Sample records for beta hydroxylase activity

  1. Genotype-independent decrease in plasma dopamine beta-hydroxylase activity in Alzheimer's disease


    Mustapić, Maja; Presečki, Paola; Pivac, Nela; Mimica, Ninoslav; Hof, Patrick R.; Šimić, Goran; Folnegović-Šmalc, Vera; Muck-Šeler, Dorotea


    The noradrenergic system is involved in the etiology and progression of Alzheimer’s disease (AD) but its role is still unclear. Dopamine beta-hydroxylase (DBH) as a catecholamine-synthesizing enzyme plays a central role in noradrenaline (NA) synthesis and turnover. Plasma DBH (pDBH) activity shows wide inheritable interindividual variability that is under genetic control. The aim of this study was to determine pDBH activity, DBH (C-970T; rs1611115) and DBH (C1603T; rs6271) gene polymorphisms ...

  2. Coexistence of dopamine-beta-hydroxylase and activated protein-2 alpha in rat cerebellar Purkinje cells

    Institute of Scientific and Technical Information of China (English)

    Kejian Wang; Wei Li; Shanquan Sun; Zhongqin Ren; Guiqiong He


    BACKGROUND:Tyrosine hydroxylase and phenylethanolamine-n-methyl transferase expression coexist in Purkinje cells of the rat cerebellum.Numerous reports have also been published addressing whether dopamine-beta-hydroxylase (DBH) expression exists in cerebellar Purkinje cells.OBJECTIVE:To investigate the coexistence of DBH and activator protein-2α expression in rat cerebellar Purkinje cells.DESIGN,TIME AND SETTING:A cell morphological study was performed at the Institute of Neuroscience,Chongqing Medical University,China in May 2007.MATERIALS:Ten healthy Wistar rats,of either gender,aged 14 weeks,served as experimental animals.Rabbit anti-mouse DBH,goat anti-mouse activator protein-2α and rabbit anti-mouse β-actin (Santa Cruz Biotechnology,Inc.,USA),horseradish peroxidase-labeled goat anti-rabbit IgG,FITC-labeled mouse anti-rabbit IgG,and Cy3-labeled mouse anti-goat IgG (Boster,Wuhan,China),were used in this study.METHODS:Immunohistochemical staining was used to measure the expression of DBH or activator protein-2α,with double-label immunofluorescence being employed to determine coexpression of both,in the cerebellum of 5 randomly selected rats.Western blot assay was utilized to determine the expression of DBH and activator protein-2α in the cerebellum of the remaining 5 rats.MAIN OUTCOME MEASURES:Expression,localization and coexistence of DBH and activator protein-2α in the cerebellum were measured separately.RESULTS:Immunohistochemical staining demonstrated that cerebellar Purkinje cells stained positive for DBH and activator protein-2α.Western blot assay also demonstrated DBH and activator protein-2α expression in the cerebellum.Double-labeling immunofluorescence showed the coexistence of DBH and activator protein-2α in cerebellar Purkinje cells.CONCLUSION:Norepinephrine and activator protein-2α coexist in rat cerebellar Purkinje cells.

  3. Dopamine beta-hydroxylase deficiency

    Directory of Open Access Journals (Sweden)

    Senard Jean-Michel


    Full Text Available Abstract Dopamine beta-hydroxylase (DβH deficiency is a very rare form of primary autonomic failure characterized by a complete absence of noradrenaline and adrenaline in plasma together with increased dopamine plasma levels. The prevalence of DβH deficiency is unknown. Only a limited number of cases with this disease have been reported. DβH deficiency is mainly characterized by cardiovascular disorders and severe orthostatic hypotension. First symptoms often start during a complicated perinatal period with hypotension, muscle hypotonia, hypothermia and hypoglycemia. Children with DβH deficiency exhibit reduced ability to exercise because of blood pressure inadaptation with exertion and syncope. Symptoms usually worsen progressively during late adolescence and early adulthood with severe orthostatic hypotension, eyelid ptosis, nasal stuffiness and sexual disorders. Limitation in standing tolerance, limited ability to exercise and traumatic morbidity related to falls and syncope may represent later evolution. The syndrome is caused by heterogeneous molecular alterations of the DBH gene and is inherited in an autosomal recessive manner. Restoration of plasma noradrenaline to the normal range can be achieved by therapy with the synthetic precursor of noradrenaline, L-threo-dihydroxyphenylserine (DOPS. Oral administration of 100 to 500 mg DOPS, twice or three times daily, increases blood pressure and reverses the orthostatic intolerance.

  4. Construction of a tissue engineered intervertebral disc with high biological activity using an allogeneic intervertebral disc supplemented with transfected nucleus pulposus cells expressing exogenous dopamine beta-hydroxylase. (United States)

    Bai, M; Wang, Y H; Yin, H P; Li, S W


    This study addressed the in vitro construction and biological activity of tissue engineered intervertebral discs with exogenous human dopamine beta-hydroxylase (DBH) nucleus pulposus cells. pSNAV2.0-DBH expression plasmids were utilized to enhance the survival rates of intervertebral disc tissue cells. Various concentrations of transfected nucleus pulposus cells were injected into the discs, and DBH mRNA expression was determined using polymerase chain reaction amplification. Polysaccharide content and total collagen protein content in the engineered disc nucleus pulposus tissue were determined. The visible fluorescence intensities of the 1 x 10(5) and 1 x 10(6) groups vs the 1 x 10(4) group were significantly increased (P 0.05) at 7 days after injection. DBH mRNA expression could be detected in the all but the EGFP control group at 14 days culture. No significant difference was observed in the protein content between the 1 x 10(4) and the control groups at various times, while the protein content was significantly higher in the 1 x 10(5) vs the control and the 1 x 10(4) groups at 7-, 14-, and 21-day cultures. These results demonstrate that a tissue engineered intervertebral disc with high biological activity can be constructed by utilizing allogeneic intervertebral discs stored in liquid nitrogen and a 1 x 10(5) transfected nucleus pulposus cell complex with in vitro culture for 14 days. This model can be used in animal experiments to study the biological activity of the engineered discs.

  5. Family studies of plasma dopamine-beta-hydroxylase thermal stability.


    Dunnette, J; Weinshilboum, R


    There are large individual variations in the thermal stability of human plasma dopamine-beta-hydroxylase (DBH). These variations are a characteristic of the DBH molecule itself. Individual subjects may be classified as those with thermolabile and those with thermostable plasma DBH. Of 362 randomly selected unrelated children, 8.01%, and of 238 randomly selected unrelated adult subjects, 5.46% had thermolabile plasma DBH. There was not a significant correlation of DBH thermolability with eithe...

  6. Dopamine-beta hydroxylase polymorphism and cocaine addiction


    Collier David; Laranjeira Ronaldo; Guindalini Camila; Messas Guilherme; Vallada Homero; Breen Gerome


    Abstract Cocaine addiction involves a number of medical, psychological and social problems. Understanding the genetic aetiology of this disorder will be essential for design of effective treatments. Dopamine-beta hydroxylase (DbH) catalyzes the conversion of dopamine to norepinephrine and could, therefore, have an influence on both cocaine action and the basal sensitivity of neurotransmitter systems to cocaine. Recently, the -1021C>T polymorphism have been found to strongly correlated with in...

  7. Dopamine-beta hydroxylase polymorphism and cocaine addiction

    Directory of Open Access Journals (Sweden)

    Collier David


    Full Text Available Abstract Cocaine addiction involves a number of medical, psychological and social problems. Understanding the genetic aetiology of this disorder will be essential for design of effective treatments. Dopamine-beta hydroxylase (DbH catalyzes the conversion of dopamine to norepinephrine and could, therefore, have an influence on both cocaine action and the basal sensitivity of neurotransmitter systems to cocaine. Recently, the -1021C>T polymorphism have been found to strongly correlated with individual variation in plasma DbH activity. To test the influence of this polymorphism on the susceptibility of cocaine addiction, we decided to genotype it in a sample of 689 cocaine addicts and 832 healthy individuals. Genotypic and allelic analyses did not show any evidence of association with cocaine addiction, even after correcting for the effect of population stratification and other possible confounders. Our results do not support a major role of the -1021C>T polymorphism or the gene itself in the development of cocaine addiction but further examination of other variants within this gene will be necessary to completely rule out an effect.

  8. Genetics Home Reference: congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency (United States)

    ... Intersex Society of North America MalaCards: adrenal hyperplasia, congenital, due to 11-beta-hydroxylase deficiency March of Dimes: Genital and Urinary Tract Defects Merck Manual Consumer Version: The Body's Control ...

  9. [From gene to disease; dopamine-beta-hydroxylase deficiency and orthostatic hypotension

    NARCIS (Netherlands)

    Deinum, J.; Meiracker, A.H. van den; Boomsma, F.; Ittersum, F.J. van; Wevers, R.A.; Lenders, J.W.M.


    The DBH gene encodes dopamine-beta-hydroxylase (DbetaH), the enzyme that catalyses the formation of norepinephrine from dopamine. Inactivation of this enzyme due to a mutation of the DBH gene causes a selective (nor)-adrenergic failure of the sympathetic nervous system. This manifests as a severe or

  10. The dopamine beta-hydroxylase -1021C/T polymorphism is associated with the risk of Alzheimer's disease in the Epistasis Project.

    NARCIS (Netherlands)

    Combarros, O.; Warden, D.R.; Hammond, N.; Cortina-Borja, M.; Belbin, O.; Lehmann, M.G.; Wilcock, G.K.; Brown, K.; Kehoe, P.G.; Barber, R.; Coto, E.; Alvarez, V.; Deloukas, P.; Gwilliam, R.; Heun, R.; Kolsch, H.; Mateo, I.; Oulhaj, A.; Arias Vasquez, A.; Schuur, M.; Aulchenko, Y.S.; Ikram, M.A.; Breteler, M.M.B.; Duijn, C.M. van; Morgan, K.; Smith, A.D.; Lehmann, D.J.


    BACKGROUND: The loss of noradrenergic neurones of the locus coeruleus is a major feature of Alzheimer's disease (AD). Dopamine beta-hydroxylase (DBH) catalyses the conversion of dopamine to noradrenaline. Interactions have been reported between the low-activity -1021T allele (rs1611115) of DBH and p

  11. Anti-dopamine beta-hydroxylase immunotoxin-induced sympathectomy in adult rats (United States)

    Picklo, M. J.; Wiley, R. G.; Lonce, S.; Lappi, D. A.; Robertson, D.


    Anti-dopamine beta-hydroxylase immunotoxin (DHIT) is an antibody-targeted noradrenergic lesioning tool comprised of a monoclonal antibody against the noradrenergic enzyme, dopamine beta-hydroxylase, conjugated to saporin, a ribosome-inactivating protein. Noradrenergic-neuron specificity and completeness and functionality of sympathectomy were assessed. Adult, male Sprague-Dawley rats were given 28.5, 85.7, 142 or 285 micrograms/kg DHIT i.v. Three days after injection, a 6% to 73% decrease in the neurons was found in the superior cervical ganglia of the animals. No loss of sensory, nodose and dorsal root ganglia, neurons was observed at the highest dose of DHIT. In contrast, the immunotoxin, 192-saporin (142 micrograms/kg), lesioned all three ganglia. To assess the sympathectomy, 2 wk after treatment (285 micrograms/kg), rats were anesthetized with urethane (1 g/kg) and cannulated in the femoral artery and vein. DHIT-treated animals' basal systolic blood pressure and heart rate were significantly lower than controls. Basal plasma norepinephrine levels were 41% lower in DHIT-treated animals than controls. Tyramine-stimulated release of norepinephrine in DHIT-treated rats was 27% of controls. Plasma epinephrine levels of DHIT animals were not reduced. DHIT-treated animals exhibited a 2-fold hypersensitivity to the alpha-adrenergic agonist phenylephrine. We conclude that DHIT selectively delivered saporin to noradrenergic neurons resulting in destruction of these neurons. Anti-dopamine beta-hydroxylase immunotoxin administration produces a rapid, irreversible sympathectomy.

  12. Dopamine beta-hydroxylase knockout mice have alterations in dopamine signaling and are hypersensitive to cocaine. (United States)

    Schank, Jesse R; Ventura, Rossella; Puglisi-Allegra, Stefano; Alcaro, Antonio; Cole, Charlene D; Liles, L Cameron; Seeman, Philip; Weinshenker, David


    Multiple lines of evidence demonstrate that the noradrenergic system provides both direct and indirect excitatory drive onto midbrain dopamine (DA) neurons. We used DA beta-hydroxylase (DBH) knockout (Dbh-/-) mice that lack norepinephrine (NE) to determine the consequences of chronic NE deficiency on midbrain DA neuron function in vivo. Basal extracellular DA levels were significantly attenuated in the nucleus accumbens (NAc) and caudate putamen (CP), but not prefrontal cortex (PFC), of Dbh-/- mice, while amphetamine-induced DA release was absent in the NAc and attenuated in the CP and PFC. The decrease in dopaminergic tone was associated with a profound increase in the density of high-affinity state D1 and D2 DA receptors in the NAc and CP, while DA receptors in the PFC were relatively unaffected. As a behavioral consequence of these neurochemical changes, Dbh-/- mice were hypersensitive to the psychomotor, rewarding, and aversive effects of cocaine, as measured by locomotor activity and conditioned place preference. Antagonists of DA, but not 5-HT, receptors attenuated the locomotor hypersensitivity to cocaine in Dbh-/- mice. As DBH activity in humans is genetically controlled and the DBH inhibitor disulfiram has shown promise as a pharmacotherapy for cocaine dependence, these results have implications for the influence of genetic and pharmacological DBH inhibition on DA system function and drug addiction. PMID:16395294

  13. Mechanism-based inactivation of dopamine beta-hydroxylase by p-cresol and related alkylphenols

    International Nuclear Information System (INIS)

    The mechanism-based inhibition of dopamine beta-hydroxylase by p-cresol (4-methylphenol) and other simple structural analogues of dopamine, which lack a basic side-chain nitrogen, is reported. p-Cresol binds DBH by a mechanism that is kinetically indistinguishable from normal dopamine substrate binding. Under conditions (pH 6.6) of random oxygen and phenethylamine substrate addition p-cresol adds randomly, whereas at pH 4.5 or in the presence of fumarate activator addition of p-cresol precedes oxygen binding as is observed with phenethylamine substrate. p-Cresol is shown to be a rapid (kinact = 2.0 min-1, pH 5.0) mechanism-based inactivator of DBH. This inactivation exhibits pseudo-first-order kinetics, is irreversible, is prevented by tyramine substrate or competitive inhibitor, and is dependent upon oxygen and ascorbic acid cosubstrates. Inhibition occurs with partial covalent incorporation of p-cresol into DBH. A plot of -log kinact vs. pH shows maximal inactivation occurs at pH 5.0 with dependence upon enzymatic groups with apparent pK values of 4.51 +/- 0.06 and 5.12 +/- 0.06. p-Cresol and related alkylphenols, unlike other mechanism-based inhibitors of DBH, lack a latent electrophile. These inhibitors are postulated to covalently modify DBH by a direct insertion of an aberrant substrate-derived benzylic radical into an active site residue

  14. Purification of recombinant flavanone 3beta-hydroxylase from petunia hybrida and assignment of the primary site of proteolytic degradation. (United States)

    Lukacin, R; Gröning, I; Schiltz, E; Britsch, L; Matern, U


    Flavanone 3beta-hydroxylase catalyzes the Fe(II)/oxoglutarate-dependent hydroxylation of (2S)-flavanones to (2R,3R)-dihydroflavonols in the course of flavonol/anthocyanin or catechin biosynthesis. The enzyme from Petunia hybrida consists of a 41,655-Da polypeptide that is prone to rapid proteolysis in crude plant extracts as well as on expression in Escherichia coli, and commercial protease inhibitors were inefficient in stopping the degradation. To pinpoint the primary site of proteolysis and to improve the activity yields, two revised schemes of purification were developed for the recombinant polypeptides. Applying a four-step protocol based on extraction and ion-exchange chromatography at pH 7.5, the primary, catalytically inactive proteolytic enzyme fragment (1.1 mg) was isolated and shown to cross-react on Western blotting as one homogeneous band of about 38 kDa. Mass spectrometric analysis assigned a mass of 37,820 +/- 100 Da to this fragment, and partial sequencing revealed an unblocked amino terminus identical to that of the native 3beta-hydroxylase. Thus, the native enzyme had been degraded by proteolysis of a small carboxy-terminal portion, and the primary site of cleavage must be assigned most likely to the Glu 337-Leu 338 bond, accounting for a loss of about 3800 Da. Alternatively, the enzyme degradation was greatly reduced when the extraction of recombinant bacteria was carried out with phosphate buffer at pH 5.5 followed by size exlusion and anion-exchange chromatography. This rapid, two-step purification resulted in a homogeneous 3beta-hydroxylase of high specific acitivity (about 32 mkat/kg) at roughly 5% yield, and the procedure is a major breakthrough in mechanistic investigations of this class of labile dioxygenases.

