing to its similarity with the Synechocystis sp. MgP(IX)MT gene. After expression in Escherichia coli, the recombinant Arabidopsis thaliana cDNA was shown to encode a protein having MgP(IX)MT activity. The full-length polypeptide exhibits a chloroplast transit peptide that is processed during import into the chloroplast. The mature protein contains two functional regions. The C-terminal part aligns with the Synechocystis full-length protein. The corresponding truncated region binds to Ado-met, as assayed by UV crosslinking, and is shown to harbor the MgP(IX)MT activity. Downstream of the cleaved transit peptide, the 40 N-terminal amino acids of the mature protein are very hydrophobic and enhance the association of the protein with the membrane. In A. thaliana and spinach, the MgP(IX)MT protein has a dual localization in chloroplast envelope membranes as well as in thylakoids. The protein is active in each membrane and has the same apparent size corresponding to the processed mature protein. The protein is very likely a monotopic membrane protein embedded within one leaflet of the membrane as indicated by ionic and alkaline extraction of each membrane. The rationale for a dual localization of the protein in the chloroplast is discussed. PMID:11784318

  7. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  8. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying the peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  9. Ares I-X USS Material Testing

    NASA Technical Reports Server (NTRS)

    Dawicke, David S.; Smith, Stephen W.; Raju, Ivatury S.

    2008-01-01

    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). Material characterization tests were conducted to quantify the material behavior for use in the CIFS analyses. Fatigue crack growth rate, Charpy impact, and fracture tests were conducted on the parent and welded A516 Grade 70 steel. The crack growth rate tests confirmed that the material behaved in agreement with literature data and that a salt water environment would not significantly degrade the fatigue resistance. The Charpy impact tests confirmed that the fracture resistance of the material did not have a significant reduction for the expected operational temperatures of the vehicle.

  10. Ares I-X Flight Test Philosophy

    NASA Technical Reports Server (NTRS)

    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.

  11. Virulence Factor-activity Relationships: Workshop Summary

    EPA Science Inventory

    The concept or notion of virulence factor–activity relationships (VFAR) is an approach for identifying an analogous process to the use of qualitative structure–activity relationships (QSAR) for identifying new microbial contaminants. In QSAR, it is hypothesized that, for new chem...

  12. Title IX and Intercollegiate Athletics in the Federal Appellate Courts: Myth vs. Reality.

    ERIC Educational Resources Information Center

    Pieronek, Catherine

    2000-01-01

    Focuses on the enforcement of the nondiscrimination provisions of Title IX in higher education, and shows how the courts have wrestled with the application of Title IX to college athletic programs over the past decade. Provides a history of Title IX enforcement and describes current approaches to evaluating Title IX compliance. (SLD)

  13. IBM PC/IX operating system evaluation plan

    NASA Technical Reports Server (NTRS)

    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.

  14. Pyrazolylbenzo[d]imidazoles as new potent and selective inhibitors of carbonic anhydrase isoforms hCA IX and XII.

    PubMed

    Kumar, Satish; Ceruso, Mariangela; Tuccinardi, Tiziano; Supuran, Claudiu T; Sharma, Pawan K

    2016-07-01

    Novel pyrazolylbenzo[d]imidazole derivatives (2a-2f) were designed, synthesized and evaluated against four human carbonic anhydrase isoforms belonging to α family comprising of two cytosolic isoforms hCA I and II as well as two transmembrane tumor associated isoforms hCA IX and XII. Starting from these derivatives that showed high potency but low selectivity in favor of tumor associated isoforms hCA IX and XII, we investigated the impact of removing the sulfonamide group. Thus, analogs 3a-3f without sulfonamide moiety were synthesized and biological assay revealed a good activity as well as an excellent selectivity as inhibitors for tumor associated hCA IX and hCA XII and the same was analyzed by molecular docking studies. PMID:27166574

  15. Prolylcarboxypeptidase Independently Activates Plasma Prekallikrein (Fletcher Factor)

    PubMed Central

    Wang, J.; Matafonov, A.; Madkhali, H.; Mahdi, F.; Watson, D.; Schmaier, A.H.; Gailani, D.; Shariat-Madar, Z.

    2015-01-01

    Prolylcarboxypeptidase isoform 1 (PRCP1) is capable of regulating numerous autocrines and hormones, such as angiotensin II, angiotensin III, αMSH1-13, and DesArg9 bradykinin. It does so by cleaving a C-terminal PRO-X bond. Recent work also indicates that the human PRCP1 activates plasma prekallikrein (PK) to kallikrein on endothelial cells through an uncharacterized mechanism. This study aims to identify PRCP1 binding interaction and cleavage site on PK. Recently, a cDNA encoding a novel splice variant of the human PRCP1 was identified. This isoform differed only in the N-terminal region of the deduced amino acid sequence. Using structural and functional studies, a combination of peptide mapping and site-directed mutagenesis approaches were employed to investigate the interaction of PRCP1 with PK. Three PRCP peptides, in decreasing order of potency, from 1) the N-terminus of the secreted protein, 2) spanning the opening of the active site pocket, and 3) in the dimerization region inhibit PRCP activation of PK on endothelial cells. Investigations also tested the hypothesis that PRCP cleavage site on PK is between its C-terminal Pro 637 (P637) and Ala 638 (A638). Recombinant forms of PK with C-terminal alanine mutagenesis or a stop codon is activated equally as wild type PK by PRCP. In conclusion, PRCP1 interacts with PK at multiple sites for PK activation. PRCP1 also enhances FXIIa activation of PK, suggesting that its activation site on PK is not identical to that of FXIIa. PMID:25324000

  16. Two Coregulated Efflux Transporters Modulate Intracellular Heme and Protoporphyrin IX Availability in Streptococcus agalactiae

    PubMed Central

    Fernandez, Annabelle; Lechardeur, Delphine; Derré-Bobillot, Aurélie; Couvé, Elisabeth; Gaudu, Philippe; Gruss, Alexandra

    2010-01-01

    Streptococcus agalactiae is a major neonatal pathogen whose infectious route involves septicemia. This pathogen does not synthesize heme, but scavenges it from blood to activate a respiration metabolism, which increases bacterial cell density and is required for full virulence. Factors that regulate heme pools in S. agalactiae are unknown. Here we report that one main strategy of heme and protoporphyrin IX (PPIX) homeostasis in S. agalactiae is based on a regulated system of efflux using two newly characterized operons, gbs1753 gbs1752 (called pefA pefB), and gbs1402 gbs1401 gbs1400 (called pefR pefC pefD), where pef stands for ‘porphyrin-regulated efflux’. In vitro and in vivo data show that PefR, a MarR-superfamily protein, is a repressor of both operons. Heme or PPIX both alleviate PefR-mediated repression. We show that bacteria inactivated for both Pef efflux systems display accrued sensitivity to these porphyrins, and give evidence that they accumulate intracellularly. The ΔpefR mutant, in which both pef operons are up-regulated, is defective for heme-dependent respiration, and attenuated for virulence. We conclude that this new efflux regulon controls intracellular heme and PPIX availability in S. agalactiae, and is needed for its capacity to undergo respiration metabolism, and to infect the host. PMID:20421944

  17. Postbiotic activities of lactobacilli-derived factors.

    PubMed

    Cicenia, Alessia; Scirocco, Annunziata; Carabotti, Marilia; Pallotta, Lucia; Marignani, Massimo; Severi, Carola

    2014-01-01

    Probiotics are alive nonpathogenic microorganisms present in the gut microbiota that confer benefits to the host for his health. They act through molecular and cellular mechanisms that contrast pathogen bacteria adhesion, enhance innate immunity, decrease pathogen-induced inflammation, and promote intestinal epithelial cell survival, barrier function, and protective responses. Some of these beneficial effects result to be determined by secreted probiotic-derived factors that recently have been identified as "postbiotic" mediators. They have been reported for several probiotic strains but most available literature concerns Lactobacilli. In this review, we focus on the reported actions of several secretory products of different Lactobacillus species highlighting the available mechanistic data. The identification of soluble factors mediating the beneficial effects of probiotics may present an opportunity not only to understand their fine mechanisms of action, but also to develop effective pharmacological strategies that could integrate the action of treatments with live bacteria. PMID:25291118

  18. Leishmania major possesses a unique HemG-type protoporphyrinogen IX oxidase

    PubMed Central

    Zwerschke, Dagmar; Karrie, Simone; Jahn, Dieter; Jahn, Martina

    2014-01-01

    Leishmania major was proposed to either utilize haem from its host or partially synthesize the tetrapyrrole from host provided precursors. However, only indirect evidence was available for this partial late haem biosynthetic pathway. Here, we demonstrate that the LMJF_06_1280 gene of L. major encodes a HemG-type PPO (protoporphyrinogen IX oxidase) catalysing the oxidation of protoporphyrinogen IX to protoporphyrin IX. Interestingly, trypanosomatids are currently the only known eukaryotes possessing HemG-type enzymes. The LMJF_06_1280 gene forms a potential transcriptional unit with LMJF_06_1270 encoding CPO (coproporphyrinogen III oxidase) and with LMJF_06_1290 for a cytochrome b5. In vivo function of the L. major hemG gene was shown by the functional complementation of the Escherichia coli ΔhemG strain LG285. Restored haem formation in E. coli was observed using HPLC analyses. Purified recombinant L. major HemG revealed PPO activity in vitro using different ubiquinones and triphenyltetrazolium as electron acceptors. FMN was identified as the L. major HemG cofactor. Active site residues were found to be essential for HemG catalysis. These data in combination with the solved crystal structures of L. major CPO and the physiological proof of a ferrochelatase activity provide clear-cut evidence for a partial haem biosynthetic pathway in L. major. PMID:24962471

  19. The relationship between inhibition of vitamin K1 2,3-epoxide reductase and reduction of clotting factor activity with warfarin.

    PubMed Central

    Choonara, I A; Malia, R G; Haynes, B P; Hay, C R; Cholerton, S; Breckenridge, A M; Preston, F E; Park, B K

    1988-01-01

    1 The effect of low dose steady state warfarin (0.2 mg and 1 mg daily) on clotting factor activity and vitamin K1 metabolism was studied in seven healthy volunteers. 2 Steady state plasma warfarin concentrations were 41-99 ng ml-1 for the 0.2 mg dose and 157-292 ng ml-1 for the 1 mg dose. 3 There was a significant prolongation of the mean prothrombin time (0.9 s) after 1 mg warfarin daily, but no significant change in prothrombin time after 0.2 mg warfarin daily. There was no significant change in individual clotting factor activity (II, VII, IX or X) with either dose of warfarin. 4 Following the administration of a pharmacological dose of vitamin K1 (10 mg), all seven volunteers had detectable levels of vitamin K1 2,3-epoxide with both doses of warfarin (Cpmax 31-409 ng ml-1). 5 Both the Cpmax and the AUC for vitamin K1 2,3-epoxide were significantly greater on 1 mg of warfarin daily than 0.2 mg daily (P less than 0.01). 6 The apparent dissociation between inhibition of vitamin K1 2,3-epoxide reductase and reduction of clotting factor activity, produced by warfarin, may reflect the insensitivity of functional clotting factor assays to a small reduction in clotting factor concentration. PMID:3370190

  20. Learning Risk Factors for Suicide: A Scenario-Based Activity

    ERIC Educational Resources Information Center

    Madson, Laura; Vas, Corey J.

    2003-01-01

    We created a classroom activity to illustrate factors that may predict suicide. In the activity, students rank 4 fictional individuals in terms of their relative risk for attempting or committing suicide. Students described the activity as "eye-opening," and students who participated in the activity learned more about the warning signs of an…

  1. Posttranslational modifications and activity of natural and recombinant tissue factor

    PubMed Central

    Butenas, Saulius; Krudysz-Amblo, Jolanta; Mann, Kenneth G

    2010-01-01

    Tissue factor is a membrane protein, which in a complex with factor VIIa initiates in vivo blood coagulation. Due to the scarcity of natural tissue factor protein, most studies have relied upon recombinant tissue factor forms. However, there have been only cursory experimental comparisons of natural and recombinant tissue factor proteins. Our preliminary data suggested that placental tissue factor in a complex with factor VIIa was more efficient activator of factor X than the recombinant protein. After deglycosylation, both forms of tissue factor showed almost an identical activity in the extrinsic factor Xase. Analyses using tryptic digestion and mass-spectrometry revealed that the levels of glycosylation and the composition of carbohydrates present in natural placental tissue factor were different than those in its recombinant counterpart. These data indicate that natural and recombinant tissue factor proteins differ in their posttranslational modifications and that these differences translate into different cofactor activity. Thus the use of recombinant tissue factor proteins for the quantitation of natural tissue factor is misleading. PMID:20138335

  2. 26. Photocopy of August 1918 photograph. Glass Negative Box IX, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    26. Photocopy of August 1918 photograph. Glass Negative Box IX, Tower Grove, Missouri Botanical Garden. ITALIAN GARDEN, LOOKING SOUTHWEST - Missouri Botanical Garden, 2345 Tower Grove Avenue, Saint Louis, Independent City, MO

  3. 29. Photocopy of 1921 photograph. Glass Negative Box IX, Tower ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    29. Photocopy of 1921 photograph. Glass Negative Box IX, Tower Grove, Missouri Botanical Garden. ITALIAN GARDEN AND NEW PALM HOUSE (DEMOLISHED), LOOKING NORTHEAST - Missouri Botanical Garden, 2345 Tower Grove Avenue, Saint Louis, Independent City, MO

  4. Reduction of salivary tissue factor (thromboplastin) activity by warfarin therapy.

    PubMed

    Zacharski, L R; Rosenstein, R

    1979-03-01

    The coagulant of normal human saliva has been identified as tissue factor (thromboplastin, TF) by virtue of its ability to cause rapid coagulation in plasmas deficient in first-stage coagulation factors and to activate factor x in the presence of factor VII and by virtue of the fact that its activity is expressed only in the presence of factor VII and is inhibited by an antibody to TF. The TF is related to cells and cell fragments in saliva. Salivary TF activity has been found to be significantly reduced in patients taking warfarin. The decline in TF activity during induction of warfarin anticoagulation occurs during the warfarin-induced decline in vitamin-K-dependent clotting factor activity, as judged by the prothrombin time. The decrease in TF activity is not related to a reduction in salivary cell count or total protein content or to a direct effect of warfarin on the assay. It is hypothesized that the mechanism by which warfarin inhibits TF activity may be related to the mechanism by which it inhibits expression of the activity of the vitamin-K-dependent clotting factors. Inhibition of the TF activity may be involved in the antithrombotic effect of warfarin. PMID:760859

  5. Influencing Factors of Thermogenic Adipose Tissue Activity

    PubMed Central

    Zhang, Guoqing; Sun, Qinghua; Liu, Cuiqing

    2016-01-01

    Obesity is an escalating public health challenge and contributes tremendously to the disease burden globally. New therapeutic strategies are required to alleviate the health impact of obesity-related metabolic dysfunction. Brown adipose tissue (BAT) is specialized for dissipating chemical energy for thermogenesis as a defense against cold environment. Intriguingly, the brown-fat like adipocytes that dispersed throughout white adipose tissue (WAT) in rodents and humans, called “brite” or “beige” adipocytes, share similar thermogenic characteristics to brown adipocytes. Recently, researchers have focused on cognition of these thermogenic adipose tissues. Some factors have been identified to regulate the development and function of thermogenic adipose tissues. Cold exposure, pharmacological conditions, and lifestyle can enhance non-shivering thermogenesis and metabolism via some mechanisms. However, environmental pollutants, such as ambient fine particulates and ozone, may impair the function of these thermogenic adipose tissues and thereby induce metabolic dysfunction. In this review, the origin, function and influencing factors of thermogenic adipose tissues were summarized and it will provide insights into identifying new therapeutic strategies for the treatment of obesity and obesity-related diseases. PMID:26903879

  6. Influencing Factors of Thermogenic Adipose Tissue Activity.

    PubMed

    Zhang, Guoqing; Sun, Qinghua; Liu, Cuiqing

    2016-01-01

    Obesity is an escalating public health challenge and contributes tremendously to the disease burden globally. New therapeutic strategies are required to alleviate the health impact of obesity-related metabolic dysfunction. Brown adipose tissue (BAT) is specialized for dissipating chemical energy for thermogenesis as a defense against cold environment. Intriguingly, the brown-fat like adipocytes that dispersed throughout white adipose tissue (WAT) in rodents and humans, called "brite" or "beige" adipocytes, share similar thermogenic characteristics to brown adipocytes. Recently, researchers have focused on cognition of these thermogenic adipose tissues. Some factors have been identified to regulate the development and function of thermogenic adipose tissues. Cold exposure, pharmacological conditions, and lifestyle can enhance non-shivering thermogenesis and metabolism via some mechanisms. However, environmental pollutants, such as ambient fine particulates and ozone, may impair the function of these thermogenic adipose tissues and thereby induce metabolic dysfunction. In this review, the origin, function and influencing factors of thermogenic adipose tissues were summarized and it will provide insights into identifying new therapeutic strategies for the treatment of obesity and obesity-related diseases. PMID:26903879

  7. Anisotropic Bianchi types VIII and IX locally rotationally symmetric cosmologies

    SciTech Connect

    Assad, M.J.D.; Soares, I.D.

    1983-10-15

    We present a class of exact cosmological solutions of Einstein-Maxwell equations, which are anisotropic and spatially homogeneous of Bianchi types VIII and IX, and class IIIb in the Stewart-Ellis classification of locally rotationally symmetric models. If we take the electromagnetic field equal to zero, a class of Bianchi types VIII/IX spatially homogeneous anisotropic cosmological solutions with perfect fluid is obtained.

  8. Factor XI and Contact Activation as Targets for Antithrombotic Therapy

    PubMed Central

    Gailani, David; Bane, Charles E.; Gruber, Andras

    2015-01-01

    Summary The most commonly used anticoagulants produce therapeutic antithrombotic effects either by inhibiting thrombin or factor Xa, or by lowering the plasma levels of the precursors of these key enzymes, prothrombin and factor X. These drugs do not distinguish between thrombin generation contributing to thrombosis from thrombin generation required for hemostasis. Thus, anticoagulants increase bleeding risk, and many patients who would benefit from therapy go untreated because of comorbidities that place them at unacceptable risk for hemorrhage. Studies in animals demonstrate that components of the plasma contact activation system contribute to experimentally-induced thrombosis, despite playing little or no role in hemostasis. Attention has focused on factor XII, the zymogen of a protease (factor XIIa) that initiates contact activation when blood is exposed to foreign surfaces; and factor XI, the zymogen of the protease factor XIa, which links contact activation to the thrombin generation mechanism. In the case of factor XI, epidemiologic data indicate this protein contributes to stroke and venous thromboembolism, and perhaps myocardial infarction, in humans. A phase 2 trial showing that reduction of factor XI may be more effective than low-molecular-weight heparin at preventing venous thrombosis during knee replacement surgery provides proof of concept for the premise that an antithrombotic effect can be uncoupled from an anticoagulant effect in humans by targeting components of contact activation. Here we review data on the role of factor XI and factor XII in thrombosis, and results of pre-clinical and human trials for therapies targeting these proteins. PMID:25976012

  9. Discovering novel carbonic anhydrase type IX (CA IX) inhibitors from seven million compounds using virtual screening and in vitro analysis.

    PubMed

    Salmas, Ramin Ekhteiari; Senturk, Murat; Yurtsever, Mine; Durdagi, Serdar

    2016-06-01

    Carbonic anhydrase type IX (CA IX) enzyme is mostly over expressed in different cancer cell lines and tumor tissues. Potent CA IX inhibitors can be effective for adjusting the pH imbalance in tumor cells. In the present work, we represented the successful application of high throughput virtual screening (HTVS) of large dataset from ZINC database included of ∼7 million compounds to discover novel inhibitors of CA IX. HTVS and molecular docking were performed using consequence Glide/standard precision (SP), extra precision (XP) and induced fit docking (IFD) molecular docking protocols. For each compound, docking code calculates a set of low-energy poses and then exhaustively scans the binding pocket of the target with small compounds. Novel CA IX inhibitor candidates were suggested based on molecular modeling studies and a few of them were tested using in vitro analysis. These compounds were determined as good inhibitors against human CA IX target with Ki in the range of 0.85-1.58 μM. In order to predict the pharmaceutical properties of the selected compounds, ADME (absorption, distribution, metabolism and excretion) analysis was also carried out. PMID:25950196

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

    PubMed

    Otsuka, Shimpei; Matsumoto, Kentaro; Nakajima, Motowo; Tanaka, Tohru; Ogura, Shun-Ichiro

    2015-01-01

    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. PMID:26717566

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

    PubMed Central

    Otsuka, Shimpei; Matsumoto, Kentaro; Nakajima, Motowo; Tanaka, Tohru; Ogura, Shun-ichiro

    2015-01-01

    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. PMID:26717566

  12. Coronal survey in X-rays of O VII and Ne IX.

    NASA Technical Reports Server (NTRS)

    Acton, L. W.; Catura, R. C.; Wolfson, C. J.; Culhane, J. L.; Meyerott, A. J.

    1972-01-01

    Results of a rocket experiment flown on Apr. 29, 1971, in which a survey of the solar corona was carried out with a pair of collimated Bragg spectrometers to study the resonance, intersystem, and forbidden line emission from the helium-like ions O vii (22 A) and Ne ix (13 A). The observed X-ray emission came from the general corona, seven plage regions, and one dynamic feature - the late stage of a small flare. From the intensity of the O vii and Ne ix resonance lines the electron temperature and emission measure of the individual emitting regions are derived on the basis of two models, one in which the region is assumed to be isothermal and another in which the emission measure decreases exponentially with increasing temperature. The latter model, which is the most adequate of the two, yields for the electron temperature of the time-varying feature 2,000,000 to 3,000,000 K, for the other active regions 1,500,000 to 2,500,000 K, and for the general corona 1,300,000 to 1,700,000 K. The Ne ix emitting regions are about 1.5 times as hot as the O vii regions.

  13. [Geriatric rehabilitation from the perspective of Book 9 of the German social code, SGB IX].

    PubMed

    Fuchs, H

    2007-10-01

    The legal foundations for provision and realization of geriatric rehabilitation benefits are contained in particular in Book 9 of the German social code, SGB IX (covering rehabilitation and participation of people with disabilities). This paper discusses claims foundations and benefit prerequisites of geriatric rehabilitation taking into consideration the relations between Book 5 (on health insurance) and Book 9 of the social code. The article includes a definition of "geriatric rehabilitation" in light of the SGB IX, describes the benefit carriers' obligations as well as the procedure in place for determining geriatric rehab need, in this context appraising the designation as "geriatric patient" in terms of its appropriateness as an identifying criterion in determining need. Provision of geriatric rehab benefits is contingent on a potential for attaining rehab goals as specified by SGB IX as well as on fulfillment of the benefit prerequisites. Responsibility for the content, extent and quality of geriatric rehabilitation lies with the benefit carriers, as is the case for the obligation to secure availability of the required numbers and quality of rehabilitation facilities and services. The article specifies the legal foundations of the various benefit types (ambulatory, mobile rehab, under a Personal Budget, integrated benefit provision, or early rehab), and discusses geriatric rehabilitation in the framework of an insurance-based medical care system as well as of activating care. PMID:17955397

  14. Operational Lessons Learned from the Ares I-X Flight Test

    NASA Technical Reports Server (NTRS)

    Davis, Stephan R.

    2010-01-01

    The Ares I-X flight test, launched in 2009, is the first test of the Ares I crew launch vehicle. This development flight test evaluated the flight dynamics, roll control, and separation events, but also provided early insights into logistical, stacking, launch, and recovery operations for Ares I. Operational lessons will be especially important for NASA as the agency makes the transition from the Space Shuttle to the Constellation Program, which is designed to be less labor-intensive. The mission team itself comprised only 700 individuals over the life of the project compared to the thousands involved in Shuttle and Apollo missions; while missions to and beyond low-Earth orbit obviously will require additional personnel, this lean approach will serve as a model for future Constellation missions. To prepare for Ares I-X, vehicle stacking and launch infrastructure had to be modified at Kennedy Space Center's Vehicle Assembly Building (VAB) as well as Launch Complex (LC) 39B. In the VAB, several platforms and other structures designed for the Shuttle s configuration had to be removed to accommodate the in-line, much taller Ares I-X. Vehicle preparation activities resulted in delays, but also in lessons learned for ground operations personnel, including hardware deliveries, cable routing, transferred work and custodial paperwork. Ares I-X also proved to be a resource challenge, as individuals and ground service equipment (GSE) supporting the mission also were required for Shuttle or Atlas V operations at LC 40/41 at Cape Canaveral Air Force Station. At LC 39B, several Shuttle-specific access arms were removed and others were added to accommodate the in-line Ares vehicle. Ground command, control, and communication (GC3) hardware was incorporated into the Mobile Launcher Platform (MLP). The lightning protection system at LC 39B was replaced by a trio of 600-foot-tall towers connected by a catenary wire to account for the much greater height of the vehicle. Like Shuttle

  15. Regional intravascular delivery of AAV-2-F.IX to skeletal muscle achieves long-term correction of hemophilia B in a large animal model.

    PubMed

    Arruda, Valder R; Stedman, Hansell H; Nichols, Timothy C; Haskins, Mark E; Nicholson, Matthew; Herzog, Roland W; Couto, Linda B; High, Katherine A

    2005-05-01

    In earlier work, we showed that adeno-associated virus-mediated delivery of a Factor IX gene to skeletal muscle by direct intramuscular injection resulted in therapeutic levels of circulating Factor IX in mice. However, achievement of target doses in humans proved impractical because of the large number of injections required. We used a novel intravascular delivery technique to achieve successful transduction of extensive areas of skeletal muscle in a large animal with hemophilia. We provide here the first report of long-term (> 3 years, with observation ongoing), robust Factor IX expression (circulating levels of 4%-14%) by muscle-directed gene transfer in a large animal, resulting in essentially complete correction of the bleeding disorder in hemophilic dogs. The results of this translational study establish an experimental basis for clinical studies of this delivery method in humans with hemophilia B. These findings also have immediate relevance for gene transfer in patients with muscular dystrophy. PMID:15479726

  16. ANALYSIS OF ARACHIDONIC ACID METABOLITE AND PLATELET ACTIVATING FACTOR PRODUCTION

    EPA Science Inventory

    Metabolites of arachidonic acid ("eicosanoids") and platelet activating factor are important bioactive lipids that may be involved in the pathobiological alterations in animals induced by pollutant exposure. nalysis of these substances in biological tissue and fluids is important...

  17. Activation of factor XII and prekallikrein with cholesterol sulfate.

    PubMed

    Shimada, T; Kato, H; Iwanaga, S; Iwamori, M; Nagai, Y

    1985-04-01

    Cholesterol sulfate was found to display a strong ability to trigger the activation of Factor XII and prekallikrein in the presence of HMW kininogen. Other sulfate ester derivatives of testosterone, estrone, pregnenolone and dehydroepiandrosterone and cholesterol tested did not show any effect on the activation of Factor XII and prekallikrein. The activity of cholesterol acetate and sulfodeoxycholic acid was very weak. Cholesterol sulfate markedly shortened the partial thromboplastin time of normal human plasma, but not plasmas deficient in Factor XII, Factor XI and HMW kininogen. Upon prolonged incubation, the partial thromboplastin time of prekallikrein-deficient plasma was also shortened. Moreover, as well as kaolin and sulfatide, cholesterol sulfate shortened the partial thromboplastin time of plasmas from monkey, dog, rat, guinea pig, sheep, cow, hog and horse, but not from duck and chicken. Since cholesterol sulfate is distributed in erythrocytes, various organs and body fluids, it may play an important role in the activation of the intrinsic blood coagulation system. PMID:3847226

  18. The dimeric structure of factor XI and zymogen activation

    PubMed Central

    Geng, Yipeng; Verhamme, Ingrid M.; Smith, Stephen B.; Sun, Mao-fu; Matafonov, Anton; Cheng, Qiufang; Smith, Stephanie A.; Morrissey, James H.

    2013-01-01

    Factor XI (fXI) is a homodimeric zymogen that is converted to a protease with 1 (1/2-fXIa) or 2 (fXIa) active subunits by factor XIIa (fXIIa) or thrombin. It has been proposed that the dimeric structure is required for normal fXI activation. Consistent with this premise, fXI monomers do not reconstitute fXI-deficient mice in a fXIIa-dependent thrombosis model. FXI activation by fXIIa or thrombin is a slow reaction that can be accelerated by polyanions. Phosphate polymers released from platelets (poly-P) can enhance fXI activation by thrombin and promote fXI autoactivation. Poly-P increased initial rates of fXI activation 30- and 3000-fold for fXIIa and thrombin, respectively. FXI monomers were activated more slowly than dimers by fXIIa in the presence of poly-P. However, this defect was not observed when thrombin was the activating protease, nor during fXI autoactivation. The data suggest that fXIIa and thrombin activate fXI by different mechanisms. FXIIa may activate fXI through a trans-activation mechanism in which the protease binds to 1 subunit of the dimer, while activating the other subunit. For activation by thrombin, or during autoactivation, the data support a cis-activation mechanism in which the activating protease binds to and activates the same fXI subunit. PMID:23515926

  19. Thermal Model Development for Ares I-X

    NASA Technical Reports Server (NTRS)

    Amundsen, Ruth M.; DelCorso, Joe

    2008-01-01

    Thermal analysis for the Ares I-X vehicle has involved extensive thermal model integration, since thermal models of vehicle elements came from several different NASA and industry organizations. Many valuable lessons were learned in terms of model integration and validation. Modeling practices such as submodel, analysis group and symbol naming were standardized to facilitate the later model integration. Upfront coordination of coordinate systems, timelines, units, symbols and case scenarios was very helpful in minimizing integration rework. A process for model integration was developed that included pre-integration runs and basic checks of both models, and a step-by-step process to efficiently integrate one model into another. Extensive use of model logic was used to create scenarios and timelines for avionics and air flow activation. Efficient methods of model restart between case scenarios were developed. Standardization of software version and even compiler version between organizations was found to be essential. An automated method for applying aeroheating to the full integrated vehicle model, including submodels developed by other organizations, was developed.