  15. Noradrenergic lesioning with an anti-dopamine beta-hydroxylase immunotoxin (United States)

    Picklo, M. J.; Wiley, R. G.; Lappi, D. A.; Robertson, D.


    Sympathectomy has been achieved by a variety of methods but each has its limitations. These include lack of tissue specificity, incomplete lesioning, and the age range of susceptibility to the lesioning. To circumvent these drawbacks, an immunotoxin was constructed using a monoclonal antibody against the noradrenergic specific enzyme dopamine beta-hydroxylase (D beta H) coupled via a disulfide bond to saporin, a ribosomal inactivating protein. Three days after intravenous injection of the anti-D beta H immunotoxin (50 micrograms) into adult Sprague-Dawley rats, 66% of neurons in the superior cervical ganglia were chromatolytic. Superior cervical ganglia neurons were poisoned in 1 day old and 1 week old (86% of neurons) neonatal rats following subcutaneous injection of 3.75 and 15 micrograms, respectively. The anti-D beta H immunotoxin will be a useful tool in the study of the peripheral noradrenergic system in adult and neonatal animals.

  16. The primary structure of human dopamine-beta-hydroxylase: insights into the relationship between the soluble and the membrane-bound forms of the enzyme.


    Lamouroux, A; Vigny, A; Faucon Biguet, N; Darmon, M C; Franck, R.; Henry, J P; Mallet, J


    A full length dopamine-beta-hydroxylase (DBH) cDNA clone was isolated from a human pheochromocytoma lambda gt11 library. Both structural and functional evidence confirms the authenticity of the clone: (i) antibodies selected with fusion proteins generated by positive clones precipitate DBH activity, (ii) the sequence of three internal DBH tryptic peptides are included in the deduced DBH sequence, (iii) the previously reported N-terminal 15 amino acids of bovine DBH exhibits a nearly complete ...

  17. Absence of steroid biosynthetic defects in heterozygote individuals for classic 11{beta}-hydroxylase deficiency due to a R448H mutation in the CYP11B1 gene

    Energy Technology Data Exchange (ETDEWEB)

    Roesler, A.; Cohen, H. [Hadassah-Hebrew Univ. Medical Center, Jerusalem (Israel)


    Steroid 11{beta}-hydroxylase deficiency (failure to convert 11-deoxycortisol to cortisol) is responsible for less than 5% of cases of classic congenital adrenal hyperplasia, but it is relatively frequent in Israel, among Jews of Moroccan origin. Affected individuals have a single base substitution in exon 8 of CYP11B1 gene, codon 448, from CGC (arginine) to CAC (histidine) (R448H), a mutation that abolishes enzyme activity completely. We studied the hormonal response to ACTH stimulation in individuals genotyped to have the R448H mutation in one allele only (heterozygotes), and who were therefore assumed to have 50% of 11{beta}-hydroxylase activity. No demonstrable hormonal abnormalities were found in the 6 adults (3 mothers and 3 fathers) and 2 sons studied, suggesting that a quantitatively reduced 11{beta}-hydroxylase is still enough for normal adrenal biosynthesis. 19 refs., 1 fig., 2 tabs.

  18. Molecular characterization of flavanone 3 beta-hydroxylases. Consensus sequence, comparison with related enzymes and the role of conserved histidine residues. (United States)

    Britsch, L; Dedio, J; Saedler, H; Forkmann, G


    A heterologous cDNA probe from Petunia hybrida was used to isolate flavanone-3 beta-hydroxylase-encoding cDNA clones from carnation (Dianthus caryophyllus), china aster (Callistephus chinensis) and stock (Matthiola incana). The deduced protein sequences together with the known sequences of the enzyme from P. hybrida, barley (Hordeum vulgare) and snapdragon (Antirrhinum majus) enabled the determination of a consensus sequence which revealed an overall 84% similarity (53% identity) of flavanone 3 beta-hydroxylases from the different sources. Alignment with the sequences of other known enzymes of the same class and to related non-heme iron-(II) enzymes demonstrated the strict genetic conservation of 14 amino acids, in particular, of three histidines and an aspartic acid. The conservation of the histidine motifs provides strong support for the possible conservation of structurally similar iron-binding sites in these enzymes. The putative role of histidines as chelators of ferrous ions in the active site of flavanone 3 beta-hydroxylases was corroborated by diethyl-pyrocarbonate modification of the partially purified recombinant Petunia enzyme.

  19. Molecular cloning of hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, from cultured roots of Hyoscyamus niger. (United States)

    Matsuda, J; Okabe, S; Hashimoto, T; Yamada, Y


    Roots of several solanaceous plants produce anticholinergic alkaloids, hyoscyamine and scopolamine. Hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase (EC, catalyzes hydroxylation of hyoscyamine in the biosynthetic pathway leading to scopolamine. We report here on the isolation of cDNA clones encoding the hydroxylase from a cDNA library made from mRNA of the cultured roots of Hyoscyamus niger. The library was screened with three synthetic oligonucleotides that encode amino acid sequences of internal peptide fragments of the purified hydroxylase. Nucleotide sequence analysis of the cloned cDNA revealed an open reading frame that encodes 344 amino acids (Mr = 38,999). All 12 internal peptide fragments determined in the purified enzyme were found in the amino acid sequence deduced from the cDNA. With computer-aided comparison to other proteins we found that the hydroxylase is homologous to two synthases involved in the biosynthesis of beta-lactam antibiotics in some microorganisms and the gene products of tomato pTOM13 cDNA and maize A2 locus which had been proposed to catalyze oxidative reactions in the biosynthesis of ethylene and anthocyan, respectively. RNA blotting hybridization showed that mRNA of the hydroxylase is abundant in cultured roots and present in plant roots, but absent in leaves, stems, and cultured cells of H. niger. PMID:2033047

  20. Mutations in the dopamine beta-hydroxylase gene are associated with human norepinephrine deficiency (United States)

    Kim, Chun-Hyung; Zabetian, Cyrus P.; Cubells, Joseph F.; Cho, Sonhae; Biaggioni, Italo; Cohen, Bruce M.; Robertson, David; Kim, Kwang-Soo


    Norepinephrine (NE), a key neurotransmitter of the central and peripheral nervous systems, is synthesized by dopamine beta-hydroxylase (DBH) that catalyzes oxidation of dopamine (DA) to NE. NE deficiency is a congenital disorder of unknown etiology, in which affected patients suffer profound autonomic failure. Biochemical features of the syndrome include undetectable tissue and circulating levels of NE and epinephrine, elevated levels of DA, and undetectable levels of DBH. Here, we report identification of seven novel variants including four potentially pathogenic mutations in the human DBH gene (OMIM 223360) from analysis of two unrelated patients and their families. Both patients are compound heterozygotes for variants affecting expression of DBH protein. Each carries one copy of a T-->C transversion in the splice donor site of DBH intron 1, creating a premature stop codon. In patient 1, there is a missense mutation in DBH exon 2. Patient 2 carries missense mutations in exons 1 and 6 residing in cis. We propose that NE deficiency is an autosomal recessive disorder resulting from heterogeneous molecular lesions at DBH. Copyright 2002 Wiley-Liss, Inc.

  1. 2,4-Dichlorophenol hydroxylase for chlorophenol removal: Substrate specificity and catalytic activity. (United States)

    Ren, Hejun; Li, Qingchao; Zhan, Yang; Fang, Xuexun; Yu, Dahai


    Chlorophenols (CPs) are common environmental pollutants. As such, different treatments have been assessed to facilitate their removal. In this study, 2,4-dichlorophenol (2,4-DCP) hydroxylase was used to systematically investigate the activity and removal ability of 19CP congeners at 25 and 0 °C. Results demonstrated that 2,4-DCP hydroxylase exhibited a broad substrate specificity to CPs. The activities of 2,4-DCP hydroxylase against specific CP congeners, including 3-CP, 2,3,6-trichlorophenol, 2-CP, and 2,3-DCP, were higher than those against 2,4-DCP, which is the preferred substrate of previously reported 2,4-DCP hydroxylase. To verify whether cofactors are necessary to promote hydroxylase activity against CP congeners, we added FAD and found that the added FAD induced a 1.33-fold to 5.13-fold significant increase in hydroxylase activity against different CP congeners. The metabolic pathways of the CP degradation in the enzymatic hydroxylation step were preliminarily proposed on the basis of the analyses of the enzymatic activities against 19CP congeners. We found that the high activity and removal rate of 2,4-DCP hydroxylase against CPs at 0 °C enhance the low-temperature-adaptability of this enzyme to the CP congeners; as such, the proposed removal process may be applied to biochemical, bioremediation, and industrial processes, particularly in cold environments. PMID:26672451

  2. The effects of the calcium ionophore, A23187, on the axoplasmic transport of dopamine beta-hydroxylase.


    Esquerro, E.; Garcia, A. G.; Sanchez-Garcia, P.


    1 The effects of the ionophore, A23187, on the intra-axonal transport of dopamine beta-hydroxylase (DBH) were investigated in the cat hypogastric nerve-inferior mesenteric ganglion preparation by monitoring, in vitro, the enzyme accumulation above a ligature, 2 to 2.5 cm distal to the ganglion. 2 DBH accumulation in the proximal segment immediately above the ligature (P1) increased linearly up to 6 h, during incubation in normal Krebs solution at 37 degrees C. The ionophore, A23187, interfere...

  3. Allelic variants from Dahlia variabilis encode flavonoid 3'-hydroxylases with functional differences in chalcone 3-hydroxylase activity. (United States)

    Schlangen, Karin; Miosic, Silvija; Halbwirth, Heidi


    In the petals of Dahlia variabilis, hydroxylation of chalcones at position 3 can be detected, except the well-known flavonoid 3'-hydroxylation. Although the reaction is well characterized at the enzymatic level, it remained unclear whether it is catalyzed by a flavonoid 3'-hydroxylase (F3'H, EC1.14.13.21, CYP75B) with broad substrate specificity. Two novel allelic variants of F3'H were cloned from D. variabilis, which differ only in three amino acids within their 508 residues. The corresponding recombinant enzymes show significant differences in their chalcone 3-hydroxylase (CH3H) activity. A substitution of alanine at position 425 with valine enables CH3H activity, whereas the reciprocal substitution leads to a loss of CH3H activity. Interaction of the valine at position 425 with not yet identified structural properties seems to be decisive for chalcone acceptance. This is the first identification of an F3'H which is able to catalyze chalcone 3-hydroxylation to a physiologically relevant extent from any plant species. PMID:19931222

  4. Congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency: functional consequences of four CYP11B1 mutations (United States)

    Menabò, Soara; Polat, Seher; Baldazzi, Lilia; Kulle, Alexandra E; Holterhus, Paul-Martin; Grötzinger, Joachim; Fanelli, Flaminia; Balsamo, Antonio; Riepe, Felix G


    Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine disease. Steroid 11β-hydroxylase deficiency (11β-OHD) is the second most common form of CAH. The aim of the study was to study the functional consequences of three novel and one previously described CYP11B1 gene mutations (p.(Arg143Trp), p.(Ala306Val), p.(Glu310Lys) and p.(Arg332Gln)) detected in patients suffering from classical and non-classical 11β-OHD. Functional analyses were performed by using a HEK293 cell in vitro expression system comparing wild type (WT) with mutant 11β-hydroxylase activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of the protein. Two mutations (p.(Ala306Val) and p.(Glu310Lys)) detected in patients with classical 11β-OHD showed a nearly complete loss of 11β-hydroxylase activity. The mutations p.(Arg143Trp) and p.(Arg332Gln) detected in patients with non-classical 11β-OHD showed a partial functional impairment with approximately 8% and 6% of WT activity, respectively. Functional mutation analysis allows the classification of novel CYP11B1 mutations as causes of classical and non-classical 11β-OHD. The detection of patients with non-classical phenotypes underscores the importance to screen patients with a phenotype comparable to non-classical 21-hydroxylase deficiency for mutations in the CYP11B1 gene in case of a negative analysis of the CYP21A2 gene. As CYP11B1 mutations are most often individual for a family, the in vitro analysis of novel mutations is essential for clinical and genetic counselling. PMID:24022297

  5. Effects of disulfiram and dopamine beta-hydroxylase knockout on cocaine-induced seizures


    Gaval-Cruz, Meriem; Schroeder, Jason P; Liles, L. Cameron; Javors, Martin A.; Weinshenker, David


    The antialcoholism drug disulfiram has shown recent promise as a pharmacotherapy for treating cocaine dependence, probably via inhibition of dopamine β-hydroxylase (DBH), the enzyme that catalyzes the conversion of dopamine (DA) to norepinephrine (NE). We previously showed that DBH knockout (Dbh -/-) mice, which lack NE, are susceptible to seizures and are hypersensitive to the psychomotor, rewarding, and aversive effects of cocaine, suggesting that disulfiram might exacerbate cocaine-induced...

  6. A unique dual activity amino acid hydroxylase in Toxoplasma gondii.

    Directory of Open Access Journals (Sweden)

    Elizabeth A Gaskell

    Full Text Available The genome of the protozoan parasite Toxoplasma gondii was found to contain two genes encoding tyrosine hydroxylase; that produces L-DOPA. The encoded enzymes metabolize phenylalanine as well as tyrosine with substrate preference for tyrosine. Thus the enzymes catabolize phenylalanine to tyrosine and tyrosine to L-DOPA. The catalytic domain descriptive of this class of enzymes is conserved with the parasite enzyme and exhibits similar kinetic properties to metazoan tyrosine hydroxylases, but contains a unique N-terminal extension with a signal sequence motif. One of the genes, TgAaaH1, is constitutively expressed while the other gene, TgAaaH2, is induced during formation of the bradyzoites of the cyst stages of the life cycle. This is the first description of an aromatic amino acid hydroxylase in an apicomplexan parasite. Extensive searching of apicomplexan genome sequences revealed an ortholog in Neospora caninum but not in Eimeria, Cryptosporidium, Theileria, or Plasmodium. Possible role(s of these bi-functional enzymes during host infection are discussed.

  7. Development of vitamin D3 25-hydroxylase activity in rat liver microsomes

    International Nuclear Information System (INIS)

    The authors have determined the ontogeny of vitamin D3 25-hydroxylase activity in rat liver microsomes. Microsomes from fetuses, neonates, and their mothers were incubated with 44 nM 3H-vitamin D3 in the presence of an NADPH generating system, oxygen, KCl, and MgCl2. Lipid extracts of the incubation samples were partially purified by thin-layer chromatography. Tritiated 25-hydroxy vitamin D3 (250HD3) was analyzed by high-pressure liquid chromatography using 94/6 hexane/isopropanol. Production rate for 250HD3 in the mothers ranged from 0.22 to 0.30 pmol/mg protein/hr. Activities in the fetuses and neonates were 2.1, 12.9, 32.0, 35.8, and 71.0% of that of their mothers at -3, 0, 2, 7, and 15 days of age. The cytosolic fraction protected the substrate from degradation, stimulated the vitamin D3 25-hydroxylase reaction in neonates and mothers (1.4 to 1.7 fold increase), and was absolutely required for 25-hydroxylase activity in fetuses. These data suggest that microsomal vitamin D3 25-hydroxylase activity develops slowly and approaches full activity near the weaning stage. A cytosolic factor present as early as -3 days of age stimulates the activity of the microsomal vitamin D3 25-hydroxylase

  8. Genotyping of the 19-bp insertion/deletion polymorphism in the 5' flank of beta-hydroxylase gene by dissociation analysis of allele-specific PCR products

    DEFF Research Database (Denmark)

    Rasmussen, Henrik Berg; Werge, Thomas


    The 19-bp insertion/deletion polymorphism in the 5' flank of the dopamine beta-hydroxylase (DBH) gene has been associated with psychiatric disorders. We have developed a simple, reliable and inexpensive closed-tube assay for genotyping of this polymorphism based upon T(m) determination of amplified...

  9. Influence of various phenolic compounds on phenol hydroxylase activity of a Trichosporon cutaneum strain. (United States)

    Gerginova, Maria; Manasiev, Jordan; Shivarova, Nedka; Alexieva, Zlatka


    The phenol-degrading strain Trichosporon cutaneum R57 utilizes various aromatic and aliphatic compounds as a sole carbon and energy source. The intracellular activities of phenol hydroxylase [EC] of a Trichosporon cutaneum R57 strain grown on phenol (0.5 g/l) were measured. Different toxic phenol derivatives (cresols, nitrophenols and hydroxyphenols) were used as substrates in the reaction mixture for determination of the enzyme activity. The data obtained showed that the investigated enzyme was capable to hydroxylate all applied aromatic substrates. The measured activities of phenol hydroxylase varied significantly depending on the aromatic compounds used as substrates. The rate of phenol hydroxylase activity with phenol as a substrate (1.0 U/mg total cell protein) was accepted as 100%.

  10. Selective estrogen receptor-beta (SERM-beta) compounds modulate raphe nuclei tryptophan hydroxylase-1 (TPH-1) mRNA expression and cause antidepressant-like effects in the forced swim test. (United States)

    Clark, J A; Alves, S; Gundlah, C; Rocha, B; Birzin, E T; Cai, S-J; Flick, R; Hayes, E; Ho, K; Warrier, S; Pai, L; Yudkovitz, J; Fleischer, R; Colwell, L; Li, S; Wilkinson, H; Schaeffer, J; Wilkening, R; Mattingly, E; Hammond, M; Rohrer, S P


    Estrogen acts through two molecularly distinct receptors termed estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) which bind estradiol with similar affinities and mediate the effects of estrogen throughout the body. ERα plays a major role in reproductive physiology and behavior, and mediates classic estrogen signaling in such tissues as the uterus, mammary gland, and skeleton. ERβ, however, modulates estrogen signaling in the ovary, the immune system, prostate, gastrointestinal tract, and hypothalamus, and there is some evidence that ERβ can regulate ERα activity. Moreover, ERβ knockout studies and receptor distribution analyses in the CNS suggest that this receptor may play a role in the modulation of mood and cognition. In recent years several ERβ-specific compounds (selective estrogen receptor beta modulators; SERM-beta) have become available, and research suggests potential utility of these compounds in menopausal symptom relief, breast cancer prevention, diseases that have an inflammatory component, osteoporosis, cardiovascular disease, and inflammatory bowel disease, as well as modulation of mood, and anxiety. Here we demonstrate an antidepressant-like effect obtained using two SERM-beta compounds, SERM-beta1 and SERM-beta2. These compounds exhibit full agonist activity at ERβ in a cell based estrogen response element (ERE) transactivation assay. SERM-beta1 and 2 are non-proliferative with respect to breast as determined using the MCF-7 breast cancer cell-based assay and non-proliferative in the uterus as determined by assessing the effects of SERM-beta compounds on immature rat uterine weight and murine uterine weight. In vivo SERM-beta1 and 2 are brain penetrant and display dose dependent efficacy in the murine dorsal raphe assays for induction of tryptophan hydroxylase mRNA and progesterone receptor protein. These compounds show activity in the murine forced swim test and promote hippocampal neurogenesis acutely in rats. Taken

  11. Analysis of Substrate Access to Active Sites in Bacterial Multicomponent Monooxygenase Hydroxylases: X-ray Crystal Structure of Xenon-Pressurized Phenol Hydroxylase from Pseudomonas sp. OX1†,‡


    McCormick, Michael S.; Lippard, Stephen J.


    In all structurally characterized bacterial multicomponent monooxygenase (BMM) hydroxylase proteins, a series of hydrophobic cavities in the α-subunit trace a conserved path from the protein exterior to the carboxylate-bridged diiron active site. The present study examines these cavities as a potential route for dioxygen transport to the active site by crystallographic characterization of a xenon-pressurized sample of the hydroxylase component of phenol hydroxylase from Pseudomonas sp. OX1. C...

  12. Association of Dopamine Beta-Hydroxylase (DBH) Polymorphisms with Susceptibility to Parkinson's Disease. (United States)

    Shao, Peng; Yu, Yun-Xia; Bao, Jing-Xi


    BACKGROUND The purpose of this study was to explore the association between 2 single-nucleotide polymorphisms (SNPs) in the dopamine β-hydroxylase (DBH) gene (rs1611115 and rs732833) and the susceptibility to Parkinson's disease (PD). MATERIAL AND METHODS Polymerase chain reaction direct sequencing (PCR-DS) was used to test the genotypes of DBH polymorphisms in 95 PD patients and 100 healthy examinees frequency-matched with the former by age and sex. The genotype and allele distribution differences between the case and control groups were analyzed by chi-square test, and the relative risk of PD in southern Chinese populations was expressed by odds ratio (OR) and 95% confidence interval (CI). Hardy-Weinberg equilibrium (HWE) was also checked by chi-square test. RESULTS The genotype and allele distribution frequencies in rs1611115 were obviously different between PD patients and the healthy control group (PDBH rs1611115 polymorphism was likely to be associated with the susceptibility to PD, but we did not find that rs732833 is a susceptibility marker for PD. PMID:27177268

  13. Stringency of the 2-His-1-Asp active-site motif in prolyl 4-hydroxylase.

    Directory of Open Access Journals (Sweden)

    Kelly L Gorres

    Full Text Available The non-heme iron(II dioxygenase family of enzymes contain a common 2-His-1-carboxylate iron-binding motif. These enzymes catalyze a wide variety of oxidative reactions, such as the hydroxylation of aliphatic C-H bonds. Prolyl 4-hydroxylase (P4H is an alpha-ketoglutarate-dependent iron(II dioxygenase that catalyzes the post-translational hydroxylation of proline residues in protocollagen strands, stabilizing the ensuing triple helix. Human P4H residues His412, Asp414, and His483 have been identified as an iron-coordinating 2-His-1-carboxylate motif. Enzymes that catalyze oxidative halogenation do so by a mechanism similar to that of P4H. These halogenases retain the active-site histidine residues, but the carboxylate ligand is replaced with a halide ion. We replaced Asp414 of P4H with alanine (to mimic the active site of a halogenase and with glycine. These substitutions do not, however, convert P4H into a halogenase. Moreover, the hydroxylase activity of D414A P4H cannot be rescued with small molecules. In addition, rearranging the two His and one Asp residues in the active site eliminates hydroxylase activity. Our results demonstrate a high stringency for the iron-binding residues in the P4H active site. We conclude that P4H, which catalyzes an especially demanding chemical transformation, is recalcitrant to change.

  14. Cloning of gibberellin 3 beta-hydroxylase cDNA and analysis of endogenous gibberellins in the developing seeds in watermelon. (United States)

    Kang, Hong-Gyu; Jun, Sung-Hoon; Kim, Joonyul; Kawaide, Hiroshi; Kamiya, Yuji; An, Gynheung


    We have isolated Cv3h, a cDNA clone from the developing seeds of watermelon, and have demonstrated significant amino acid homology with gibberellin (GA) 3 beta-hydroxylases. This cDNA clone was expressed in Escherichia coli as a fusion protein that oxidized GA(9) and GA(12) to GA(4) and GA(14), respectively. The Cv3h protein had the highest similarity with pumpkin GA 2 beta,3 beta-hydroxylase, but did not possess 2 beta-hydroxylation function. RNA blot analysis showed that the gene was expressed primarily in the inner parts of developing seeds, up to 10 d after pollination (DAP). In the parthenocarpic fruits induced by treatment with 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), the embryo and endosperm of the seeds were undeveloped, whereas the integumental tissues, of maternal origin, showed nearly normal development. Cv3h mRNA was undetectable in the seeds of CPPU-treated fruits, indicating that the GA 3 beta-hydroxylase gene was expressed in zygotic cells. In our analysis of endogenous GAs from developing seeds, GA(9) and GA(4) were detected at high levels but those of GA(20) and GA(1) were very low. This demonstrates that GA biosynthesis in seeds prefers a non-13-hydroxylation pathway over an early 13-hydroxylation pathway. We also analyzed endogenous GAs from seeds of the parthenocarpic fruits. The level of bioactive GA(4 )was much lower there than in normal seeds, indicating that bioactive GAs, unconnected with Cv3h, exist in integumental tissues during early seed development. PMID:11867694

  15. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases


    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette


    Catechol oxidases and tyrosinases belong to the family of polyphenol oxidases (PPOs). In contrast to tyrosinases, catechol oxidases were so far defined to lack hydroxylase activity toward monophenols. Aurone synthase (AUS1) is a plant catechol oxidase that specializes in the conversion of chalcones to aurones (flower pigments). We evidence for the first time, to our knowledge, hydroxylase activity for a catechol oxidase (AUS1) toward its natural monophenolic substrate (chalcone). The presente...

  16. A Preliminary Study of DBH (Encoding Dopamine Beta-Hydroxylase) Genetic Variation and Neural Correlates of Emotional and Motivational Processing in Individuals With and Without Pathological Gambling. (United States)

    Yang, Bao-Zhu; Balodis, Iris M; Lacadie, Cheryl M; Xu, Jiansong; Potenza, Marc N


    Background and aims Corticostriatal-limbic neurocircuitry, emotional and motivational processing, dopaminergic and noradrenergic systems and genetic factors have all been implicated in pathological gambling (PG). However, allelic variants of genes influencing dopaminergic and noradrenergic neurotransmitters have not been investigated with respect to the neural correlates of emotional and motivational states in PG. Dopamine beta-hydroxylase (DBH) converts dopamine to norepinephrine; the T allele of a functional single-nucleotide polymorphism rs1611115 (C-1021T) in the DBH gene is associated with less DBH activity and has been linked to emotional processes and addiction. Here, we investigate the influence of rs1611115 on the neural correlates of emotional and motivational processing in PG and healthy comparison (HC) participants. Methods While undergoing functional magnetic resonance imaging, 18 PG and 25 HC participants, all European Americans, viewed gambling-, sad-, and cocaine-related videotapes. Analyses focused on brain activation differences related to DBH genotype (CC/T-carrier [i.e., CT and TT]) and condition (sad/gambling/cocaine). Results CC participants demonstrated greater recruitment of corticostriatal-limbic regions, relative to T-carriers. DBH variants were also associated with altered corticostriatal-limbic activations across the different videotape conditions, and this association appeared to be driven by greater activation in CC participants relative to T-carriers during the sad condition. CC relative to T-carrier subjects also reported greater subjective sadness to the sad videotapes. Conclusions Individual differences in genetic composition linked to aminergic function contribute significantly to emotional regulation across diagnostic groups and warrant further investigation in PG.

  17. A Preliminary Study of DBH (Encoding Dopamine Beta-Hydroxylase) Genetic Variation and Neural Correlates of Emotional and Motivational Processing in Individuals With and Without Pathological Gambling. (United States)

    Yang, Bao-Zhu; Balodis, Iris M; Lacadie, Cheryl M; Xu, Jiansong; Potenza, Marc N


    Background and aims Corticostriatal-limbic neurocircuitry, emotional and motivational processing, dopaminergic and noradrenergic systems and genetic factors have all been implicated in pathological gambling (PG). However, allelic variants of genes influencing dopaminergic and noradrenergic neurotransmitters have not been investigated with respect to the neural correlates of emotional and motivational states in PG. Dopamine beta-hydroxylase (DBH) converts dopamine to norepinephrine; the T allele of a functional single-nucleotide polymorphism rs1611115 (C-1021T) in the DBH gene is associated with less DBH activity and has been linked to emotional processes and addiction. Here, we investigate the influence of rs1611115 on the neural correlates of emotional and motivational processing in PG and healthy comparison (HC) participants. Methods While undergoing functional magnetic resonance imaging, 18 PG and 25 HC participants, all European Americans, viewed gambling-, sad-, and cocaine-related videotapes. Analyses focused on brain activation differences related to DBH genotype (CC/T-carrier [i.e., CT and TT]) and condition (sad/gambling/cocaine). Results CC participants demonstrated greater recruitment of corticostriatal-limbic regions, relative to T-carriers. DBH variants were also associated with altered corticostriatal-limbic activations across the different videotape conditions, and this association appeared to be driven by greater activation in CC participants relative to T-carriers during the sad condition. CC relative to T-carrier subjects also reported greater subjective sadness to the sad videotapes. Conclusions Individual differences in genetic composition linked to aminergic function contribute significantly to emotional regulation across diagnostic groups and warrant further investigation in PG. PMID:27194378

  18. Expression of tyrosine hydroxylase and dopamine beta hydroxylase in lumbar sympathetic ganglia neurons in patients with Buerger disease of lower limb%下肢Buerger病患者腰交感神经节神经元TH和DBH的表达

    Institute of Scientific and Technical Information of China (English)

    李德卫; 赵渝; 洪云; 时德


    目的:研究下肢Buerger病患者腰交感神经节神经元中去甲肾上腺素合成酶酪氨酸羟化酶(tyrosine hydroxylase, TH)和多巴胺-β-羟化酶(dopamine beta hydroxylase, DBH)的表达变化. 方法:采用免疫组织化学SP法检测14例Buerger病患者腰交感神经节内神经元中TH和DBH的表达水平,11例非Buerger病患者腰交感神经节作为对照. 结果:Buerger病患者腰交感神经节神经元内TH和DBH的表达有高于正常人的总体趋势,差异具有统计学意义(P<0.01和P<0.05).结论:Buerger病患者腰交感神经节神经元TH和DBH表达增强,可使血管更多的处于痉挛状态,促进了疾病的发生发展.

  19. Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site. (United States)

    Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F


    Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

  20. Activation of retinal tyrosine hydroxylase: tolerance induced by chronic treatment with haloperidol does not modify response to light

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, J.; Neff, N.H.


    A single dose of haloperidol administered to rats in the dark increases the activity of retinal tyrosine hydroxylase. The ability of haloperidol to activate the enzyme is diminished 24 hr after terminating 22 to 30 days of treatment with haloperidol. The retinal enzyme is also tolerant to activation by treatment with chlorpromazine. In contrast, exposure of the animals to light activates the enzyme to the same extent in chronic haloperidol-treated and control animals. Thus, chronic haloperidol treatment does not modify the ability of the retinal enzyme system to respond to the physiological stimulus, light. Apparently, activation of retinol tyrosine hydroxylase by haloperidol and light occurs by independent mechanisms.

  1. Human Collagen Prolyl 4-Hydroxylase Is Activated by Ligands for Its Iron Center. (United States)

    Vasta, James D; Raines, Ronald T


    Collagen is the most abundant protein in animals. The posttranslational hydroxylation of proline residues in collagen contributes greatly to its conformational stability. Deficient hydroxylation is associated with a variety of disease states, including scurvy. The hydroxylation of proline residues in collagen is catalyzed by an Fe(II)- and α-ketoglutarate-dependent dioxygenase, collagen prolyl 4-hydroxylase (CP4H). CP4H has long been known to suffer oxidative inactivation during catalysis, and the cofactor ascorbate (vitamin C) is required to reactivate the enzyme by reducing its iron center from Fe(III) to Fe(II). Herein, we report on the discovery of the first synthetic activators of CP4H. Specifically, we find that 2,2'-bipyridine-4-carboxylate and 2,2'-bipyridine-5-carboxylate serve as ligands for the iron center in human CP4H that enhance the rate of ascorbate-dependent reactivation. This new mode of CP4H activation is available to other biheteroaryl compounds but does not necessarily extend to other prolyl 4-hydroxylases. As collagen is weakened in many indications, analogous activators of CP4H could have therapeutic benefits. PMID:27183028

  2. Immunoreactive atrial natriuretic peptide and dopamine beta-hydroxylase in myocytes and chromaffin cells of the heart of the African lungfish, Protopterus aethiopicus. (United States)

    Larsen, T H; Helle, K B; Saetersdal, T


    The heart of the African lungfish, Protopterus aethiopicus, was examined for immunoreactive atrial natriuretic peptide (ANP) and dopamine beta-hydroxylase (D beta H) as markers for hormone secreting myocytes and chromaffin cells, respectively. Specific antibodies raised against rat alpha-ANP and rat D beta H were used for immunofluorescence microscopy and immunogold electron microscopy. D beta H-immunoreactive cells were restricted to subendocardial areas of the atrium whereas ANP immunoreactivity occurred throughout both the atrial and the ventricular myocardium, showing particularly strong staining intensity in the atrial myocytes. The granular ANP immunostaining in the atrial myocytes was frequently accumulated in the sarcoplasm. In the ventricular myocytes ANP immunoreactivity occurred as scattered granular staining throughout the sarcoplasm. ANP and D beta H immunofluorescence staining coincided with the presence of immunoreactive specific granules and secretory vesicles in the cardiac myocytes and chromaffin cells, respectively, as revealed by electron microscopy. The number of ANP-containing specific granules was generally high in the atrial myocytes, and they were frequently observed in clusters in subsarcolemmal areas. Granular frequency was considerably lower and the mean granular diameter was smaller (0.142 +/- 0.045 micron versus 0.213 +/- 0.049 micron) in the ventricular than in the atrial myocytes. The present results indicate that ANP and D beta H are phylogenetically highly conserved proteins from the dipnoi to the rat. The large amounts of ANP and of specific granules are consistent with an endocrine myocardium in the Protopterus heart. The presence of D beta H and secretory vesicles in the subendocardial chromaffin cells of the atrium suggests a local production of catecholamines from dopamine in the heart of this dipnoan. PMID:7926645

  3. Experimentally calibrated computational chemistry of tryptophan hydroxylase: Trans influence, hydrogen-bonding, and 18-electron rule govern O-2-activation

    DEFF Research Database (Denmark)

    Haahr, Lærke Tvedebrink; Kepp, Kasper Planeta; Boesen, Jane;


    Insight into the nature of oxygen activation in tryptophan hydroxylase has been obtained from density functional computations. Conformations of O2-bound intermediates have been studied with oxygen trans to glutamate and histidine, respectively. An O2-adduct with O2 trans to histidine (Ohis) and a...