  20. A systematic quantitative approach to rational drug design and discovery of novel human carbonic anhydrase IX inhibitors.

    PubMed

    Sethi, Kalyan K; Verma, Saurabh M

    2014-08-01

    Drug design involves the design of small molecules that are complementary in shape and charge to the biomolecular target with which they interact and therefore will bind to it. Three-dimensional quantitative structure-activity relationship (3D-QSAR) studies were performed for a series of carbonic anhydrase IX inhibitors using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) techniques with the help of SYBYL 7.1 software. The large set of 36 different aromatic/heterocyclic sulfamates carbonic anhydrase (CA, EC 4.2.1.1) inhibitors, such as hCA IX, was chosen for this study. The conventional ligand-based 3D-QSAR studies were performed based on the low energy conformations employing database alignment rule. The ligand-based model gave q(2) values 0.802 and 0.829 and r(2) values 1.000 and 0.994 for CoMFA and CoMSIA, respectively, and the predictive ability of the model was validated. The predicted r(2) values are 0.999 and 0.502 for CoMFA and CoMSIA, respectively. SEA (steric, electrostatic, hydrogen bond acceptor) of CoMSIA has the significant contribution for the model development. The docking of inhibitors into hCA IX active site using Glide XP (Schrödinger) software revealed the vital interactions and binding conformation of the inhibitors. The CoMFA and CoMSIA field contour maps are well in agreement with the structural characteristics of the binding pocket of hCA IX active site, which suggests that the information rendered by 3D-QSAR models and the docking interactions can provide guidelines for the development of improved hCA IX inhibitors as leads for various types of metastatic cancers including those of cervical, renal, breast and head and neck origin. PMID:24090419

  1. 5-Aryl-1H-pyrazole-3-carboxylic acids as selective inhibitors of human carbonic anhydrases IX and XII.

    PubMed

    Cvijetić, Ilija N; Tanç, Muhammet; Juranić, Ivan O; Verbić, Tatjana Ž; Supuran, Claudiu T; Drakulić, Branko J

    2015-08-01

    Inhibitory activity of a congeneric set of 23 phenyl-substituted 5-phenyl-pyrazole-3-carboxylic acids toward human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms I, II, IX and XII was evaluated by a stopped-flow CO2 hydrase assay. These compounds exerted a clear, selective inhibition of hCA IX and XII over hCAI and II, with Ki in two to one digit micromolar concentrations (4-50 μM). Derivatives bearing bulkier substituents in para-position of the phenyl ring inhibited hCA XII at one-digit micromolar concentrations, while derivatives having alkyl substituents in both ortho- and meta-positions inhibited hCA IX with Kis ranging between 5 and 25 μM. Results of docking experiments offered a rational explanation on the selectivity of these compounds toward CA IX and XII, as well as on the substitution patterns leading to best CA IX or CA XII inhibitors. By examining the active sites of these four isoforms with GRID generated molecular-interaction fields, striking differences between hCA XII and the other three isoforms were observed. The field of hydrophobic probe (DRY) appeared significantly different in CA XII active site, comparing to other three isoforms studied. To the best of our knowledge such an observation was not reported in literature so far. Considering the selectivity of these carboxylates towards membrane-associated over cytosolic CA isoforms, the title compounds could be useful for the development of isoform-specific non-sulfonamide CA inhibitors. PMID:26088336

  2. Ares I-X Flight Test - The Future Begins Here

    NASA Technical Reports Server (NTRS)

    Davis, Stephan R.

    2008-01-01

    In less than two years, the National Aeronautics and Space Administration (NASA) will launch the Ares I-X mission. This will be the first flight of the Ares I crew launch vehicle, which, together with the Ares V cargo launch vehicle, will eventually send humans to the Moon, Mars, and beyond. As the countdown to this first Ares mission continues, personnel from across the Ares I-X Mission Management Office (MMO) are finalizing designs and fabricating vehicle hardware for an April 2009 launch. This paper will discuss the hardware and programmatic progress of the Ares I-X mission. Like the Apollo program, the Ares launch vehicles will rely upon extensive ground, flight, and orbital testing before sending the Orion crew exploration vehicle into space with humans on board. The first flight of Ares I, designated Ares I-X, will be a suborbital development flight test. Ares I-X gives NASA its first opportunity to gather critical data about the flight dynamics of the integrated launch vehicle stack; understand how to control its roll during flight; better characterize the severe stage separation environments that the upper stage engine will experience during future operational flights; and demonstrate the first stage recovery system. NASA also will begin modifying the launch infrastructure and fine-tuning ground and mission operations, as the agency makes the transition from the Space Shuttle to the Ares/Orion system.

  3. Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin

    SciTech Connect

    Hill-Eubanks, D.C.; Parker, C.G.; Lollar, P. )

    1989-09-01

    Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. The authors isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO{sub 4}/PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated {sup 125}I-fVIII to function as a cofactor in the activation of factor X by factor IXa, they found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by light-chain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function.

  4. Potential Role of Activating Transcription Factor 5 during Osteogenesis.

    PubMed

    Vicari, Luisa; Calabrese, Giovanna; Forte, Stefano; Giuffrida, Raffaella; Colarossi, Cristina; Parrinello, Nunziatina Laura; Memeo, Lorenzo

    2016-01-01

    Human adipose-derived stem cells are an abundant population of stem cells readily isolated from human adipose tissue that can differentiate into connective tissue lineages including bone, cartilage, fat, and muscle. Activating transcription factor 5 is a transcription factor of the ATF/cAMP response element-binding protein (CREB) family. It is transcribed in two types of mRNAs (activating transcription factor 5 isoform 1 and activating transcription factor 5 isoform 2), encoding the same single 30-kDa protein. Although it is well demonstrated that it regulates the proliferation, differentiation, and apoptosis, little is known about its potential role in osteogenic differentiation. The aim of this study was to evaluate the expression levels of the two isoforms and protein during osteogenic differentiation of human adipose-derived stem cells. Our data indicate that activating transcription factor 5 is differentially expressed reaching a peak of expression at the stage of bone mineralization. These findings suggest that activating transcription factor 5 could play an interesting regulatory role during osteogenesis, which would provide a powerful tool to study bone physiology. PMID:26770207

  5. Ares I-X: First Flight of a New Era

    NASA Technical Reports Server (NTRS)

    Davis, Stephen R.; Askins, Bruce R.

    2010-01-01

    Since 2005, NASA s Constellation Program has been designing, building, and testing the next generation of launch and space vehicles to carry humans beyond low-Earth orbit (LEO). The Ares Projects at Marshall Space Flight Center (MSFC) are developing the Ares I crew launch vehicle and Ares V cargo launch vehicle. On October 28, 2009, the first development flight test of the Ares I crew launch vehicle, Ares I-X, lifted off from a launch pad at Kennedy Space Center (KSC) on successful suborbital flight. Basing exploration launch vehicle designs on Ares I-X information puts NASA one step closer to full-up "test as you fly," a best practice in vehicle design. Although the final Constellation Program architecture is under review, the Ares I-X data and experience in vehicle design and operations can be applied to any launch vehicle. This paper presents the mission background as well as results and lessons learned from the flight.

  6. Is carbonic anhydrase IX a validated target for molecular imaging of cancer and hypoxia?

    PubMed Central

    Li, Jianbo; Zhang, Guojian; Wang, Xuemei; Li, Xiao-Feng

    2015-01-01

    ABSTRACT  The presence of hypoxia is a general feature of most solid malignancies, and hypoxia is considered as one of major factors for anticancer therapy failure. Carbonic anhydrase IX (CAIX) has been reported to be an endogenous hypoxia marker, CAIX monoclonal antibodies, their segments and inhibitors are developed for CAIX imaging. However, growing evidence indicates that CAIX expression under hypoxia condition may be cancer cell lines or cancer-type dependent. Here we review the current literature on CAIX and discuss the advantage and limitation of CAIX as a target for tumor hypoxia imaging. Accordingly, CAIX would be unreliable as a universal target for cancer and tumor hypoxia visualization. PMID:25963430

  7. 75 FR 18245 - Public Federal Regulatory Enforcement Fairness Hearing Region IX Regulatory Fairness Board

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-09

    ... From the Federal Register Online via the Government Publishing Office SMALL BUSINESS 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...

  8. Mesenchymal stem cells inhibit complement activation by secreting factor H.

    PubMed

    Tu, Zhidan; Li, Qing; Bu, Hong; Lin, Feng

    2010-11-01

    Mesenchymal stem cells (MSCs) possess potent and broad immunosuppressive capabilities, and have shown promise in clinical trials treating many inflammatory diseases. Previous studies have found that MSCs inhibit dendritic cell, T-cell, and B-cell activities in the adaptive immunity; however, whether MSCs inhibit complement in the innate immunity, and if so, by which mechanism, have not been established. In this report, we found that MSCs constitutively secrete factor H, which potently inhibits complement activation. Depletion of factor H in the MSC-conditioned serum-free media abolishes their complement inhibitory activities. In addition, production of factor H by MSCs is augmented by inflammatory cytokines TNF-α and interferon-γ (IFN-γ) in dose- and time-dependent manners, while IL-6 does not have a significant effect. Furthermore, the factor H production from MSCs is significantly suppressed by the prostaglandin E2 (PGE2) synthesis inhibitor indomethacin and the indoleamine 2,3-dioxygenase (IDO) inhibitor 1-methyl-d-tryptophan (1-MT), both of which inhibitors are known to efficiently dampen MSCs immunosuppressive activity. These results indicate that MSCs inhibit complement activation by producing factor H, which could be another mechanism underlying MSCs broad immunosuppressive capabilities. PMID:20163251

  9. Ares I-X: First Flight of a New Generation

    NASA Technical Reports Server (NTRS)

    Davis, Stephan R.; Askins, Bruce R.

    2010-01-01

    The Ares I-X suborbital development flight test demonstrated NASA s ability to design, develop, launch and control a new human-rated launch vehicle (Figure 14). This hands-on missions experience will provide the agency with necessary skills and insights regardless of the future direction of space exploration. The Ares I-X team, having executed a successful launch, will now focus on analyzing the flight data and extracting lessons learned that will be used to support the development of future vehicles.

  10. Star Formation Rate in Holmberg IX Dwarf Galaxy

    NASA Astrophysics Data System (ADS)

    Andjelic, M. M.

    2011-12-01

    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 3.4×10-4M_{⊙} 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.

  11. Formation of tissue factor activity following incubation of recombinant human tissue factor apoprotein with plasma lipoproteins

    SciTech Connect

    Sakai, T.; Kisiel, W. )

    1990-11-01

    Incubation of recombinant human tissue factor apoprotein (Apo-TF) with human plasma decreased the recalcified clotting time of this plasma in a time-and dose-dependent manner suggesting relipidation of the Apo-TF by plasma lipoproteins. Incubation of Apo-TF with purified preparations of human very low density, low density and high density lipoproteins resulted in tissue factor activity in a clotting assay. The order of effectiveness was VLDL greater than LDL much greater than HDL. Tissue factor activity generated by incubation of a fixed amount of Apo-TF with plasma lipoproteins was lipoprotein concentration-dependent and saturable. The association of Apo-TF with lipoprotein particles was supported by gel filtration studies in which {sup 125}I-Apo-TF coeluted with the plasma lipoprotein in the void volume of a Superose 6 column in the presence and absence of calcium ions. In addition, void-volume Apo-TF-lipoprotein fractions exhibited tissue factor activity. These results suggest that the factor VIII-bypassing activity of bovine Apo-TF observed in a canine hemophilic model may be due, in part, to its association with plasma lipoproteins and expression of functional tissue factor activity.

  12. Genetic incorporation of HSV-1 thymidine kinase into the adenovirus protein IX for functional display on the virion

    SciTech Connect

    Li Jing; Le, Long; Sibley, Don A.; Mathis, J. Michael; Curiel, David T. . E-mail: david.curiel@ccc.uab.edu

    2005-08-01

    Adenoviral vectors have been exploited for a wide range of gene therapy applications. Direct genetic modification of the adenovirus capsid proteins has been employed to achieve alteration of vector tropism. We have defined the carboxy-terminus of the minor capsid protein pIX as a locus capable of presenting incorporated ligands on the virus capsid surface. Thus, we sought to exploit the possibility of incorporating functional proteins at pIX. In our current study, we incorporated the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) within pIX to determine if a larger protein of this type could retain functionality in this context. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional HSV-1 TK as a component of their capsid surface. DNA packaging and cytopathic effect were not affected by this genetic modification to the virus, while CAR-dependent binding was only marginally affected. Using an in vitro [{sup 3}H]-thymidine phosphorylation assay, we demonstrated that the kinase activity of the protein IX-TK fusion protein incorporated into adenoviral virions is functional. Analysis of cell killing after adenovirus infection showed that the protein IX-TK fusion protein could also serve as a therapeutic gene by rendering transduced cells sensitive to gancyclovir. Using 9-[4-[{sup 18}F]-fluoro-3-(hydroxymethyl)butyl]guanine ([{sup 18}F]-FHBG; a positron-emitting TK substrate), we demonstrated that we could detect specific cell binding and uptake of adenoviral virions containing the protein IX-TK fusion protein at 1 h post-infection. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional HSV-1 TK as a component of their capsid surface. The alternative display of HSV-1 TK on the capsid may offer advantages with respect to direct functional applications of this gene product. In addition, the determination of an expanded upper limit of incorporable

  13. Changes in CVD risk factors in the activity counseling trial

    PubMed Central

    Baruth, Meghan; Wilcox, Sara; Sallis, James F; King, Abby C; Marcus, Bess H; Blair, Steven N

    2011-01-01

    Primary care facilities may be a natural setting for delivering interventions that focus on behaviors that improve cardiovascular disease (CVD) risk factors. The purpose of this study was to examine the 24-month effects of the Activity Counseling Trial (ACT) on CVD risk factors, to examine whether changes in CVD risk factors differed according to baseline risk factor status, and to examine whether changes in fitness were associated with changes in CVD risk factors. ACT was a 24-month multicenter randomized controlled trial to increase physical activity. Participants were 874 inactive men and women aged 35–74 years. Participants were randomly assigned to one of three arms that varied by level of counseling, intensity, and resource requirements. Because there were no significant differences in change over time between arms on any of the CVD risk factors examined, all arms were combined, and the effects of time, independent of arm, were examined separately for men and women. Time × Baseline risk factor status interactions examined whether changes in CVD risk factors differed according to baseline risk factor status. Significant improvements in total cholesterol, high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol, the ratio of total cholesterol to HDL-C, and triglycerides were seen in both men and women who had high (or low for HDL-C) baseline levels of risk factors, whereas significant improvements in diastolic blood pressure were seen only in those men with high baseline levels. There were no improvements in any risk factors among participants with normal baseline levels. Changes in fitness were associated with changes in a number of CVD risk factors. However, most relationships disappeared after controlling for changes in body weight. Improvements in lipids from the ACT interventions could reduce the risk of coronary heart disease in people with already high levels of lipids by 16%–26% in men and 11%–16% in women

  14. 45 CFR 83.5 - Effect of title IX of the Education Amendments of 1972.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 45 Public Welfare 1 2012-10-01 2012-10-01 false Effect of title IX of the Education Amendments of... Purposes; Definitions; Coverage § 83.5 Effect of title IX of the Education Amendments of 1972. The obligations imposed by this part are independent of obligations imposed by or pursuant to title IX of...

  15. 45 CFR 83.5 - Effect of title IX of the Education Amendments of 1972.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 45 Public Welfare 1 2013-10-01 2013-10-01 false Effect of title IX of the Education Amendments of... Purposes; Definitions; Coverage § 83.5 Effect of title IX of the Education Amendments of 1972. The obligations imposed by this part are independent of obligations imposed by or pursuant to title IX of...

  16. 45 CFR 83.5 - Effect of title IX of the Education Amendments of 1972.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 45 Public Welfare 1 2014-10-01 2014-10-01 false Effect of title IX of the Education Amendments of... Purposes; Definitions; Coverage § 83.5 Effect of title IX of the Education Amendments of 1972. The obligations imposed by this part are independent of obligations imposed by or pursuant to title IX of...

  17. Reduced serum inhibition of platelet-activating factor activity in preeclampsia.

    PubMed

    Benedetto, C; Massobrio, M; Bertini, E; Abbondanza, M; Enrieu, N; Tetta, C

    1989-01-01

    We determined in normal nonpregnant (group 1) women, normal pregnant (group 2) women, and patients with preeclampsia (group 3) the serum inhibition of platelet-activating factor activity, the presence of detectable amounts of platelet-activating factor in the blood, and platelet responsiveness in vitro to platelet-activating factor, and to other agonists (adenosine diphosphate, collagen, and ristocetin), and prostacyclin (prostaglandin I2). In patients with preeclampsia (group 3) the serum inhibition of platelet-activating factor activity was significantly lower than that in groups 1 and 2. However, no detectable amounts of platelet-activating factor were observed. The mean values of platelet aggregation induced by platelet-activating factor, adenosine diphosphate, collagen and ristocetin, and the prostaglandin I2-inhibitory concentration of 50% which is inversely correlated with platelet sensitivity to prostaglandin I2, were not significantly different between groups 2 and 3. It is suggested that in preeclampsia the defect in serum inhibitory potential of platelet-activating factor--induced platelet aggregation may contribute to the disturbance in the homeostatic balance between proaggregant and antiaggregant substances. PMID:2912073

  18. 78 FR 46558 - Request for Information Regarding Nondiscrimination in Certain Health Programs or Activities

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-01

    ..., sex, age, or disability in certain health programs and activities. Section 1557(c) of the Affordable... origin), Title IX of the Education Amendments of 1972 (Title IX), 20 U.S.C. 1681 et seq. (sex), the Age... prohibition of sex discrimination in Title IX applies only to education programs and activities of...

  19. In vivo activation and functions of the protease factor XII.

    PubMed

    Björkqvist, Jenny; Nickel, Katrin F; Stavrou, Evi; Renné, Thomas

    2014-11-01

    Combinations of proinflammatory and procoagulant reactions are the unifying principle for a variety of disorders affecting the cardiovascular system. Factor XII (FXII, Hageman factor) is a plasma protease that initiates the contact system. The biochemistry of the contact system in vitro is well understood; however, its in vivo functions are just beginning to emerge. The current review concentrates on activators and functions of the FXII-driven contact system in vivo. Elucidating its physiologic activities offers the exciting opportunity to develop strategies for the safe interference with both thrombotic and inflammatory diseases. PMID:25187064

  20. Protease-induced immunoregulatory activity of platelet factor 4.

    PubMed Central

    Katz, I R; Thorbecke, G J; Bell, M K; Yin, J Z; Clarke, D; Zucker, M B

    1986-01-01

    Intravenous injection of human or mouse serum or platelet material secreted from appropriately stimulated platelets ("releasate") together with antigen alleviates the immunosuppression in SJL/J mice induced by injection of irradiated lymphoma cells or in (CB6)F1 mice induced by injection of concanavalin A. We now report that injection of releasate from 10(6) human platelets restores plaque-forming cells to the unsuppressed number; greater amounts increase responses further. Immunoregulatory activity is released from platelets exposed to thrombin in parallel with other alpha-granule components. Heparin-agarose absorbs activity. Purified platelet factor 4 (PF4) has activity; beta-thromboglobulin and platelet-derived growth factor have little or none. Activity in serum is neutralized by goat anti-human PF4. An enzymatic step is necessary for production of immunoregulatory activity. Releasates boiled immediately after platelet aggregation with 250 nM A23187 or those produced by adding A23187 in the presence of 100 microM serine protease inhibitor (p-amidinophenyl)methanesulfonyl fluoride (APMSF) are ineffective, whereas releasates boiled or mixed with APMSF after incubation for 60 min are active. Activity is generated by incubating a mixture of heparin-absorbed releasate (as enzyme source) and heparin-agarose eluate of releasate made in the presence of APMSF (as substrate source). The enzymatic step does not alter the heparin-neutralizing activity of PF4. Apparently a secreted platelet protease converts PF4 to a form with immunoregulatory activity. PMID:3517862

  1. Activated factor XI increases the procoagulant activity of the extrinsic pathway by inactivating tissue factor pathway inhibitor

    PubMed Central

    Tucker, Erik I.; Matafonov, Anton; Cheng, Qiufang; Zientek, Keith D.; Gailani, Dave; Gruber, András; McCarty, Owen J. T.

    2015-01-01

    Activation of coagulation factor XI (FXI) may play a role in hemostasis. The primary substrate of activated FXI (FXIa) is FIX, leading to FX activation (FXa) and thrombin generation. However, recent studies suggest the hemostatic role of FXI may not be restricted to the activation of FIX. We explored whether FXI could interact with and inhibit the activity of tissue factor pathway inhibitor (TFPI). TFPI is an essential reversible inhibitor of activated factor X (FXa) and also inhibits the FVIIa-TF complex. We found that FXIa neutralized both endothelium- and platelet-derived TFPI by cleaving the protein between the Kunitz (K) 1 and K2 domains (Lys86/Thr87) and at the active sites of the K2 (Arg107/Gly108) and K3 (Arg199/Ala200) domains. Addition of FXIa to plasma was able to reverse the ability of TFPI to prolong TF-initiated clotting times in FXI- or FIX-deficient plasma, as well as FXa-initiated clotting times in FX-deficient plasma. Treatment of cultured endothelial cells with FXIa increased the generation of FXa and promoted TF-dependent fibrin formation in recalcified plasma. Together, these results suggest that the hemostatic role of FXIa may be attributed not only to activation of FIX but also to promoting the extrinsic pathway of thrombin generation through inactivation of TFPI. PMID:25587039

  2. Activated factor XI increases the procoagulant activity of the extrinsic pathway by inactivating tissue factor pathway inhibitor.

    PubMed

    Puy, Cristina; Tucker, Erik I; Matafonov, Anton; Cheng, Qiufang; Zientek, Keith D; Gailani, Dave; Gruber, András; McCarty, Owen J T

    2015-02-26

    Activation of coagulation factor XI (FXI) may play a role in hemostasis. The primary substrate of activated FXI (FXIa) is FIX, leading to FX activation (FXa) and thrombin generation. However, recent studies suggest the hemostatic role of FXI may not be restricted to the activation of FIX. We explored whether FXI could interact with and inhibit the activity of tissue factor pathway inhibitor (TFPI). TFPI is an essential reversible inhibitor of activated factor X (FXa) and also inhibits the FVIIa-TF complex. We found that FXIa neutralized both endothelium- and platelet-derived TFPI by cleaving the protein between the Kunitz (K) 1 and K2 domains (Lys86/Thr87) and at the active sites of the K2 (Arg107/Gly108) and K3 (Arg199/Ala200) domains. Addition of FXIa to plasma was able to reverse the ability of TFPI to prolong TF-initiated clotting times in FXI- or FIX-deficient plasma, as well as FXa-initiated clotting times in FX-deficient plasma. Treatment of cultured endothelial cells with FXIa increased the generation of FXa and promoted TF-dependent fibrin formation in recalcified plasma. Together, these results suggest that the hemostatic role of FXIa may be attributed not only to activation of FIX but also to promoting the extrinsic pathway of thrombin generation through inactivation of TFPI. PMID:25587039

  3. Factors limiting microbial activity in volcanic tuff at Yucca Mountain

    SciTech Connect

    Kieft, T.L.; Kovacik, W.P.; Taylor, J.

    1996-09-01

    Samples of tuff aseptically collected from 10 locations in the Exploratory Shaft Facility at the site of the proposed high-level nuclear waste repository at Yucca Mountain, Nevada Test Site were analyzed for microbiological populations, activities, and factors limiting microbial activity. Radiotracer assays ({sup 14}C-labeled organic substrate mineralization), direct microscopic counts, and plate counts were used. Radiolabeled substrates were glucose, acetate, and glutamate. Radiotracer experiments were carried out with and without moisture and inorganic nutrient amendments to determine factors limiting to microbial activities. Nearly all samples showed the presence of microorganisms with the potential to mineralize organic substrates. Addition of inorganic nutrients stimulated activities in a small number of samples. The presence of viable microbial communities within the tuff has implications for transport of contaminants.

  4. Hearing on Title IX of the Education Amendments of 1972. Hearing before the Subcommittee on Postsecondary Education, Training and Life-Long Learning of the Committee on Economic and Educational Opportunities. House of Representatives, One Hundred Fourth Congress, First Session (May 9, 1995).

    ERIC Educational Resources Information Center

    Congress of the U.S., Washington, DC. House Committee on Economic and Educational Opportunities.

    The subcommittee met to discuss and hear testimony on Title IX of the Education Amendments of 1972, a law designed to prohibit sex discrimination in intercollegiate athletics at federally funded education programs or activities. Among issues addressed were the means by which universities demonstrate that they are in compliance with Title IX as…

  5. EspP, an Extracellular Serine Protease from Enterohemorrhagic E. coli, Reduces Coagulation Factor Activities, Reduces Clot Strength, and Promotes Clot Lysis

    PubMed Central

    Rand, Margaret L.; Mian, Hira S.; Brnjac, Elena; Sandercock, Linda E.; Akula, Indira; Julien, Jean-Philippe; Pai, Emil F.; Chesney, Alden E.

    2016-01-01

    Background EspP (E. coli secreted serine protease, large plasmid encoded) is an extracellular serine protease produced by enterohemorrhagic E. coli (EHEC) O157:H7, a causative agent of diarrhea-associated Hemolytic Uremic Syndrome (D+HUS). The mechanism by which EHEC induces D+HUS has not been fully elucidated. Objectives We investigated the effects of EspP on clot formation and lysis in human blood. Methods Human whole blood and plasma were incubated with EspPWT at various concentrations and sampled at various time points. Thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (aPTT), coagulation factor activities, and thrombelastgraphy (TEG) were measured. Results and Conclusions Human whole blood or plasma incubated with EspPWT was found to have prolonged PT, aPTT, and TT. Furthermore, human whole blood or plasma incubated with EspPWT had reduced activities of coagulation factors V, VII, VIII, and XII, as well as prothrombin. EspP did not alter the activities of coagulation factors IX, X, or XI. When analyzed by whole blood TEG, EspP decreased the maximum amplitude of the clot, and increased the clot lysis. Our results indicate that EspP alters hemostasis in vitro by decreasing the activities of coagulation factors V, VII, VIII, and XII, and of prothrombin, by reducing the clot strength and accelerating fibrinolysis, and provide further evidence of a functional role for this protease in the virulence of EHEC and the development of D+HUS. PMID:26934472

  6. Surface-Energy Dependent Contact Activation of Blood Factor XII

    PubMed Central

    Golas, Avantika; Parhi, Purnendu; Dimachkie, Ziad O.; Siedlecki, Christopher A.; Vogler, Erwin A.

    2009-01-01

    Contact activation of blood factor XII (FXII, Hageman factor) in neat-buffer solution exhibits a parabolic profile when scaled as a function of silanized-glass-particle activator surface energy (measured as advancing water adhesion tension τao=γlvocosθ in dyne/cm, where γlvo is water interfacial tension in dyne/cm and θ is the advancing contact angle). Nearly equal activation is observed at the extremes of activator water-wetting properties −36<τao<72 dyne/cm (0° ≤ θ < 120°), falling sharply through a broad minimum within the 20<τao<40 dyne/cm (55° < θ < 75°) range over which activation yield (putatively FXIIa) rises just above detection limits. Activation is very rapid upon contact with all activators tested and did not significantly vary over 30 minutes of continuous FXII-procoagulant contact. Results suggest that materials falling within the 20<τao<40 dyne/cm surface-energy range should exhibit minimal activation of blood-plasma coagulation through the intrinsic pathway. Surface chemistries falling within this range are, however, a perplexingly difficult target for surface engineering because of the critical balance that must be struck between hydrophobicity and hydrophilicity. Results are interpreted within the context of blood plasma coagulation and the role of water and proteins at procoagulant surfaces. PMID:19892397

  7. Leadership in Honor Societies: The Effect of Title IX.

    ERIC Educational Resources Information Center

    Earwood-Smith, Glenda

    1985-01-01

    Single-sex honor societies changed membership requirements to admit members of both sexes in response to Title IX of the 1972 Education amendments. A 1982 study examining the effect of this transition on the sex make-up of members, officers, and faculty advisers revealed that women students were competing equally with men students for leadership…

  8. Title IX and Educational Equity: What Difference Does It Make?

    ERIC Educational Resources Information Center

    Golombisky, Kim

    Title IX, the 1972 United States federal law forbidding sex discrimination in education, has a rarely-talked-about but surprisingly tenuous history which illustrates how discourses of equality come to mean political powerlessness for diverse girls and women in school. Unfortunately, "sexual" debates such as women's sports and sexual harassment…

  9. Title IX and Sexual Harassment of Student Athletes.

    ERIC Educational Resources Information Center

    Wolohan, John T.

    1995-01-01

    This article reviews what constitutes sexual harassment in sports by examining Title IX of the Education Amendments of 1972 and the effect it has had on charges of sexual harrassment in educational institutions. Athletic administrators are provided with strategies and recommendations to help schools and athletic departments develop sexual…

  10. Ares I-X Flight Test Vehicle Modal Test

    NASA Technical Reports Server (NTRS)

    Buehrle, Ralph D.; Templeton, Justin D.; Reaves, Mercedes C.; Horta, Lucas G.; Gaspar, James L.; Bartolotta, Paul A.; Parks, Russel A.; Lazor, Daniel R.

    2010-01-01

    The first test flight of NASA's Ares I crew launch vehicle, called Ares I-X, was launched on October 28, 2009. Ares I-X used a 4-segment reusable solid rocket booster from the Space Shuttle heritage with mass simulators for the 5th segment, upper stage, crew module and launch abort system. Flight test data will provide important information on ascent loads, vehicle control, separation, and first stage reentry dynamics. As part of hardware verification, a series of modal tests were designed to verify the dynamic finite element model (FEM) used in loads assessments and flight control evaluations. Based on flight control system studies, the critical modes were the first three free-free bending mode pairs. Since a test of the free-free vehicle was not practical within project constraints, modal tests for several configurations during vehicle stacking were defined to calibrate the FEM. Test configurations included two partial stacks and the full Ares I-X flight test vehicle on the Mobile Launcher Platform. This report describes the test requirements, constraints, pre-test analysis, test execution and results for the Ares I-X flight test vehicle modal test on the Mobile Launcher Platform. Initial comparisons between pre-test predictions and test data are also presented.