  4. Non-hypoxic activation of the negative regulatory feedback loop of prolyl-hydroxylase oxygen sensors. (United States)

    Tug, Suzan; Delos Reyes, Buena; Fandrey, Joachim; Berchner-Pfannschmidt, Utta


    Hypoxia inducible factors (HIF) coordinate cellular responses towards hypoxia. HIFs are mainly regulated by a group of prolyl-hydroxylases (PHDs) that in the presence of oxygen, target the HIFalpha subunit for degradation. Herein, we studied the role of nitric oxide (NO) in regulating PHD activities under normoxic conditions. In the present study we show that different NO-donors initially inhibited endogenous PHD2 activity which led to accumulation of HIF-1alpha subsequently to enhance HIF-1 dependent increased PHD2 promoter activity. Consequently PHD2 abundance and activity were strongly induced which caused downregulation of HIF-1alpha. Interestingly, upregulation of endogenous PHD2 activity by NO was not found in cells that lack an intact pVHL dependent degradation pathway. Recovery of PHD activity required intact cells and was not observed in cell extracts or recombinant PHD2. In conclusion induction of endogenous PHD2 activity by NO is dependent on a feedback loop initiated despite normoxic conditions. PMID:19427832

  5. Allosteric Regulation of Phenylalanine Hydroxylase


    Fitzpatrick, Paul F.


    The liver enzyme phenylalanine hydroxylase is responsible for conversion of excess phenylalanine in the diet to tyrosine. Phenylalanine hydroxylase is activated by phenylalanine; this activation is inhibited by the physiological reducing substrate tetrahydrobiopterin. Phosphorylation of Ser16 lowers the concentration of phenylalanine for activation. This review discusses the present understanding of the molecular details of the allosteric regulation of the enzyme.

  6. Tissue Specific Expression of Cre in Rat Tyrosine Hydroxylase and Dopamine Active Transporter-Positive Neurons.

    Directory of Open Access Journals (Sweden)

    Zhenyi Liu

    Full Text Available The rat is a preferred model system over the mouse for neurological studies, and cell type-specific Cre expression in the rat enables precise ablation of gene function in neurons of interest, which is especially valuable for neurodegenerative disease modeling and optogenetics. Yet, few such Cre rats are available. Here we report the characterization of two Cre rats, tyrosine hydroxylase (TH-Cre and dopamine active transporter (DAT or Slc6a3-Cre, by using a combination of immunohistochemistry (IHC and mRNA fluorescence in situ hybridization (FISH as well as a fluorescent reporter for Cre activity. We detected Cre expression in expected neurons in both Cre lines. Interestingly, we also found that in Th-Cre rats, but not DAT-Cre rats, Cre is expressed in female germ cells, allowing germline excision of the floxed allele and hence the generation of whole-body knockout rats. In summary, our data demonstrate that targeted integration of Cre cassette lead to faithful recapitulation of expression pattern of the endogenous promoter, and mRNA FISH, in addition to IHC, is an effective method for the analysis of the spatiotemporal gene expression patterns in the rat brain, alleviating the dependence on high quality antibodies that are often not available against rat proteins. The Th-Cre and the DAT-Cre rat lines express Cre in selective subsets of dopaminergic neurons and should be particularly useful for researches on Parkinson's disease.

  7. Tissue Specific Expression of Cre in Rat Tyrosine Hydroxylase and Dopamine Active Transporter-Positive Neurons. (United States)

    Liu, Zhenyi; Brown, Andrew; Fisher, Dan; Wu, Yumei; Warren, Joe; Cui, Xiaoxia


    The rat is a preferred model system over the mouse for neurological studies, and cell type-specific Cre expression in the rat enables precise ablation of gene function in neurons of interest, which is especially valuable for neurodegenerative disease modeling and optogenetics. Yet, few such Cre rats are available. Here we report the characterization of two Cre rats, tyrosine hydroxylase (TH)-Cre and dopamine active transporter (DAT or Slc6a3)-Cre, by using a combination of immunohistochemistry (IHC) and mRNA fluorescence in situ hybridization (FISH) as well as a fluorescent reporter for Cre activity. We detected Cre expression in expected neurons in both Cre lines. Interestingly, we also found that in Th-Cre rats, but not DAT-Cre rats, Cre is expressed in female germ cells, allowing germline excision of the floxed allele and hence the generation of whole-body knockout rats. In summary, our data demonstrate that targeted integration of Cre cassette lead to faithful recapitulation of expression pattern of the endogenous promoter, and mRNA FISH, in addition to IHC, is an effective method for the analysis of the spatiotemporal gene expression patterns in the rat brain, alleviating the dependence on high quality antibodies that are often not available against rat proteins. The Th-Cre and the DAT-Cre rat lines express Cre in selective subsets of dopaminergic neurons and should be particularly useful for researches on Parkinson's disease.

  8. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases. (United States)

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette


    Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze theo-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme's interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate-enzyme complexes were performed, and a key residue was identified that influences the plant PPO's acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their--so far unknown--natural substrates in vivo. PMID:26976571

  9. Effect of water miscible organic solvents on p-nitrophenol hydroxylase (CYP2E1 activity in rat liver microsomes

    Directory of Open Access Journals (Sweden)

    Pranali G Patil


    Full Text Available Organic solvents used for solubilization of the substrates/NCEs are known to affect the activity of cytochrome P450 enzymes. Further, this effect varies with the solvents used, the substrates and CYP450 isoforms in question. In the present study, we have investigated the effect of ten commonly used water miscible organic solvents (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, acetone, dimethyl sulphoxide, N,N-dimethyl formamide, dioxane and polyethylene glycol 400 on p-nitrophenol hydroxylase activity at 0, 0.1, 0.25, 0.5, 0.75 and 1% v/v concentration in rat liver microsomes. All the solvents studied showed concentration dependent inhibition of the p-nitrophenol hydroxylase activity except acetonitrile which showed activation of the activity at concentration range studied. Out of ten solvents studied, dioxane was found to be the most inhibitory solvent (inhibition >90% at 0.25% v/v concentration. Overall, solvents like dimethyl sulphoxide, dimethyl formamide and dioxane appeared to be unsuitable for characterizing p-nitrophenol hydroxylase (CYP2E1-mediated reactions due to a high degree of inhibition. On the other hand, methanol and acetonitrile at concentrations <0.5% v/v appeared to be appropriate solvents for substrate solubilization while evaluating CYP2E1-mediated catalysis. The results of this study imply that caution should be exercised while choosing solvents for dissolution of substrate during enzyme studies in liver microsomes.

  10. Generation of Two Noradrenergic-Specific Dopamine-Beta-Hydroxylase-FLPo Knock-In Mice Using CRISPR/Cas9-Mediated Targeting in Embryonic Stem Cells. (United States)

    Sun, Jenny J; Ray, Russell


    CRISPR/Cas9 mediated DNA double strand cutting is emerging as a powerful approach to increase rates of homologous recombination of large targeting vectors, but the optimization of parameters, equipment and expertise required remain barriers to successful mouse generation by single-step zygote injection. Here, we sought to apply CRISPR/Cas9 methods to traditional embryonic stem (ES) cell targeting followed by blastocyst injection to overcome the common issues of difficult vector construction and low targeting efficiency. To facilitate the study of noradrenergic function, which is implicated in myriad behavioral and physiological processes, we generated two different mouse lines that express FLPo recombinase under control of the noradrenergic-specific Dopamine-Beta-Hydroxylase (DBH) gene. We found that by co-electroporating a circular vector expressing Cas9 and a locus-specific sgRNA, we could target FLPo to the DBH locus in ES cells with shortened 1 kb homology arms. Two different sites in the DBH gene were targeted; the translational start codon with 6-8% targeting efficiency, and the translational stop codon with 75% targeting efficiency. Using this approach, we established two mouse lines with DBH-specific expression of FLPo in brainstem catecholaminergic populations that are publically available on MMRRC (MMRRC_041575-UCD and MMRRC_041577-UCD). Altogether, this study supports simplified, high-efficiency Cas9/CRISPR-mediated targeting in embryonic stem cells for production of knock-in mouse lines in a wider variety of contexts than zygote injection alone. PMID:27441631

  11. Generation of Two Noradrenergic-Specific Dopamine-Beta-Hydroxylase-FLPo Knock-In Mice Using CRISPR/Cas9-Mediated Targeting in Embryonic Stem Cells.

    Directory of Open Access Journals (Sweden)

    Jenny J Sun

    Full Text Available CRISPR/Cas9 mediated DNA double strand cutting is emerging as a powerful approach to increase rates of homologous recombination of large targeting vectors, but the optimization of parameters, equipment and expertise required remain barriers to successful mouse generation by single-step zygote injection. Here, we sought to apply CRISPR/Cas9 methods to traditional embryonic stem (ES cell targeting followed by blastocyst injection to overcome the common issues of difficult vector construction and low targeting efficiency. To facilitate the study of noradrenergic function, which is implicated in myriad behavioral and physiological processes, we generated two different mouse lines that express FLPo recombinase under control of the noradrenergic-specific Dopamine-Beta-Hydroxylase (DBH gene. We found that by co-electroporating a circular vector expressing Cas9 and a locus-specific sgRNA, we could target FLPo to the DBH locus in ES cells with shortened 1 kb homology arms. Two different sites in the DBH gene were targeted; the translational start codon with 6-8% targeting efficiency, and the translational stop codon with 75% targeting efficiency. Using this approach, we established two mouse lines with DBH-specific expression of FLPo in brainstem catecholaminergic populations that are publically available on MMRRC (MMRRC_041575-UCD and MMRRC_041577-UCD. Altogether, this study supports simplified, high-efficiency Cas9/CRISPR-mediated targeting in embryonic stem cells for production of knock-in mouse lines in a wider variety of contexts than zygote injection alone.

  12. Generation of Two Noradrenergic-Specific Dopamine-Beta-Hydroxylase-FLPo Knock-In Mice Using CRISPR/Cas9-Mediated Targeting in Embryonic Stem Cells (United States)

    Sun, Jenny J.


    CRISPR/Cas9 mediated DNA double strand cutting is emerging as a powerful approach to increase rates of homologous recombination of large targeting vectors, but the optimization of parameters, equipment and expertise required remain barriers to successful mouse generation by single-step zygote injection. Here, we sought to apply CRISPR/Cas9 methods to traditional embryonic stem (ES) cell targeting followed by blastocyst injection to overcome the common issues of difficult vector construction and low targeting efficiency. To facilitate the study of noradrenergic function, which is implicated in myriad behavioral and physiological processes, we generated two different mouse lines that express FLPo recombinase under control of the noradrenergic-specific Dopamine-Beta-Hydroxylase (DBH) gene. We found that by co-electroporating a circular vector expressing Cas9 and a locus-specific sgRNA, we could target FLPo to the DBH locus in ES cells with shortened 1 kb homology arms. Two different sites in the DBH gene were targeted; the translational start codon with 6–8% targeting efficiency, and the translational stop codon with 75% targeting efficiency. Using this approach, we established two mouse lines with DBH-specific expression of FLPo in brainstem catecholaminergic populations that are publically available on MMRRC (MMRRC_041575-UCD and MMRRC_041577-UCD). Altogether, this study supports simplified, high-efficiency Cas9/CRISPR-mediated targeting in embryonic stem cells for production of knock-in mouse lines in a wider variety of contexts than zygote injection alone. PMID:27441631

  13. PPARα activators down-regulate CYP2C7, a retinoic acid and testosterone hydroxylase

    International Nuclear Information System (INIS)

    Peroxisome proliferators (PP) are a large class of structurally diverse chemicals that mediate their effects in the liver mainly through the peroxisome proliferator-activated receptor α (PPARα). Exposure to PP results in down-regulation of CYP2C family members under control of growth hormone and sex steroids including CYP2C11 and CYP2C12. We hypothesized that PP exposure would also lead to similar changes in CYP2C7, a retinoic acid and testosterone hydroxylase. CYP2C7 gene expression was dramatically down-regulated in the livers of rats treated for 13 weeks by WY-14,643 (WY; 500 ppm) or gemfibrozil (GEM; 8000 ppm). In the same tissues, exposure to WY and GEM and to a lesser extent di-n-butyl phthalate (20 000 ppm) led to decreases in CYP2C7 protein levels in both male and female rats. An examination of the time and dose dependence of CYP2C7 protein changes after PP exposure revealed that CYP2C7 was more sensitive to compound exposure compared to other CYP2C family members. Protein expression was decreased after 1, 5 and 13 weeks of PP treatment. CYP2C7 protein expression was completely abolished at 5 ppm WY, the lowest dose tested. GEM and DBP exhibited dose-dependent decreases in CYP2C7 protein expression, becoming significant at 1000 ppm or 5000 ppm and above, respectively. These results show that PP exposure leads to changes in CYP2C7 mRNA and protein levels. Thus, in addition to known effects on steroid metabolism, exposure to PP may alter retinoic acid metabolism

  14. The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers. (United States)

    Zhang, Shengnan; Hinck, Andrew P; Fitzpatrick, Paul F


    Liver phenylalanine hydroxylase is allosterically activated by phenylalanine. The structural changes that accompany activation have not been identified, but recent studies of the effects of phenylalanine on the isolated regulatory domain of the enzyme support a model in which phenylalanine binding promotes regulatory domain dimerization. Such a model predicts that compounds that stabilize the regulatory domain dimer will also activate the enzyme. Nuclear magnetic resonance spectroscopy and analytical ultracentrifugation were used to determine the ability of different amino acids and phenylalanine analogues to stabilize the regulatory domain dimer. The abilities of these compounds to activate the enzyme were analyzed by measuring their effects on the fluorescence change that accompanies activation and on the activity directly. At concentrations of 10-50 mM, d-phenylalanine, l-methionine, l-norleucine, and (S)-2-amino-3-phenyl-1-propanol were able to activate the enzyme to the same extent as 1 mM l-phenylalanine. Lower levels of activation were seen with l-4-aminophenylalanine, l-leucine, l-isoleucine, and 3-phenylpropionate. The ability of these compounds to stabilize the regulatory domain dimer agreed with their ability to activate the enzyme. These results support a model in which allosteric activation of phenylalanine hydroxylase is linked to dimerization of regulatory domains.

  15. The dopamine beta-hydroxylase gene polymorphism rs1611114 is associated with schizophrenia in the Chinese Zhuang but not Chinese Han population. (United States)

    Long, Jianxiong; Huang, Guifeng; Liang, Baoyun; Ling, Weijun; Guo, Xiaojing; Jiang, Juan; Su, Li


    Schizophrenia (SCZ) is a devastating neurodevelopmental disorder. However, the mechanism underlying this highly heritable disorder remains unclear. The dopamine beta-hydroxylase (DBH) gene encodes a key metabolic enzyme of dopamine. Consequently, DBH is considered a candidate gene for SCZ. However, previous studies on its association with SCZ susceptibility have shown conflicting results. Here, we examined association between the rs1611114 polymorphism of DBH and SCZ susceptibility and related clinical symptoms. A total of 691 SCZ patients and 698 age- and gender-matched healthy controls were examined. mRNA expression levels of DBH were measured by quantitative real-time polymerase chain reaction, and the rs1611114 polymorphism was genotyped using the Sequenom MassARRAY platform. Also, the Positive and Negative Syndrome Scale (PANSS) was used to assess SCZ clinical symptoms. Our results show lower DBH mRNA expression levels in SCZ patients than healthy controls (Zhuang: p = 0.000; Han: p = 0.037). Interestingly, the rs1611114 polymorphism was significantly associated with SCZ susceptibility (overdominant model: p = 0.010) in only the Chinese Zhuang population. Furthermore, the rs1611114 polymorphism was associated with PANSS total score (allele T/C: p = 0.015) and general psychopathology score (allele T/C: p = 0.027) in Chinese Zhuang SCZ patients. These results suggest that the DBH gene may play an important role in the occurrence of SCZ. Also, rs1611114 may be associated with SCZ susceptibility and related clinical symptoms in the Chinese Zhuang but not Han Chinese population. Further studies with larger samples of different ethnicities are needed to confirm the role of DBH in SCZ. PMID:27236774

  16. 福尔马林致痛大鼠脊髓去甲肾上腺素能神经元及其激活蛋白2α的表达变化%Changes of dopamine-beta-hydroxylase and activator protein-2α expression in spinal cord of formalin-induced rat pain model

    Institute of Scientific and Technical Information of China (English)

    汪克建; 孙善全; 贺桂琼; 陈海; 余维华


    目的 观察疼痛应激时,脊髓去甲肾上腺素(noradrenaline, NA)能神经元的形态、分布、数量和激活蛋白2α(AP-2α)的表达变化,以探讨NA参与痛觉调制的分子机制.方法 应用免疫组化、免疫荧光双标、Western blotting和计算机图像分析方法检测福尔马林诱导致痛模型大鼠脊髓多巴胺-β-羟化酶(dopamine-β-hydroxylase, DBH)和AP-2α的表达变化.结果 正常脊髓内NA能神经元主要分布于脊髓前角,模型鼠脊髓前角、中间带和后角均出现大量深染的DBH阳性神经元,3 d时达到高峰,至第7天时DBH阳性神经元数有所下降,但仍高于正常水平.图像分析和统计学分析表明,DBH阳性神经元的数量和染色强度明显增加,与对照组相比具有显著性差异(P<0.05);Western blotting结果证实DBH不同时段的表达变化规律与上述形态学变化一致.AP-2α的表达变化与DBH的表达变化规律具有显著的相似性.免疫荧光双标显示DBH和AP-2α共存于脊髓神经元内.结论 在疼痛等应激状态下,脊髓前角NA能神经元内DBH表达明显增强;且中间带和后角新出现大量DBH阳性神经元,推测这些区域内一些神经元可能发生了化学性质的改变,由非NA能神经元转化为NA能神经元;而上述区域内AP-2α表达随着DBH表达增强而增加,提示NA与AP-2α在痛觉的调制和调控中发挥重要作用.

  17. Embryotoxicity, teratogenicity, and aryl hydrocarbon hydroxylase activity in Forster's terns on Green Bay, Lake Michigan

    Energy Technology Data Exchange (ETDEWEB)

    Hoffman, D.J.; Rattner, B.A.; Sileo, L.; Docherty, D.; Kubiak, T.J.


    Known reproductive problems, including congenital malformations and poor hatching success, exist for the state endangered Forster's tern (Sterna forsteri) in Green Bay, Wisconsin. Twenty Forster's tern eggs were collected from separate nests at (i) a natural colony with documented reproductive problems, situated at Green Bay, Lake Michigan, and (ii) an inland colony at Lake Poygan (control) where reproduction was documented as normal. Eggs from the two locations were placed in the same laboratory incubator and candled throughout incubation. Hatching success of Green Bay eggs was 52% of that for controls. Several early embryonic deaths occurred, but most mortality occurred close to the time of hatching. Liver microsomal aryl hydrocarbon hydroxylase activity was elevated approximately threefold in Green Bay hatchlings compared to controls. Green Bay terns that hatched weighed less than controls, had an increased liver to body weight ratio, and had a shorter femur length. Two Green Bay embryos that failed to hatch had anomalies, one with a crossed beak and one with poor ossification of the foot. One Green Bay hatchling had an abnormally ossified ilium. These effects were observed in eggs where there were measurable levels of aryl hydrocarbon hydroxylase inducers including polychlorinated biphenyls and polychlorinated dibenzo-p-dioxins.

  18. Pollination-, development-, and auxin-specific regulation of gibberellin 3beta-hydroxylase gene expression in pea fruit and seeds. (United States)

    Ozga, Jocelyn A; Yu, Jody; Reinecke, Dennis M


    To understand further how pollination, seeds, auxin (4-chloroindole-3-acetic acid [4-Cl-IAA]), and gibberellins (GAs) regulate GA biosynthesis in pea (Pisum sativum) fruit, we studied expression of the gene PsGA3ox1 that codes for the enzyme that converts GA(20) to biologically active GA(1) using real-time reverse transcription-polymerase chain reaction analysis. PsGA3ox1 mRNA levels were minimally detectable in prepollinated pericarps and ovules (-2 d after anthesis [DAA]), increased dramatically after pollination (0 DAA), then decreased by 1 DAA. Seed PsGA3ox1 mRNA levels increased at 4 DAA and again 8 to 12 DAA, when seed development was rapid. Pericarp PsGA3ox1 mRNA levels peaked coincidentally with rapid pod diameter expansion (6-10 DAA) to accommodate the growing seeds. The effects of seeds and hormones on the expression of pericarp PsGA3ox1 were investigated over a 24-h treatment period. Pericarp PsGA3ox1 mRNA levels gradually increased from 2 to 3 DAA when seeds were present; however, when the seeds were removed, the pericarp transcript levels dramatically declined. When 2-DAA deseeded pericarps were treated with 4-Cl-IAA, PsGA3ox1 mRNA levels peaked 4 h after hormone treatment (270-fold increase), then decreased. PsGA3ox1 mRNA levels in deseeded pericarps treated with indole-3-acetic acid or GA(3) were the same or lower than deseeded controls. These data show that PsGA3ox1 is expressed and developmentally regulated in pea pericarps and seeds. These data also show that pericarp PsGA3ox1 expression is hormonally regulated and suggest that the conversion of GA(20) to GA(1) occurs in the pericarp and is regulated by the presence of seeds and 4-Cl-IAA for fruit growth.

  19. Possible errors in assay for beta-glycosidase activity.


    Chadwick, R W; Allison, J C; Talley, D L; George, S.E.


    Cecal homogenates were assayed for the enzymes beta-glucosidase, beta-glucuronidase, and beta-galactosidase. Anaerobic incubation with the addition of excess 3,4-dichloronitrobenzene, a substrate for nitroreductase, significantly increased the detection of the beta-glycosidase enzymes' activities.

  20. Genetic analysis of beta1 integrin "activation motifs" in mice

    DEFF Research Database (Denmark)

    Czuchra, Aleksandra; Meyer, Hannelore; Legate, Kyle R;


    /beta tails, leading to tail separation and integrin activation. We analyzed mice in which we mutated the tyrosines of the beta1 tail and the membrane-proximal aspartic acid required for the salt bridge. Tyrosine-to-alanine substitutions abolished beta1 integrin functions and led to a beta1 integrin...... and the membrane-proximal salt bridge between alpha and beta1 tails have no apparent function under physiological conditions in vivo....

  1. In Vitro Interaction of 5-Hydroxytrptamine with Cytosolic Molybdenum Hydroxylases as a Potential Inhibitor for Initial Rates Activities

    Directory of Open Access Journals (Sweden)

    Abdullah M. Al-Mohizea


    Full Text Available Problem statement: The role of 5-HT has been investigated in many behavioral activities. Thus, studies using raphe lesion showed that 5-HT is involved in sleep, general activity levels, habituation, aggression, pain sensitivity and morphine analgesia, avoidance behavior, self-stimulation and water consumption. Approach: The metabolic interaction between serotonin (5- hydroxytrptamine and indole-3-aldehyde and xanthine via aldehyde oxidase (EC and xanthine oxidase (EC, respectively, were studied in liver tissue homogenate of Dunkin-Hartley guinea pigs by following the decrease in substrate concentration using spectrophotometer. Homogenates of liver were incubated with indole-3-aldehyde in the presence and absence of serotonin or (chlorpromazine and allopurinol a potent and selective inhibitors for aldehyde oxidase and xanthine oxidase, respectively. Oxidation of indole-3-aldehyde to indole-3-acetic acid was reduced up to 63.2% in the presence of serotonin (100 µM, while oxidation of xanthine to uric acid was reduced up to 51.6% under the same conditions. Results: In comparison, incubation of the substrates with their specific inhibitors (100 µM of chlorpromazine and 100 µM allopurinol give almost complete inhibition. These results demonstrate that in the guinea pig liver a metabolic interaction between serotonin and indole-3-aldehyde or xanthine via molybdenum hydroxylases system may take place in liver, which is the main tissue for xenobiotics detoxification. Conclusion: The overall conclusion from this research is that serotonin could be a protector for neurons and other tissue from the insult of oxidation of aldehydes and xanthines by molybdenum hydroxylases.

  2. Complement activation by the amyloid proteins A beta peptide and beta 2-microglobulin

    DEFF Research Database (Denmark)

    Nybo, Mads; Nielsen, E H; Svehag, S E


    Complement activation (CA) has been reported to play a role in the pathogenesis of Alzheimer's disease (AD). To investigate whether CA may contribute to amyloidogenesis in general, the CA potential of different amyloid fibril proteins was tested. CA induced by A beta preparations containing soluble...... protein, protofilaments and some fibrils or only fibrils in a solid phase system (ELISA) was modest with a slow kinetics compared to the positive delta IgG control. Soluble A beta induced no detectable CA in a liquid phase system (complement consumption assay) while fibrillar A beta caused CA at 200 mg....../ml and higher concentrations. Soluble beta 2-microglobulin (beta 2M) purified from peritoneal dialysates was found to be as potent a complement activator as A beta in both solid and liquid phase systems while beta 2M purified from urine exhibited lower activity, a difference which may be explained...

  3. Polymorphism in the tyrosine hydroxylase (TH gene is associated with activity-impulsivity in German Shepherd Dogs.

    Directory of Open Access Journals (Sweden)

    Eniko Kubinyi

    Full Text Available We investigated the association between repeat polymorphism in intron 4 of the tyrosine hydroxylase (TH gene and two personality traits, activity-impulsivity and inattention, in German Shepherd Dogs. The behaviour of 104 dogs was characterized by two instruments: (1 the previously validated Dog-Attention Deficit Hyperactivity Disorder Rating Scale (Dog-ADHD RS filled in by the dog owners and (2 the newly developed Activity-impulsivity Behavioural Scale (AIBS containing four subtests, scored by the experimenters. Internal consistency, inter-observer reliability, test-retest reliability and convergent validity were demonstrated for AIBS. Dogs possessing at least one short allele were proved to be more active-impulsive by both instruments, compared to dogs carrying two copies of the long allele (activity-impulsivity scale of Dog-ADHD RS: p = 0.007; AIBS: p = 0.023. The results have some potential to support human studies; however, further research should reveal the molecular function of the TH gene variants, and look for the effect in more breeds.

  4. Mapping of the Allosteric Site in Cholesterol Hydroxylase CYP46A1 for Efavirenz, a Drug That Stimulates Enzyme Activity. (United States)

    Anderson, Kyle W; Mast, Natalia; Hudgens, Jeffrey W; Lin, Joseph B; Turko, Illarion V; Pikuleva, Irina A


    Cytochrome P450 46A1 (CYP46A1) is a microsomal enzyme and cholesterol 24-hydroxylase that controls cholesterol elimination from the brain. This P450 is also a potential target for Alzheimer disease because it can be activated pharmacologically by some marketed drugs, as exemplified by efavirenz, the anti-HIV medication. Previously, we suggested that pharmaceuticals activate CYP46A1 allosterically through binding to a site on the cytosolic protein surface, which is different from the enzyme active site facing the membrane. Here we identified this allosteric site for efavirenz on CYP46A1 by using a combination of hydrogen-deuterium exchange coupled to MS, computational modeling, site-directed mutagenesis, and analysis of the CYP46A1 crystal structure. We also mapped the binding region for the CYP46A1 redox partner oxidoreductase and found that the allosteric and redox partner binding sites share a common border. On the basis of the data obtained, we propose the mechanism of CYP46A1 allostery and the pathway for the signal transmission from the P450 allosteric site to the active site. PMID:27056331

  5. AGN 2979 [3(3-methoxyphenyl)-3-(3-dimethylaminopropyl)-4, 4-dimethylpiperidine-2,6-dione]. An inhibitor of the activation of tryptophan hydroxylase. (United States)

    Boadle-Biber, M C; Phan, T H


    AGN 2979 [3-(3-methoxyphenyl)-3-(3-dimethylaminopropyl)-4, 4-dimethylpiperidine-2,6-dione] blocked the increase in tryptophan hydroxylase activity that occurred when slices of brainstem were exposed to a depolarizing medium or to agents that mobilize intracellular pools of calcium, but it had no effect on the activity of enzyme prepared from slices of brainstem incubated in control medium. AGN 2979 also blocked the calcium-calmodulin-dependent activation of tryptophan hydroxylase that was seen when supernatant preparations of the enzyme were exposed to phosphorylating conditions but not the activation induced by calcium-dependent proteases that was triggered by millimolar calcium concentrations. An identical pattern of inhibition has been found with the antipsychotic drugs, haloperidol and fluphenazine [Boadle-Biber, Biochem. Pharmac. 31, 2495 (1982)]. The sensitivity to the same inhibitors of both the activation of tryptophan hydroxylase produced by pretreatment of brainstem slices and that induced by incubation of supernatant preparations of enzyme under phosphorylating conditions suggests involvement of a common mechanism of enzyme activation in response to these different treatments.

  6. Radiation-induced polymerization of {beta}(+)-pinene and synthesis of optically active {beta}(+)/{beta}(-)pinene polymers and copolymers

    Energy Technology Data Exchange (ETDEWEB)

    Cataldo, Franco, E-mail: franco.cataldo@fastwebnet.i [Lupi Chemical Research, Via Casilina 1626/A, 00133 Rome (Italy); Lilla, Edo; Ursini, Ornella [Institute of Chemical Methodologies, CNR, Via Salaria Km. 29300, Monterotondo Stazione 00016, Rome (Italy)


    Poly-{beta}(+)-pinene (pB(+)p) was synthesized with {gamma} irradiation of the monomer {beta}(+)-pinene in bulk under vacuum at 1181 kGy. Also scalemic mixtures of {beta}(+)-pinene and {beta}(-)-pinene were irradiated at 1181 kGy to obtain synthetic copolymers of pB(+)/B(-)p. For comparison also {beta}(-)-pinene was converted into poly-{beta}(-)-pinene (pB(-)p) under the identical conditions adopted for its enantiomer. Furthermore pB(+)p and pB(-)p were also synthesized by thermal processing under the action of a chemical free radical initiator. The optical rotatory dispersion (ORD) of all pBp resins synthesized were accurately studied in the spectral range comprised between 375 and 625 nm and a curious asymmetry in the ORD of pB(+)p versus the ORD of pB(-)p is reported. Furthermore, it is shown that (+)-p-menth-1-ene and (-)-p-menth-1-ene are useful as a model compounds for the pBp resins and for the explanation of the amplification of the optical activity of the {beta}(+)-pinene and {beta}(-)-pinene after their ring-opening polymerization to pB(+)p and pB(-)p. The pBp resins were studied also by FT-IR spectroscopy and by thermal analysis (TGA and DTG).