  11. Methods of producing protoporphyrin IX and bacterial mutants therefor

    DOEpatents

    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.

  12. Gender Equity in Intercollegiate Athletics: Determinants of Title IX Compliance

    ERIC Educational Resources Information Center

    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…

  13. A Guide for Title IX Self-Study.

    ERIC Educational Resources Information Center

    Boesdorfer, Kent; And Others

    This document is a set of guidelines on how to conduct the self-evaluation required by Title IX, the amendment prohibiting discrimination on the basis of sex. Its purpose is to help school districts comply with the necessity to evaluate policies and practices by providing a data base for decision-making. The paper is divided into these sections:…

  14. National Environmental/Energy Workforce Assessment for Region IX.

    ERIC Educational Resources Information Center

    National Field Research Center Inc., Iowa City, IA.

    This report represents a detailed summation of existing workforce levels, training programs, career potential, and staffing level projections through 1981 for EPA Region IX. This region serves the states of Arizona, California, Hawaii, and Nevada. The specific pollution programs considered include air, noise, pesticides, potable water, radiation…

  15. Ares I-X Malfunction Turn Range Safety Analysis

    NASA Technical Reports Server (NTRS)

    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.

  16. Ares I-X Launch Vehicle Modal Test Overview

    NASA Technical Reports Server (NTRS)

    Buehrle, Ralph D.; Bartolotta, Paul A.; Templeton, Justin D.; Reaves, Mercedes C.; Horta, Lucas G.; Gaspar, James L.; Parks, Russell A.; Lazor, Daniel R.

    2010-01-01

    The first test flight of NASA's Ares I crew launch vehicle, called Ares I-X, is scheduled for launch in 2009. Ares IX will use a 4-segment reusable solid rocket booster from the Space Shuttle heritage with mass simulators for the 5th segment, upper stage, crew module and launch abort system. Flight test data will provide important information on ascent loads, vehicle control, separation, and first stage reentry dynamics. As part of hardware verification, a series of modal tests were designed to verify the dynamic finite element model (FEM) used in loads assessments and flight control evaluations. Based on flight control system studies, the critical modes were the first three free-free bending mode pairs. Since a test of the free-free vehicle is not practical within project constraints, modal tests for several configurations in the nominal integration flow were defined to calibrate the FEM. A traceability study by Aerospace Corporation was used to identify the critical modes for the tested configurations. Test configurations included two partial stacks and the full Ares I-X launch vehicle on the Mobile Launcher Platform. This paper provides an overview for companion papers in the Ares I-X Modal Test Session. The requirements flow down, pre-test analysis, constraints and overall test planning are described.

  17. Title VI and Title IX Compliance by the Office for Civil Rights in State-Operated Special Purpose and Vocational Schools Pursuant to Adams v. Mathews.

    ERIC Educational Resources Information Center

    McClure, Phyllis

    This paper examines the compliance activities of the Office for Civil Rights (OCR) undertaken to eliminate racial and sex discrimination in special purpose schools administered by State departments of education. After reviewing background to non-enforcement of Title VI, and briefly explaining Title IX, the activities of OCR with regard to special…

  18. Factors Influencing Teachers' Engagement in Informal Learning Activities

    ERIC Educational Resources Information Center

    Lohman, Margaret C.

    2006-01-01

    Purpose: The purpose of this study is to examine factors influencing the engagement of public school teachers in informal learning activities. Design/methodology/approach: This study used a survey research design. Findings: Analysis of the data found that teachers rely to a greater degree on interactive than on independent informal learning…

  19. Total Chemical Synthesis of Biologically Active Vascular Endothelial Growth Factor

    SciTech Connect

    Mandal, Kalyaneswar; Kent, Stephen B.H.

    2011-09-15

    The 204-residue covalent-dimer vascular endothelial growth factor (VEGF, see picture) with full mitogenic activity was prepared from three unprotected peptide segments by one-pot native chemical ligations. The covalent structure of the synthetic VEGF was confirmed by precise mass measurement, and the three-dimensional structure of the synthetic protein was determined by high-resolution X-ray crystallography.

  20. Factors Influencing Active Learning in Small Enterprises. Working Paper.

    ERIC Educational Resources Information Center

    Hawke, Geof

    The factors influencing active learning in small enterprises were examined. Data from earlier Australian studies were examined in an attempt to provide a framework that might inform the relationship between educational systems and small enterprises. Special attention was paid to a 1988 study of systematic differences between small businesses that…

  1. Characterization and estrogen regulation of uterine growth factor activity

    SciTech Connect

    Beck, C.A.

    1988-01-01

    Acid extracts of rat, bovine and rabbit uterus stimulated glucose transport, measured by phosphorylation of 2-deoxyglucose and DNA synthesis, measured by {sup 3}H-thymidne incorporation, in uterine tumor cells and in primary cultures of rat uterine cells. The stimulation of glucose transport was of the same magnitude and followed the same time course as estradiol stimulation in vivo. Uteri from estradiol-treated rat uteri contained 4 times more glucose transport-stimulating activity as control uteri. DNA synthetic activity in rat uterine homogenates was elevated 3-fold within 18-24 h after estradiol injection. Gel filtration showed molecular weight heterogeneity with activity eluting between 10-30 kDA. Both activities were acid and heat stable, were reduced by trypsin but not by dextran-coated charcoal. The effect of purified growth factors on DNA synthesis in primary cultures of rat uterine cells was examined. Epidermal growth factor (EGF), basic fibroblasts growth factor (bFGF), and transforming growth factor-{beta} (TGF{beta}) had no effect on {sup 3}H-thymidine incorporation.

  2. The Relevant Factors in Promoting Reading Activities in Elementary Schools

    ERIC Educational Resources Information Center

    Huang, Han-Chen; Tsai, Yao-Hsu; Huang, Shih-Hsiang

    2015-01-01

    In order to help students absorb knowledge, schools often conduct reading activities. Thorough planning and strategies, however, are needed to insure the effect of reading promotions, and make them a deeply-rooted part of life. This study adopted the analytic hierarchy process (AHP) to discuss the relevant factors in promoting reading activities…

  3. Ares I-X Flight Data Evaluation: Executive Overview

    NASA Technical Reports Server (NTRS)

    Huebner, Lawrence D.; Waits, David A.; Lewis, Donny L.; Richards, James S.; Coates, R. H., Jr.; Cruit, Wendy D.; Bolte, Elizabeth J.; Bangham, Michal E.; Askins, Bruce R.; Trausch, Ann N.

    2011-01-01

    NASA's Constellation Program (CxP) successfully launched the Ares I-X flight test vehicle on October 28, 2009. The Ares I-X flight was a developmental flight test to demonstrate that this very large, long, and slender vehicle could be controlled successfully. The flight offered a unique opportunity for early engineering data to influence the design and development of the Ares I crew launch vehicle. As the primary customer for flight data from the Ares I-X mission, the Ares Projects Office (APO) established a set of 33 flight evaluation tasks to correlate flight results with prospective design assumptions and models. The flight evaluation tasks used Ares I-X data to partially validate tools and methodologies in technical disciplines that will ultimately influence the design and development of Ares I and future launch vehicles. Included within these tasks were direct comparisons of flight data with preflight predictions and post-flight assessments utilizing models and processes being applied to design and develop Ares I. The benefits of early development flight testing were made evident by results from these flight evaluation tasks. This overview provides summary information from assessment of the Ares I-X flight test data and represents a small subset of the detailed technical results. The Ares Projects Office published a 1,600-plus-page detailed technical report that documents the full set of results. This detailed report is subject to the International Traffic in Arms Regulations (ITAR) and is available in the Ares Projects Office archives files.

  4. Platelet activating factor: regulation by mast cells and aspirin.

    PubMed

    Denburg, J A; Williams, D B; Kinlough-Rathbone, R L; Cazenave, J P; Bienenstock, J

    1984-02-01

    We have investigated some aspects of the regulation of production of rat platelet activating factor (PAF)2 in vitro. Suspensions of unseparated (PLC1), mast cell-depleted (PLC2), or mast cell (MC)-enriched rat peritoneal lavage cells (PLC) were analyzed for PAF content by extraction at alkaline pH. PAF activity extracted from PLC1 varied inversely with viable cell concentration: at 1 X 10(6) cells/ml, 32 +/- 9.3 PAF units, decreasing to 11.2 +/- 9.5 units at 10 X 10(6) cells/ml, and no activity at higher concentrations. Incubation of PLC1 in Tyrode's buffer or acetylsalicylic acid (ASA), but not salicylate, resulted in a time-dependent loss of PAF activity. Mean PAF activity of PLC2 was similar to that in PLC1, while no PAF activity was extractable from MC. Co-incubation with MC extracts inhibited PAF activity of PLC1 extracts in a dose-dependent fashion. Ultracentrifugation of PAF-containing samples led to a loss of all PAF activity in PLC1 extracts, suggesting the association of PAF activity with subcellular components. PAF appears to be derived from a non-MC population of rat PLC, is not extractable from rat PLC in the presence of ASA and is inhibited by MC extracts. These studies suggest that ASA regulates PAF availability unrelated to its effect on cyclooxygenase and that MC membrane products directly inhibit PAF activity from rat PLC. PMID:6711391

  5. Defective Association of the Platelet Glycoprotein Ib-IX Complex with the Glycosphingolipid-Enriched Membrane Domain Inhibits Murine Thrombus and Atheroma Formation.

    PubMed

    Zhou, Hao; Ran, Yali; Da, Qi; Shaw, Tanner S; Shang, Dan; Reddy, Anilkumar K; López, José A; Ballantyne, Christie M; Ware, Jerry; Wu, Huaizhu; Peng, Yuandong

    2016-07-01

    Localization of the platelet glycoprotein Ib-IX complex to the membrane lipid domain is essential for platelet adhesion to von Willebrand factor and subsequent platelet activation in vitro. Yet, the in vivo importance of this localization has never been addressed. We recently found that the disulfide linkage between Ibα and Ibβ is critical for the association of Ibα with the glycosphingolipid-enriched membrane domain; in this study, we established a transgenic mouse model expressing this mutant human Ibα that is also devoid of endogenous Ibα (HαSSMα(-/-)). Characterization of this model demonstrated a similar dissociation of Ibα from murine platelet glycosphingolipid-enriched membrane to that expressed in Chinese hamster ovary cells, which correlates well with the impaired adhesion of the transgenic platelets to von Willebrand factor ex vivo and in vivo. Furthermore, we bred our transgenic mice into an atherosclerosis-prone background (HαSSMα(-/-)ApoE(-/-) and HαWTMα(-/-)ApoE(-/-)). We observed that atheroma formation was significantly inhibited in mutant mice where fewer platelet-bound CD11c(+) leukocytes were circulating (CD45(+)/CD11c(+)/CD41(+)) and residing in atherosclerotic lesions (CD45(+)/CD11c(+)), suggesting that platelet-mediated adhesion and infiltration of CD11c(+) leukocytes may be one of the mechanisms. To our knowledge, these observations provide the first in vivo evidence showing that the membrane GEM is physiologically and pathophysiologically critical in the function of the glycoprotein Ib-IX complex. PMID:27206768

  6. Psychosocial Factors and Theory in Physical Activity Studies in Minorities

    PubMed Central

    Mama, Scherezade K.; McNeill, Lorna H.; McCurdy, Sheryl A.; Evans, Alexandra E.; Diamond, Pamela M.; Adamus-Leach, Heather J.; Lee, Rebecca E.

    2015-01-01

    Objectives To summarize the effectiveness of interventions targeting psychosocial factors to increase physical activity (PA) among ethnic minority adults and explore theory use in PA interventions. Methods Studies (N = 11) were identified through a systematic review and targeted African American/Hispanic adults, specific psychosocial factors, and PA. Data were extracted using a standard code sheet and the Theory Coding Scheme. Results Social support was the most common psychosocial factor reported, followed by motivational readiness, and self-efficacy, as being associated with increased PA. Only 7 studies explicitly reported using a theoretical framework. Conclusions Future efforts should explore theory use in PA interventions and how integration of theoretical constructs, including psychosocial factors, increases PA. PMID:25290599

  7. LPS-inducible factor(s) from activated macrophages mediates cytolysis of Naegleria fowleri amoebae

    SciTech Connect

    Cleary, S.F.; Marciano-Cabral, F.

    1986-03-01

    Soluble cytolytic factors of macrophage origin have previously been described with respect to their tumoricidal activity. The purpose of this study was to investigate the mechanism and possible factor(s) responsible for cytolysis of the amoeba Naegleria fowleri by activated peritoneal macrophages from B6C3F1 mice. Macrophages or conditioned medium (CM) from macrophage cultures were incubated with /sup 3/H-Uridine labeled amoebae. Percent specific release of label served as an index of cytolysis. Bacille Calmette-Guerin (BCG) and Corynebacterium parvum macrophages demonstrated significant cytolysis of amoebae at 24 h with an effector to target ratio of 10:1. Treatment of macrophages with inhibitors of RNA or protein synthesis blocked amoebicidal activity. Interposition of a 1 ..mu..m pore membrane between macrophages and amoebae inhibited killing. Inhibition in the presence of the membrane was overcome by stimulating the macrophages with LPS. CM from SPS-stimulated, but not unstimulated, cultures of activated macrophages was cytotoxic for amoebae. The activity was heat sensitive and was recovered from ammonium sulfate precipitation of the CM. Results indicate that amoebicidal activity is mediated by a protein(s) of macrophage origin induced by target cell contact or stimulation with LPS.

  8. 40 CFR Appendix Ix to Part 268 - Extraction Procedure (EP) Toxicity Test Method and Structural Integrity Test (Method 1310B)

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 27 2014-07-01 2014-07-01 false Extraction Procedure (EP) Toxicity Test Method and Structural Integrity Test (Method 1310B) IX Appendix IX to Part 268 Protection of.... 268, App. IX Appendix IX to Part 268—Extraction Procedure (EP) Toxicity Test Method and...

  9. 40 CFR Appendix Ix to Part 268 - Extraction Procedure (EP) Toxicity Test Method and Structural Integrity Test (Method 1310B)

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 28 2012-07-01 2012-07-01 false Extraction Procedure (EP) Toxicity Test Method and Structural Integrity Test (Method 1310B) IX Appendix IX to Part 268 Protection of.... 268, App. IX Appendix IX to Part 268—Extraction Procedure (EP) Toxicity Test Method and...

  10. Factors affecting daily activities of patients with cerebral infarction

    PubMed Central

    Liu, Peng; Zhou, Cheng-ye; Zhang, Ying; Wang, Yun-feng; Zou, Chang-lin

    2010-01-01

    BACKGROUND: Stroke is the leading cause of death and long-term disability. This study was undertaken to investigate the factors influencing daily activities of patients with cerebral infarction so as to take interventional measures earlier to improve their daily activities. METHODS: A total of 149 patients with first-episode cerebral infarction were recruited into this prospective study. They were admitted to the Encephalopathy Center, Department of Neurology, the First Affiliated Hospital of Wenzhou Medical College in Zhejiang Province from August 2008 to December 2008. The baseline characteristics of the patients and cerebral infarction risk factors on the first day of admission were recorded. White blood cell (WBC) count, plasma glucose (PG), and many others of laboratory targets were collected in the next morning. Barthel index (BI) was calculated at 2 weeks and 3 months respectively after onset of the disease at the outpatient clinic or by telephone call. Lung infection, urinary tract infection and atrial fibrillation if any were recorded on admission. The National Institute of Health Stroke Scale (NIHSS) scores and the GCS scores were recorded within 24 hours on and after admission, at the second week, and at the third month after the onset of cerebral infarction respectively. RESULTS: The factors of BI at 2 weeks and 3 months after onset were the initial PG level, WBC count and initial NIHSS scores. Besides, urinary tract infection on admission was also the factor for BI at 3 months. CONCLUSION: Active measures should be taken to control these factors to improve the daily activities of patients with cerebral infarction. PMID:25214953

  11. Biosynthesis of nitric oxide activates iron regulatory factor in macrophages.

    PubMed Central

    Drapier, J C; Hirling, H; Wietzerbin, J; Kaldy, P; Kühn, L C

    1993-01-01

    Biosynthesis of nitric oxide (NO) from L-arginine modulates activity of iron-dependent enzymes, including mitochondrial acontiase, an [Fe-S] protein. We examined the effect of NO on the activity of iron regulatory factor (IRF), a cytoplasmic protein which modulates both ferritin mRNA translation and transferrin receptor mRNA stability by binding to specific mRNA sequences called iron responsive elements (IREs). Murine macrophages were activated with interferon-gamma and lipopolysaccharide to induce NO synthase activity and cultured in the presence or absence of NG-substituted analogues of L-arginine which served as selective inhibitors of NO synthesis. Measurement of the nitrite concentration in the culture medium was taken as an index of NO production. Mitochondria-free cytosols were then prepared and aconitase activity as well as IRE binding activity and induction of IRE binding activity were correlated and depended on NO synthesis after IFN-gamma and/or LPS stimulation. Authentic NO gas as well as the NO-generating compound 3-morpholinosydnonimine (SIN-1) also conversely modulated aconitase and IRE binding activities of purified recombinant IRF. These results provide evidence that endogenously produced NO may modulate the post-transcriptional regulation of genes involved in iron homeostasis and support the hypothesis that the [Fe-S] cluster of IRF mediates iron-dependent regulation. Images PMID:7504626

  12. Losac, the First Hemolin that Exhibits Procogulant Activity through Selective Factor X Proteolytic Activation*

    PubMed Central

    Alvarez-Flores, Miryam Paola; Furlin, Daniel; Ramos, Oscar H. P.; Balan, Andrea; Konno, Katsuhiro; Chudzinski-Tavassi, Ana Marisa

    2011-01-01

    Envenoming by the contact of human skin with Lonomia obliqua caterpillars promotes a hemorrhagic syndrome characterized by a consumptive coagulopathy. Losac (Lonomia obliqua Stuart factor activator) is a component of the bristle of L. obliqua that is probably partially responsible for the observed syndrome because it activates factor X and is recognized by an effective antilonomic serum. Here we unveil the proteolytic activity of Losac and demonstrate the feasibility of its recombinant production. On the other hand, Losac has no homology to known proteases, but it can be inhibited by PMSF, a serine protease inhibitor. Instead, it shows closer homology to members of the hemolin family of proteins, a group of cell adhesion molecules. The recombinant protein (rLosac) shortened the coagulation time of normal and deficient plasmas, whereas it was ineffective in factor X-deficient plasma unless reconstituted with this protein. rLosac was able to activate factor X in a dose- and time-dependent manner but not γ-carboxyglutamic acid domainless factor X. Moreover, phospholipids and calcium ions increased rLosac activity. Also, rLosac had no effect on fibrin or fibrinogen, indicating its specificity for blood coagulation activation. Linear double reciprocal plots indicate that rLosac follows a Michaelis-Menten kinetics. Cleavage of factor X by rLosac resulted in fragments that are compatible with those generated by RVV-X (a well known factor X activator). Together, our results validate Losac as the first protein from the hemolin family exhibiting procoagulant activity through selective proteolysis on coagulation factor X. PMID:21177860

  13. The Development of the Ares I-X Flight Test

    NASA Technical Reports Server (NTRS)

    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

  14. Selective Activation of Transcription by a Novel CCAAT Binding Factor

    NASA Astrophysics Data System (ADS)

    Maity, Sankar N.; Golumbek, Paul T.; Karsenty, Gerard; de Crombrugghe, Benoit

    1988-07-01

    A novel CCAAT binding factor (CBF) composed of two different subunits has been extensively purified from rat liver. Both subunits are needed for specific binding to DNA. Addition of this purified protein to nuclear extracts of NIH 3T3 fibroblasts stimulates transcription from several promoters including the α 2(I) collagen, the α 1(I) collagen, the Rous sarcoma virus long terminal repeat (RSV-LTR), and the adenovirus major late promoter. Point mutations in the CCAAT motif that show either no binding or a decreased binding of CBF likewise abolish or reduce activation of transcription by CBF. Activation of transcription requires, therefore, the specific binding of CBF to its recognition sites.

  15. Factors affecting the behavior of unburned carbon upon steam activation

    NASA Astrophysics Data System (ADS)

    Lu, Zhe

    The main objective of this study is to investigate the factors that could affect the behavior of unburned carbon samples upon steam activation. Through this work, the relationships among the factors that could influence the carbon-steam reaction with the surface area of the produced activated carbon were explored. Statistical analysis was used to relate the chemical and physical properties of the unburned carbon to the surface area of the activated carbon. Six unburned carbons were selected as feedstocks for activated carbon, and marked as UCA through UCF. The unburned carbons were activated using steam at 850°C for 90 minutes, and the surface areas of their activated counterparts were measured using N2 adsorption isotherms at 77K. The activated carbons produced from different unburned carbon precursors presented different surface areas at similar carbon burn-off levels. Moreover, in different carbon burn-off regions, the sequences for surface area of activated carbons from different unburned carbon samples were different. The factors that may affect the carbon-steam gasification reactions, including the concentration of carbon active sites, the crystallite size of the carbon, the intrinsic porous structure of carbon, and the inorganic impurities, were investigated. All unburned carbons investigated in this study were similar in that they showed the very broad (002) and (10 ) carbon peaks, which are characteristic of highly disordered carbonaceous materials. In this study, the unburned carbon samples contained about 17--48% of inorganic impurities. Compared to coals, the unburned carbon samples contain a larger amount of inorganic impurities as a result of the burn-off, or at lease part, of the carbon during the combustion process. These inorganic particles were divided into two groups in terms of the way they are associated with carbon particles: free single particles, and particles combined with carbon particles. As indicated from the present work, unburned

  16. The dust covering factor in active galactic nuclei

    NASA Astrophysics Data System (ADS)

    Stalevski, Marko; Ricci, Claudio; Ueda, Yoshihiro; Lira, Paulina; Fritz, Jacopo; Baes, Maarten

    2016-05-01

    The primary source of emission of active galactic nuclei (AGNs), the accretion disc, is surrounded by an optically and geometrically thick dusty structure (`the so-called dusty torus'). The infrared radiation emitted by the dust is nothing but a reprocessed fraction of the accretion disc emission, so the ratio of the torus to the AGN luminosity (Ltorus/LAGN) should corresponds to the fraction of the sky obscured by dust, i.e. the covering factor. We undertook a critical investigation of the Ltorus/LAGN as the dust covering factor proxy. Using state-of-the-art 3D Monte Carlo radiative transfer code, we calculated a grid of spectral energy distributions (SEDs) emitted by the clumpy two-phase dusty structure. With this grid of SEDs, we studied the relation between Ltorus/LAGN and the dust covering factor for different parameters of the torus. We found that in the case of type 1 AGNs the torus anisotropy makes Ltorus/LAGN underestimate low covering factors and overestimate high covering factors. In type 2 AGNs Ltorus/LAGN always underestimates covering factors. Our results provide a novel easy-to-use method to account for anisotropy and obtain correct covering factors. Using two samples from the literature, we demonstrated the importance of our result for inferring the obscured AGN fraction. We found that after the anisotropy is properly accounted for, the dust covering factors show very weak dependence on LAGN, with values in the range of ≈0.6-0.7. Our results also suggest a higher fraction of obscured AGNs at high luminosities than those found by X-ray surveys, in part owing to the presence of a Compton-thick AGN population predicted by population synthesis models.

  17. Immunoregulatory activity of peptides related to platelet factor 4.

    PubMed Central

    Zucker, M B; Katz, I R; Thorbecke, G J; Milot, D C; Holt, J

    1989-01-01

    Platelet factor 4 (PF4), a secreted platelet protein, alleviates concanavalin A-induced immunosuppression in mice. We now find that activity also resides in (i) the C-terminal tridecapeptide of PF4 (P13S), (ii) an analog of this in which arginine replaces the lysine residues and in which the last two amino acids are absent, (iii) the C-terminal 18 amino acids of low-affinity platelet factor 4, which is very similar to P13S, and (iv) peptide fragments of P13S that contain only 5-9 amino acids. P13S treated with fluorescamine to derivatize the free amino groups retained immunoregulatory activity but did not bind to heparin-agarose. The N-terminal and middle portions of PF4, polylysine, protamine, and three unrelated peptides were inactive in this assay. PMID:2678107

  18. Reliable and Affordable Control Systems Active Combustor Pattern Factor Control

    NASA Technical Reports Server (NTRS)

    McCarty, Bob; Tomondi, Chris; McGinley, Ray

    2004-01-01

    Active, closed-loop control of combustor pattern factor is a cooperative effort between Honeywell (formerly AlliedSignal) Engines and Systems and the NASA Glenn Research Center to reduce emissions and turbine-stator vane temperature variations, thereby enhancing engine performance and life, and reducing direct operating costs. Total fuel flow supplied to the engine is established by the speed/power control, but the distribution to individual atomizers will be controlled by the Active Combustor Pattern Factor Control (ACPFC). This system consist of three major components: multiple, thin-film sensors located on the turbine-stator vanes; fuel-flow modulators for individual atomizers; and control logic and algorithms within the electronic control.

  19. Stellar Activity and CMEs: Important Factors of Planetary Evolution

    NASA Astrophysics Data System (ADS)

    Khodachenko, Maxim L.

    CME activity of the Sun is known to be an important impacting factor for the magnetospheres, atmospheres, and surfaces of solar system planets. Following an idea of a solar-stellar analogy, CME phenomena are expected on other stars as well. The main planetary impact factors of the stellar CMEs include the associated interplanetary shocks, plasma density and velocity disturbances, energetic particles accelerated in the shock regions, as well as distortions of the magnetic field direction and modulus. All these factors should be properly taken into account during the study of evolutionary processes on exoplanets and their atmospheric and plasma environments. The planetary impact of the stellar CME activity may vary depending on stellar age, stellar spectral type and the orbital distance of a planet. Because of the relatively short range of propagation of the majority of CMEs, they affect most strongly the magnetospheres and atmospheres of close-orbit ( < 0.1 AU) exoplanets. In this chapter we discuss an issue of the stellar CME activity in the context of several actual problems of modern exoplanetology, including planetary atmosphere mass loss, planet survival at close orbits, and definition of a criterion for habitability.

  20. The Potential of Liposomes with Carbonic Anhydrase IX to Deliver Anticancer Ingredients to Cancer Cells in Vivo

    PubMed Central

    Ng, Huei Leng Helena; Lu, Aiping; Lin, Ge; Qin, Ling; Yang, Zhijun

    2014-01-01

    Drug delivery nanocarriers, especially targeted drug delivery by liposomes are emerging as a class of therapeutics for cancer. Early research results suggest that liposomal therapeutics enhanced efficacy, while simultaneously reducing side effects, owing to properties such as more targeted localization in tumors and active cellular uptake. Here, we highlight the features of immunoliposomes that distinguish them from previous anticancer therapies, and describe how these features provide the potential for therapeutic effects that are not achievable with other modalities. While a large number of studies has been published, the emphasis here is placed on the carbonic anhydrase IX (CA-IX) and the conjugated liposomes that are likely to open a new chapter on drug delivery system by using immunoliposomes to deliver anticancer ingredients to cancer cells in vivo. PMID:25547490

  1. Andrographolide stimulates p38 mitogen-activated protein kinase-nuclear factor erythroid-2-related factor 2-heme oxygenase 1 signaling in primary cerebral endothelial cells for definite protection against ischemic stroke in rats.

    PubMed

    Yen, Ting-Lin; Chen, Ray-Jade; Jayakumar, Thanasekaran; Lu, Wan-Jung; Hsieh, Cheng-Ying; Hsu, Ming-Jen; Yang, Chih-Hao; Chang, Chao-Chien; Lin, Yen-Kuang; Lin, Kuan-Hung; Sheu, Joen-Rong

    2016-04-01

    Stroke pathogenesis involves complex oxidative stress-related pathways. The nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) pathways have been considered molecular targets in pharmacologic intervention for ischemic diseases. Andrographolide, a labdane diterpene, has received increasing attention in recent years because of its various pharmacologic activities. We determined that andrographolide modulates the mitogen-activated protein kinase (MAPK)-Nrf2-HO-1 signaling cascade in primary cerebral endothelial cells (CECs) to provide positive protection against middle cerebral artery occlusion (MCAO)-induced ischemic stroke in rats. In the present study, andrographolide (10 μM) increased HO-1 protein and messenger RNA expressions, Nrf2 phosphorylation, and nuclear translocation in CECs, and these activities were disrupted by a p38 MAPK inhibitor, SB203580, but not by the extracellular signal-regulated kinase inhibitor PD98059 or c-Jun amino-terminal kinase inhibitor SP600125. Similar results were observed in confocal microscopy analysis. Moreover, andrographolide-induced Nrf2 and HO-1 protein expressions were significantly inhibited by Nrf2 small interfering RNA. Moreover, HO-1 knockdown attenuated the protective effect of andrographolide against oxygen-glucose deprivation-induced CEC death. Andrographolide (0.1 mg/kg) significantly suppressed free radical formation, blood-brain barrier disruption, and brain infarction in MCAO-insulted rats, and these effects were reversed by the HO-1 inhibitor zinc protoporphyrin IX. The mechanism is attributable to HO-1 activation, as directly evidenced by andrographolide-induced pronounced HO-1 expression in brain tissues, which was highly localized in the cerebral capillary. In conclusion, andrographolide increased Nrf2-HO-1 expression through p38 MAPK regulation, confirming that it provides protection against MCAO-induced brain injury. These findings provide strong evidence that andrographolide could

  2. A NEW CALCULATION OF Ne IX LINE DIAGNOSTICS

    SciTech Connect

    Smith, Randall K.; Chen Guoxin; Kirby, Kate; Brickhouse, Nancy S.

    2009-07-20

    We describe the effect that new atomic calculations, including fully relativistic R-matrix calculations of collisional excitation rates and level-specific dielectronic and radiative recombination rates, have on line ratios from the astrophysically significant ion Ne IX. The new excitation rates systematically change some predicted Ne IX line ratios by 25% at temperatures at or below the temperature of maximum emissivity (4 x 10{sup 6} K), while the new recombination rates lead to systematic changes at higher temperatures. The new line ratios are shown to agree with observations of Capella and {sigma}{sup 2} CrB significantly better than older line ratios, showing that 25%-30% accuracy in atomic rates is inadequate for high-resolution X-ray observations from existing spectrometers.

  3. Ares I-X Thermal Model Correlation and Lessons Learned

    NASA Technical Reports Server (NTRS)

    Amundsen, Ruth M.

    2010-01-01

    The Ares I-X vehicle launched and flew successfully on October 28, 2009. This paper will describe the correlation of the vehicle thermal model to both ground testing and flight data. A main purpose of the vehicle model and ground testing was to ensure that the avionics within the vehicle were held within their thermal limits prior to launch and during flight. The correlation of the avionics box temperatures will be shown. Also, the lessons learned in the thermal discipline during the modeling, test, correlation to test, and flight of the Ares I-X flight test vehicle will be described. Lessons learned will cover thermal modeling, as well as management of the thermal discipline, thermal team, and thermal-related actions in design, testing, and flight.

  4. Ares I-X Best Estimated Trajectory Analysis and Results

    NASA Technical Reports Server (NTRS)

    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.

  5. Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease

    PubMed Central

    Naranjo, José R.; Zhang, Hongyu; Villar, Diego; González, Paz; Dopazo, Xose M.; Morón-Oset, Javier; Higueras, Elena; Oliveros, Juan C.; Arrabal, María D.; Prieto, Angela; Cercós, Pilar; González, Teresa; De la Cruz, Alicia; Casado-Vela, Juan; Rábano, Alberto; Valenzuela, Carmen; Gutierrez-Rodriguez, Marta; Li, Jia-Yi; Mellström, Britt

    2016-01-01

    Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD. PMID:26752648

  6. Parental Factors in Children’s Active Transport to School

    PubMed Central

    Henne, Heather M.; Tandon, Pooja S.; Frank, Larry D.; Saelens, Brian E.

    2014-01-01

    Objective Identify non-distance factors related to children’s active transport (AT) to school, including parental, home, and environment characteristics. Understanding the factors related to children’s AT to school, beyond distance to school, could inform interventions to increase AT and children’s overall physical activity. Study Design Participants were in the Neighborhood Impact on Kids Study, a longitudinal, observational cohort study of children aged 6 - 11 and their parents in King County, WA and San Diego County, CA between 2007-2009. Parents reported frequency and mode of child transport to school, perceived neighborhood, home and family environments, parental travel behaviors, and sociodemographics. Methods Children living less than a 20 minute walk to school were in this analysis. Children classified as active transporters (walked/bicycled to or from school at least once per week) were compared with those not using AT as often. Results Children using AT were older and had parents who reported themselves using active transport. Having a family rule that restricts the child to stay within sight of the parent or home and more parent working hours was related to lower odds of a child using AT. Conclusions Children’s AT to school is associated with parental AT to work and other locations. Interventions should be considered that enable whole family AT, ameliorate safety concerns and decrease the need for parental supervision, such as walking school buses. PMID:24999161

  7. Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease.

    PubMed

    Naranjo, José R; Zhang, Hongyu; Villar, Diego; González, Paz; Dopazo, Xose M; Morón-Oset, Javier; Higueras, Elena; Oliveros, Juan C; Arrabal, María D; Prieto, Angela; Cercós, Pilar; González, Teresa; De la Cruz, Alicia; Casado-Vela, Juan; Rábano, Alberto; Valenzuela, Carmen; Gutierrez-Rodriguez, Marta; Li, Jia-Yi; Mellström, Britt

    2016-02-01

    Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD. PMID:26752648

  8. Transcriptional activation in yeast cells lacking transcription factor IIA.

    PubMed Central

    Chou, S; Chatterjee, S; Lee, M; Struhl, K

    1999-01-01

    The general transcription factor IIA (TFIIA) forms a complex with TFIID at the TATA promoter element, and it inhibits the function of several negative regulators of the TATA-binding protein (TBP) subunit of TFIID. Biochemical experiments suggest that TFIIA is important in the response to transcriptional activators because activation domains can interact with TFIIA, increase recruitment of TFIID and TFIIA to the promoter, and promote isomerization of the TFIID-TFIIA-TATA complex. Here, we describe a double-shut-off approach to deplete yeast cells of Toa1, the large subunit of TFIIA, to <1% of the wild-type level. Interestingly, such TFIIA-depleted cells are essentially unaffected for activation by heat shock factor, Ace1, and Gal4-VP16. However, depletion of TFIIA causes a general two- to threefold decrease of transcription from most yeast promoters and a specific cell-cycle arrest at the G2-M boundary. These results indicate that transcriptional activation in vivo can occur in the absence of TFIIA. PMID:10581267

  9. Equal Educational Opportunity and Nondiscrimination for Girls in Advanced Mathematics, Science, and Technology Education: Federal Enforcement of Title IX. Equal Educational Opportunity Project Series, Volume V.

    ERIC Educational Resources Information Center

    Aneckstein, Laura; Baird, Andrea; Butler, Margaret; Chambers, David; Johnson, Wanda; Kraus, Rebecca; Mann, Eric; Trost, Tami; Zalokar, Nadja; Zieseniss, Mireille

    This report focuses on the Office for Civil Rights' (OCR's) activities relating to Title IX and advanced mathematics, science, and technology education for girls. It examines some of the barriers and inequities that undermine girls' opportunities to choose college majors and enter careers in the advanced mathematics, science, and technology…

  10. Overexpression of Plastidic Protoporphyrinogen IX Oxidase Leads to Resistance to the Diphenyl-Ether Herbicide Acifluorfen1

    PubMed Central

    Lermontova, Inna; Grimm, Bernhard

    2000-01-01

    The use of herbicides to control undesirable vegetation has become a universal practice. For the broad application of herbicides the risk of damage to crop plants has to be limited. We introduced a gene into the genome of tobacco (Nicotiana tabacum) plants encoding the plastid-located protoporphyrinogen oxidase of Arabidopsis, the last enzyme of the common tetrapyrrole biosynthetic pathway, under the control of the cauliflower mosaic virus 35S promoter. The transformants were screened for low protoporphyrin IX accumulation upon treatment with the diphenyl ether-type herbicide acifluorfen. Leaf disc incubation and foliar spraying with acifluorfen indicated the lower susceptibility of the transformants against the herbicide. The resistance to acifluorfen is conferred by overexpression of the plastidic isoform of protoporphyrinogen oxidase. The in vitro activity of this enzyme extracted from plastids of selected transgenic lines was at least five times higher than the control activity. Herbicide treatment that is normally inhibitory to protoporphyrinogen IX oxidase did not significantly impair the catalytic reaction in transgenic plants and, therefore, did not cause photodynamic damage in leaves. Therefore, overproduction of protoporphyrinogen oxidase neutralizes the herbicidal action, prevents the accumulation of the substrate protoporphyrinogen IX, and consequently abolishes the light-dependent phytotoxicity of acifluorfen. PMID:10631251

  11. Efficient Expression and Crystallization System of Cancer-Associated Carbonic Anhydrase Isoform IX.

    PubMed

    Leitans, Janis; Kazaks, Andris; Balode, Agnese; Ivanova, Jekaterina; Zalubovskis, Raivis; Supuran, Claudiu T; Tars, Kaspars

    2015-11-25

    Human carbonic anhydrase IX (CA IX) is overexpressed in a number of solid tumors and is considered to be a marker for cellular hypoxia that it is not produced in most normal tissues. CA IX contributes to the acidification of the extracellular matrix, which, in turn, favors tumor growth and metastasis. Therefore, CA IX is considered to be a promising anti-cancer drug target. However, the ability to specifically target CA IX is challenging due to the fact that the human genome encodes 15 different carbonic anhydrase isoforms that have a high degree of homology. Furthermore, structure-based drug design of CA IX inhibitors so far has been largely unsuccessful due to technical difficulties regarding the expression and crystallization of the enzyme. Currently, only one baculovirus-produced CA IX structure in complex with a nonspecific CA inhibitor, acetazolamide, is available in Protein Data Bank. We have developed an efficient system for the production of the catalytic domain of CA IX in methylotrophic yeast Pichia pastoris. The produced protein can be easily crystallized in the presence of inhibitors, as we have demonstrated for several 2-thiophene-sulfonamide compounds. We have also observed significant differences in the binding mode of chemically identical compounds to CA IX and CA II, which can be further exploited in the design of CA IX-specific inhibitors. PMID:26522624

  12. Macrophage Migration Inhibitory Factor (MIF) Enzymatic Activity and Lung Cancer

    PubMed Central

    Mawhinney, Leona; Armstrong, Michelle E; O’ Reilly, Ciaran; Bucala, Richard; Leng, Lin; Fingerle-Rowson, Gunter; Fayne, Darren; Keane, Michael P; Tynan, Aisling; Maher, Lewena; Cooke, Gordon; Lloyd, David; Conroy, Helen; Donnelly, Seamas C

    2014-01-01

    The cytokine macrophage migration inhibitory factor (MIF) possesses unique tautomerase enzymatic activity, which contributes to the biological functional activity of MIF. In this study, we investigated the effects of blocking the hydrophobic active site of the tautomerase activity of MIF in the pathogenesis of lung cancer. To address this, we initially established a Lewis lung carcinoma (LLC) murine model in Mif-KO and wild-type (WT) mice and compared tumor growth in a knock-in mouse model expressing a mutant MIF lacking enzymatic activity (Mif P1G). Primary tumor growth was significantly attenuated in both Mif-KO and Mif P1G mice compared with WT mice. We subsequently undertook a structure-based, virtual screen to identify putative small molecular weight inhibitors specific for the tautomerase enzymatic active site of MIF. From primary and secondary screens, the inhibitor SCD-19 was identified, which significantly attenuated the tautomerase enzymatic activity of MIF in vitro and in biological functional screens. In the LLC murine model, SCD-19, given intraperitoneally at the time of tumor inoculation, was found to significantly reduce primary tumor volume by 90% (p < 0.001) compared with the control treatment. To better replicate the human disease scenario, SCD-19 was given when the tumor was palpable (at d 7 after tumor inoculation) and, again, treatment was found to significantly reduce tumor volume by 81% (p < 0.001) compared with the control treatment. In this report, we identify a novel inhibitor that blocks the hydrophobic pocket of MIF, which houses its specific tautomerase enzymatic activity, and demonstrate that targeting this unique active site significantly attenuates lung cancer growth in in vitro and in vivo systems. PMID:25826675

  13. Loop quantum cosmology of Bianchi type IX models

    SciTech Connect

    Wilson-Ewing, Edward

    2010-08-15

    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.

  14. Time-activity relationships to VOC personal exposure factors

    NASA Astrophysics Data System (ADS)

    Edwards, Rufus D.; Schweizer, Christian; Llacqua, Vito; Lai, Hak Kan; Jantunen, Matti; Bayer-Oglesby, Lucy; Künzli, Nino

    Social and demographic factors have been found to play a significant role in differences between time-activity patterns of population subgroups. Since time-activity patterns largely influence personal exposure to compounds as individuals move across microenvironments, exposure subgroups within the population may be defined by factors that influence daily activity patterns. Socio-demographic and environmental factors that define time-activity subgroups also define quantifiable differences in VOC personal exposures to different sources and individual compounds in the Expolis study. Significant differences in exposures to traffic-related compounds ethylbenzene, m- and p-xylene and o-xylene were observed in relation to gender, number of children and living alone. Categorization of exposures further indicated time exposed to traffic at work and time in a car as important determinants. Increased exposures to decane, nonane and undecane were observed for males, housewives and self-employed. Categorization of exposures indicated exposure subgroups related to workshop use and living downtown. Higher exposures to 3-carene and α-pinene commonly found in household cleaning products and fragrances were associated with more children, while exposures to traffic compounds ethylbenzene, m- and p-xylene and o-xylene were reduced with more children. Considerable unexplained variation remained in categorization of exposures associated with home product use and fragrances, due to individual behavior and product choice. More targeted data collection methods in VOC exposure studies for these sources should be used. Living alone was associated with decreased exposures to 2-methyl-1-propanol and 1-butanol, and traffic-related compounds. Identification of these subgroups may help to reduce the large amount of unexplained variation in VOC exposure studies. Further they may help in assessing impacts of urban planning that result in changes in behavior of individuals, resulting in shifts in

  15. Atomic data and spectral line intensities for S IX

    NASA Astrophysics Data System (ADS)

    Bhatia, A. K.; Landi, E.

    2003-09-01

    Electron impact collision strengths, energy levels, oscillator strengths, and spontaneous radiative decay rates are calculated for the O-like ion S IX. The configurations used are 2s 22p 4, 2s2p 5, 2p 6, 2s 22p 33s, 2s 22p 33p, and 2s 22p 33d giving rise to 86 fine-structure levels in intermediate coupling. Collision strengths are calculated at five incident energies, 25, 50, 75, 100, and 125 Ry. Excitation rate coefficients are calculated by assuming a Maxwellian electron velocity distribution at several electron temperatures in the 5.6 ⩽log Te (K)⩽6.2 range, where S IX is formed. Using the excitation rate coefficients and the radiative transition rates, statistical equilibrium equations for level populations are solved at electron densities covering the range of 10 8-10 14 cm -3. Relative spectral line intensities are calculated. Proton excitation rates among the lowest three levels have been included in the statistical equilibrium equations. The predicted S IX line intensities are compared with SUMER (SOHO) observations of the quiet Sun.

  16. Bladder tissue diagnostics utilizing Protoporphyrin IX fluorescence detection

    NASA Astrophysics Data System (ADS)

    Stepp, Herbert G.; Baumgartner, Reinhold; Beyer, Wolfgang; Knuechel, Ruth; Rick, Kai; Steinbach, Pia; Kriegmair, M.

    1995-01-01

    Instillation of a solution of 5-aminolevulinic acid (5-ALA) into the urinary bladder leads to a tumorselective accumulation of fluorescing Protoporphyrin IX (PpIX) within hours. Upon fluorescence excitation using a Kr+- laser, cystoscopy provides high contrast images even of early stage tumors, that are invisible or hardly detectable by routine white light cystoscopy. Fluorescence can simply be judged by naked eyes or recorded with a target integrating camera in real color. Histological and fluorescence data of 91 patients were evaluated statistically, showing a sensitivity of 97% and a specificity of 68% for the detection of dysplastic lesions or malignant tumors. The detectability of a sufficient fluorescence contrast of suspicious versus normal tissue is not affected significantly by either short incubation times of less than 1 hour or prolonged retention times without 5-ALA in the instillation liquid of up to about 6 hours. The fluorescence intensity detected from the tissue surface is not only dependent on PpIX concentration. The additional influence of optical parameters of tissue and fluorochrome distribution on the fluorescence signal was determined using Monte Carlo computer simulations. Results show that 5-ALA induced fluorochrome detection is superior to the detection of fluorochromes that do not exclusively stain the epithelium. Using the ratio of fluorescence intensity to backscattered excitation light corrects for geometrical and absorption effects but would introduce a dependence on the scattering coefficient.

  17. ALA-PpIX variability quantitatively imaged in A431 epidermoid tumors using in vivo ultrasound fluorescence tomography and ex vivo assay

    NASA Astrophysics Data System (ADS)

    DSouza, Alisha V.; Flynn, Brendan P.; Gunn, Jason R.; Samkoe, Kimberley S.; Anand, Sanjay; Maytin, Edward V.; Hasan, Tayyaba; Pogue, Brian W.

    2014-03-01

    Treatment monitoring of Aminolevunilic-acid (ALA) - Photodynamic Therapy (PDT) of basal-cell carcinoma (BCC) calls for superficial and subsurface imaging techniques. While superficial imagers exist for this purpose, their ability to assess PpIX levels in thick lesions is poor; additionally few treatment centers have the capability to measure ALA-induced PpIX production. An area of active research is to improve treatments to deeper and nodular BCCs, because treatment is least effective in these. The goal of this work was to understand the logistics and technical capabilities to quantify PpIX at depths over 1mm, using a novel hybrid ultrasound-guided, fiber-based fluorescence molecular spectroscopictomography system. This system utilizes a 633nm excitation laser and detection using filtered spectrometers. Source and detection fibers are collinear so that their imaging plane matches that of ultrasound transducer. Validation with phantoms and tumor-simulating fluorescent inclusions in mice showed sensitivity to fluorophore concentrations as low as 0.025μg/ml at 4mm depth from surface, as presented in previous years. Image-guided quantification of ALA-induced PpIX production was completed in subcutaneous xenograft epidermoid cancer tumor model A431 in nude mice. A total of 32 animals were imaged in-vivo, using several time points, including pre-ALA, 4-hours post-ALA, and 24-hours post-ALA administration. On average, PpIX production in tumors increased by over 10-fold, 4-hours post-ALA. Statistical analysis of PpIX fluorescence showed significant difference among all groups; p<0.05. Results were validated by exvivo imaging of resected tumors. Details of imaging, analysis and results will be presented to illustrate variability and the potential for imaging these values at depth.

  18. Transcription Factor Arabidopsis Activating Factor1 Integrates Carbon Starvation Responses with Trehalose Metabolism1[OPEN

    PubMed Central

    Garapati, Prashanth; Feil, Regina; Lunn, John Edward; Van Dijck, Patrick; Balazadeh, Salma; Mueller-Roeber, Bernd

    2015-01-01

    Plants respond to low carbon supply by massive reprogramming of the transcriptome and metabolome. We show here that the carbon starvation-induced NAC (for NO APICAL MERISTEM/ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR/CUP-SHAPED COTYLEDON) transcription factor Arabidopsis (Arabidopsis thaliana) Transcription Activation Factor1 (ATAF1) plays an important role in this physiological process. We identified TREHALASE1, the only trehalase-encoding gene in Arabidopsis, as a direct downstream target of ATAF1. Overexpression of ATAF1 activates TREHALASE1 expression and leads to reduced trehalose-6-phosphate levels and a sugar starvation metabolome. In accordance with changes in expression of starch biosynthesis- and breakdown-related genes, starch levels are generally reduced in ATAF1 overexpressors but elevated in ataf1 knockout plants. At the global transcriptome level, genes affected by ATAF1 are broadly associated with energy and carbon starvation responses. Furthermore, transcriptional responses triggered by ATAF1 largely overlap with expression patterns observed in plants starved for carbon or energy supply. Collectively, our data highlight the existence of a positively acting feedforward loop between ATAF1 expression, which is induced by carbon starvation, and the depletion of cellular carbon/energy pools that is triggered by the transcriptional regulation of downstream gene regulatory networks by ATAF1. PMID:26149570

  19. Transcription Factor Arabidopsis Activating Factor1 Integrates Carbon Starvation Responses with Trehalose Metabolism.

    PubMed

    Garapati, Prashanth; Feil, Regina; Lunn, John Edward; Van Dijck, Patrick; Balazadeh, Salma; Mueller-Roeber, Bernd

    2015-09-01

    Plants respond to low carbon supply by massive reprogramming of the transcriptome and metabolome. We show here that the carbon starvation-induced NAC (for NO APICAL MERISTEM/ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR/CUP-SHAPED COTYLEDON) transcription factor Arabidopsis (Arabidopsis thaliana) Transcription Activation Factor1 (ATAF1) plays an important role in this physiological process. We identified TREHALASE1, the only trehalase-encoding gene in Arabidopsis, as a direct downstream target of ATAF1. Overexpression of ATAF1 activates TREHALASE1 expression and leads to reduced trehalose-6-phosphate levels and a sugar starvation metabolome. In accordance with changes in expression of starch biosynthesis- and breakdown-related genes, starch levels are generally reduced in ATAF1 overexpressors but elevated in ataf1 knockout plants. At the global transcriptome level, genes affected by ATAF1 are broadly associated with energy and carbon starvation responses. Furthermore, transcriptional responses triggered by ATAF1 largely overlap with expression patterns observed in plants starved for carbon or energy supply. Collectively, our data highlight the existence of a positively acting feedforward loop between ATAF1 expression, which is induced by carbon starvation, and the depletion of cellular carbon/energy pools that is triggered by the transcriptional regulation of downstream gene regulatory networks by ATAF1. PMID:26149570

  20. Overexpression of Intrinsic Hypoxia Markers HIF1{alpha} and CA-IX Predict for Local Recurrence in Stage T1-T2 Glottic Laryngeal Carcinoma Treated With Radiotherapy

    SciTech Connect

    Schrijvers, M.L.; Laan, B.F.A.M. van der; Bock, G.H. de; Pattje, W.J.; Mastik, M.F.; Menkema, L.; Langendijk, J.A.; Kluin, P.M.; Schuuring, E.; Wal, J.E. van der

    2008-09-01

    Purpose: To examine the prognostic value of three endogenous hypoxia markers (hypoxia inducible factor 1 {alpha} subunit [HIF1{alpha}], carbonic anhydrase IX [CA-IX], and glucose transporter type 1 [GLUT-1]) on the clinical outcome in patients with early-stage glottic carcinoma primarily treated with radiotherapy (RT) and to determine the predictive hypoxic profile to choose the optimal treatment of early-stage laryngeal carcinoma. Methods and Materials: Immunohistochemistry for HIF1{alpha}, CA-IX, and GLUT-1 was performed on formalin-fixed, paraffin-embedded, pretreatment tissue samples of 91 glottic squamous cell carcinoma specimens. The patient group consisted only of those with early-stage (T1-T2) glottic carcinoma, and all patients were treated with RT only. Relative tumor staining was scored on the tissue samples. Receiver operating curve analysis was performed to determine the optimal cutoff value for each tumor marker. Cox regression analyses for the variables HIF1{alpha}, CA-IX, GLUT-1, gender, age, hemoglobin level, T category, N category, tobacco use, and alcohol use were performed with local control and overall survival as endpoints. Results: HIF1{alpha} overexpression in early-stage glottic carcinoma correlated significantly with worse local control (hazard ratio [HR], 3.05; p = 0.021) and overall survival (HR, 2.92; p = 0.016). CA-IX overexpression correlated significantly with worse local control (HR, 2.93; p = 0.020). GLUT-1 overexpression did not show any correlation with the clinical outcome parameters. Tumors with a nonhypoxic profile (defined as low HIF1{alpha} and low CA-IX expression) had significantly better local control (HR, 6.32; p 0.013). Conclusion: The results of our study have shown that early-stage glottic laryngeal carcinomas with low HIF1{alpha} and CA-IX expression are highly curable with RT. For this group, RT is a good treatment option. For tumors with HIF1{alpha} or CA-IX overexpression, hypoxic modification before RT or primary

  1. GUN4-Protoporphyrin IX Is a Singlet Oxygen Generator with Consequences for Plastid Retrograde Signaling.

    PubMed

    Tarahi Tabrizi, Shabnam; Sawicki, Artur; Zhou, Shuaixiang; Luo, Meizhong; Willows, Robert D

    2016-04-22

    The genomes uncoupled 4 (GUN4) protein is a nuclear-encoded, chloroplast-localized, porphyrin-binding protein implicated in retrograde signaling between the chloroplast and nucleus, although its exact role in this process is still unclear. Functionally, it enhances Mg-chelatase activity in the chlorophyll biosynthesis pathway. Because GUN4 is present only in organisms that carry out oxygenic photosynthesis and because it binds protoporphyrin IX (PPIX) and Mg-PPIX, it has been suggested that it prevents production of light- and PPIX- or Mg-PPIX-dependent reactive oxygen species. A chld-1/GUN4 mutant with elevated PPIX has a light-dependent up-regulation of GUN4, implicating this protein in light-dependent sensing of PPIX, with the suggestion that GUN4 reduces PPIX-generated singlet oxygen, O2(a(1)Δg), and subsequent oxidative damage (Brzezowski, P., Schlicke, H., Richter, A., Dent, R. M., Niyogi, K. K., and Grimm, B. (2014) Plant J. 79, 285-298). In direct contrast, our results show that purified GUN4 and oxidatively damaged ChlH increase the rate of PPIX-generated singlet oxygen production in the light, by a factor of 5 and 10, respectively, when compared with PPIX alone. Additionally, the functional GUN4-PPIX-ChlH complex and ChlH-PPIX complexes generate O2(a(1)Δg) at a reduced rate when compared with GUN4-PPIX. As O2(a(1)Δg) is a potential plastid-to-nucleus signal, possibly through second messengers, light-dependent O2(a(1)Δg) generation by GUN4-PPIX is proposed to be part of a signal transduction pathway from the chloroplast to the nucleus. GUN4 thus senses the availability and flux of PPIX through the chlorophyll biosynthetic pathway and also modulates Mg-chelatase activity. The light-dependent O2(a(1)Δg) generation from GUN4-PPIX is thus proposed as the first step in retrograde signaling from the chloroplast to the nucleus. PMID:26969164

  2. Factors associated with active commuting to work among women.

    PubMed

    Bopp, Melissa; Child, Stephanie; Campbell, Matthew

    2014-01-01

    Active commuting (AC), the act of walking or biking to work, has notable health benefits though rates of AC remain low among women. This study used a social-ecological framework to examine the factors associated with AC among women. A convenience sample of employed, working women (n = 709) completed an online survey about their mode of travel to work. Individual, interpersonal, institutional, community, and environmental influences were assessed. Basic descriptive statistics and frequencies described the sample. Simple logistic regression models examined associations with the independent variables with AC participation and multiple logistic regression analysis determined the relative influence of social ecological factors on AC participation. The sample was primarily middle-aged (44.09±11.38 years) and non-Hispanic White (92%). Univariate analyses revealed several individual, interpersonal, institutional, community and environmental factors significantly associated with AC. The multivariable logistic regression analysis results indicated that significant factors associated with AC included number of children, income, perceived behavioral control, coworker AC, coworker AC normative beliefs, employer and community supports for AC, and traffic. The results of this study contribute to the limited body of knowledge on AC participation for women and may help to inform gender-tailored interventions to enhance AC behavior and improve health. PMID:24512572

  3. The Education Innovator. Volume IX, No. 3

    ERIC Educational Resources Information Center

    Herbert, Doug, Ed.

    2010-01-01

    The purpose of the U.S. Department of Education's online newsletter "The Education Innovator" is to promote innovative practices in education; to offer features on promising programs and practices; to provide information on innovative research, schools, policies, and trends; and to keep readers informed of key Department priorities and activities.…

  4. The Education Innovator. Volume IX, No. 4

    ERIC Educational Resources Information Center

    Herbert, Doug, Ed.

    2010-01-01

    The purpose of the U.S. Department of Education's online newsletter "The Education Innovator" is to promote innovative practices in education; to offer features on promising programs and practices; to provide information on innovative research, schools, policies, and trends; and to keep readers informed of key Department priorities and activities.…

  5. The Education Innovator. Volume IX, No. 2

    ERIC Educational Resources Information Center

    Herbert, Doug, Ed.

    2010-01-01

    The purpose of the U.S. Department of Education's online newsletter "The Education Innovator" is to promote innovative practices in education; to offer features on promising programs and practices; to provide information on innovative research, schools, policies, and trends; and to keep readers informed of key Department priorities and activities.…

  6. Secretion of platelet-activating factor by periovulatory ovine follicles

    SciTech Connect

    Alexander, B.M.; Van Kirk, E.A.; Murdoch, W.J. )

    1990-01-01

    Secretion of platelet-activating factor (PAF) in vitro by ovine follicles and ovarian interstitium obtained at various times before, during and after the endogenous preovulatory surge of luteinizing hormone (LH) and ovulation was quantified by radioimmunoassay. Release of PAF by the preovulatory follicle increased within 2 h after initiation of the surge of LH. Capacity for secretion of PAF was greatest at the time of ovulation, then declined thereafter. Production of PAF by ovarian interstitium throughout the periovulatory period was relatively low and did not change with time. It appears that PAF could act as an intrafollicular mediator in the mechanisms of ovulation and(or) luteinization.

  7. Platelet-Activating Factor-Receptor and Tumor Immunity

    PubMed Central

    Sahu, Ravi P; Konger, Raymond L.; Travers, Jeffrey B.

    2016-01-01

    First described in 1972 by Benveniste and colleagues, platelet-activating factor (PAF) remains one of the potent phospholipid known to date. The role of PAF produced enzymatically in mediating diverse biological and pathophysiological processes including inflammatory and allergic diseases and cancers in response to various stimuli has been extensively studied. However, little is known about the role of non-enzymatically-generated PAF-like lipids produced in response to pro-oxidative stressors, particularly in modulating the host immune responses to tumor immunity, which is the focus of this review.

  8. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1.

    PubMed Central

    Forsythe, J A; Jiang, B H; Iyer, N V; Agani, F; Leung, S W; Koos, R D; Semenza, G L

    1996-01-01

    Expression of vascular endothelial growth factor (VEGF) is induced in cells exposed to hypoxia or ischemia. Neovascularization stimulated by VEGF occurs in several important clinical contexts, including myocardial ischemia, retinal disease, and tumor growth. Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix protein that activates transcription of the human erythropoietin gene in hypoxic cells. Here we demonstrate the involvement of HIF-1 in the activation of VEGF transcription. VEGF 5'-flanking sequences mediated transcriptional activation of reporter gene expression in hypoxic Hep3B cells. A 47-bp sequence located 985 to 939 bp 5' to the VEGF transcription initiation site mediated hypoxia-inducible reporter gene expression directed by a simian virus 40 promoter element that was otherwise minimally responsive to hypoxia. When reporters containing VEGF sequences, in the context of the native VEGF or heterologous simian virus 40 promoter, were cotransfected with expression vectors encoding HIF-1alpha and HIF-1beta (ARNT [aryl hydrocarbon receptor nuclear translocator]), reporter gene transcription was much greater in both hypoxic and nonhypoxic cells than in cells transfected with the reporter alone. A HIF-1 binding site was demonstrated in the 47-bp hypoxia response element, and a 3-bp substitution eliminated the ability of the element to bind HIF-1 and to activate transcription in response to hypoxia and/or recombinant HIF-1. Cotransfection of cells with an expression vector encoding a dominant negative form of HIF-1alpha inhibited the activation of reporter transcription in hypoxic cells in a dose-dependent manner. VEGF mRNA was not induced by hypoxia in mutant cells that do not express the HIF-1beta (ARNT) subunit. These findings implicate HIF-1 in the activation of VEGF transcription in hypoxic cells. PMID:8756616

  9. A novel library of saccharin and acesulfame derivatives as potent and selective inhibitors of carbonic anhydrase IX and XII isoforms.