  7. Latency and activation in the control of TGF-beta (United States)

    Barcellos-Hoff, M. H.; Chatterjee, A. (Principal Investigator)


    The biological activity of the transforming growth factor-beta's (TGF-beta)3 is tightly controlled by their persistence in the extracellular compartment as latent complexes. Each of the three mammalian isoform genes encodes a product that is cleaved intracellularly to form two polypeptides, each of which dimerizes. Mature TGF-beta, a 24 kD homodimer, is noncovalently associated with the 80 kD latency-associated peptide (LAP). LAP is a fundamental component of TGF-beta that is required for its efficient secretion, prevents it from binding to ubiquitous cell surface receptors, and maintains its availability in a large extracellular reservoir that is readily accessed by activation. This latent TGF-beta complex (LTGF-beta) is secreted by all cells and is abundant both in circulating forms and bound to the extracellular matrix. Activation describes the collective events leading to the release of TGF-beta. Despite the importance of TGF-beta regulation of growth and differentiation in physiological and malignant tissue processes, remarkably little is known about the mechanisms of activation in situ. Recent studies of irradiated mammary gland reveal certain features of TGF-beta 1 activation that may shed light on its regulation and potential roles in the normal and neoplastic mammary gland.

  8. Genetics Home Reference: tyrosine hydroxylase deficiency (United States)

    Skip to main content Your Guide to Understanding Genetic Conditions Enable Javascript for addthis links to activate. ... Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Home Health Conditions TH deficiency tyrosine hydroxylase deficiency ...

  9. HYSCORE Analysis of the Effects of Substrates on Coordination of Water to the Active Site Iron in Tyrosine Hydroxylase. (United States)

    McCracken, John; Eser, Bekir E; Mannikko, Donald; Krzyaniak, Matthew D; Fitzpatrick, Paul F


    Tyrosine hydroxylase is a mononuclear non-heme iron monooxygenase found in the central nervous system that catalyzes the hydroxylation of tyrosine to yield L-3,4-dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. Catalysis requires the binding of tyrosine, a tetrahydropterin, and O₂ at an active site that consists of a ferrous ion coordinated facially by the side chains of two histidines and a glutamate. We used nitric oxide as a surrogate for O₂ to poise the active site iron in an S = ³/₂ {FeNO}⁷ form that is amenable to electron paramagnetic resonance (EPR) spectroscopy. The pulsed EPR method of hyperfine sublevel correlation (HYSCORE) spectroscopy was then used to probe the ligands at the remaining labile coordination sites on iron. For the complex formed by the addition of tyrosine and nitric oxide, TyrH/NO/Tyr, orientation-selective HYSCORE studies provided evidence of the coordination of one H₂O molecule characterized by proton isotropic hyperfine couplings (A(iso) = 0.0 ± 0.3 MHz) and dipolar couplings (T = 4.4 and 4.5 ± 0.2 MHz). These data show complex HYSCORE cross peak contours that required the addition of a third coupled proton, characterized by an A(iso) of 2.0 MHz and a T of 3.8 MHz, to the analysis. This proton hyperfine coupling differed from those measured previously for H₂O bound to {FeNO}⁷ model complexes and was assigned to a hydroxide ligand. For the complex formed by the addition of tyrosine, 6-methyltetrahydropterin, and NO, TyrH/NO/Tyr/6-MPH₄, the HYSCORE cross peaks attributed to H₂O and OH⁻ for the TyrH/NO/Tyr complex were replaced by a cross peak due to a single proton characterized by an A(iso) of 0.0 MHz and a dipolar coupling (T = 3.8 MHz). This interaction was assigned to the N₅ proton of the reduced pterin.

  10. Furosemide and 11beta-hydroxysteroid dehydrogenase activity, in man. (United States)

    Palermo, M; Armanini, D; Shackleton, C H L; Sorba, G; Cossu, M; Roitman, E; Scaroni, C; Delitala, G


    Mineralocorticoid receptors possess the same affinity for aldosterone and for cortisol and preferential binding of aldosterone is modulated by the 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) enzyme, which converts cortisol to its inactive metabolite cortisone. Several endogenous or exogenous compounds able to inhibit the enzyme have been described and, as a consequence, produce the syndrome of apparent mineralocorticoid excess (AME) characterized by hypertension, hypokalemia, volume repletion and suppression of the renin-angiotensin-aldosterone system. High doses of furosemide, a diuretic that works in the luminal surface of the thick ascending limb of Henle's loop, have been reported to inhibit 11 beta-OHSD activity to the same extent as licorice in vivo and in vitro, in rat. The aim of our study was to verify the effect of the drug on 11 beta-OHSD activity in man at the doses currently used in clinical practice. We tested the activity of 11 beta-OHSD following both acute and protracted administration of furosemide. In the acute study, the drug was administered at low (40 mg i.v. in bolo) and high doses (infusion of 10 mg/kg bw i.v for six hours); the protracted furosemide administration consisted in 50 mg/day for 20 days, by mouth. The ratios between the cortisol metabolites tetrahydrocortisol plus allo-tetrahydrocortisol to tetra-hydrocortisone and urinary free cortisol to urinary free cortisone were used to measure the activity of 11 beta-OHSD. Urinary cortisol, cortisone and their metabolites were tested by a gas-chromatographic/mass spectrometric method. Neither acute nor prolonged administration of furosemide did affect the activity of 11 beta-OHSD although the drug was able to modify plasma aldosterone and PRA secretion and to determine hypokalemia. Our results suggest that furosemide does not play a significant role in 11 beta-OHSD modulation in humans, at least at the dosage used in clinical practice. PMID:12373630

  11. Bile acids activate fibroblast growth factor 19 signaling in human hepatocytes to inhibit cholesterol 7α-hydroxylase gene expression


    Song, Kwang-Hoon; Li, Tiangang; Owsley, Erika; Strom, Stephen; Chiang, John Y. L.


    Mouse fibroblast growth factor 15 (FGF15) and human ortholog FGF19 have been identified as the bile acid-induced intestinal factors that mediate bile acid feedback inhibition of cholesterol 7α-hydroxylase gene transcription in mouse liver. The mechanism underlying FGF15/FGF19 inhibition of bile acid synthesis in hepatocytes remains unclear. Chenodeoxycholic acid (CDCA) and a farnesoid X receptor (FXR)-specific agonist GW4064 strongly induced FGF19 but inhibited CYP7A1 mRNA levels in primary h...

  12. Peripherally administered tetrahydrobiopterin increases in vivo tryptophan hydroxylase activity in the striatum after transplantation of fetal ventral mesencephalon in six hydroxydopamine lesioned rats. (United States)

    Ishida, Y; Todaka, K; Kuwahara, I; Hashiguchi, H; Ishizuka, Y; Nakane, H; Mitsuyama, Y


    The intraperitoneal administration of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH4), a natural cofactor for tyrosine hydroxylase and tryptophan hydroxylase (TRH), dose-dependently increased the extracellular concentration of 6R-BH4 itself in rat striatum. The concentration was investigated by in vivo microdialysis and measured simultaneously with 5-hydroxytryptophan (5-HTP), a precursor of serotonin, by high performance liquid chromatography with electrochemical detection. The 6R-BH4 (50 mg/kg, i.p.) administration increased the accumulation of 5-HTP as an index of in vivo TRH activity under the inhibition of aromatic L-amino acid decarboxylase by NSD-1015 in the striatum of both normal control and 6-hydroxydopamine lesioned rats with intrastriatal transplants of fetal ventral mesencephalon (VM). The results suggest that TRH in the striatum of both control and VM-grafted rats is activated by 6R-BH4 penetrating into the brain from the blood. PMID:9754801

  13. Novel regulator MphX represses activation of phenol hydroxylase genes caused by a XylR/DmpR-type regulator MphR in Acinetobacter calcoaceticus.

    Directory of Open Access Journals (Sweden)

    Haiying Yu

    Full Text Available Acinetobacter calcoaceticus PHEA-2 utilizes phenol as its sole carbon and energy source and has a multi-component phenol hydroxylase-encoding gene operon (mphKLMNOP for phenol degradation. Two additional genes, mphR and mphX, were found upstream and downstream of mphKLMNOP, respectively. The mphR gene encodes a XylR/DmpR-type regulator-like protein and is transcribed in the opposite direction to mphKLMNOP. The mphX gene is transcribed in the same direction as mphKLMNOP and encodes a protein with 293 amino acid residues showing weak identity with some unknown proteins encoded in the meta-cleavage pathway gene clusters for aromatic compound degradation. Disruption of mphR by homologous recombination resulted in the loss of phenol degradation while disruption of mphX caused significantly faster phenol degradation than in the wild type strain. Transcriptional assays for mphK, mphR, and mphX revealed that mphR activated mphKLMNOP transcription in the presence of phenol, but mphX partially repressed this activation. Gel mobility-shift assay demonstrated a direct interaction of MphR with the mphK promoter region. These results indicate the involvement of a novel repressor protein MphX in transcriptional regulation of phenol hydroxylase genes caused by a XylR/DmpR-type regulator MphR.

  14. Potent and Selective Triazole-Based Inhibitors of the Hypoxia-Inducible Factor Prolyl-Hydroxylases with Activity in the Murine Brain.

    Directory of Open Access Journals (Sweden)

    Mun Chiang Chan

    Full Text Available As part of the cellular adaptation to limiting oxygen availability in animals, the expression of a large set of genes is activated by the upregulation of the hypoxia-inducible transcription factors (HIFs. Therapeutic activation of the natural human hypoxic response can be achieved by the inhibition of the hypoxia sensors for the HIF system, i.e. the HIF prolyl-hydroxylases (PHDs. Here, we report studies on tricyclic triazole-containing compounds as potent and selective PHD inhibitors which compete with the 2-oxoglutarate co-substrate. One compound (IOX4 induces HIFα in cells and in wildtype mice with marked induction in the brain tissue, revealing that it is useful for studies aimed at validating the upregulation of HIF for treatment of cerebral diseases including stroke.

  15. In vitro activities of 15 oral beta-lactams against Klebsiella pneumoniae harboring new extended-spectrum beta-lactamases.


    Kitzis, M D; Liassine, N; Ferré, B.; Gutmann, L; Acar, J F; Goldstein, F.


    The activities of 15 oral beta-lactams against Klebsiella pneumoniae harboring new extended-spectrum beta-lactamases were studied. All compounds were affected by these enzymes, especially by the SHV derivatives. Except for ceftibuten, the compounds with the greatest intrinsic activity were more affected by the presence of these enzymes than were older compounds with moderate intrinsic activity.

  16. Chemical inhibition of potato ABA-8'-hydroxylase activity alters in vitro and in vivo ABA metabolism and endogenous ABA levels but does not affect potato microtuber dormancy duration. (United States)

    Suttle, Jeffrey C; Abrams, Suzanne R; De Stefano-Beltrán, Luis; Huckle, Linda L


    The effects of azole-type P450 inhibitors and two metabolism-resistant abscisic acid (ABA) analogues on in vitro ABA-8'-hydroxylase activity, in planta ABA metabolism, endogenous ABA content, and tuber meristem dormancy duration were examined in potato (Solanum tuberosum L. cv. Russet Burbank). When functionally expressed in yeast, three potato CYP707A genes were demonstrated to encode enzymatically active ABA-8'-hydroxylases with micromolar affinities for (+)-ABA. The in vitro activity of the three enzymes was inhibited by the P450 azole-type inhibitors ancymidol, paclobutrazol, diniconazole, and tetcyclasis, and by the 8'-acetylene- and 8'-methylene-ABA analogues, with diniconazole and tetcyclasis being the most potent inhibitors. The in planta metabolism of [(3)H](±)-ABA to phaseic acid and dihydrophaseic acid in tuber meristems was inhibited by diniconazole, tetcyclasis, and to a lesser extent by 8'-acetylene- and 8'-methylene-ABA. Continuous exposure of in vitro generated microtubers to diniconazole resulted in a 2-fold increase in endogenous ABA content and a decline in dihydrophaseic acid content after 9 weeks of development. Similar treatment with 8'-acetylene-ABA had no effects on the endogenous contents of ABA or phaseic acid but reduced the content of dihydrophaseic acid. Tuber meristem dormancy progression was determined ex vitro in control, diniconazole-, and 8'-acetylene-ABA-treated microtubers following harvest. Continuous exposure to diniconazole during microtuber development had no effects on subsequent sprouting at any time point. Continuous exposure to 8'-acetylene-ABA significantly increased the rate of microtuber sprouting. The results indicate that, although a decrease in ABA content is a hallmark of tuber dormancy progression, the decline in ABA levels is not a prerequisite for dormancy exit and the onset of tuber sprouting. PMID:22664582

  17. Immunocytochemical localization of latent transforming growth factor-beta1 activation by stimulated macrophages (United States)

    Chong, H.; Vodovotz, Y.; Cox, G. W.; Barcellos-Hoff, M. H.; Chatterjee, A. (Principal Investigator)


    Transforming growth factor-beta1 (TGF-beta) is secreted in a latent form consisting of mature TGF-beta noncovalently associated with its amino-terminal propeptide, which is called latency associated peptide (LAP). Biological activity depends upon the release of TGF-beta from the latent complex following extracellular activation, which appears to be the key regulatory mechanism controlling TGF-beta action. We have identified two events associated with latent TGF-beta (LTGF-beta) activation in vivo: increased immunoreactivity of certain antibodies that specifically detect TGF-beta concomitant with decreased immunoreactivity of antibodies to LAP. Macrophages stimulated in vitro with interferon-gamma and lipopolysaccharide reportedly activate LTGF-beta via cell membrane-bound protease activity. We show through dual immunostaining of paraformaldehyde-fixed macrophages that such physiological TGF-beta activation is accompanied by a loss of LAP immunoreactivity with concomitant revelation of TGF-beta epitopes. The induction of TGF-beta immunoreactivity colocalized with immunoreactive betaglycan/RIII in activated macrophages, suggesting that LTGF-beta activation occurs on the cell surface. Confocal microscopy of metabolically active macrophages incubated with antibodies to TGF-beta and betaglycan/RIII prior to fixation supported the localization of activation to the cell surface. The ability to specifically detect and localize LTGF-beta activation provides an important tool for studies of its regulation.

  18. Vaccinia virus recombinants expressing an 11-kilodalton beta-galactosidase fusion protein incorporate active beta-galactosidase in virus particles. (United States)

    Huang, C; Samsonoff, W A; Grzelecki, A


    Recombinant plasmids in which vaccinia virus transcriptional regulatory sequences were fused to the Escherichia coli lacZ gene were constructed for insertion of the lacZ gene into the vaccinia virus genome. beta-Galactosidase (beta-gal) was found in some purified recombinant vaccinia virions. By enzyme activity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and microscopic techniques, the evidence suggested that beta-gal accounted for 5% of the total protein in the virion. These recombinant viruses were constructed so that a portion of the coding sequences of a late vaccinia virus structural polypeptide was fused to the amino terminus of beta-gal to produce the fusion protein. Removal of the coding sequences resulted in the complete loss of beta-gal activity. This demonstrated that a vaccinia virus DNA segment from a late structural gene is responsible for the incorporation of beta-gal into the virion.