    PubMed

    Carradori, Simone; Secci, Daniela; De Monte, Celeste; Mollica, Adriano; Ceruso, Mariangela; Akdemir, Atilla; Sobolev, Anatoly P; Codispoti, Rossella; De Cosmi, Federica; Guglielmi, Paolo; Supuran, Claudiu T

    2016-03-01

    Small libraries of N-substituted saccharin and N-/O-substituted acesulfame derivatives were synthesized and tested as atypical and selective inhibitors of four different isoforms of human carbonic anhydrase (hCA I, II, IX and XII, EC 4.2.1.1). Most of them inhibited hCA XII in the low nanomolar range, hCA IX with KIs ranging between 19 and 2482nM, whereas they were poorly active against hCA II (KIs >10μM) and hCA I (KIs ranging between 318nM and 50μM). Since hCA I and II are ubiquitous off-target isoforms, whereas the cancer-related isoforms hCA IX and XII were recently validated as drug targets, these results represent an encouraging achievement in the development of new anticancer candidates. Moreover, the lack of a classical zinc binding group in the structure of these inhibitors opens innovative, yet unexplored scenarios for different mechanisms of inhibition that could explain the high inhibitory selectivity. A computational approach has been carried out to further rationalize the biological data and to characterize the binding mode of some of these inhibitors. PMID:26810710

  10. [The SGB IX, book 9 of the German social code, in the development of social security law].

    PubMed

    Welti, F

    2002-08-01

    The SGB IX is the legal basis of rehabilitation in Germany since 2001. It is embedded in the codification of the German social code, the Sozialgesetzbuch (SGB). There it has an intermediate function between the general social law and the special laws of the social insurance and social security institutions. Rehabilitation in Germany is working in a system of several different institutions with different legal roots. In special these are compensation of damages caused by employment accident and war and the avoidance of disability benefits by rehabilitation and activation. Inequality of treatment of disabled people according to the cause of their disability and to their employment status are accepted less; common principles of rehabilitation are being searched. Important reforms of rehabilitation law have taken place in 1974 and 1975. Since then problems have become visible in implementation and in the coordination of the complex system. SGB IX is a new effort for a general rehabilitation law. Open questions remain in handling the relations between rehabilitation and sickness treatment, long term care and prevention. Important are the newly defined terms for participation (Teilhabe) and disability (Behinderung), which build the linkage of SGB IX with constitutional law and with the health sciences. A weak point is still the relation of disability and age. Political attempts for unequal benefits in health insurance endanger the now reached level of rehabilitation law. PMID:12168151

  11. The effect of ALA/PpIX PDT on putative cancer stem cells in tumor side populations

    NASA Astrophysics Data System (ADS)

    Morgan, Janet; Petrucci, Cara M.

    2009-06-01

    Protoporphyrin IX (PpIX) synthesized endogenously from 5-aminolevulinic acid (ALA), is effluxed from cells expressing the ATP-dependent transporter ABCG2. Side population (SP) cells (named for their low red/blue fluorescence distribution in flow cytometry plots with ABCG2 substrates such as Hoechst) are postulated to contain cancer stem cells (CSC). The SP in U87 (human gliblastoma cell line) were more resistant to ALA-PDT than NON-SP cells. Inhibiting ABCG2 activity with the tyrosine kinase inhibitor imatinib mesylate (IM, Gleevec) during incubation with ALA increased PpIX in the SP by preventing its efflux and decreased the SP after subsequent PDT, enhancing phototoxicity. Evasion of SP cells from ALA-PDT could cause tumor recurrence from CSC. Manipulation of ABCG2 levels on the SP with small molecule modulators may be a potential strategy for enhancing PDT by decreasing the amount of substrate photosensitizer extruded from cells and lowering the threshold for phototoxicity.

  12. The Application of Lean Thinking Principles and Kaizen Practices for the Successful Development and Implementation of the Ares I-X Flight Test Rocket and Mission

    NASA Technical Reports Server (NTRS)

    Askins, B. R.; Davis, S. R.; Heitzman, K. S.; Olsen, R. A.

    2011-01-01

    On October 28, 2009 the Ares I-X flight test rocket launched from Kennedy Space Center and flew its suborbital trajectory as designed. The mission was successfully completed as data from the test, and associated development activities were analyzed, transferred to stakeholders, and well documented. Positive lessons learned from Ares I-X were that the application of lean thinking principles and kaizen practices are effective in streamlining development activities. Ares I-X, like other historical rocket development projects, was hampered by technical, cost, and schedule challenges and if not addressed boldly could have resulted in cancellation of the test. The mission management team conducted nine major meetings, referred to as lean events, across its elements to assess plans, procedures, processes, requirements, controls, culture, organization, use of resources, and anything that could be changed to optimize schedule or reduce risk. The preeminent aspect of the lean events was the focus on value added activities and the removal or at least reduction in non-value activities. Trained Lean Six Sigma facilitators assisted the Ares I-X developers in conducting the lean events. They indirectly helped formulate the mission s own unique methodology for assessing schedule. A core team was selected to lead the events and report to the mission manager. Each activity leveraged specialized participants to analyze the subject matter and its related processes and then recommended alternatives and solutions. Stakeholders were the event champions. They empowered and encouraged the team to succeed. The keys to success were thorough preparation, honest dialog, small groups, adherence to the Ares I-X ground rules, and accountability through disciplined reporting and tracking of actions. This lean event formula was game-changing as demonstrated by the success of Ares I-X. It is highly recommended as a management tool to help develop other complex systems efficiently. The key benefits

  13. The Application of Lean Thinking Principles and Kaizen Practices for the Successful Development and Implementation of the Ares I-X Flight Test Rocket and Mission

    NASA Technical Reports Server (NTRS)

    Askins, B. R.; Davis, S. R.; Heitzman, K. S.; Olsen, R. A.

    2011-01-01

    On October 28, 2009 the Ares I-X flight test rocket launched from Kennedy Space Center and flew its suborbital trajectory as designed. The mission was successfully completed as data from the test, and associated development activities were analyzed, transferred to stakeholders, and well documented. A positive lesson learned from Ares I-X was that the application of lean thinking principles and kaizen practices was very effective in streamlining development activities. Ares I-X, like other historical rocket development projects, was hampered by technical, cost, and schedule challenges and if not addressed boldly could have resulted in cancellation of the test. The mission management team conducted nine major meetings, referred to as lean events, across its elements to assess plans, procedures, processes, requirements, controls, culture, organization, use of resources, and anything that could be changed to optimize schedule or reduce risk. The preeminent aspect of the lean events was the focus on value added activities and the removal or at least reduction in non-value added activities. Trained Lean Six Sigma facilitators assisted the Ares I-X developers in conducting the lean events. They indirectly helped formulate the mission s own unique methodology for assessing schedule. A core team was selected to lead the events and report to the mission manager. Each activity leveraged specialized participants to analyze the subject matter and its related processes and then recommended alternatives and solutions. Stakeholders were the event champions. They empowered and encouraged the team to succeed. The keys to success were thorough preparation, honest dialog, small groups, adherence to the Ares I-X ground rules, and accountability through disciplined reporting and tracking of actions. This lean event formula was game-changing as demonstrated by Ares I-X. It is highly recommended as a management tool to help develop other complex systems efficiently. The key benefits for

  14. Nuclear factor Y regulates ancient budgerigar hepadnavirus core promoter activity.

    PubMed

    Shen, Zhongliang; Liu, Yanfeng; Luo, Mengjun; Wang, Wei; Liu, Jing; Liu, Wei; Pan, Shaokun; Xie, Youhua

    2016-09-16

    Endogenous viral elements (EVE) in animal genomes are the fossil records of ancient viruses and provide invaluable information on the origin and evolution of extant viruses. Extant hepadnaviruses include avihepadnaviruses of birds and orthohepadnaviruses of mammals. The core promoter (Cp) of hepadnaviruses is vital for viral gene expression and replication. We previously identified in the budgerigar genome two EVEs that contain the full-length genome of an ancient budgerigar hepadnavirus (eBHBV1 and eBHBV2). Here, we found eBHBV1 Cp and eBHBV2 Cp were active in several human and chicken cell lines. A region from nt -85 to -11 in eBHBV1 Cp was critical for the promoter activity. Bioinformatic analysis revealed a putative binding site of nuclear factor Y (NF-Y), a ubiquitous transcription factor, at nt -64 to -50 in eBHBV1 Cp. The NF-Y core binding site (ATTGG, nt -58 to -54) was essential for eBHBV1 Cp activity. The same results were obtained with eBHBV2 Cp and duck hepatitis B virus Cp. The subunit A of NF-Y (NF-YA) was recruited via the NF-Y core binding site to eBHBV1 Cp and upregulated the promoter activity. Finally, the NF-Y core binding site is conserved in the Cps of all the extant avihepadnaviruses but not of orthohepadnaviruses. Interestingly, a putative and functionally important NF-Y core binding site is located at nt -21 to -17 in the Cp of human hepatitis B virus. In conclusion, our findings have pinpointed an evolutionary conserved and functionally critical NF-Y binding element in the Cps of avihepadnaviruses. PMID:27501758

  15. 46 CFR 57.02-2 - Adoption of section IX of the ASME Code.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 2 2011-10-01 2011-10-01 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... in this part. Table 57.02-1(a)—Limitations and Modifications to the Adoption of section IX of...

  16. Combinatorial influence of environmental parameters on transcription factor activity

    PubMed Central

    Knijnenburg, T.A.; Wessels, L.F.A.; Reinders, M.J.T.

    2008-01-01

    Motivation: Cells receive a wide variety of environmental signals, which are often processed combinatorially to generate specific genetic responses. Changes in transcript levels, as observed across different environmental conditions, can, to a large extent, be attributed to changes in the activity of transcription factors (TFs). However, in unraveling these transcription regulation networks, the actual environmental signals are often not incorporated into the model, simply because they have not been measured. The unquantified heterogeneity of the environmental parameters across microarray experiments frustrates regulatory network inference. Results: We propose an inference algorithm that models the influence of environmental parameters on gene expression. The approach is based on a yeast microarray compendium of chemostat steady-state experiments. Chemostat cultivation enables the accurate control and measurement of many of the key cultivation parameters, such as nutrient concentrations, growth rate and temperature. The observed transcript levels are explained by inferring the activity of TFs in response to combinations of cultivation parameters. The interplay between activated enhancers and repressors that bind a gene promoter determine the possible up- or downregulation of the gene. The model is translated into a linear integer optimization problem. The resulting regulatory network identifies the combinatorial effects of environmental parameters on TF activity and gene expression. Availability: The Matlab code is available from the authors upon request. Contact: t.a.knijnenburg@tudelft.nl Supplementary information: Supplementary data are available at Bioinformatics online. PMID:18586711

  17. Epidermal Platelet-activating Factor Receptor Activation and Ultraviolet B Radiation Result in Synergistic Tumor Necrosis Factor-alpha Production

    PubMed Central

    Wolverton, Jay E.; Al-Hassani, Mohammed; Yao, Yongxue; Zhang, Qiwei; Travers, Jeffrey B.

    2010-01-01

    Ultraviolet B radiation (UVB) is a potent stimulator of epidermal cytokine production which has been implicated in photoaggravated dermatoses. In addition to cytokines such as tumor necrosis factor-α (TNF-α), UVB generates bioactive lipids including platelet-activating factor (PAF). Our previous studies have demonstrated that UVB-mediated production of keratinocyte TNF-α is in part due to PAF. The current studies use a human PAF-receptor (PAF-R) negative epithelial cell line transduced with PAF-Rs and PAF–R-deficient mice to demonstrate that activation of the epidermal PAF-R along with UVB irradiation results in a synergistic production of TNF-α. It should be noted that PAF-R effects are mimicked by the protein kinase C (PKC) agonist phorbol myristic acetate, and are inhibited by pharmacological antagonists of the PKC gamma isoenzyme. These studies suggest that concomitant PAF-R activation and UVB irradiation results in a synergistic production of the cytokine TNF-α which is mediated in part via PKC. These studies provide a novel potential mechanism for photosensitivity responses. PMID:19769579

  18. Thyroid Transcription Factor 1 Reprograms Angiogenic Activities of Secretome

    PubMed Central

    Wood, Lauren W.; Cox, Nicole I.; Phelps, Cody A.; Lai, Shao-Chiang; Poddar, Arjun; Talbot, Conover; Mu, David

    2016-01-01

    Through both gain- and loss-of-TTF-1 expression strategies, we show that TTF-1 positively regulates vascular endothelial growth factor (VEGF) and that the VEGF promoter element contains multiple TTF-1-responsive sequences. The major signaling receptor for VEGF, i.e VEGFR2, also appears to be under a direct and positive regulation of TTF-1. The TTF-1-dependent upregulation of VEGF was moderately sensitive to rapamycin, implicating a partial involvement of mammalian target of rapamycin (mTOR). However, hypoxia did not further increase the secreted VEGF level of the TTF-1+ lung cancer cells. The TTF-1-induced VEGF upregulation occurs in both compartments (exosomes and exosome-depleted media (EDM)) of the conditioned media. Surprisingly, the EDM of TTF-1+ lung cancer cells (designated EDM-TTF-1+) displayed an anti-angiogenic activity in the endothelial cell tube formation assay. Mechanistic studies suggest that the increased granulocyte-macrophage colony-stimulating factor (GM-CSF) level in the EDM-TTF-1+ conferred the antiangiogenic activities. In human lung cancer, the expression of TTF-1 and GM-CSF exhibits a statistically significant and positive correlation. In summary, this study provides evidence that TTF-1 may reprogram lung cancer secreted proteome into an antiangiogenic state, offering a novel basis to account for the long-standing observation of favorable prognosis associated with TTF-1+ lung adenocarcinomas. PMID:26912193

  19. Thyroid Transcription Factor 1 Reprograms Angiogenic Activities of Secretome.

    PubMed

    Wood, Lauren W; Cox, Nicole I; Phelps, Cody A; Lai, Shao-Chiang; Poddar, Arjun; Talbot, Conover; Mu, David

    2016-01-01

    Through both gain- and loss-of-TTF-1 expression strategies, we show that TTF-1 positively regulates vascular endothelial growth factor (VEGF) and that the VEGF promoter element contains multiple TTF-1-responsive sequences. The major signaling receptor for VEGF, i.e VEGFR2, also appears to be under a direct and positive regulation of TTF-1. The TTF-1-dependent upregulation of VEGF was moderately sensitive to rapamycin, implicating a partial involvement of mammalian target of rapamycin (mTOR). However, hypoxia did not further increase the secreted VEGF level of the TTF-1(+) lung cancer cells. The TTF-1-induced VEGF upregulation occurs in both compartments (exosomes and exosome-depleted media (EDM)) of the conditioned media. Surprisingly, the EDM of TTF-1(+) lung cancer cells (designated EDM-TTF-1(+)) displayed an anti-angiogenic activity in the endothelial cell tube formation assay. Mechanistic studies suggest that the increased granulocyte-macrophage colony-stimulating factor (GM-CSF) level in the EDM-TTF-1(+) conferred the antiangiogenic activities. In human lung cancer, the expression of TTF-1 and GM-CSF exhibits a statistically significant and positive correlation. In summary, this study provides evidence that TTF-1 may reprogram lung cancer secreted proteome into an antiangiogenic state, offering a novel basis to account for the long-standing observation of favorable prognosis associated with TTF-1(+) lung adenocarcinomas. PMID:26912193

  20. The photospheric filling factor of the active binary II Pegasi

    NASA Astrophysics Data System (ADS)

    Marino, G.; Rodonó, M.; Leto, G.; Cutispoto, G.

    1999-12-01

    UBV and JHK photometry of the active single-lined binary II Peg, we performed in 1995, is presented. A method to determine the fraction of the photosphere covered by spots (filling factor) and to check the accuracy of generally assumed values of photospheric parameters has been developed. The procedure is based on the comparison between multiband fluxes and low resolution synthetic spectra weighted on the base of the spot filling factor and scaled with the ratio between the star radius and distance (R/d), so that we can also estimate the R/d ratio. A chi 2 fit has been performed for II Peg observations close to the light maximum and minimum by assuming reliable values of the photospheric parameters. Although a unique solution cannot be reached, we found clear indication for a spot filling factor at light maximum >= 40%. We find that the same set of parameters that gives us the best fit solutions at light maximum also provides the best fit at light minimum. The resulting solutions are consistent with the observed amplitude of the photometric wave, and with the commonly accepted value of R, unspotted V magnitude and spectral classification for II Pegasi.

  1. DeltaPhage--a novel helper phage for high-valence pIX phagemid display.

    PubMed

    Nilssen, Nicolay R; Frigstad, Terje; Pollmann, Sylvie; Roos, Norbert; Bogen, Bjarne; Sandlie, Inger; Løset, Geir Å

    2012-09-01

    Phage display has been instrumental in discovery of novel binding peptides and folded domains for the past two decades. We recently reported a novel pIX phagemid display system that is characterized by a strong preference for phagemid packaging combined with low display levels, two key features that support highly efficient affinity selection. However, high diversity in selected repertoires are intimately coupled to high display levels during initial selection rounds. To incorporate this additional feature into the pIX display system, we have developed a novel helper phage termed DeltaPhage that allows for high-valence display on pIX. This was obtained by inserting two amber mutations close to the pIX start codon, but after the pVII translational stop, conditionally inactivating the helper phage encoded pIX. Until now, the general notion has been that display on pIX is dependent on wild-type complementation, making high-valence display unachievable. However, we found that DeltaPhage does facilitate high-valence pIX display when used with a non-suppressor host. Here, we report a side-by-side comparison with pIII display, and we find that this novel helper phage complements existing pIX phagemid display systems to allow both low and high-valence display, making pIX display a complete and efficient alternative to existing pIII phagemid display systems. PMID:22539265

  2. Evaluation of PpIX formation in Cervical Intraepithelial Neoplasia I (CIN) using widefield fluorescence images

    NASA Astrophysics Data System (ADS)

    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.

  3. Saccharin: a Lead Compound for Structure-Based Drug Design of Carbonic Anhydrase IX Inhibitors

    PubMed Central

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

    2015-01-01

    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. PMID:25614109

  4. Loop Dynamics of the Extracellular Domain of Human Tissue Factor and Activation of Factor VIIa

    PubMed Central

    Minazzo, Agnese S.; Darlington, Reuben C.; Ross, J.B. Alexander

    2009-01-01

    Abstract In the crystal structure of the complex between the soluble extracellular domain of tissue factor (sTF) and active-site-inhibited VIIa, residues 91 and 92 in the Pro79-Pro92 loop of sTF interact with the catalytic domain of VIIa. It is not known, however, whether this loop has a role in allosteric activation of VIIa. Time-resolved fluorescence anisotropy measurements of probes covalently bound to sTF mutants E84C and T121C show that binding uninhibited Factor VIIa affects segmental motions in sTF. Glu84 resides in the Pro79-Pro92 loop, and Thr121 resides in the turn between the first and second antiparallel β-strands of the sTF subdomain that interacts with the Gla and EGF1 domains of VIIa; neither Glu84 nor Thr121 makes direct contact with VIIa. Probes bound to T121C report limited segmental flexibility in free sTF, which is lost after VIIa binding. Probes bound to E84C report substantial segmental flexibility in the Pro79-Pro92 loop in free sTF, which is greatly reduced after VIIa binding. Thus, VIIa binding reduces dynamic motions in sTF. In particular, the decrease in the Pro79-Pro92 loop motions indicates that loop entropy has a role in the thermodynamics of the protein-protein interactions involved in allosteric control of VIIa activation. PMID:19167313

  5. Purification of human plasma platelet-activating factor acetylhydrolase

    SciTech Connect

    Stafforini, D.M.; Prescott, S.M.; McIntyre, T.M.

    1986-05-01

    Platelet-activating factor (PAF;1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine is synthesized by a variety of cells. It induces hypotension, and activates platelets, neutrophils, and macrophages at nanomolar concentrations. Removal of the acetate abolishes biological activity, and is catalyzed by a specific PAF acetylhydrolase present in plasma and tissues. The authors developed a rapid assay, based on separation of (/sup 3/H)acetate from (/sup 3/H-acetyl)PAF by reversed-phase chromatography. In human plasma the enzyme exhibits an apparent Km of 5.7..mu..M, with a Vmax of 0.027..mu..mol/h/mg. Ultracentrifugation in density gradients showed that 30% of the activity is associated with high density lipoproteins (HDL) and 70% with low density lipoproteins (LDL). The enzyme was purified from LDL by precipitation with Na phosphotungstate and MgCl/sub 2/, solubilization with Tween 20, column chromatography and electrophoresis. This procedure resulted in a preparation that was 21,000-fold purified from plasma (spec. act. 575..mu..mol/h/mg) with a recovery of 10%. The purified enzyme has a molecular weight of about 43,000, a broad pH optimum (peak 7.5-8.0), and a pl of 4.6. It has greater activity when PAF is in a micellar, as compared to monomeric, and exhibits surface dilution kinetics, which may be important in vivo. The purification and characterization of this enzyme will allow detailed studies of its role in PAF metabolism.

  6. Crystal Structure of Human Plasma Platelet-Activating Factor Acetylhydrolase

    SciTech Connect

    Samanta, U.; Bahnson, B

    2008-01-01

    Human plasma platelet-activating factor (PAF) acetylhydrolase functions by reducing PAF levels as a general anti-inflammatory scavenger and is linked to anaphylactic shock, asthma, and allergic reactions. The enzyme has also been implicated in hydrolytic activities of other pro-inflammatory agents, such as sn-2 oxidatively fragmented phospholipids. This plasma enzyme is tightly bound to low and high density lipoprotein particles and is also referred to as lipoprotein-associated phospholipase A{sub 2}. The crystal structure of this enzyme has been solved from x-ray diffraction data collected to a resolution of 1.5{angstrom}. It has a classic lipase {alpha}/{beta}-hydrolase fold, and it contains a catalytic triad of Ser{sup 273}, His{sup 351}, and Asp{sup 296}. Two clusters of hydrophobic residues define the probable interface-binding region, and a prediction is given of how the enzyme is bound to lipoproteins. Additionally, an acidic patch of 10 carboxylate residues and a neighboring basic patch of three residues are suggested to play a role in high density lipoprotein/low density lipoprotein partitioning. A crystal structure is also presented of PAF acetylhydrolase reacted with the organophosphate compound paraoxon via its active site Ser{sup 273}. The resulting diethyl phosphoryl complex was used to model the tetrahedral intermediate of the substrate PAF to the active site. The model of interface binding begins to explain the known specificity of lipoprotein-bound substrates and how the active site can be both close to the hydrophobic-hydrophilic interface and at the same time be accessible to the aqueous phase.

  7. Involvement of an Skp-Like Protein, PGN_0300, in the Type IX Secretion System of Porphyromonas gingivalis

    PubMed Central

    Taguchi, Yuko; Sato, Keiko; Yukitake, Hideharu; Inoue, Tetsuyoshi; Nakayama, Masaaki; Naito, Mariko; Kondo, Yoshio; Kano, Konami; Hoshino, Tomonori; Nakayama, Koji; Takashiba, Shogo

    2015-01-01

    The oral Gram-negative anaerobic bacterium Porphyromonas gingivalis is an important pathogen involved in chronic periodontitis. Among its virulence factors, the major extracellular proteinases, Arg-gingipain and Lys-gingipain, are of interest given their abilities to degrade host proteins and process other virulence factors. Gingipains possess C-terminal domains (CTDs) and are translocated to the cell surface or into the extracellular milieu by the type IX secretion system (T9SS). Gingipains contribute to the colonial pigmentation of the bacterium on blood agar. In this study, Omp17, the PGN_0300 gene product, was found in the outer membrane fraction. A mutant lacking Omp17 did not show pigmentation on blood agar and showed reduced proteolytic activity of the gingipains. CTD-containing proteins were released from bacterial cells without cleavage of the CTDs in the omp17 mutant. Although synthesis of the anionic polysaccharide (A-LPS) was not affected in the omp17 mutant, the processing of and A-LPS modification of CTD-containing proteins was defective. PorU, a C-terminal signal peptidase that cleaves the CTDs of other CTD-containing proteins, was not detected in any membrane fraction of the omp17 mutant, suggesting that the defective maturation of CTD-containing proteins by impairment of Omp17 is partly due to loss of function of PorU. In the mouse subcutaneous infection experiment, the omp17 mutant was less virulent than the wild type. These results suggested that Omp17 is involved in P. gingivalis virulence. PMID:26502912

  8. Deficiency of platelet-activating factor acetylhydrolase is a severity factor for asthma

    PubMed Central

    Stafforini, Diana M.; Numao, Toshio; Tsodikov, Alexander; Vaitkus, Darius; Fukuda, Takeshi; Watanabe, Naoto; Fueki, Naoto; McIntyre, Thomas M.; Zimmerman, Guy A.; Makino, Sohei; Prescott, Stephen M.

    1999-01-01

    Asthma, a family of airway disorders characterized by airway inflammation, has an increasing incidence worldwide. Platelet-activating factor (PAF) may play a role in the pathophysiology of asthma. Its proinflammatory actions are antagonized by PAF acetylhydrolase. A missense mutation (V279F) in the PAF acetylhydrolase gene results in the complete loss of activity, which occurs in 4% of the Japanese population. We asked if PAF acetylhydrolase deficiency correlates with the incidence and severity of asthma in Japan. We found that the prevalence of PAF acetylhydrolase deficiency is higher in Japanese asthmatics than healthy subjects and that the severity of this syndrome is highest in homozygous-deficient subjects. We conclude that the PAF acetylhydrolase gene is a modulating locus for the severity of asthma. PMID:10194471

  9. Associations between Socio-Motivational Factors, Physical Education Activity Levels and Physical Activity Behavior among Youth

    ERIC Educational Resources Information Center

    Ning, Weihong; Gao, Zan; Lodewyk, Ken

    2012-01-01

    This study examined the relationships between established socio-motivational factors and children's physical activity levels daily and during physical education classes. A total of 307 middle school students (149 boys, 158 girls) from a suburban public school in the Southern United States participated in this study. Participants completed…

  10. Atrial natriuretic factor-like activity in rat posterior pituitary

    SciTech Connect

    Gutkowska, J.; Debinski, W.; Racz, K.; Thibault, G.; Garcia, R.; Kuchel, O.; Genest, J.; Cantin, M.

    1986-03-05

    The presence of a biologically active peptide: Atrial Natriuretic Factor (ANF) has been demonstrated in rat and human circulation and ANF is considered now as a new hormone. ANF may be involved in body fluid regulation. A very sensitive radioimmunoassay for rat ANF allowed the authors to search for immunoreactive ANF (IR-ANF) in rat posterior pituitary. Serial dilutions of homogenates of rat posterior pituitary showed a good parallelism with a reference curve in a radioimmunoassay system. The IR-ANF was extracted from rat posterior pituitary homogenates by activated Vycor glass beads. The lyophilized extract was purified by HPLC on C/sub 18/ ..mu.. Bondapak column. The HPLC yielded two IR-ANF peaks. Both isolated ANF-like material showed biological activity. The IR-ANF eluted with 33% acetonitrile, inhibited ACTH-stimulated aldosterone secretion with a similar potency as synthetic (Arg 101 - Tyr 126) ANF (0.7 x 10/sup -10/M). A much less potent ANF-like material was found in the second peak eluted with 36% acetonitrile. They conclude that ANF-like material is present in rat posterior pituitary and this suggest a possible role in ANF on AVP secretion directly in situ.

  11. Constellation's First Flight Test: Ares I-X

    NASA Technical Reports Server (NTRS)

    Davis, Stephan R.; Askins, Bruce R.

    2010-01-01

    On October 28, 2009, NASA launched Ares I-X, the first flight test of the Constellation Program that will send human beings to the Moon and beyond. This successful test is the culmination of a three-and-a-half-year, multi-center effort to design, build, and fly the first demonstration vehicle of the Ares I crew launch vehicle, the successor vehicle to the Space Shuttle. The suborbital mission was designed to evaluate the atmospheric flight characteristics of a vehicle dynamically similar to Ares I; perform a first stage separation and evaluate its effects; characterize and control roll torque; stack, fly, and recover a solid-motor first stage testing the Ares I parachutes; characterize ground, flight, and reentry environments; and develop and execute new ground hardware and procedures. Built from existing flight and new simulator hardware, Ares I-X integrated a Shuttle-heritage four-segment solid rocket booster for first stage propulsion, a spacer segment to simulate a five-segment booster, Peacekeeper axial engines for roll control, and Atlas V avionics, as well as simulators for the upper stage, crew module, and launch abort system. The mission leveraged existing logistical and ground support equipment while also developing new ones to accommodate the first in-line rocket for flying astronauts since the Saturn IB last flew from Kennedy Space Center (KSC) in 1975. This paper will describe the development and integration of the various vehicle and ground elements, from conception to stacking in KSC s Vehicle Assembly Building; hardware performance prior to, during, and after the launch; and preliminary lessons and data gathered from the flight. While the Constellation Program is currently under review, Ares I-X has and will continue to provide vital lessons for NASA personnel in taking a vehicle concept from design to flight.

  12. Carbonic anhydrase IX is a clinically significant tissue and serum biomarker associated with renal cell carcinoma

    PubMed Central

    TAKACOVA, MARTINA; BARTOSOVA, MARIA; SKVARKOVA, LUCIA; ZATOVICOVA, MIRIAM; VIDLICKOVA, IVANA; CSADEROVA, LUCIA; BARATHOVA, MONIKA; BREZA, JAN; BUJDAK, PETER; PASTOREK, JAROMIR; BREZA, JAN; PASTOREKOVA, SILVIA

    2013-01-01

    Carbonic anhydrase IX (CA IX) is regarded as one of the most prominent markers of tumor hypoxia with potential to serve as a diagnostic biomarker, prognostic indicator as well as tumor therapeutic target. The aim of the present study was to perform an in-depth analysis of CA IX expression in blood and tissue samples and to evaluate the significance of CA IX status for different renal cell carcinomas (RCCs). The expression of CA IX was determined in blood and tissue samples from 74 kidney cancer patients using reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), Western blotting (WB) and immunohistochemistry (IHC). The CA IX status was correlated with RCC type and tumor stage. IHC and WB provided evidence for a significantly higher expression of CA IX in clear cell RCC (CCRCC) specimens compared to other RCCs. RT-PCR assay revealed that 32.42% of all RCC patients possess CA9-positive cells in peripheral blood and three-quarters of CA9-positive patients were diagnosed with CCRCC. When the patients were subdivided according to tumor stage, decreased positivity was observed with higher tumor stage (50% in T1 vs. 17% in T3). Serum CA IX levels determined by ELISA were significantly higher in CCRCC patients than in non-CCRCC. A significant association between s-CA IX and CCRCC tumor stage was also determined (T1-87.51 vs. T3-341.98 pg/ml, p=0.046). We demonstrated that the CA IX expression profiles in blood and tissue samples from 74 kidney cancer patients are closely correlated with their histological subtypes. This is the first study reporting CA IX expression in blood and tissue samples from kidney cancer patients determined by four different methods. PMID:23255918

  13. Oscillator strengths for Ar VII, Ca IX and Fe XV

    NASA Technical Reports Server (NTRS)

    Tayal, S. S.

    1986-01-01

    The excitation energies and oscillator strengths are calculated for electric-dipole-allowed and intercombination transitions between 3s2 1S, 3s3p(1,3)P0, 3p2 3P, 1D, 1S and 3s3d(1,3)D states in Ar VII, Ca IX, and Fe XV ions of the magnesium sequence. These states are represented by the fairly large configuration-interaction expansions. The calculations have been carried out in both LS and intermediate coupling schemes. The relativistic corrections have been included through the Breit-Pauli Hamiltonian. The results are compared with previous theoretical calculations and with measurements.

  14. Ares I-X Test Flight Reference Trajectory Development

    NASA Technical Reports Server (NTRS)

    Starr, Brett R.; Gumbert, Clyde R.; Tartabini, Paul V.

    2011-01-01

    Ares I-X was the first test flight of NASA's Constellation Program's Ares I crew launch vehicle. Ares I is a two stage to orbit launch vehicle that provides crew access to low Earth orbit for NASA's future manned exploration missions. The Ares I first stage consists of a Shuttle solid rocket motor (SRM) modified to include an additional propellant segment and a liquid propellant upper stage with an Apollo J2X engine modified to increase its thrust capability. The modified propulsion systems were not available for the first test flight, thus the test had to be conducted with an existing Shuttle 4 segment reusable solid rocket motor (RSRM) and an inert Upper Stage. The test flight's primary objective was to demonstrate controllability of an Ares I vehicle during first stage boost and the ability to perform a successful separation. In order to demonstrate controllability, the Ares I-X ascent control algorithms had to maintain stable flight throughout a flight environment equivalent to Ares I. The goal of the test flight reference trajectory development was to design a boost trajectory using the existing RSRM that results in a flight environment equivalent to Ares I. A trajectory similarity metric was defined as the integrated difference between the Ares I and Ares I-X Mach versus dynamic pressure relationships. Optimization analyses were performed that minimized the metric by adjusting the inert upper stage weight and the ascent steering profile. The sensitivity of the optimal upper stage weight and steering profile to launch month was also investigated. A response surface approach was used to verify the optimization results. The analyses successfully defined monthly ascent trajectories that matched the Ares I reference trajectory dynamic pressure versus Mach number relationship to within 10% through Mach 3.5. The upper stage weight required to achieve the match was found to be feasible and varied less than 5% throughout the year. The paper will discuss the flight

  15. Human factors in remote control engineering development activities

    SciTech Connect

    Clarke, M.M.; Hamel, W.R.; Draper, J.V.

    1983-01-01

    Human factors engineering, which is an integral part of the advanced remote control development activities at the Oak Ridge National Laboratory, is described. First, work at the Remote Systems Development Facility (RSDF) has shown that operators can perform a wide variety of tasks, some of which were not specifically designed for remote systems, with a dextrous electronic force-reflecting servomanipulator and good television remote viewing capabilities. Second, the data collected during mock-up remote maintenance experiments at the RSDF have been analyzed to provide guidelines for the design of human interfaces with an integrated advanced remote maintenance system currently under development. Guidelines have been provided for task allocation between operators, remote viewing systems, and operator controls. 6 references, 5 figures, 2 tables.

  16. Platelet activating factor as a mediator of equine cell locomotion.

    PubMed

    Dawson, J; Lees, P; Sedgwick, A D

    1988-01-01

    Equine polymorphonuclear (PMN) and mononuclear (MN) leucocytes were separated on Percoll gradients and used to study the chemoattractant properties of the polar ether-linked phospholipid, platelet activating factor (PAF). Six concentrations of PAF ranging from 1 ng/ml to 100 micrograms/ml were studied in each of two in vitro assay systems, the agarose microdroplet and a microfilter technique. Very significant (p less than 0.01) increases in the movement of both PMN and MN cells were obtained with most concentrations of PAF. In two instances there was no apparent concentration-response relationship, although the action of PAF was approximately bell-shaped in two others. The possible significance of these findings for equine inflammatory conditions is discussed. PMID:3188378

  17. Phylogenomics of caspase-activated DNA fragmentation factor

    SciTech Connect

    Eckhart, Leopold . E-mail: leopold.eckhart@meduniwien.ac.at; Fischer, Heinz; Tschachler, Erwin

    2007-04-27

    The degradation of nuclear DNA by DNA fragmentation factor (DFF) is a key step in apoptosis of mammalian cells. Using comparative genomics, we have here determined the evolutionary history of the genes encoding the two DFF subunits, DFFA (also known as ICAD) and DFFB (CAD). Orthologs of DFFA and DFFB were identified in Nematostella vectensis, a representative of the primitive metazoan clade cnidarians, and in various vertebrates and insects, but not in representatives of urochordates, echinoderms, and nematodes. The domains mediating the interaction of DFFA and DFFB, a caspase cleavage site in DFFA, and the amino acid residues critical for endonuclease activity of DFFB were conserved in Nematostella. These findings suggest that DFF has been a part of the primordial apoptosis system of the eumetazoan common ancestor and that the ancient cell death machinery has degenerated in several evolutionary lineages, including the one leading to the prototypical apoptosis model, Caenorhabditis elegans.

  18. The Regulatory Role of Activating Transcription Factor 2 in Inflammation

    PubMed Central

    Yu, Tao; Li, Yong Jun; Bian, Ai Hong; Zuo, Hui Bin; Zhu, Ti Wen; Ji, Sheng Xiang; Kong, Fanming; Yin, De Qing; Wang, Chuan Bao; Wang, Zi Fu; Wang, Hong Qun; Yang, Yanyan; Yoo, Byong Chul

    2014-01-01

    Activating transcription factor 2 (ATF2) is a member of the leucine zipper family of DNA-binding proteins and is widely distributed in tissues including the liver, lung, spleen, and kidney. Like c-Jun and c-Fos, ATF2 responds to stress-related stimuli and may thereby influence cell proliferation, inflammation, apoptosis, oncogenesis, neurological development and function, and skeletal remodeling. Recent studies clarify the regulatory role of ATF2 in inflammation and describe potential inhibitors of this protein. In this paper, we summarize the properties and functions of ATF2 and explore potential applications of ATF2 inhibitors as tools for research and for the development of immunosuppressive and anti-inflammatory drugs. PMID:25049453

  19. Control of mechanically activated polymersome fusion: Factors affecting fusion

    DOE PAGESBeta

    Henderson, Ian M.; Paxton, Walter F.

    2014-12-15

    Previously we have studied the mechanically-activated fusion of extruded (200 nm) polymer vesicles into giant polymersomes using agitation in the presence of salt. In this study we have investigated several factors contributing to this phenomenon, including the effects of (i) polymer vesicle concentration, (ii) agitation speed and duration, and iii) variation of the salt and its concentration. It was found that increasing the concentration of the polymer dramatically increases the production of giant vesicles through the increased collisions of polymersomes. Our investigations also found that increasing the frequency of agitation increased the efficiency of fusion, though ultimately limited the sizemore » of vesicle which could be produced due to the high shear involved. Finally it was determined that salt-mediation of the fusion process was not limited to NaCl, but is instead a general effect facilitated by the presence of solvated ionic compounds, albeit with different salts initiating fusion at different concentration.« less

  20. Control of mechanically activated polymersome fusion: Factors affecting fusion

    SciTech Connect

    Henderson, Ian M.; Paxton, Walter F.

    2014-12-15

    Previously we have studied the mechanically-activated fusion of extruded (200 nm) polymer vesicles into giant polymersomes using agitation in the presence of salt. In this study we have investigated several factors contributing to this phenomenon, including the effects of (i) polymer vesicle concentration, (ii) agitation speed and duration, and iii) variation of the salt and its concentration. It was found that increasing the concentration of the polymer dramatically increases the production of giant vesicles through the increased collisions of polymersomes. Our investigations also found that increasing the frequency of agitation increased the efficiency of fusion, though ultimately limited the size of vesicle which could be produced due to the high shear involved. Finally it was determined that salt-mediation of the fusion process was not limited to NaCl, but is instead a general effect facilitated by the presence of solvated ionic compounds, albeit with different salts initiating fusion at different concentration.

  1. Cooperative Regulation of the Activity of Factor Xa within Prothrombinase by Discrete Amino Acid Regions from Factor Va Heavy Chain†

    PubMed Central

    2008-01-01

    The prothrombinase complex catalyzes the activation of prothrombin to α-thrombin. We have repetitively shown that amino acid region 695DYDY698 from the COOH terminus of the heavy chain of factor Va regulates the rate of cleavage of prothrombin at Arg271 by prothrombinase. We have also recently demonstrated that amino acid region 334DY335 is required for the optimal activity of prothrombinase. To assess the effect of these six amino acid residues on cofactor activity, we created recombinant factor Va molecules combining mutations at amino acid regions 334–335 and 695−698 as follows: factor V3K (334DY335 → KF and 695DYDY698 → KFKF), factor VKF/4A (334DY335 → KF and 695DYDY698 → AAAA), and factor V6A (334DY335 → AA and 695DYDY698 → AAAA). The recombinant factor V molecules were expressed and purified to homogeneity. Factor Va3K, factor VaK4/4A, and factor Va6A had reduced affinity for factor Xa, when compared to the affinity of the wild-type molecule (factor VaWt) for the enzyme. Prothrombinase assembled with saturating concentrations of factor Va3K had a 6-fold reduced second-order rate constant for prothrombin activation compared to the value obtained with prothrombinase assembled with factor VaWt, while prothrombinase assembled with saturating concentrations of factor VaKF/4A and factor Va6A had approximately 1.5-fold reduced second-order rate constants. Overall, the data demonstrate that amino acid region 334–335 together with amino acid region 695−698 from factor Va heavy chain are part of a cooperative mechanism within prothrombinase regulating cleavage and activation of prothrombin by factor Xa. PMID:18991406

  2. Transcription factor PIF4 controls the thermosensory activation of flowering.

    PubMed

    Kumar, S Vinod; Lucyshyn, Doris; Jaeger, Katja E; Alós, Enriqueta; Alvey, Elizabeth; Harberd, Nicholas P; Wigge, Philip A

    2012-04-12

    Plant growth and development are strongly affected by small differences in temperature. Current climate change has already altered global plant phenology and distribution, and projected increases in temperature pose a significant challenge to agriculture. Despite the important role of temperature on plant development, the underlying pathways are unknown. It has previously been shown that thermal acceleration of flowering is dependent on the florigen, FLOWERING LOCUS T (FT). How this occurs is, however, not understood, because the major pathway known to upregulate FT, the photoperiod pathway, is not required for thermal acceleration of flowering. Here we demonstrate a direct mechanism by which increasing temperature causes the bHLH transcription factor PHYTOCHROME INTERACTING FACTOR4 (PIF4) to activate FT. Our findings provide a new understanding of how plants control their timing of reproduction in response to temperature. Flowering time is an important trait in crops as well as affecting the life cycles of pollinator species. A molecular understanding of how temperature affects flowering will be important for mitigating the effects of climate change. PMID:22437497

  3. Allosteric activation of ADAMTS13 by von Willebrand factor

    PubMed Central

    Muia, Joshua; Zhu, Jian; Gupta, Garima; Haberichter, Sandra L.; Friedman, Kenneth D.; Feys, Hendrik B.; Deforche, Louis; Vanhoorelbeke, Karen; Westfield, Lisa A.; Roth, Robyn; Tolia, Niraj Harish; Heuser, John E.

    2014-01-01

    The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) within endovascular platelet aggregates, and ADAMTS13 deficiency causes fatal microvascular thrombosis. The proximal metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains of ADAMTS13 recognize a cryptic site in VWF that is exposed by tensile force. Another seven T and two complement C1r/C1s, sea urchin epidermal growth factor, and bone morphogenetic protein (CUB) domains of uncertain function are C-terminal to the MDTCS domains. We find that the distal T8-CUB2 domains markedly inhibit substrate cleavage, and binding of VWF or monoclonal antibodies to distal ADAMTS13 domains relieves this autoinhibition. Small angle X-ray scattering data indicate that distal T-CUB domains interact with proximal MDTCS domains. Thus, ADAMTS13 is regulated by substrate-induced allosteric activation, which may optimize VWF cleavage under fluid shear stress in vivo. Distal domains of other ADAMTS proteases may have similar allosteric properties. PMID:25512528

  4. Increased activity of coagulation factor XII (Hageman factor) causes hereditary angioedema type III.

    PubMed

    Cichon, Sven; Martin, Ludovic; Hennies, Hans Christian; Müller, Felicitas; Van Driessche, Karen; Karpushova, Anna; Stevens, Wim; Colombo, Roberto; Renné, Thomas; Drouet, Christian; Bork, Konrad; Nöthen, Markus M

    2006-12-01

    Hereditary angioedema (HAE) is characterized clinically by recurrent acute skin swelling, abdominal pain, and potentially life-threatening laryngeal edema. Three forms of HAE have been described. The classic forms, HAE types I and II, occur as a consequence of mutations in the C1-inhibitor gene. In contrast to HAE types I and II, HAE type III has been observed exclusively in women, where it appears to be correlated with conditions of high estrogen levels--for example, pregnancy or the use of oral contraceptives. A recent report proposed two missense mutations (c.1032C-->A and c.1032C-->G) in F12, the gene encoding human coagulation factor XII (FXII, or Hageman factor) as a possible cause of HAE type III. Here, we report the occurrence of the c.1032C-->A (p.Thr328Lys) mutation in an HAE type III-affected family of French origin. Investigation of the F12 gene in a large German family did not reveal a coding mutation. Haplotype analysis with use of microsatellite markers is compatible with locus heterogeneity in HAE type III. To shed more light on the pathogenic relevance of the HAE type III-associated p.Thr328Lys mutation, we compared FXII activity and plasma levels in patients carrying the mutation with that of healthy control individuals. Our data strongly suggest that p.Thr328Lys is a gain-of-function mutation that markedly increases FXII amidolytic activity but that does not alter FXII plasma levels. We conclude that enhanced FXII enzymatic plasma activity in female mutation carriers leads to enhanced kinin production, which results in angioedema. Transcription of F12 is positively regulated by estrogens, which may explain why only women are affected with HAE type III. The results of our study represent an important step toward an understanding of the molecular processes involved in HAE type III and provide diagnostic and possibly new therapeutic opportunities. PMID:17186468

  5. Grazing by protozoa as selection factor for activated sludge bacteria.

    PubMed

    Güde, H

    1979-09-01

    In continuous culture enrichments that were inoculated with activated sludge and were fed with polymeric substrates, freely dispersed single-celled bacteria belonging to theCytophaga group dominated among the initial populations, irrespective of the activated sludge source. These populations were grazed by flagellated protozoa which after several days reached high cell densities. Other morphologic bacterial groups such as spiral-shaped or filamentous bacteria then became dominant. In defined mixed culture experiments with bacterial isolates from the enrichment cultures, it was shown that a "grazing-resistant"Microcyclus strain outgrew aCytophaga strain in the presence of grazing protozoa. In contrast, theCytophaga strain competed successfully with theMicrocyclus strain and with other "grazing-resistant" strains under protozoa-free conditions. Furthermore, it was demonstrated that assumed grazing resistance factors such as floccing or filamentous growth were lost by some of the strains when they were grown for several generations in continuous culture under the same conditions, but in the absence of protozoa. PMID:24232496

  6. Radiation therapy generates platelet-activating factor agonists

    PubMed Central

    Sahu, Ravi P.; Harrison, Kathleen A.; Weyerbacher, Jonathan; Murphy, Robert C.; Konger, Raymond L.; Garrett, Joy Elizabeth; Chin-Sinex, Helen Jan; Johnston, Michael Edward; Dynlacht, Joseph R.; Mendonca, Marc; McMullen, Kevin; Li, Gengxin; Spandau, Dan F.; Travers, Jeffrey B.

    2016-01-01

    Pro-oxidative stressors can suppress host immunity due to their ability to generate oxidized lipid agonists of the platelet-activating factor-receptor (PAF-R). As radiation therapy also induces reactive oxygen species, the present studies were designed to define whether ionizing radiation could generate PAF-R agonists and if these lipids could subvert host immunity. We demonstrate that radiation exposure of multiple tumor cell lines in-vitro, tumors in-vivo, and human subjects undergoing radiation therapy for skin tumors all generate PAF-R agonists. Structural characterization of radiation-induced PAF-R agonistic activity revealed PAF and multiple oxidized glycerophosphocholines that are produced non-enzymatically. In a murine melanoma tumor model, irradiation of one tumor augmented the growth of the other (non-treated) tumor in a PAF-R-dependent process blocked by a cyclooxygenase-2 inhibitor. These results indicate a novel pathway by which PAF-R agonists produced as a byproduct of radiation therapy could result in tumor treatment failure, and offer important insights into potential therapeutic strategies that could improve the overall antitumor effectiveness of radiation therapy regimens. PMID:26959112

  7. Radiation therapy generates platelet-activating factor agonists.

    PubMed

    Sahu, Ravi P; Harrison, Kathleen A; Weyerbacher, Jonathan; Murphy, Robert C; Konger, Raymond L; Garrett, Joy Elizabeth; Chin-Sinex, Helen Jan; Johnston, Michael Edward; Dynlacht, Joseph R; Mendonca, Marc; McMullen, Kevin; Li, Gengxin; Spandau, Dan F; Travers, Jeffrey B

    2016-04-12

    Pro-oxidative stressors can suppress host immunity due to their ability to generate oxidized lipid agonists of the platelet-activating factor-receptor (PAF-R). As radiation therapy also induces reactive oxygen species, the present studies were designed to define whether ionizing radiation could generate PAF-R agonists and if these lipids could subvert host immunity. We demonstrate that radiation exposure of multiple tumor cell lines in-vitro, tumors in-vivo, and human subjects undergoing radiation therapy for skin tumors all generate PAF-R agonists. Structural characterization of radiation-induced PAF-R agonistic activity revealed PAF and multiple oxidized glycerophosphocholines that are produced non-enzymatically. In a murine melanoma tumor model, irradiation of one tumor augmented the growth of the other (non-treated) tumor in a PAF-R-dependent process blocked by a cyclooxygenase-2 inhibitor. These results indicate a novel pathway by which PAF-R agonists produced as a byproduct of radiation therapy could result in tumor treatment failure, and offer important insights into potential therapeutic strategies that could improve the overall antitumor effectiveness of radiation therapy regimens. PMID:26959112

  8. 32 CFR 2003.9 - Reports to the President (Article IX).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 32 National Defense 6 2013-07-01 2013-07-01 false Reports to the President (Article IX). 2003.9... PANEL (ISCAP) BYLAWS, RULES, AND APPEAL PROCEDURES Bylaws § 2003.9 Reports to the President (Article IX... President of the United States. The Panel also includes such information in any reports it may make to...

  9. Not Second-Class: Title IX, Equity, and Girls' High School Sports

    ERIC Educational Resources Information Center

    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…

  10. 46 CFR 57.02-2 - Adoption of section IX of the ASME Code.

    Code of Federal Regulations, 2010 CFR

    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 57.02-2 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING WELDING... accordance with section IX of the ASME (American Society of Mechanical Engineers) Code, as limited,...

  11. 46 CFR 57.02-2 - Adoption of section IX of the ASME Code.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 2 2013-10-01 2013-10-01 false Adoption of section IX of the ASME Code. 57.02-2 Section 57.02-2 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING WELDING... accordance with section IX of the ASME (American Society of Mechanical Engineers) Code, as limited,...

  12. 46 CFR 57.02-2 - Adoption of section IX of the ASME Code.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 2 2012-10-01 2012-10-01 false Adoption of section IX of the ASME Code. 57.02-2 Section 57.02-2 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING WELDING... accordance with section IX of the ASME (American Society of Mechanical Engineers) Code, as limited,...

  13. Sex Discrimination Against Students: Implications of Title IX of the Education Amendments of 1972.

    ERIC Educational Resources Information Center

    Dunkle, Margaret C.; Sandler, Bernice

    Title IX of the Education Amendments of 1972 mandates that sex discrimination be eliminated in federally assisted education programs. Title IX has significant implications for a variety of issues including recruiting, admissions, financial aid, student rules and regulations, housing rules, health care and insurance benefits, student employment,…

  14. Almost As Fairly: The First Year of Title IX Implementation in Six Southern States. A Report.

    ERIC Educational Resources Information Center

    American Friends Service Committee, Columbia, SC. Southeastern Public Education Program.

    Volunteers from community organizations in six southern states monitored 21 school districts to find their districts' initial answer to Title IX, federal legislation barring sex discrimination. The actual monitoring of the 21 districts was completed in the late spring of 1976, with data covering the first year of Title IX implementation. The…

  15. "What Do I Think about Title IX?" Voices from a University Community

    ERIC Educational Resources Information Center

    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…

  16. The Impact of Implementing Title IX in a Predominantly Black Public University.

    ERIC Educational Resources Information Center

    Simmons, Gertrude L.

    Information on the impact of implementing Title IX of the 1972 Education Amendments at Florida A and M University, a predominantly Black public university, is presented. Title IX assures everyone regardless of sex an equal opportunity to learn a skill, choose a course of study, advance in status, participate in a sport, receive a scholarship, or…

  17. A Place on the Team: The Triumph and Tragedy of Title IX

    ERIC Educational Resources Information Center

    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 opportunities for…

  18. Derivation of a triple mosaic adenovirus based on modification of the minor capsid protein IX

    SciTech Connect

    Tang Yizhe; Le, Long P.; Matthews, Qiana L.; Han Tie; Wu Hongju; Curiel, David T.

    2008-08-01

    Adenoviral capsid protein IX (pIX) has been shown to be a potential locale to insert targeting, imaging-related and therapeutic modalities by genetic modification. Recent evidences suggested that capsid protein mosaicism could be a promising strategy for improving the utility of Ad vector. In this study, we explored a method to genetically generate triple pIX mosaic Ad serotype 5 (Ad5) displaying three types of pIX on a single virion. pIXs were modified at their carboxy termini with a Flag sequence, a hexahistidine sequence (His{sub 6}) or a monomeric red fluorescent protein (mRFP1), respectively. Western blotting analysis and fluorescence microscopy of the purified recombinant viruses indicated that all three modified pIXs were incorporated into the viral particles. Immuno-gold electron microscopy (EM) further confirmed that three types of pIX indeed co-existed on an individual virion. These results firstly validated a triple mosaic capsid configuration on pIX, and demonstrated the possibility of further radical design.

  19. Plasma protoporphyrin IX following administration of 5-aminolevulinic acid as a potential tumor marker

    PubMed Central

    OTA, URARA; FUKUHARA, HIDEO; ISHIZUKA, MASAHIRO; ABE, FUMINORI; KAWADA, CHIAKI; TAMURA, KENJI; TANAKA, TOHRU; INOUE, KEIJI; OGURA, SHUN-ICHIRO; SHUIN, TARO

    2015-01-01

    Exogenously administered 5-aminolevulinic acid (ALA) is metabolized to protoporphyrin IX (PpIX), which specifically accumulates in cancer cells and emits red fluorescence by blue light irradiation. These phenomena are applied for the intraoperative diagnosis of cancer. Based on the fact that accumulated PpIX in cancer cells is exported extracellularly via the ATP-binding cassette transporter G2, we hypothesized that the measurement of plasma PpIX concentrations could be applied as a tumor marker for cancer screening. In the present study, the use of plasma samples from bladder cancer patients were evaluated as a tumor marker. ALA, 1.0 g, was orally administered to bladder cancer patients and healthy adults. The plasma concentration of PpIX was measured using a high-performance liquid chromatography system. The plasma PpIX concentration following ALA administration was significantly higher in bladder cancer patients than that in the healthy adults, suggesting the effectiveness of plasma PpIX analysis following ALA administration for cancer screening. Additionally, 4 h after ALA administration, plasma PpIX showed high sensitivity (94.4%) and high specificity (80.0%). PMID:26171183

  20. Perceptions of Women's Teams Coaches Regarding Gender Equity and Title IX Compliance in Community Colleges

    ERIC Educational Resources Information Center

    Kenney, Cynthia A.

    2013-01-01

    Title IX was enacted over 40 years ago, and although there have been marked increases in the number of girls and women participating in athletics at every level, gender equity in athletics continues to be a concern. This is especially evident at the community college level. Title IX requires equity in the areas of opportunities for participation,…

  1. Title IX Compliance at Two-Year Colleges: An Analysis of Perceived Barriers and Strategies

    ERIC Educational Resources Information Center

    Causby, Cory Scott

    2010-01-01

    Although Title IX legislation has been in effect since 1972 and has created unprecedented positive change on intercollegiate athletics, educational institutions have still had difficulty meeting the basic requirements set forth by Title IX and ensuring gender equity in their athletic programs. Additionally, specific research has been largely…

  2. Title IX and Employment Discrimination: A Wrong in Search of a Remedy.

    ERIC Educational Resources Information Center

    Salomone, Rosemary C.

    1980-01-01

    Focuses on the litigation challenging the authority of the Department of Health, Education, and Welfare to promulgate regulations governing employment discrimination pursuant to Title IX. Despite compelling challenges, interest in Title IX continues because its penalties are more extensive than those available under Title VII. (IRT)

  3. 40 CFR Appendix Ix to Part 266 - Methods Manual for Compliance With the BIF Regulations

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... (CONTINUED) SOLID WASTES (CONTINUED) STANDARDS FOR THE MANAGEMENT OF SPECIFIC HAZARDOUS WASTES AND SPECIFIC TYPES OF HAZARDOUS WASTE MANAGEMENT FACILITIES Pt. 266, App. IX Appendix IX to Part 266—Methods Manual... Protection Agency regulations for boilers and industrial furnaces (BIFs) burning hazardous waste (see 40...

  4. 40 CFR Appendix Ix to Part 266 - Methods Manual for Compliance With the BIF Regulations

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... (CONTINUED) SOLID WASTES (CONTINUED) STANDARDS FOR THE MANAGEMENT OF SPECIFIC HAZARDOUS WASTES AND SPECIFIC TYPES OF HAZARDOUS WASTE MANAGEMENT FACILITIES Pt. 266, App. IX Appendix IX to Part 266—Methods Manual... Protection Agency regulations for boilers and industrial furnaces (BIFs) burning hazardous waste (see 40...

  5. Franklin v. Gwinnett County Public Schools and Its Impact on Title IX Enforcement.

    ERIC Educational Resources Information Center

    Vargyas, Ellen J.

    1993-01-01

    A court decision allowing monetary damages for intentional violations of Title IX of the Education Amendments of 1972 is seen as dramatically changing enforcement of the principal federal law against sex discrimination. Its treatment of sexual harassment is also considered. Implications for Title IX enforcement in colleges and universities are…

  6. Sexual Harassment and Student Rights: The Supreme Court Expands Title IX Remedies.

    ERIC Educational Resources Information Center

    Russo, Charles J.; And Others

    1992-01-01

    Ruling in "Franklin," the Supreme Court found in favor of a high school student who alleged that she had been subjected to sexual harassment in violation of Title IX of the Educational Amendments of 1972. Inquires about the nature and scope of damages available under Title IX. Concludes with policy considerations for administrators. (MLF)

  7. Prediction of Pathway Activation by Xenobiotic-Responsive Transcription Factors in the Mouse Liver

    EPA Science Inventory

    Many drugs and environmentally-relevant chemicals activate xenobioticresponsive transcription factors (TF). Identification of target genes of these factors would be useful in predicting pathway activation in in vitro chemical screening. Starting with a large compendium of Affymet...

  8. Atomic Data and Spectral Line Intensities for Ca IX

    NASA Technical Reports Server (NTRS)

    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.

  9. Ares I-X Separation and Reentry Trajectory Analyses

    NASA Technical Reports Server (NTRS)

    Tartabini, Paul V.; Starr, Brett R.