  19. Differential activation and tyrosine hydroxylase distribution in the hippocampal, pallial and midbrain brain regions in response to cognitive performance in Indian house crows exposed to abrupt light environment. (United States)

    Taufique, S K Tahajjul; Kumar, Vinod


    Disruption of the cyclic feature of the day-night environment can cause negative effects on daily activity and advanced brain functions such as learning, memory and decision-making behaviour. These functions in songbirds, including corvids, involve the hippocampus, pallium and midbrain, as revealed by ZENK (a neuronal activation marker) and tyrosine hydroxylase (TH) expressions. TH is rate-limiting marker enzyme of the biosynthesis of dopamine, widely implicated in learning and memory. Here, we measured ZENK and TH immunoreactivity in the hippocampal, pallial and midbrain regions in response to cognitive performance (learning-memory retrieval) tests in Indian house crows (Corvus splendens) exposed to constant light environment (LL) with controls on 12h light:12h darkness. Along with the decay of circadian rhythm in activity behaviour, LL caused a significant decline in the cognitive performance. There was also a decrease under LL in the activity of neurons in the hippocampus, medial and central caudal nidopallium, and hyperpallium apicale, which are widely distributed with TH-immunoreactive fibres. Further, under LL, TH- immunoreactive neurons were reduced in number in midbrain dopamine synthesis sites, the venteral tegmental area (VTA) and substantia nigra (SN), with a negative correlation of co-localized ZENK/TH- immunoreactive cells on errors during the association tasks. These results show decreased activity of learning and memory neural systems, and underscore the role of dopamine in reduced cognitive performance of diurnal corvids with disrupted circadian rhythms under an abrupt light environment. PMID:27478138

  20. Variations in tryptophan hydroxylase 2 linked to decreased serotonergic activity are associated with elevated risk for metabolic syndrome in depression. (United States)

    Kloiber, S; Kohli, M A; Brueckl, T; Ripke, S; Ising, M; Uhr, M; Menke, A; Unschuld, P G; Horstmann, S; Salyakina, D; Muller-Myhsok, B; Binder, E B; Holsboer, F; Lucae, S


    Major depression and the metabolic syndrome (MetS) are interacting clinical conditions influenced by genetic susceptibility. For both disorders, impaired serotonergic neurotransmission in specific brain areas has been suggested. This led us to investigate whether variants in the gene coding for tryptophan hydroxylase 2 (TPH2), the brain-specific and rate-limiting enzyme for serotonin biosynthesis, might be predictive for an increased liability for the development of MetS in depressed patients. In a case-control study consisting of 988 patients with recurrent unipolar depression (RUD) and 1023 psychiatric healthy controls, MetS components were ascertained according to the International Diabetes Foundation criteria. A total of 41 single nucleotide polymorphisms fully covering the TPH2 gene region were genotyped in stage 1 (300 patients/300 controls), resulting in significant genetic associations of polymorphisms located in exon 7 and intron 8 of TPH2 and the occurrence of MetS in depressed patients after correction for age, gender and multiple testing (51 RUD-MetS/179 RUD-non-MetS). We were able to confirm the significant association of rs17110690 in stage 2 (688 patients/723 controls; 110 RUD-MetS/549 RUD-non-MetS) and to link risk-genotypes and risk-haplotypes for MetS to lower TPH2 mRNA expression and to lower 5-hydroxyindoleacetic acid levels in cerebrospinal fluid previously reported in functional studies. Our findings suggest that TPH2 polymorphisms characterize a subgroup of depressed patients who are especially prone to develop metabolic disorders induced by a genotype-dependent impairment of serotonergic neurotransmission. Identifying depressed patients at high risk for MetS using genetic variants could have direct clinical impact on individualized disease management and prevention strategies. PMID:19125159

  1. 11 beta-Hydroxysteroid dehydrogenase activity in hypothalamic obesity. (United States)

    Tiosano, Dov; Eisentein, Israel; Militianu, Daniela; Chrousos, George P; Hochberg, Ze'ev


    After extensive suprasellar operations for hypothalamic tumor removal, some patients develop Cushing-like morbid obesity while they receive replacement doses of glucocorticoids. In this study, we examined the hypothesis that target tissue conversion of inactive 11-ketosteroids to active 11 beta-OH glucocorticoids might explain the obesity of some patients with hypothalamic lesions. Toward this aim, we studied 10 patients with hypothalamic obesity and secondary adrenal insufficiency and 6 control Addisonian patients while they were on glucocorticoid replacement therapy. Pituitary hormone deficiencies were replaced when medically indicated. Twenty-four-hour urine was collected after a single oral dose of 12 mg/m(2) hydrocortisone acetate. The ratios of free and conjugated cortisol (F) to cortisone (E) and their metabolites, [tetrahydrocortisol (THF)+5 alpha THF]/tetrahyrdocortisone (THE), dihydrocortisols/dihydrocortisones, cortols/cortolones, and (F+E)/(THF+THE+5 alpha THF), were considered to represent 11 beta-hydroxysteroid dehydrogenase (HSD) activity. The 11-OH/11-oxo ratios were significantly higher in the urine of patients with hypothalamic obesity. The 11-OH/11-oxo ratios, however, did not correlate with the degree of obesity, yet a significant correlation was found between conjugated F/E and the ratio of visceral fat to sc fat measured by computerized tomography at the umbilical level. The consequence of increased 11 beta-HSD1 activity and the shift of the interconversion toward cortisol may contribute to the effects of the latter in adipose tissue. We propose that deficiency of hypothalamic messengers after surgical injury induces a paracrine/autocrine effect of enhanced glucocorticoid activity due to up-regulated 11 beta-HSD1 activity. PMID:12519880

  2. Antidepressant-like action of AGN 2979, a tryptophan hydroxylase activation inhibitor, in a chronic mild stress model of depression in rats. (United States)

    Gittos, M W; Papp, M


    Chronic mild stress (CMS) procedure was used to study an antidepressant-like activity of AGN 2979, a selective inhibitor of tryptophan hydroxylase (TH) activation. At the dose of 4 mg/kg, AGN 2979 fully reversed the CMS-induced reduction in the consumption of 1% sucrose solution. This effect was maintained for at least 1 week after cessation of treatment and no signs of withdrawal were observed in either stressed or control animals receiving AGN 2979. The lower (1 mg/kg) and higher (16 mg/kg) doses were ineffective. The magnitude of action of AGN 2979 in the CMS model was comparable to that of imipramine (10 mg/kg) but its onset of action appears to be faster since the inhibition of sucrose intake in stressed animals was already reversed after the 1st week of AGN 2979 administration while imipramine required 3 weeks of treatment to cause similar effect. These results provide support for the hypothesis that inhibition of TH activation may result in a potent antidepressant activity.

  3. Neural oscillations: beta band activity across motor networks. (United States)

    Khanna, Preeya; Carmena, Jose M


    Local field potential (LFP) activity in motor cortical and basal ganglia regions exhibits prominent beta (15-40Hz) oscillations during reaching and grasping, muscular contraction, and attention tasks. While in vitro and computational work has revealed specific mechanisms that may give rise to the frequency and duration of this oscillation, there is still controversy about what behavioral processes ultimately drive it. Here, simultaneous behavioral and large-scale neural recording experiments from non-human primate and human subjects are reviewed in the context of specific hypotheses about how beta band activity is generated. Finally, a new experimental paradigm utilizing operant conditioning combined with motor tasks is proposed as a way to further investigate this oscillation. PMID:25528615

  4. Lactoferrin from bovine colostrum regulates prolyl hydroxylase 2 activity and prevents prion protein-mediated neuronal cell damage via cellular prion protein. (United States)

    Park, Y-G; Moon, J-H; Park, S-Y


    Prion disorders are associated with the conversion of normal cellular prion protein (PrPc) to the abnormal scrapie isoform of prion protein (PrPsc). Recent studies have shown that expression of normal PrPc is regulated by hypoxia-inducible factor-1 alpha (HIF-1α), and that lactoferrin increases full-length PrPc on the cell surface. Lactoferrin is an 80-kDa iron-binding glycoprotein with various biological activities, including iron-chelating ability. HIF-1α and the associated ubiquitin-proteasome pathway are regulated by HIF prolyl-hydroxylases 2 (PHD2). We hypothesized that lactoferrin regulates PHD2 expression and enzymatic activity, and the PHD2 regulation promotes HIF-1α stability and prevention of neuronal cell death mediated by prion protein (PrP) residues (106-126). Lactoferrin prevented PrP (106-126)-induced neurotoxicity by the induction of PrPc expression via promoting HIF-1α stability in neuronal cells. Our results demonstrated that lactoferrin prevented PrP (106-126)-induced neurotoxicity via the up-regulation of HIF-1α stability determined by PHD2 expression and enzymatic activity. These findings suggest that possible therapies such as PHD2 inhibition, or promotion of lactoferrin secretion, may have clinical benefits in neurodegenerative diseases, including prion disease. PMID:24875174

  5. The tryptophan hydroxylase activation inhibitor, AGN-2979, decreases regional 5-HT synthesis in the rat brain measured with alpha-[14C]methyl-L-tryptophan: an autoradiographic study. (United States)

    Hasegawa, Shu; Kanemaru, Kazuya; Gittos, Maurice; Diksic, Mirko


    Many experimental conditions are stressful for animals. It is well known that stress induces tryptophan hydroxylase (TPH) activation, resulting in increased serotonin (5-HT) synthesis. In our experimental procedure to measure 5-HT synthesis using alpha-[(14)C]methyl-L-tryptophan (alpha-MTrp) autoradiographic method, the hind limbs of animals are restrained using a loose-fitted plaster cast such that the forelimbs of the animal remain free. The objective of the present investigation was to evaluate the changes, if any, in 5-HT synthesis, after injecting these restrained rats with the TPH activation inhibitor AGN-2979. The effect on regional 5-HT synthesis was studied using the alpha-MTrp autoradiographic method. The hypothesis was that the TPH activation inhibitor would reduce 5-HT synthesis, if TPH activation was induced by this restraint. The rats received injection of AGN-2979 (10 mg/kg, i.p.) or distilled water vehicle (1 mL/kg, i.p.) 1 h prior to tracer administration. The free- and total tryptophan concentrations were not significantly different between the treatment and control groups. The results demonstrate that 5-HT synthesis in AGN-2979 treated rats is significantly decreased (-12 to -35%) in both the raphe nuclei and their terminal areas when compared to the control rats. These findings suggest that restrained conditions, such as those used in our experimental protocol, induce TPH activation resulting in an increased 5-HT synthesis throughout the brain. The reduction in 5-HT synthesis in the AGN-2979 group is not related to a change in the plasma tryptophan. Because there was no activation in the pineal body, the structure having a different isoform of TPH, we can propose that it is only the brain TPH that becomes activated with this specific restraint.

  6. Endocrine expression of the active form of TGF-beta1 in the TGF-beta1 null mice fails to ameliorate lethal phenotype. (United States)

    Longenecker, Glenn; Thyagarajan, Tamizchelvi; Nagineni, Chandrasekharam N; Flanders, Kathleen C; Factor, Valentina; Miller, Georgina; Ward, Jerrold M; Nalca, Aysegul; Rangnekar, Vivek M; Thorgeirsson, Snorri; Kulkarni, Ashok B


    TGF-beta1 null mice die by 3 to 4 weeks of age due to a severe autoimmune-mediated multifocal inflammation resulting in multi-organ failure. To assess the therapeutic potential of circulating levels of active TGF-beta1, we generated mice with endocrine expression of active TGF-beta1 on a TGF-beta1 null background (TGF-beta1 (-/-/TG)) by crossing TGF-beta1(+/-) mice with transgenic mice (TG) that express recombinant TGF-beta1 specifically in the liver and secrete it in the blood. The TGF-beta1 (-/-/TG) mice exhibit a survival profile similar to the TGF-beta1 (-/-) mice indicating a failure to rescue the lethal phenotype. However, serum TGF-beta1 levels in the TGF-beta1 (-/-/TG) mice were restored to near normal levels with expression in all the tissues, notably in the kidney and spleen. Histopathology showed reduced inflammation in the target tissues, especially in the heart. Interestingly, unlike TGF-beta1 (-/-) mice, the TGF-beta1 (-/-/TG) mice have glomerulonephritis in their kidneys similar to the TG mice. Thus, the phenotype of TGF-beta1 (-/-/TG) animal model indicates the potential role of circulating active-TGF-beta1 in reducing inflammation, but its failure to rescue lethality in TGF-beta1 null mice indicates a critical autocrine role of TGF-beta1.

  7. Immunohistochemical detection of active transforming growth factor-beta in situ using engineered tissue (United States)

    Barcellos-Hoff, M. H.; Ehrhart, E. J.; Kalia, M.; Jirtle, R.; Flanders, K.; Tsang, M. L.; Chatterjee, A. (Principal Investigator)


    The biological activity of transforming growth factor-beta 1 (TGF-beta) is governed by dissociation from its latent complex. Immunohistochemical discrimination of active and latent TGF-beta could provide insight into TGF-beta activation in physiological and pathological processes. However, evaluation of immunoreactivity specificity in situ has been hindered by the lack of tissue in which TGF-beta status is known. To provide in situ analysis of antibodies to differentiate between these functional forms, we used xenografts of human tumor cells modified by transfection to overexpress latent TGF-beta or constitutively active TGF-beta. This comparison revealed that, whereas most antibodies did not differentiate between TGF-beta activation status, the immunoreactivity of some antibodies was activation dependent. Two widely used peptide antibodies to the amino-terminus of TGF-beta, LC(1-30) and CC(1-30) showed marked preferential immunoreactivity with active TGF-beta versus latent TGF-beta in cryosections. However, in formalin-fixed, paraffin-embedded tissue, discrimination of active TGF-beta by CC(1-30) was lost and immunoreactivity was distinctly extracellular, as previously reported for this antibody. Similar processing-dependent extracellular localization was found with a neutralizing antibody raised to recombinant TGF-beta. Antigen retrieval recovered cell-associated immunoreactivity of both antibodies. Two antibodies to peptides 78-109 showed mild to moderate preferential immunoreactivity with active TGF-beta only in paraffin sections. LC(1-30) was the only antibody tested that discriminated active from latent TGF-beta in both frozen and paraffin-embedded tissue. Thus, in situ discrimination of active versus latent TGF-beta depends on both the antibody and tissue preparation. We propose that tissues engineered to express a specific form of a given protein provide a physiological setting in which to evaluate antibody reactivity with specific functional forms of a

  8. Study of RNA interference inhibiting rat ovarian androgen biosynthesis by depressing 17alpha-hydroxylase/17, 20-lyase activity in vivo

    Directory of Open Access Journals (Sweden)

    Yang Xing


    Full Text Available Abstract Background 17alpha-hydroxylase/17, 20-lyase encoded by CYP17 is the key enzyme in androgen biosynthesis pathway. Previous studies demonstrated the accentuation of the enzyme in patients with polycystic ovary syndrome (PCOS was the most important mechanism of androgen excess. We chose CYP17 as the therapeutic target, trying to suppress the activity of 17alpha-hydroxylase/17, 20-lyase and inhibit androgen biosynthesis by silencing the expression of CYP17 in the rat ovary. Methods Three CYP17-targeting and one negative control oligonucleotides were designed and used in the present study. The silence efficiency of lentivirus shRNA was assessed by qRT-PCR, Western blotting and hormone assay. After subcapsular injection of lentivirus shRNA in rat ovary, the delivery efficiency was evaluated by GFP fluorescence and qPCR. Total RNA was extracted from rat ovary for CYP17 mRNA determination and rat serum was collected for hormone measurement. Results In total, three CYP17-targeting lentivirus shRNAs were synthesized. The results showed that all of them had a silencing effect on CYP17 mRNA and protein. Moreover, androstenedione secreted by rat theca interstitial cells (TIC in the RNAi group declined significantly compared with that in the control group. Two weeks after rat ovarian subcapsular injection of chosen CYP17 shRNA, the GFP fluorescence of frozen ovarian sections could be seen clearly under fluorescence microscope. It also showed that the GFP DNA level increased significantly, and its relative expression level was 7.42 times higher than that in the control group. Simultaneously, shRNA treatment significantly decreased CYP17 mRNA and protein levels at 61% and 54%, respectively. Hormone assay showed that all the levels of androstenedione, 17-hydroxyprogesterone and testosterone declined to a certain degree, but progesterone levels declined significantly. Conclusion The present study proves for the first time that ovarian androgen

  9. XAFS of human tyrosine hydroxylase (United States)

    Meyer, W.; Haavik, J.; Winkler, H.; Trautwein, A. X.; Nolting, H.-F.


    Tyrosine hydroxylase (TH) catalyses the rate-limiting step (hydroxylation of tyrosine to form dihydroxyphenylalanine) in the biosynthetic pathway leading to the catecholamines dopamine, noradrenaline and adrenaline. The human enzyme (hTH) is present in four isoforms, generated by splicing of pre-mRNA. The purified apoenzyme (metal free) binds stoichiometric amounts of iron. The incorporation of Fe(II) results in a rapid and up to 40-fold increase of activity [1]. Besides the coordination of the metal centers in native enzyme we studied the purported inhibition of TH by its immediate products. So we analysed Fe-hTH isoform 1 native as well as oxidized with dopamine and Co-hTH isoform 2.

  10. Resting-state beta and gamma activity in Internet addiction. (United States)

    Choi, Jung-Seok; Park, Su Mi; Lee, Jaewon; Hwang, Jae Yeon; Jung, Hee Yeon; Choi, Sam-Wook; Kim, Dai Jin; Oh, Sohee; Lee, Jun-Young


    Internet addiction is the inability to control one's use of the Internet and is related to impulsivity. Although a few studies have examined neurophysiological activity as individuals with Internet addiction engage in cognitive processing, no information on spontaneous EEG activity in the eyes-closed resting-state is available. We investigated resting-state EEG activities in beta and gamma bands and examined their relationships with impulsivity among individuals with Internet addiction and healthy controls. Twenty-one drug-naïve patients with Internet addiction (age: 23.33 ± 3.50 years) and 20 age-, sex-, and IQ-matched healthy controls (age: 22.40 ± 2.33 years) were enrolled in this study. Severity of Internet addiction was identified by the total score on Young's Internet Addiction Test. Impulsivity was measured with the Barratt Impulsiveness Scale-11 and a stop-signal task. Resting-state EEG during eyes closed was recorded, and the absolute/relative power of beta and gamma bands was analyzed. The Internet addiction group showed high impulsivity and impaired inhibitory control. The generalized estimating equation showed that the Internet-addiction group showed lower absolute power on the beta band than did the control group (estimate = -3.370, p Internet-addiction group showed higher absolute power on the gamma band than did the control group (estimate = 0.434, p Internet addiction as well as with the extent of impulsivity. The present study suggests that resting-state fast-wave brain activity is related to the impulsivity characterizing Internet addiction. These differences may be neurobiological markers for the pathophysiology of Internet addiction.