    2011-01-01

    The Ares I-X Flight Test Vehicle was launched on October 28, 2009 and was the first and only test flight of NASA s two-stage Ares I launch vehicle design. The launch was successful and the flight test met all of its primary and secondary objectives. This paper discusses the stage separation and reentry trajectory analysis that was performed in support of the Ares I-X test flight. Pre-flight analyses were conducted to assess the risk of stage recontact during separation, to evaluate the first stage flight dynamics during reentry, and to define the range safety impact ellipses of both stages. The results of these pre-flight analyses were compared with available flight data. On-board video taken during flight showed that the flight test vehicle successfully separated without any recontact. Reconstructed trajectory data also showed that first stage flight dynamics were well characterized by pre-flight Monte Carlo results. In addition, comparisons with flight data indicated that the complex interference aerodynamic models employed in the reentry simulation were effective in capturing the flight dynamics during separation. Finally, the splash-down locations of both stages were well within predicted impact ellipses.

  10. Anisotropic cosmology and inflation from a tilted Bianchi IX model

    NASA Astrophysics Data System (ADS)

    Sundell, P.; Koivisto, T.

    2015-12-01

    The dynamics of the tilted axisymmetric Bianchi IX cosmological models are explored allowing energy flux in the source fluid. The Einstein equations and the continuity equation are presented treating the equation of state w and the tilt angle of the fluid λ as time-dependent functions, but when analyzing the phase space w and λ are considered free parameters and the shear, the vorticity and the curvature of the spacetime span a three-dimensional phase space that contains seven fixed points. One of them is an attractor that inflates the universe anisotropically, thus providing a counterexample to the cosmic no-hair conjecture. Also, examples of realistic though fine-tuned cosmologies are presented wherein the rotation can become significant towards the present epoch but the shear stays within the observational bounds. The examples suggest that the model used here can explain the parity-violating anomalies of the cosmic microwave background. The result significantly differs from an earlier study, where a nonaxisymmetric Bianchi IX type model with a tilted perfect dust source was found to induce too much shear for observationally significant vorticity.

  11. Ares I-X Upper Stage Simulator Residual Stress Analysis

    NASA Technical Reports Server (NTRS)

    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.

  12. Releasing the brakes in coagulation Factor IXa by co-operative maturation of the substrate-binding site.

    PubMed

    Kristensen, Line Hyltoft; Olsen, Ole H; Blouse, Grant E; Brandstetter, Hans

    2016-08-01

    Coagulation Factor IX is positioned at the merging point of the intrinsic and extrinsic blood coagulation cascades. Factor IXa (activated Factor IX) serves as the trigger for amplification of coagulation through formation of the so-called Xase complex, which is a ternary complex of Factor IXa, its substrate Factor X and the cofactor Factor VIIIa on the surface of activated platelets. Within the Xase complex the substrate turnover by Factor IXa is enhanced 200000-fold; however, the mechanistic and structural basis for this dramatic enhancement remains only partly understood. A multifaceted approach using enzymatic, biophysical and crystallographic methods to evaluate a key set of activity-enhanced Factor IXa variants has demonstrated a delicately balanced bidirectional network. Essential molecular interactions across multiple regions of the Factor IXa molecule co-operate in the maturation of the active site. This maturation is specifically facilitated by long-range communication through the Ile(212)-Ile(213) motif unique to Factor IXa and a flexibility of the 170-loop that is further dependent on the conformation in the Cys(168)-Cys(182) disulfide bond. Ultimately, the network consists of compensatory brakes (Val(16) and Ile(213)) and accelerators (Tyr(99) and Phe(174)) that together allow for a subtle fine-tuning of enzymatic activity. PMID:27208168

  13. Determination of factor X activator in the venom of the saw-scaled viper (Echis carinatus).

    PubMed

    Stocker, K; Fischer, H; Brogli, M

    1986-01-01

    Factor X activator in Echis carinatus venom was determined by incubating the zymogen 'factor X' with venom, interrupting the activation process by ethylenediaminetetraacetic acid and measuring the generated proteinase 'factor Xa' by means of a synthetic chromogenic substrate. A comparison of factor X- and prothrombin-activating potencies in E. carinatus venoms of five different geographic origins revealed no correlation between these two procoagulant activities. PMID:3715901

  14. "What do I think about Title IX?" Voices from a university community.

    PubMed

    Paule-Koba, Amanda L; Harris, Othello; Freysinger, Valeria J

    2013-03-01

    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 changes it incurred. Using a gendered lens (Birrell, 2000) and qualitative interviews with 13 individuals connected to the university community, insight was gained into their perceptions of Title IX and women's and men's athletic opportunities. The theme of opportunity gained and lost was central to how these individuals perceive Title IX and resonates with the liberal (feminist) concerns out of which Title IX was born. PMID:23611015

  15. Ares I-X Flight Test Vehicle: Stack 5 Modal Test

    NASA Technical Reports Server (NTRS)

    Buehrle, Ralph D.; Templeton, Justin D.; Reaves, Mercedes C.; Horta, Lucas G.; Gaspar, James L.; Bartolotta, Paul A.; Parks, Russel A.; Lazor, Danel R.

    2010-01-01

    Ares I-X was the first flight test vehicle used in the development of NASA's Ares I crew launch vehicle. The Ares I-X used a 4-segment reusable solid rocket booster from the Space Shuttle heritage with mass simulators for the 5th segment, upper stage, crew module and launch abort system. Three modal tests were defined to verify the dynamic finite element model of the Ares I-X flight test vehicle. Test configurations included two partial stacks and the full Ares I-X flight test vehicle on the Mobile Launcher Platform. This report focuses on the first modal test that was performed on the top section of the vehicle referred to as Stack 5, which consisted of the spacecraft adapter, service module, crew module and launch abort system simulators. This report describes the test requirements, constraints, pre-test analysis, test operations and data analysis for the Ares I-X Stack 5 modal test.

  16. Collagen type IX from human cartilage: a structural profile of intermolecular cross-linking sites.

    PubMed Central

    Diab, M; Wu, J J; Eyre, D R

    1996-01-01

    Type IX collagen, a quantitatively minor collagenous component of cartilage, is known to be associated with and covalently cross-linked to type II collagen fibrils in chick and bovine cartilage. Type IX collagen molecules have also been shown to form covalent cross-links with each other in bovine cartilage. In the present study we demonstrate by structural analysis and location of cross-linking sites that, in human cartilage, type IX collagen is covalently cross-linked to type II collagen and to other molecules of type IX collagen. We also present evidence that, if the proteoglycan form of type IX collagen is present in human cartilage, it can only be a minor component of the matrix, similar to findings with bovine cartilage. PMID:8660302

  17. Ares I-X Flight Test Vehicle:Stack 1 Modal Test

    NASA Technical Reports Server (NTRS)

    Buehrle, Ralph D.; Templeton, Justin D.; Reaves, Mercedes C.; Horta, Lucas G.; Gaspar, James L.; Bartolotta, Paul A.; Parks, Russel A.; Lazor, Daniel R.

    2010-01-01

    Ares I-X was the first flight test vehicle used in the development of NASA s Ares I crew launch vehicle. The Ares I-X used a 4-segment reusable solid rocket booster from the Space Shuttle heritage with mass simulators for the 5th segment, upper stage, crew module and launch abort system. Three modal tests were defined to verify the dynamic finite element model of the Ares I-X flight test vehicle. Test configurations included two partial stacks and the full Ares I-X flight test vehicle on the Mobile Launcher Platform. This report focuses on the second modal test that was performed on the middle section of the vehicle referred to as Stack 1, which consisted of the subassembly from the 5th segment simulator through the interstage. This report describes the test requirements, constraints, pre-test analysis, test operations and data analysis for the Ares I-X Stack 1 modal test.

  18. Water Activity Limits the Hygroscopic Growth Factor of Organic Aerosols

    NASA Astrophysics Data System (ADS)

    Rodriguez, L. I.; Cabrera, J. A.; Golden, D.; Tabazadeh, A.

    2007-12-01

    In this work we study the hygroscopic behavior of organic aerosols, which has important implications for Earth's climate. The hygroscopic growth factor (HGF) is defined as the ratio of the diameter of a spherical particle when it is exposed to dry conditions to that at humid conditions. We present a new formulation to express the HGF of an aerosol particle as a function of water activity (aw) in the aqueous phase. This new formulation matches reported HGFs for common inorganic salts and water-miscible organic particles that are known to deliquesce into aqueous drops at high relative humidities (RH). Many studies use tandem differential mobility analyzers (TDMA) to determine the HGF of organic aerosols. For example, Brooks et al. used a TDMA to measure a HGF of 1.2 for 2 μm phthalic acid (PA) particles at 90% RH (aw= 0.9). However, water activity limits the growth of a particle that can be attributed to water uptake. We have assembled a vapor pressure apparatus to measure aw of aqueous solutions at room temperature. Measured water activities for PA, used in our growth formulation, yield a HGF of ~ 1.0005 for 2 μm PA particles at 90% RH. Comparing our results against Brooks et al. suggests that TDMA experiments may grossly overestimate the HGF of PA particles since water activity limits this growth to below 1.0005. Alternatively, we suggest that the adsorption of a negligible mass of water by a highly porous PA particle can lead to an apparent growth in particle size by changing its morphology. Other studies also use TDMAs to measure HGFs of secondary organic aerosols (SOAs). HGFs reported for SOAs are very similar to PA, suggesting that the observed growth may be due to morphological changes in particle size rather than water uptake as commonly assumed. We built a smog chamber where an organic precursor, such as d-limonene, reacts with nitrogen oxides under UV radiation to produce SOAs. We compare the HGFs for SOAs obtained with our method to those obtained with

  19. Biochemistry of platelet-activating factor: A unique class of biologically active phospholipids

    SciTech Connect

    Snyder, F. )

    1989-01-01

    This brief overview describes the chemical features of this unique bioactive phospholipid that possesses biologic properties identical to platelet-activating factor (PAF) and an antihypertensive polar renal lipid (APRL). The current understanding of PAF metabolism and its regulation are emphasized, particularly in the context of explaining the enzymatic source of PAF in physiologic vs pharmacologic processes. Also included are brief accounts of the biologic properties, structural-functional relationships, antagonists, receptors and mode of action of PAF.

  20. Comparative Evaluation of the Antibacterial Efficacy of Type II Glass lonomer Cement, Type IX Glass lonomer Cement, and AMALGOMER™ Ceramic Reinforcement by Modified “Direct Contact Test”: An in vitro Study

    PubMed Central

    Assudani, Harsha G; Patil, Vidyavathi; Kukreja, Pratibha; Uppin, Chaitanya; Thakkar, Prachi

    2016-01-01

    ABSTRACT Background: Streptococcus mutans (ATCC25175) has a profound effect on the incidence of dental decay in the human population. Many studies have been performed to assess the antimicrobial activity of different cements. However, little or no information is available about the antibacterial properties of Type II glass ionomer cement (GIC), Type IX GIC, and AMALGOMER™ ceramic reinforcement (CR). Aim: To comparatively evaluate the antibacterial activity of Type II GIC, Type IX GIC, and AMALGOMER™ CR by modified direct contact test. Materials and methods: The total sample size was 72 which was divided into four study groups. Six wells were coated by each: Type II GIC, Type IX GIC, AMALGOMER™ CR, and control group (only S. mutans). Statistical analysis was done using analysis of variance and the intergroup comparison was done using post hoc Tukey test. Results: AMALGOMER™ CR was found to have a better antibacterial effect as compared with Type II and IX GIC. Conclusion: AMALGOMER™ CR can serve as a valuable cement in pediatric dentistry due to its anticariogenic property. How to cite this article: Hugar SM, Assudani HG, Patil V, Kukreja P, Uppin C, Thakkar P. Comparative Evaluation of the Antibacterial Efficacy of Type II Glass lonomer Cement, Type IX Glass Ionomer Cement, and AMALGOMER™ Ceramic Reinforcement by Modified “Direct Contact Test”: An in vitro Study. Int J Clin Pediatr Dent 2016;9(2):114-117. PMID:27365930

  1. 5-Aminolevulinic Acid-Induced Protoporphyrin IX Fluorescence in Meningioma: Qualitative and Quantitative Measurements In Vivo

    PubMed Central

    Valdes, Pablo A.; Bekelis, Kimon; Harris, Brent T.; Wilson, Brian C.; Leblond, Frederic; Kim, Anthony; Simmons, Nathan E.; Erkmen, Kadir; Paulsen, Keith D.; Roberts, David W.

    2014-01-01

    BACKGROUND The use of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence has shown promise as a surgical adjunct for maximizing the extent of surgical resection in gliomas. To date, the clinical utility of 5-ALA in meningiomas is not fully understood, with most descriptive studies using qualitative approaches to 5-ALA-PpIX. OBJECTIVE To assess the diagnostic performance of 5-ALA-PpIX fluorescence during surgical resection of meningioma. METHODS ALA was administered to 15 patients with meningioma undergoing PpIX fluorescence-guided surgery at our institution. At various points during the procedure, the surgeon performed qualitative, visual assessments of fluorescence by using the surgical microscope, followed by a quantitative fluorescence measurement by using an intra-operative probe. Specimens were collected at each point for subsequent neuropathological analysis. Clustered data analysis of variance was used to ascertain a difference between groups, and receiver operating characteristic analyses were performed to assess diagnostic capabilities. RESULTS Red-pink fluorescence was observed in 80% (12/15) of patients, with visible fluorescence generally demonstrating a strong, homogenous character. Quantitative fluorescence measured diagnostically significant PpIX concentrations (CPpIx) in both visibly and nonvisibly fluorescent tissues, with significantly higher CPpIx in both visibly fluorescent (P < .001) and tumor tissue (P = .002). Receiver operating characteristic analyses also showed diagnostic accuracies up to 90% for differentiating tumor from normal dura. CONCLUSION ALA-induced PpIX fluorescence guidance is a potential and promising adjunct in accurately detecting neoplastic tissue during meningioma resective surgery. These results suggest a broader reach for PpIX as a biomarker for meningiomas than was previously noted in the literature. PMID:23887194

  2. Supreme Court Says Title IX Covers Employment but Raises a Serious Question about the Future Impact of the Law.

    ERIC Educational Resources Information Center

    Flygare, Thomas J.

    1982-01-01

    The Supreme Court recently decided that Title IX covers employment practices in schools and colleges; however, portions of the Court's decision (whether Title IX provides "institutionwide" or "program-specific" coverage) raise serious questions about the future of Title IX as a force for sex equity in education. (Author/MLF)

  3. 12 CFR 900.3 - Terms relating to other entities and concepts used throughout 12 CFR chapter IX.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... used throughout 12 CFR chapter IX. 900.3 Section 900.3 Banks and Banking FEDERAL HOUSING FINANCE BOARD... to other entities and concepts used throughout 12 CFR chapter IX. As used throughout this chapter, the following terms relating to other entities and concepts used throughout 12 CFR chapter IX have...

  4. 12 CFR 900.3 - Terms relating to other entities and concepts used throughout 12 CFR chapter IX.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... used throughout 12 CFR chapter IX. 900.3 Section 900.3 Banks and Banking FEDERAL HOUSING FINANCE BOARD... to other entities and concepts used throughout 12 CFR chapter IX. As used throughout this chapter, the following terms relating to other entities and concepts used throughout 12 CFR chapter IX have...

  5. 12 CFR 900.3 - Terms relating to other entities and concepts used throughout 12 CFR chapter IX.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... used throughout 12 CFR chapter IX. 900.3 Section 900.3 Banks and Banking FEDERAL HOUSING FINANCE BOARD... to other entities and concepts used throughout 12 CFR chapter IX. As used throughout this chapter, the following terms relating to other entities and concepts used throughout 12 CFR chapter IX have...

  6. 12 CFR 900.3 - Terms relating to other entities and concepts used throughout 12 CFR chapter IX.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... used throughout 12 CFR chapter IX. 900.3 Section 900.3 Banks and Banking FEDERAL HOUSING FINANCE BOARD... to other entities and concepts used throughout 12 CFR chapter IX. As used throughout this chapter, the following terms relating to other entities and concepts used throughout 12 CFR chapter IX have...

  7. Longer-acting clotting factor concentrates for hemophilia.

    PubMed

    Powell, J S

    2015-06-01

    Hemophilia, when severe, leads to spontaneous life-threatening bleeding episodes. Current therapy requires frequent intravenous infusions. Most patients must limit their physical activities to avoid bleeding when the factor activity levels are below normal. In 2014, new therapeutic factor VIII and IX products were approved in Canada and the U.S. Over the next couple of years, other new factor products will likely be approved. These new factors have been engineered to have improved pharmacokinetic properties, including extended half-life in circulation, thus providing major therapeutic advances for patients with hemophilia. In the completed clinical trials, over 700 patients have successfully used these longer acting products regularly for more than one year. These promising new therapies should allow patients with hemophilia to use fewer infusions to prevent spontaneous bleeding or to treat bleeding episodes, and to provide appropriate clotting factor levels for different physical activities. PMID:26149018

  8. Platelet-activating factor and laser trauma of the iris

    SciTech Connect

    Verbey, N.L.; Van Delft, J.L.; Van Haeringen, N.J.; Braquet, P.

    1989-06-01

    Local application of platelet-activating factor (PAF) on the rabbit eye caused a dose-dependent significant increase in intraocular pressure (IOP). After laser irradiation of the iris the IOP showed a hypertensive phase of about 3 hr. Prophylactic treatment with the PAF antagonist BN 52021 but not with indomethacin abolished the hypertensive phase. Elevated levels of protein (10.6 +/- 0.9 g/l) and prostaglandin E2 (PGE2, 1.7 +/- 0.2 ng/ml) were measured in the aqueous humor 2 hr after laser irradiation of the iris. Prophylactic treatment with BN 52021 showed lower levels of protein (6.1 +/- 0.7) and PGE2 (1.1 +/- 0.02); with indomethacin pretreatment the level of protein was 3.4 +/- 0.7 g/l and of PGE2 0.10 +/- 0.02 ng/ml. A role of PAF as a mediator in ocular inflammatory response is suggested.

  9. Formaldehyde activation factor, tetrahydromethanopterin, a coenzyme of methanogenesis

    SciTech Connect

    Escalante-Semerena, J.C.; Leigh, J.A.; Rinehart, K.L. Jr.; Wolfe, R.S.

    1984-04-01

    An oxygen-labile formaldehyde activation factor (FAF) was isolated in highly purified form by use of anoxic fractionation procedures. The molecular weight of FAF was determined to be 776 and that of methanopterin (MPT) 772 by fast-atom-bombardment mass spectrometry (FABMS). High-resolution FABMS measurements on MPT and FAF indicated molecular formulas of C/sub 30/H/sub 41/N/sub 6/O/sub 16/P and C/sub 30/H/sub 45/N/sub 6/O/sub 16/P, respectively. The presence of phosphorus was confirmed by 100-MHz /sup 31/P NMR. The 360-MHz /sup 1/H NMR spectrum of FAF in deuterium oxide was similar to that of MPT. A functional relationship between MPT and FAF was documented; both compounds stimulated the reductive demethylation of 2-(methylthio)ethanesulfonic acid (CH/sub 3/-S-CoM) to CH/sub 4/ when formaldehyde oxidation provided a source of electrons, and FAF replaced MPT in the CH/sub 3/-S-CoM-stimulated conversion of CO/sub 2/ to CH/sub 4/ under H/sub 2/ (the RPG effect). MPT was enzymically converted to FAF during the reduction of CH/sub 3/-S-CoM, and HCHO to CH/sub 4/ under H/sub 2/. Evidence indicates that FAF is tetrahydromethanopterin. 14 references, 8 figures.

  10. Recombinant activated factor VII in post partum haemorrhage

    PubMed Central

    Magon, Navneet; Babu, K. M.; Kapur, Krishan; Chopra, Sanjiv; Joneja, Gurdarshan Singh

    2013-01-01

    Post-partum haemorrhage (PPH) is a life-threatening obstetric complication and the leading cause of maternal death. Any bleeding that results in or could result in haemodynamic instability, if untreated, must be considered as PPH. There is no controversy about the need for prevention and treatment of PPH. The keystone of management of PPH entails first, non-invasive and nonsurgical methods and then invasive and surgical methods. However, mortality remains high. Therefore, new advancements in the treatment are most crucial. One such advancement has been the use of recombinant activated factor VII (rFVIIa) in PPH. First used 12 years back in PPH, this universal haemostatic agent has been effectively used in controlling PPH. The best available indicator of rFVIIa efficacy is the arrest of haemorrhage, which is judged by visual evidence and haemodynamic stabilization. It also reduces costs of therapy and the use of blood components in massive PPH. In cases of intractable PPH with no other obvious indications for hysterectomy, administration of rFVIIa should be considered before surgery. We share our experience in a series of cases of PPH, successfully managed using rFVIIa. PMID:24403703

  11. Regulating the regulators: modulators of transcription factor activity.

    PubMed

    Everett, Logan; Hansen, Matthew; Hannenhalli, Sridhar

    2010-01-01

    Gene transcription is largely regulated by DNA-binding transcription factors (TFs). However, the TF activity itself is modulated via, among other things, post-translational modifications (PTMs) by specific modification enzymes in response to cellular stimuli. TF-PTMs thus serve as "molecular switchboards" that map upstream signaling events to the downstream transcriptional events. An important long-term goal is to obtain a genome-wide map of "regulatory triplets" consisting of a TF, target gene, and a modulator gene that specifically modulates the regulation of the target gene by the TF. A variety of genome-wide data sets can be exploited by computational methods to obtain a rough map of regulatory triplets, which can guide directed experiments. However, a prerequisite to developing such computational tools is a systematic catalog of known instances of regulatory triplets. We first describe PTM-Switchboard, a recent database that stores triplets of genes such that the ability of one gene (the TF) to regulate a target gene is dependent on one or more PTMs catalyzed by a third gene, the modifying enzyme. We also review current computational approaches to infer regulatory triplets from genome-wide data sets and conclude with a discussion of potential future research. PTM-Switchboard is accessible at http://cagr.pcbi.upenn.edu/PTMswitchboard / PMID:20827600

  12. Influence of Environmental Factors on Feammox Activity in Soil Environments

    NASA Astrophysics Data System (ADS)

    Huang, S.; Jaffe, P. R.

    2015-12-01

    The oxidation of ammonium (NH4+) under iron reducing conditions, referred to as Feammox, has been described in recent years by several investigators. The environmental characteristics in which the Feammox process occurs need to be understood in order to determine its contribution to the nitrogen cycle. In this study, a total of 66 locations were selected covering 4 different types of soils/sediments: wetland soils (W), river sediments (R), forest soils (F), and paddy soils (P) from several locations in central New Jersey, at Tims Branch at Savannah River in South Carolina, both in the Unities States, and at several locations in the Guangdong province in China. Though soil chemical analyses, serial culturing experiments, analysis of microbial communities, and using a canonical correspondence analysis, the occurrence of the Feammox reaction and the presence of Acidimicrobiaceae bacterium A6, which plays a key role in the Feammox process(1), were found in 17 samples. Analyses showed that the soil pH, as well as its Fe(III) and NH4+ content were the most important factors controlling the distribution of these Feammox microorganisms. Based on the results, soils in the subtropical forests and soils that are near agricultural areas could be Feammox hotspot. Under the conditions that favor the presence and activity of Feammox microorganisms and their oxidation of NH4+, denitrification bacteria were also active. However, the presence of nitrous oxide (N2O) reducers was limited under these conditions, implying that at locations where the Feammox process is active, conditions are favoring a higher ratio of N2O: N2 as the nitrogen (N) end products. Incubations of soils where the presence of Acidimicrobiaceae bacterium A6 was detected, were conducted for 120 days under two different DO levels (DO < 0.02 mg/L and DO = 0.8~1.0 mg/L) showing comparable amounts of NH4+ oxidation. In the incubations with DO < 0.02 mg/L, the proportion of Acidimicrobiaceae bacteria increased and

  13. Ethylene Response Factors Are Controlled by Multiple Harvesting Stresses in Hevea brasiliensis.

    PubMed

    Putranto, Riza-Arief; Duan, Cuifang; Kuswanhadi; Chaidamsari, Tetty; Rio, Maryannick; Piyatrakul, Piyanuch; Herlinawati, Eva; Pirrello, Julien; Dessailly, Florence; Leclercq, Julie; Bonnot, François; Tang, Chaorong; Hu, Songnian; Montoro, Pascal

    2015-01-01

    Tolerance of recurrent mechanical wounding and exogenous ethylene is a feature of the rubber tree. Latex harvesting involves tapping of the tree bark and ethephon is applied to increase latex flow. Ethylene is an essential element in controlling latex production. The ethylene signalling pathway leads to the activation of Ethylene Response Factor (ERF) transcription factors. This family has been identified in Hevea brasiliensis. This study set out to understand the regulation of ERF genes during latex harvesting in relation to abiotic stress and hormonal treatments. Analyses of the relative transcript abundance were carried out for 35 HbERF genes in latex, in bark from mature trees and in leaves from juvenile plants under multiple abiotic stresses. Twenty-one HbERF genes were regulated by harvesting stress in laticifers, revealing an overrepresentation of genes in group IX. Transcripts of three HbERF-IX genes from HbERF-IXc4, HbERF-IXc5 and HbERF-IXc6 were dramatically accumulated by combining wounding, methyl jasmonate and ethylene treatments. When an ethylene inhibitor was used, the transcript accumulation for these three genes was halted, showing ethylene-dependent induction. Subcellular localization and transactivation experiments confirmed that several members of HbERF-IX are activator-type transcription factors. This study suggested that latex harvesting induces mechanisms developed for the response to abiotic stress. These mechanisms probably depend on various hormonal signalling pathways. Several members of HbERF-IX could be essential integrators of complex hormonal signalling pathways in Hevea. PMID:25906196

  14. Ixora coccinea Enhances Cutaneous Wound Healing by Upregulating the Expression of Collagen and Basic Fibroblast Growth Factor.

    PubMed

    Upadhyay, Aadesh; Chattopadhyay, Pronobesh; Goyary, Danswrang; Mitra Mazumder, Papiya; Veer, Vijay

    2014-01-01

    Background. Ixora coccinea L. (Rubiaceae) has been documented for traditional use in hypertension, menstrual irregularities, sprain, chronic ulcer, and skin diseases. In the present study, I. coccinea was subjected to in vitro and in vivo wound healing investigation. Methods. Petroleum ether, chloroform, methanol, and water sequential I. coccinea leaves extracts were evaluated for in vitro antioxidant, antimicrobial, and fibroblast proliferation activities. The promising I. coccinea methanol extract (IxME) was screened for in vivo wound healing activity in Wistar rat using circular excision model. Wound contraction measurement, hydroxyproline quantification, and western blot for collagen type III (COL3A1), basic fibroblast growth factor (bFGF), and Smad-2, -3, -4, and -7 was performed with 7-day postoperative wound granulation tissue. Gentamicin sulfate (0.01% w/w) hydrogel was used as reference standard. Results. IxME showed the potent antimicrobial, antioxidant activities, with significant fibroblast proliferation inducing activity, as compared to all other extracts. In vivo study confirmed the wound healing accelerating potential of IxME, as evidenced by faster wound contraction, higher hydroxyproline content, and improved histopathology of granulation tissue. Western blot analysis revealed that the topical application of I. coccinea methanol extract stimulates the fibroblast growth factor and Smad mediated collagen production in wound tissue. PMID:24624303

  15. Ixora coccinea Enhances Cutaneous Wound Healing by Upregulating the Expression of Collagen and Basic Fibroblast Growth Factor

    PubMed Central

    Upadhyay, Aadesh; Chattopadhyay, Pronobesh; Goyary, Danswrang; Mitra Mazumder, Papiya; Veer, Vijay

    2014-01-01

    Background. Ixora coccinea L. (Rubiaceae) has been documented for traditional use in hypertension, menstrual irregularities, sprain, chronic ulcer, and skin diseases. In the present study, I. coccinea was subjected to in vitro and in vivo wound healing investigation. Methods. Petroleum ether, chloroform, methanol, and water sequential I. coccinea leaves extracts were evaluated for in vitro antioxidant, antimicrobial, and fibroblast proliferation activities. The promising I. coccinea methanol extract (IxME) was screened for in vivo wound healing activity in Wistar rat using circular excision model. Wound contraction measurement, hydroxyproline quantification, and western blot for collagen type III (COL3A1), basic fibroblast growth factor (bFGF), and Smad-2, -3, -4, and -7 was performed with 7-day postoperative wound granulation tissue. Gentamicin sulfate (0.01% w/w) hydrogel was used as reference standard. Results. IxME showed the potent antimicrobial, antioxidant activities, with significant fibroblast proliferation inducing activity, as compared to all other extracts. In vivo study confirmed the wound healing accelerating potential of IxME, as evidenced by faster wound contraction, higher hydroxyproline content, and improved histopathology of granulation tissue. Western blot analysis revealed that the topical application of I. coccinea methanol extract stimulates the fibroblast growth factor and Smad mediated collagen production in wound tissue. PMID:24624303

  16. ALA-mediated photodynamic therapy of experimental malignant glioma in the BD-IX rat model

    NASA Astrophysics Data System (ADS)

    Hirschberg, Henry; Angell-Petersen, Even; Peng, Qian; Sun, Chung-Ho; Sorensen, Dag R.; Carper, Steven W.; Madsen, Steen J.

    2005-04-01

    Introduction: Failure of treatment for high grade gliomas is usually due to local recurrence at the site of surgical resec-tion indicating that a more aggressive form of local therapy could be of benefit. Photodynamic therapy (PDT) is a local form of treatment involving the administration of a tumor-localizing photosensitizing drug that is activated by light of a specific wavelength The results of in vitro experiments indicated that PDT, given at low fluence rates was substantially more effective at inhibiting glioma spheroid growth than short term high fluence rate regimes. This prompted the initia-tion of in vivo studies of low fluence rate 5-aminolevulinic acid (ALA) PDT in a rat glioma model. Methods:BT4C cell line tumors were established in the brains of inbred BD- IX rats. Eighteen days following tumor induction the animals were injected with 125 mg/kg ALA ip. and four hours later light treatment at various fluences and fluence rates were given after the introduction of an optical fiber. Tumor histology and animal survival were examined. Results: In vitro experiments verified that the cell line was sensitive to ALA PDT. Microfluorometry of frozen tissue sections showed that PpIX is produced with a greater than 20:1 tumor to normal tissue selectivity ratio four hours after ALA injection. Histological examination demonstrated neutrophil infiltration and tumor central necrosis in low fluence rate treated tumors. Conclusions: Low fluence rate long term ALA mediated PDT had a more pronounced effect on tumor histology than single shot short duration treatments at similar total fluence levels.

  17. Hepatocyte growth factor, hepatocyte growth factor activator and arginine in a rat fulminant colitis model

    PubMed Central

    Zwintscher, Nathan P.; Shah, Puja M.; Salgar, Shashikumar K.; Newton, Christopher R.; Maykel, Justin A.; Samy, Ahmed; Jabir, Murad; Steele, Scott R.

    2016-01-01

    Introduction Dextran sodium sulfate (DSS) is commonly used to induce a murine fulminant colitis model. Hepatocyte growth factor (HGF) has been shown to decrease the symptoms of inflammatory bowel disease (IBD) but the effect of its activator, HGFA, is not well characterized. Arginine reduces effects of oxidative stress but its effect on IBD is not well known. The primary aim is to determine whether HGF and HGFA, or arginine will decrease IBD symptoms such as pain and diarrhea in a DSS-induced fulminant colitis murine model. Methods A severe colitis was induced in young, male Fischer 344 rats with 4% (w/v) DSS oral solution for seven days; rats were sacrificed on day 10. Rats were divided into five groups of 8 animals: control, HGF (700 mcg/kg/dose), HGF and HGFA (10 mcg/dose), HGF and arginine, and high dose HGF (2800 mcg/kg/dose). Main clinical outcomes were pain, diarrhea and weight loss. Blinded pathologists scored the terminal ileum and distal colon. Results DSS reliably induced severe active colitis in 90% of animals (n = 36/40). There were no differences in injury scores between control and treatment animals. HGF led to 1.38 fewer days in pain (p = 0.036), while arginine led to 1.88 fewer days of diarrhea (P = 0.017) compared to controls. 88% of HGFA-treated rats started regaining weight (P < 0.001). Discussion/Conclusion Although treatment was unable to reverse fulminant disease, HGF and arginine were associated with decreased days of pain and diarrhea. These clinical interventions may reduce associated symptoms for severe IBD patients, even when urgent surgical intervention remains the only viable option. PMID:27144006

  18. The molecular biology and nomenclature of the activating transcription factor/cAMP responsive element binding family of transcription factors: activating transcription factor proteins and homeostasis.

    PubMed

    Hai, T; Hartman, M G

    2001-07-25

    The mammalian ATF/CREB family of transcription factors represents a large group of basic region-leucine zipper (bZip) proteins which was originally defined in the late 1980s by their ability to bind to the consensus ATF/CRE site 'TGACGTCA'. Over the past decade, cDNA clones encoding identical or homologous proteins have been isolated by different laboratories and given different names. These proteins can be grouped into subgroups according to their amino acid similarity. In this review, we will briefly describe the classification of these proteins with a historical perspective of their nomenclature. We will then review three members of the ATF/CREB family of proteins: ATF3, ATF4 and ATF6. We will address four issues for each protein: (a) homologous proteins and alternative names, (b) dimer formation with other bZip proteins, (c) transcriptional activity, and (d) potential physiological functions. Although the name Activating Transcription Factor (ATF) implies that they are transcriptional activators, some of these proteins are transcriptional repressors. ATF3 homodimer is a transcriptional repressor and ATF4 has been reported to be either an activator or a repressor. We will review the reports on the transcriptional activities of ATF4, and propose potential explanations for the discrepancy. Although the physiological functions of these proteins are not well understood, some clues can be gained from studies with different approaches. When the data are available, we will address the following questions. (a) How is the expression (at the mRNA level or protein level) regulated? (b) How are the transcriptional activities regulated? (c) What are the interacting proteins (other than bZip partners)? (d) What are the consequences of ectopically expressing the gene (gain-of-function) or deleting the gene (loss-of-function)? Although answers to these questions are far from being complete, together they provide clues to the functions of these ATF proteins. Despite the

  19. Mobilization of hepatic calcium pools by platelet activating factor

    SciTech Connect

    Lapointe, D.S.; Hanahan, D.J.; Olson, M.S.

    1987-03-24

    In the perfused rat liver, platelet activating factor, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC), infusion produces an extensive but transient glycogenolytic response which at low AGEPC concentrations is markedly dependent upon the perfusate calcium levels. The role of calcium in the glycogenolytic response of the liver to AGEPC was investigated by assessing the effect of AGEPC on various calcium pools in the intact liver. Livers from fed rats were equilibrated with /sup 45/Ca/sup 2 +/, and the kinetics of /sup 45/Ca/sup 2 +/ efflux were determined in control, AGEPC-stimulated, and phenylephrine-stimulated livers during steady-state washout of /sup 45/Ca/sup 2 +/. AGEPC treatment had only a slight if any effect on the pattern of steady-state calcium efflux from the liver, as opposed to major perturbations in the pattern of calcium efflux effected by the ..cap alpha..-adrenergic agonist phenylephrine. Infusion of short pulses of AGEPC during the washout of /sup 45/Ca/sup 2 +/ from labeled livers caused a transient release of /sup 45/Ca/sup 2 +/ which was not abolished at low calcium concentrations in the perfusate. Infusion of latex beads, which are removed by the reticuloendothelial cells, caused the release of hepatic /sup 45/Ca/sup 2 +/ in a fashion similar to the case with AGEPC. The findings indicate that AGEPC does not perturb a major pool of calcium within the liver as occurs upon ..cap alpha..-adrenergic stimulation; it is likely that AGEPC mobilizes calcium from a smaller yet very important pool, very possibly from nonparenchymal cells in the liver.

  20. Activation of G Proteins by Guanine Nucleotide Exchange Factors Relies on GTPase Activity

    PubMed Central

    Stanley, Rob J.; Thomas, Geraint M. H.

    2016-01-01

    G proteins are an important family of signalling molecules controlled by guanine nucleotide exchange and GTPase activity in what is commonly called an ‘activation/inactivation cycle’. The molecular mechanism by which guanine nucleotide exchange factors (GEFs) catalyse the activation of monomeric G proteins is well-established, however the complete reversibility of this mechanism is often overlooked. Here, we use a theoretical approach to prove that GEFs are unable to positively control G protein systems at steady-state in the absence of GTPase activity. Instead, positive regulation of G proteins must be seen as a product of the competition between guanine nucleotide exchange and GTPase activity—emphasising a central role for GTPase activity beyond merely signal termination. We conclude that a more accurate description of the regulation of G proteins via these processes is as a ‘balance/imbalance’ mechanism. This result has implications for the understanding of intracellular signalling processes, and for experimental strategies that rely on modulating G protein systems. PMID:26986850

  1. Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

    SciTech Connect

    Morrison, W.J.

    1988-01-01

    The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. ({sup 3}H)PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 {mu}M. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF or thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRP{gamma}S and GDP{beta}S, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA).

  2. Quantum Bianchi Type IX Cosmology in K-Essence Theory

    NASA Astrophysics Data System (ADS)

    Espinoza-García, Abraham; Socorro, J.; Pimentel, Luis O.

    2014-09-01

    We use one of the simplest forms of the K-essence theory and apply it to the anisotropic Bianchi type IX cosmological model, with a barotropic perfect fluid modeling the usual matter content. We show that the most important contribution of the scalar field occurs during a stiff matter phase. Also, we present a canonical quantization procedure of the theory which can be simplified by reinterpreting the scalar field as an exotic part of the total matter content. The solutions to the Wheeler-DeWitt equation were found using the Bohmian formulation Bohm (Phys. Rev. 85(2):166, 1952) of quantum mechanics, employing the amplitude-real-phase approach Moncrief and Ryan (Phys. Rev. D 44:2375, 1991), where the ansatz for the wave function is of the form Ψ( ℓ μ )= χ( ϕ) W( ℓ μ ), where S is the superpotential function, which plays an important role in solving the Hamilton-Jacobi equation.

  3. Protoporphyrin IX: the Good, the Bad, and the Ugly.

    PubMed

    Sachar, Madhav; Anderson, Karl E; Ma, Xiaochao

    2016-02-01

    Protoporphyrin IX (PPIX) is ubiquitously present in all living cells in small amounts as a precursor of heme. PPIX has some biologic functions of its own, and PPIX-based strategies have been used for cancer diagnosis and treatment (the good). PPIX serves as the substrate for ferrochelatase, the final enzyme in heme biosynthesis, and its homeostasis is tightly regulated during heme synthesis. Accumulation of PPIX in human porphyrias can cause skin photosensitivity, biliary stones, hepatobiliary damage, and even liver failure (the bad and the ugly). In this work, we review the mechanisms that are associated with the broad aspects of PPIX. Because PPIX is a hydrophobic molecule, its disposition is by hepatic rather than renal excretion. Large amounts of PPIX are toxic to the liver and can cause cholestatic liver injury. Application of PPIX in cancer diagnosis and treatment is based on its photodynamic effects. PMID:26588930

  4. Characterization and standardization of tissue-simulating protoporphyrin IX optical phantoms

    NASA Astrophysics Data System (ADS)

    Marois, Mikael; Bravo, Jaime; Davis, Scott C.; Kanick, Stephen Chad

    2016-03-01

    Optical devices for measuring protoporphryin IX (PpIX) fluorescence in tissue are routinely validated by measurements in optical phantoms. Yet there exists limited data to form a consensus on the recipe for phantoms that both mimic the optical properties found in tissue and yield a reliable and stable relationship between PpIX concentration and the fluorescence remission intensity. This study characterizes the influence of multiple phantom components on PpIX fluorescence emission intensity, using Intralipid as the scattering source, bovine whole blood as the background absorber, and Tween as a surfactant to prevent PpIX aggregation. Optical measurements showed a linear proportionality (r>0.99) between fluorescence intensity and PpIX concentration (0.1 to 10 μg/mL) over a range of Intralipid (1 to 2%) and whole blood (0.5 to 3%) for phantoms containing low surfactant (≤0.1%), with fluorescence intensities and scattering and absorption properties stable for 5 h after mixing. The role of surfactant in PpIX phantoms was found to be complex, as aggregation was evident in aqueous nonturbid phantoms with no surfactant (0% Tween), and avoided in phantoms containing Intralipid as the scattering source with no additional or low amounts of added surfactant (≤0.1% Tween). Conversely, phantoms containing higher surfactant content (>0.1% Tween) and whole blood showed interactions that distorted the fluorescence emissions.

  5. Atomic data and spectral line intensities for Ca IX

    SciTech Connect

    Landi, E.; Bhatia, A.K.

    2014-11-15

    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, and 5 complexes, corresponding to 283 fine-structure levels in the 3l3l{sup ′}, 3l4l{sup ″}, and 3l5l{sup ‴} configurations, where l,l{sup ′}=s,p,d, l{sup ″}=s,p,d,f and l{sup ‴}=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 range of 10{sup 8}–10{sup 14}  cm{sup −3} and at an electron temperature of logT{sub e}(K)=5.8, corresponding to the maximum abundance of Ca IX. Spectral line intensities are calculated, and their diagnostic relevance is discussed.

  6. Ares I-X Range Safety Flight Envelope Analysis

    NASA Technical Reports Server (NTRS)

    Starr, Brett R.; Olds, Aaron D.; Craig, Anthony S.

    2011-01-01

    Ares I-X was the first test flight of NASA's Constellation Program's Ares I Crew Launch Vehicle designed to provide manned access to low Earth orbit. As a one-time test flight, the Air Force's 45th Space Wing required a series of Range Safety analysis data products to be developed for the specified launch date and mission trajectory prior to granting flight approval on the Eastern Range. The range safety data package is required to ensure that the public, launch area, and launch complex personnel and resources are provided with an acceptable level of safety and that all aspects of prelaunch and launch operations adhere to applicable public laws. The analysis data products, defined in the Air Force Space Command Manual 91-710, Volume 2, consisted of a nominal trajectory, three sigma trajectory envelopes, stage impact footprints, acoustic intensity contours, trajectory turn angles resulting from potential vehicle malfunctions (including flight software failures), characterization of potential debris, and debris impact footprints. These data products were developed under the auspices of the Constellation's Program Launch Constellation Range Safety Panel and its Range Safety Trajectory Working Group with the intent of beginning the framework for the operational vehicle data products and providing programmatic review and oversight. A multi-center NASA team in conjunction with the 45th Space Wing, collaborated within the Trajectory Working Group forum to define the data product development processes, performed the analyses necessary to generate the data products, and performed independent verification and validation of the data products. This paper outlines the Range Safety data requirements and provides an overview of the processes established to develop both the data products and the individual analyses used to develop the data products, and it summarizes the results of the analyses required for the Ares I-X launch.

  7. Ares I-X Range Safety Analyses Overview

    NASA Technical Reports Server (NTRS)

    Starr, Brett R.; Gowan, John W., Jr.; Thompson, Brian G.; Tarpley, Ashley W.

    2011-01-01

    Ares I-X was the first test flight of NASA's Constellation Program's Ares I Crew Launch Vehicle designed to provide manned access to low Earth orbit. As a one-time test flight, the Air Force's 45th Space Wing required a series of Range Safety analysis data products to be developed for the specified launch date and mission trajectory prior to granting flight approval on the Eastern Range. The range safety data package is required to ensure that the public, launch area, and launch complex personnel and resources are provided with an acceptable level of safety and that all aspects of prelaunch and launch operations adhere to applicable public laws. The analysis data products, defined in the Air Force Space Command Manual 91-710, Volume 2, consisted of a nominal trajectory, three sigma trajectory envelopes, stage impact footprints, acoustic intensity contours, trajectory turn angles resulting from potential vehicle malfunctions (including flight software failures), characterization of potential debris, and debris impact footprints. These data products were developed under the auspices of the Constellation's Program Launch Constellation Range Safety Panel and its Range Safety Trajectory Working Group with the intent of beginning the framework for the operational vehicle data products and providing programmatic review and oversight. A multi-center NASA team in conjunction with the 45th Space Wing, collaborated within the Trajectory Working Group forum to define the data product development processes, performed the analyses necessary to generate the data products, and performed independent verification and validation of the data products. This paper outlines the Range Safety data requirements and provides an overview of the processes established to develop both the data products and the individual analyses used to develop the data products, and it summarizes the results of the analyses required for the Ares I-X launch.

  8. Ares I-X Flight Test Vehicle Similitude to the Ares I Crew Launch Vehicle

    NASA Technical Reports Server (NTRS)

    Huebner, Lawrence D.; Smith, R. Marshall; Campbell, John R.; Taylor, Terry L.

    2009-01-01

    The Ares I-X Flight Test Vehicle is the first in a series of flight test vehicles that will take the Ares I Crew Launch Vehicle design from development to operational capability. Ares I-X is scheduled for a 2009 flight date, early enough in the Ares I design and development process so that data obtained from the flight can impact the design of Ares I before its Critical Design Review. Decisions on Ares I-X scope, flight test objectives, and FTV fidelity were made prior to the Ares I systems requirements being baselined. This was necessary in order to achieve a development flight test to impact the Ares I design. Differences between the Ares I-X and the Ares I configurations are artifacts of formulating this experimental project at an early stage and the natural maturation of the Ares I design process. This paper describes the similarities and differences between the Ares I-X Flight Test Vehicle and the Ares I Crew Launch Vehicle. Areas of comparison include the outer mold line geometry, aerosciences, trajectory, structural modes, flight control architecture, separation sequence, and relevant element differences. Most of the outer mold line differences present between Ares I and Ares I-X are minor and will not have a significant effect on overall vehicle performance. The most significant impacts are related to the geometric differences in Orion Crew Exploration Vehicle at the forward end of the stack. These physical differences will cause differences in the flow physics in these areas. Even with these differences, the Ares I-X flight test is poised to meet all five primary objectives and six secondary objectives. Knowledge of what the Ares I-X flight test will provide in similitude to Ares I - as well as what the test will not provide - is important in the continued execution of the Ares I-X mission leading to its flight and the continued design and development of Ares I.

  9. Ares I-X Flight Test Vehicle similitude to the Ares I Crew Launch Vehicle

    NASA Astrophysics Data System (ADS)

    Huebner, Lawrence D.; Smith, R. Marshall; Campbell, John R.; Taylor, Terry L.

    2009-12-01

    The Ares I-X Flight Test Vehicle is the first in a series of flight test vehicles that will take the Ares I Crew Launch Vehicle design from development to operational capability. Ares I-X is scheduled for a 2009 flight date, early enough in the Ares I design and development process so that data obtained from the flight can impact the design of Ares I before its Critical Design Review. Decisions on Ares I-X scope, flight test objectives, and FTV fidelity were made prior to the Ares I systems requirements being baselined. This was necessary in order to achieve a development flight test to impact the Ares I design. Differences between the Ares I-X and the Ares I configurations are artifacts of formulating this experimental project at an early stage and the natural maturation of the Ares I design process. This paper describes the similarities and differences between the Ares I-X Flight Test Vehicle and the Ares I Crew Launch Vehicle. Areas of comparison include the outer mold line geometry, aerosciences, trajectory, structural modes, flight control architecture, separation sequence, and relevant element differences. Most of the outer mold line differences present between Ares I and Ares I-X are minor and will not have a significant effect on overall vehicle performance. The most significant impacts are related to the geometric differences in Orion Crew Exploration Vehicle at the forward end of the stack. These physical differences will cause differences in the flow physics in these areas. Even with these differences, the Ares I-X flight test is poised to meet all five primary objectives and six secondary objectives. Knowledge of what the Ares I-X flight test will provide in similitude to Ares I—as well as what the test will not provide—is important in the continued execution of the Ares I-X mission leading to its flight and the continued design and development of Ares I.

  10. Evaluation of ALA-induced PpIX as a photosensitizer for PDT in cats

    NASA Astrophysics Data System (ADS)

    Lucroy, Michael D.; Edwards, Benjamin F.; Peavy, George M.; Krasieva, Tatiana B.; Griffey, Stephen M.; Madewell, Bruce R.

    1998-07-01

    Given exogenously, ALA defeats intrinsic regulatory feedback mechanisms allowing intracellular accumulation of protoporphyrin IX (PpIX), a highly efficient photosensitizer. In vivo, PpIX synthesis in neoplastic mammary tissues averages 20-fold higher than in normal mammary tissues. PpIX is retained intracellularly, unlike perivascular localization of other photosensitizers, and it is then cleared quickly from the body. In vitro, ALA induced PpIX production in our laboratory in 6 cell lines tested, including an established feline kidney cell line and dermal fibroblasts from primary skin biopsy explant, resulting in photosensitization. Fluorescent microscopy confirmed PpIX production in skin adnexae following ALA administration in a normal cat. To evaluate toxicity, three cats were treated with a single i.v. dose of ALA (either 100, 200, of 400 mg/kg) and followed for 7 days. Cats receiving 100 or 200 mg/kg ALA i.v. had elevated liver enzymes and bilirubin within 24 hours. Histopathology revealed hydropic changes in the liver and renal fibrosis. The cat receiving 400 mg/kg ALA intravenously had cutaneous flush, bradycardia and apnea associated with ALA administration; within 24 hours the cat was lethargic, anorectic and icteric. ALT, AST and bilirubin concentrations had increased significantly. At necropsy the liver had a prominent lobular pattern; histopathology revealed severe periportal hepatitis and splenic necrosis. Systemically administered ALA induces PpIX production, but toxicity may preclude its clinical application in the cat. PpIX levels seem to be more time dependent than those dependent at these three ALA doses and they are well beyond the saturation point for adequate PpIX conversion. The literature is scant regarding toxicity associated with parenteral administration of ALA.

  11. Adolescent Sexual Activity: An Ecological, Risk-Factor Approach.

    ERIC Educational Resources Information Center

    Small, Stephen A.; Luster, Tom

    1994-01-01

    Examined relationship between adolescent sexual intercourse and history of physical abuse, neighborhood monitoring, and adolescent's attachment to school. Findings from 2,108 adolescents suggest that there are many significant risk factors related to whether adolescents are sexually experienced and that importance of some factors vary by gender.…

  12. Hardware and Programmatic Progress on the Ares I-X Flight Test

    NASA Technical Reports Server (NTRS)

    Davis, Stephan R.

    2008-01-01

    In less than two years, the National Aeronautics and Space Administration (NASA) will execute the Ares I-X mission. This will be the first flight of the Ares I crew launch vehicle; which, together with the Ares V cargo launch vehicle (Figure 1), will eventually send humans to the Moon, Mars, and beyond. As the countdown to this first Ares mission continues, personnel from across the Ares I-X Mission Management Office (MMO) are finalizing designs and, in some cases, already fabricating vehicle hardware in preparation for an April 2009 launch. This paper will discuss the hardware and programmatic progress of the Ares I-X mission.

  13. The nuclear factor SPBP contains different functional domains and stimulates the activity of various transcriptional activators.

    PubMed

    Rekdal, C; Sjøttem, E; Johansen, T

    2000-12-22

    SPBP (stromelysin-1 platelet-derived growth factor-responsive element binding protein) was originally cloned from a cDNA expression library by virtue of its ability to bind to a platelet-derived growth factor-responsive element in the human stromelysin-1 promoter. A 937-amino acid-long protein was deduced from a 3995-nucleotide murine cDNA sequence. By analyses of both human and murine cDNAs, we now show that SPBP is twice as large as originally found. The human SPBP gene contains six exons and is located on chromosome 22q13.1-13.3. Two isoforms differing in their C termini are expressed due to alternative splicing. PCR analyses of multitissue cDNA panels showed that SPBP is expressed in most tissues except for ovary and prostate. Functional mapping revealed that SPBP is a nuclear, multidomain protein containing an N-terminal region with transactivating ability, a novel type of DNA-binding domain containing an AT hook motif, and a bipartite nuclear localization signal as well as a C-terminal zinc finger domain. This type of zinc finger domain is also found in the trithorax family of chromatin-based transcriptional regulator proteins. Using cotransfection experiments, we find that SPBP enhances the transcriptional activity of various transcription factors such as c-Jun, Ets1, Sp1, and Pax6. Hence, SPBP seems to act as a transcriptional coactivator. PMID:10995766

  14. Recombinant factor VIIa.

    PubMed

    Aitken, Michael G

    2004-01-01

    Human coagulation factor (F) VII is a single chain protease that circulates in the blood as a weakly active zymogen at concentrations of approximately 10 nmol/L. When converted to the active 2 chain form (FVIIa), it is a powerful initiator of haemostasis. Recombinant factor VIIa (rFVIIa, eptacog alfa, NovoSeven) is a genetically engineered product that was first introduced in 1988 for the treatment of patients with haemophilia A and B with high inhibitory antibody titres to factors VIII and IX. Recent reports in the form of case studies and series, and early trial data, have suggested a role for rFVIIa across a diverse range of indications including bleeding associated with trauma, surgery, thrombocytopaenia, liver disease and oral anticoagulant toxicity. This review describes the physiology of the coagulation pathway and in particular the role of recombinant factor VIIa. It will also focus on the emerging role of rFVIIa in both trauma and non-trauma bleeding and its potential use in the ED. PMID:15537408

  15. The immunological generation of a platelet-activating factor and a platet-lytic factor in the rat.

    PubMed Central

    Valone, F H; Whitmer, D I; Pickett, W C; Austen, K F; Goetzl, E J

    1979-01-01

    Antigen challenge of the rat peritoneal cavity which had been prepared with IgGa-rich antiserum generated activities which released [14C]-serotonin from pre-labelled human platelets. After adsorption of these activities onto Amberlite XAD-8 and elution in 80% ethanol, two factors of differing polarity were resolved by chromatography on diethylaminoethyl cellulose in organic solvents. The activity eluting in the 7:1 chloroform:methanol solvent contained a platelet-lytic factor (PLF) assessed by the parallel release of lactic acid dehydrogenase and [14C]-serotonin; the cytotoxicity of this fraction was confirmed by phase-contrast microscopy examination which demonstrated fragmentation of the exposed platelets. The activity eluting in the 1:1 methanol: aqueous 1.0 M ammonium carbonate solvent was a platelet-activating factor (PAF) as defined by release of [14C]-serotonin without lactic acid dehydrogenase. Both the lytic and the activating principles were separable from slow reacting substance of anaphylaxis and polymorphonuclear leucocyte chemotactic activity, and each presented a single activity peak of differing mobility when chromatographed on silica gel H plates. Human eosinophil phospholipase D inactivated the lytic factor by more than 85% in 2 h at 37 degrees without affecting the activity of the activating factor. The release of [14C]-serotonin induced by the PAF was not affected by the absence of calcium from the medium or by elevations in the platelet concentrations of cyclic AMP or cyclic GMP that resulted from pre-incubation of platelets with prostaglandin D2 or sodium ascorbate, respectively. PMID:227784

  16. Mg-chelatase of tobacco: The role of the subunit CHL D in the chelation step of protoporphyrin IX

    PubMed Central

    Gräfe, Susanna; Saluz, Hans-Peter; Grimm, Bernhard; Hänel, Frank

    1999-01-01

    The Mg-chelation is found to be a prerequisite to direct protoporphyrin IX into the chlorophyll (Chl)-synthesizing branch of the tetrapyrrol pathway. The ATP-dependent insertion of magnesium into protoporphyrin IX is catalyzed by the enzyme Mg-chelatase, which consists of three protein subunits (CHL D, CHL I, and CHL H). We have chosen the Mg-chelatase from tobacco to obtain more information about the mode of molecular action of this complex enzyme by elucidating the interactions in vitro and in vivo between the central subunit CHL D and subunits CHL I and CHL H. We dissected CHL D in defined peptide fragments and assayed for the essential part of CHL D for protein–protein interaction and enzyme activity. Surprisingly, only a small part of CHL D, i.e., 110 aa, was required for interaction with the partner subunits and maintenance of the enzyme activity. In addition, it could be demonstrated that CHL D is capable of forming homodimers. Moreover, it interacted with both CHL I and CHL H. Our data led to the outline of a two-step model based on the cooperation of the subunits for the chelation process. PMID:10051574

  17. ComIXS on BACH: a compact soft x-ray spectrometer operating at Elettra

    SciTech Connect

    Cocco, Daniele; Matteucci, Maurizio; Zangrando, Marco; Bondino, Federica; Zacchigna, Michele; Plate, Mauro; Parmigiani, Fulvio; Nelles, Bruno; Prince, Kevin C.

    2004-05-12

    To accommodate increasing interest in soft x-ray inelastic scattering, a new spectrometer has been designed, constructed and commissioned at Elettra. This instrument uses as the dispersive element one of two interchangeable Variable Line Spacing (VLS) spherical gratings. The energy scan is performed by a 7 cm linear translation of a back illuminated CCD which also collects the zero order light, facilitating alignment and calibration. The two gratings have the same radius of curvature while the groove densities and the groove density variations differ by a factor four. Thus the energies focused by the gratings at a particular position differ by a factor of four. The total length of the instrument is 60 cm, the energy range covered is roughly 25-1000 eV and the expected resolving power ranges from 1000 to 5000. The spectrometer is now operating on the beamline Bach. It takes advantage of the small size of the photon spot in the experimental chamber and of the possibility to control the polarization of the incoming radiation. The small spot constitutes the virtual entrance slit, and the spectrometer collects the photons emitted in a solid angle of about 30x10 mrad2. The instrument, named ComIXS (Compact Inelastic X-ray Spectrometer), has been routinely operating since October 2002. Several experiments have already been carried out, and some results illustrating the characteristics of the instrument are described. The manufacture and testing of the blaze gratings are also discussed.

  18. ComIXS on BACH: a compact soft x-ray spectrometer operating at Elettra

    NASA Astrophysics Data System (ADS)

    Cocco, Daniele; Zangrando, Marco; Matteucci, Maurizio; Bondino, Federica; Platè, Mauro; Zacchigna, Michele; Parmigiani, Fulvio; Nelles, Bruno; Prince, Kevin C.

    2004-05-01

    To accommodate increasing interest in soft x-ray inelastic scattering, a new spectrometer has been designed, constructed and commissioned at Elettra. This instrument uses as the dispersive element one of two interchangeable Variable Line Spacing (VLS) spherical gratings. The energy scan is performed by a 7 cm linear translation of a back illuminated CCD which also collects the zero order light, facilitating alignment and calibration. The two gratings have the same radius of curvature while the groove densities and the groove density variations differ by a factor four. Thus the energies focused by the gratings at a particular position differ by a factor of four. The total length of the instrument is 60 cm, the energy range covered is roughly 25-1000 eV and the expected resolving power ranges from 1000 to 5000. The spectrometer is now operating on the beamline Bach. It takes advantage of the small size of the photon spot in the experimental chamber and of the possibility to control the polarization of the incoming radiation. The small spot constitutes the virtual entrance slit, and the spectrometer collects the photons emitted in a solid angle of about 30×10 mrad2. The instrument, named ComIXS (Compact Inelastic X-ray Spectrometer), has been routinely operating since October 2002. Several experiments have already been carried out, and some results illustrating the characteristics of the instrument are described. The manufacture and testing of the blaze gratings are also discussed.

  19. Factors Shaping Students' Opportunities to Engage in Argumentative Activity

    ERIC Educational Resources Information Center

    Ayalon, Michal; Even, Ruhama

    2016-01-01

    This study examines how students' opportunities to engage in argumentative activity are shaped by the teacher, the class, and the mathematical topic. It compares the argumentative activity between two classes taught by the same teacher using the same textbook and across two beginning algebra topics--investigating algebraic expressions and…

  20. Social and Environmental Factors Associated with Preschoolers' Nonsedentary Physical Activity

    ERIC Educational Resources Information Center

    Brown, William H.; Pfeiffer, Karin A.; McIver, Kerry L.; Dowda, Marsha; Addy, Cheryl L.; Pate, Russell R.

    2009-01-01

    The twofold purposes of the investigation were (a) to describe with direct observation data the physical activity behaviors and the accompanying social and environmental events of those behaviors for children in preschools and (b) to determine which contextual conditions were predictors of moderate to vigorous physical activity (MVPA) and…