  11. Overview of total beta activity index and beta rest in surface waters of the Spanish rivers; Vision general del indice de actividad beta total y beta resto en las aguas superficiales de los rios espanoles

    Energy Technology Data Exchange (ETDEWEB)

    Pujol, L.; Payeras, J.; Pablo, M. A. de


    This work aims to give an overview of the index of total beta activity and the activity index beta rest in surface waters of the main Spanish rivers. These indices are a parameter over water quality that CEDEX comes determined by order of the Ministry of Agriculture, Food and Environment, in water policy. (Author)

  12. Glucose activates prenyltransferases in pancreatic islet {beta}-cells

    Energy Technology Data Exchange (ETDEWEB)

    Goalstone, Marc [Department of Medicine, University of Colorado, VA Medical Center, Denver, CO 80220 (United States); Kamath, Vasudeva [Department of Pharmaceutical Sciences, Wayne State University, VA Medical Center, Detroit, MI 48201 (United States); Kowluru, Anjaneyulu, E-mail: [Department of Pharmaceutical Sciences, Wayne State University, VA Medical Center, Detroit, MI 48201 (United States)


    A growing body of evidence implicates small G-proteins [e.g., Cdc42 and Rac1] in glucose-stimulated insulin secretion [GSIS] in the islet {beta}-cell. These signaling proteins undergo post-translational modifications [e.g., prenylation] at their C-terminal cysteine residue and appear to be essential for the transport and fusion of insulin-containing secretory granules with the plasma membrane and the exocytotic secretion of insulin. However, potential regulation of the prenylating enzymes by physiological insulin secretogues [e.g., glucose] has not been investigated thus far. Herein, we report immunological localization, sub-cellular distribution and regulation of farnesyltransferases [FTases] and geranylgeranyltransferase [GGTase] by glucose in insulin-secreting INS 832/13 {beta}-cells and normal rat islets. Our findings suggest that an insulinotropic concentration of glucose [20 mM] markedly stimulated the expression of the {alpha}-subunits of FTase/GGTase-1, but not the {beta}-subunits of FTase or GGTase-1 without significantly affecting the predominantly cytosolic distribution of these holoenzymes in INS 832/13 cells and rodent islets. Under these conditions, glucose significantly stimulated [2.5- to 4.0-fold over basal] the activities of both FTase and GGTase-1 in both cell types. Together, these findings provide the first evidence to suggest that GSIS involves activation of the endogenous islet prenyltransferases by glucose, culminating in the activation of their respective G-protein substrates, which is necessary for cytoskeletal rearrangement, vesicular transport, fusion and secretion of insulin.

  13. Evaluation of in vivo partial beta 1/beta 2-agonist activity: a dose-ranging study with carteolol.


    Wheeldon, N M; McDevitt, D G; Lipworth, B J


    1. The aims of this study were to investigate the partial agonist profile of carteolol and evaluate methodology for differentiating relative beta 1 and beta 2 partial agonist activity (PAA) in vivo. 2. Eight normal subjects received single oral doses of carteolol 10 mg, 30 mg and 60 mg; nadolol 40 mg; pindolol 30 mg and placebo, given in a single-blind, randomised crossover design. 3. beta 1-PAA was demonstrated with carteolol by dose-related increases in resting heart rate and systolic blood...

  14. CK1epsilon is required for breast cancers dependent on beta-catenin activity.

    Directory of Open Access Journals (Sweden)

    So Young Kim

    Full Text Available Aberrant beta-catenin signaling plays a key role in several cancer types, notably colon, liver and breast cancer. However approaches to modulate beta-catenin activity for therapeutic purposes have proven elusive to date.To uncover genetic dependencies in breast cancer cells that harbor active beta-catenin signaling, we performed RNAi-based loss-of-function screens in breast cancer cell lines in which we had characterized beta-catenin activity. Here we identify CSNK1E, the gene encoding casein kinase 1 epsilon (CK1epsilon as required specifically for the proliferation of breast cancer cells with activated beta-catenin and confirm its role as a positive regulator of beta-catenin-driven transcription. Furthermore, we demonstrate that breast cancer cells that harbor activated beta-catenin activity exhibit enhanced sensitivity to pharmacological blockade of Wnt/beta-catenin signaling. We also find that expression of CK1epsilon is able to promote oncogenic transformation of human cells in a beta-catenin-dependent manner.These studies identify CK1epsilon as a critical contributor to activated beta-catenin signaling in cancer and suggest it may provide a potential therapeutic target for cancers that harbor active beta-catenin. More generally, these observations delineate an approach that can be used to identify druggable synthetic lethal interactions with signaling pathways that are frequently activated in cancer but are difficult to target with the currently available small molecule inhibitors.

  15. Antibacterial activity of Beta vulgaris L. pomace extract

    Directory of Open Access Journals (Sweden)

    Velićanski Aleksandra S.


    Full Text Available Antibacterial activity of Beta vulgaris L. (beetroot pomace extract (concentration 100 mg/ml was tested against five Gram positive and seven Gram negative bacterial strains (reference cultures and natural isolates. Disc diffusion method with 15 µl of extract and agar-well diffusion method with 50 and 100 µl were used. Antibiotic (cefotaxime/clavulanic acid was used as a control sample. The tested extract showed the highest activity against Staphylococcus aureus and Bacillus cereus, where clear zones (without growth appeared. There was no any activity against other tested Gram-positive bacteria, except for Staphylococcus epidermidis, with a small zone of reduced growth. Growth of all tested Gram-negative bacteria was inhibited usually with 100 µl of extract. The most susceptible were Citrobacter freundii and Salmonella typhymurium. The tested antibiotic gave clear, usually large zones for all tested strains except for Staphylococcus cohni spp. cohni, where only a zone of reduced growth appeared.

  16. Vasoactive intestinal peptide-induced expression of cytochrome P450 cholesterol side-chain cleavage and 17 alpha-hydroxylase enzyme activity in hen granulosa cells. (United States)

    Johnson, A L; Li, Z; Gibney, J A; Malamed, S


    Experiments were conducted to determine whether vasoactive intestinal peptide (VIP) can regulate expression of cytochrome P450 side-chain cleavage (P450scc) and P450 17 alpha-hydroxylase (P450 17 alpha-OH) mRNA levels and enzyme activity in granulosa cells from nonhierarchal (6-8-mm) follicles. Initial studies demonstrated that immunoreactive VIP is localized within the theca (but not granulosa) layer of both resting (< 0.5-mm follicles) and 6-8-mm follicles, thus providing a potential paracrine mechanism of action for VIP. While short-term (3 h) incubation of granulosa cells with VIP (0.001-1.0 microM) failed to stimulate progesterone production from 6-8-mm follicle granulosa cells, a 4-h culture period in the presence of VIP resulted in increased cyclic AMP (cAMP) accumulation, and a 24-h culture period resulted in progesterone synthesis and increased P450scc mRNA levels; control levels of each endpoint measurement were not altered within the period observed. By contrast, culture with the growth factor transforming growth factor alpha (TGF alpha) in the presence of VIP (1 microM) prevented increases in P450scc mRNA levels and progesterone production. Similar effects of VIP and TGF alpha in the presence of VIP were demonstrated for P450 17 alpha-OH mRNA levels and enzyme activity. Finally, there was an additive effect of VIP (0.1 microM) plus recombinant human (rh) FSH (100 mIU) on the initiation of progesterone production in cultured 6-8-mm follicle granulosa cells compared to the addition of VIP or rhFSH alone.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. IFN-beta inhibits T cell activation capacity of central nervous system APCs

    DEFF Research Database (Denmark)

    Teige, Ingrid; Liu, Yawei; Issazadeh-Navikas, Shohreh


    We have previously investigated the physiological effects of IFN-beta on chronic CNS inflammation and shown that IFN-beta(-/-) mice develop a more severe experimental autoimmune encephalomyelitis than their IFN-beta(+/-) littermates. This result was shown to be associated with a higher activation...

  18. Proteolytic activation of latent TGF-beta precedes caspase-3 activation and enhances apoptotic death of lung epithelial cells. (United States)

    Solovyan, Victor T; Keski-Oja, Jorma


    Transforming growth factors beta (TGF-betas) are multifunctional cytokines, which are secreted in latent forms in large latent TGF-beta complexes (LL-TGF-beta) with subsequent deposition to the extracellular matrix (ECM). While a variety of mechanisms capable of activating latent TGF-beta in vitro have been described, the physiological conditions, which promote the activation of TGF-beta in vivo are poorly understood. Mink lung epithelial cells (Mv1Lu) are a widely used model for evaluation of the effects of exogenous TGF-beta both in transcriptional and growth inhibitor assays. We find here that apoptosis of Mv1Lu cells, induced either by staurosporine or serum deprivation, is accompanied by proteolytic processing of LL-TGF-beta and the activation of endogenous TGF-beta. Activation of TGF-beta preceded caspase-3 activation and was almost completely suppressed by the serine protease inhibitor, AEBSF. Both exogenous and endogenously activated TGF-betas were able to enhance the apoptotic response of Mv1Lu cells leading to potentiation of cell death. Potentiation of cell death by activated TGF-beta was associated with downregulation of Akt and p38 MAPK, which were both activated at the initial stages of Mv1Lu apoptosis and were suppressed by exogenous TGF-beta. Pharmacological interruption of either phosphoinositide-3-kinase (PI-3K)/Akt or p38 MAPK signaling by the specific inhibitors mimicked the effect of TGF-beta leading to potentiation of cell death. Current results suggest that proteolytic activation of endogenous TGF-beta is a component of the apoptotic response, capable of modulating the death of Mv1Lu cells by inhibition of both PI-3K/Akt and p38 MAPK-dependent survival pathways. PMID:16447253

  19. Characterisation of two novel CYP4 genes from the marine polychaete Nereis virens and their involvement in pyrene hydroxylase activity

    DEFF Research Database (Denmark)

    Jørgensen, Anne; Rasmussen, Lene Juel; Andersen, Ole


    genes isolated from N. virens gut tissue are reported. One named CYP342A1, the first member of a new family and the other named CYP4BB1, the first member of a new subfamily. This is the first investigation of specific CYP enzymes from marine polychaetes in which catalytic activity has been determined......Cytochrome P450 enzymes (CYP enzymes) catalyse the initial step in biotransformation of xenobiotics like polycyclic aromatic hydrocarbons (PAHs). The marine polychaete Nereis virens has a high capacity for biotransformation of PAHs. In the present study, the complete cDNA sequences of two novel CYP....... Both CYP enzymes had monooxygenase activity and catalysed hydroxylation of pyrene to 1-hydroxypyrene. Based on the present results it is likely that both CYP4BB1 and CYP342A1 are involved in xenobiotic biotransformation. Furthermore, site-directed mutagenesis of the conserved cysteine residue...

  20. Beta-glucosidase enzymatic activity of crystal polypeptide of the Bacillus thuringiensis strain 1.1. (United States)

    Papalazaridou, A; Charitidou, L; Sivropoulou, A


    The crystals of Bacillus thuringiensis strain 1.1 consist of the 140 kDa delta-endotoxin, which exhibits beta-glucosidase enzymatic activity, based on the following data. (i) Purified crystals exhibit beta-glucosidase enzymatic activity. When the crystals are reacted with specific antibodies directed either against the commercial (almond purified) beta-glucosidase or against the 140 kDa polypeptide, then considerable reduction of enzymatic activity is observed almost at the same level with both antibodies. (ii) Commercial beta-glucosidase and the 140 kDa crystal polypeptide share antigenic similarities; in Western immunoblots, the 140 kDa crystal polypeptide is recognized by anti-beta-glucosidase antibodies, and commercial beta-glucosidase is recognized by anti-140-kDa antibodies. (iii) The enzymatic properties of commercial beta-glucosidase and that resident in the crystals of B. thuringiensis strain 1.1 are very similar. Thus, both enzymes hydrolyze a wide range of substrates (aryl-beta-glucosides, disaccharides with alpha- or beta-linkage polysaccharides) and have an optimum activity at 40 degrees C and pH 5. Both enzymes are relatively thermostable and are resistant to end-product inhibition by glucose. Additionally, they show the same pattern of inhibition or activation by several chemical compounds. (iv) The crystals and commercial beta-glucosidase show almost equivalent levels of insecticidal activity against Drosophila melanogaster larvae and, furthermore, cause reduction in adult flies that emerge from larvae surviving treatment.

  1. Structural analysis of a phosphonate hydroxylase with an access tunnel at the back of the active site. (United States)

    Li, Changqing; Junaid, Muhammad; Almuqri, Eman Abdullah; Hao, Shiguang; Zhang, Houjin


    FrbJ is a member of the Fe(2+)/α-ketoglutarate-dependent dioxygenase family which hydroxylates the natural product FR-900098 of Streptomyces rubellomurinus, yielding the phosphonate antibiotic FR-33289. Here, the crystal structure of FrbJ, which shows structural homology to taurine dioxygenase (TauD), a key member of the same family, is reported. Unlike other members of the family, FrbJ has an unusual lid structure which consists of two β-strands with a long loop between them. To investigate the role of this lid motif, a molecular-dynamics simulation was performed with the FrbJ structure. The molecular-dynamics simulation analysis implies that the lid-loop region is highly flexible, which is consistent with the fact that FrbJ has a relatively broad spectrum of substrates with different lengths. Interestingly, an access tunnel is found at the back of the active site which connects the putative binding site of α-ketoglutarate to the solvent outside. PMID:27139827

  2. P53 and Beta-Catenin Activity during Estrogen treatment of Osteoblasts

    Directory of Open Access Journals (Sweden)

    Kolman Kevin


    Full Text Available Abstract Background This study was undertaken to examine the relationship between the tumor suppressor gene p53 and the nuclear signaling protein beta-catenin during bone differentiation. Cross talk between p53 and beta-catenin pathways has been demonstrated and is important during tumorigenesis and DNA damage, where deregulation of beta catenin activates p53. In this study, we used estrogen treatment of osteoblasts as a paradigm to study the relationship between the two proteins during osteoblast differentiation. Results We exposed osteoblast-like ROS17/2.8 cells to 17-beta estradiol (E2, in a short term assay, and studied the cellular distribution and expression of beta-catenin. We found beta-catenin to be up regulated several fold following E2 treatment. Levels of p53 and its functional activity mirrored the quantitative changes seen in beta-catenin. Alkaline phosphatase, an early marker of osteoblast differentiation, was increased in a manner similar to beta-catenin and p53. In order to determine if there was a direct relationship between alkaline phosphatase expression and beta-catenin, we used two different approaches. In the first approach, treatment with LiCl, which is known to activate beta-catenin, caused a several fold increase in alkaline phosphatase activity. In the second approach, transient transfection of wild type beta-catenin into osteoblasts increased alkaline phosphatase activity two fold over basal levels, showing that beta catenin expression can directly affect alkaline phosphatase expression. However increase in beta catenin activity was not associated with an increase in its signaling activity through TCF/LEF mediated transcription. Immunofluorescence analyses of p53 and beta-catenin localization showed that E2 first caused an increase in cytosolic beta-catenin followed by the accumulation of beta-catenin in the nucleus. Nuclear p53 localization was detected in several cells. Expression of p53 was accompanied by

  3. Evaluation of the beta 2 adrenoceptor agonist/antagonist activity of formoterol and salmeterol.


    Grove, A.; Lipworth, B J


    BACKGROUND: Salmeterol and formoterol have a lower intrinsic activity at beta 2 receptors than isoprenaline in human bronchus in vitro. The aim of the present study was to evaluate in vivo the beta 2 agonist/antagonist activity of salmeterol and formoterol at rest with low endogenous adrenergic tone, on exercise with raised endogenous adrenergic tone, and in the presence of fenoterol, an exogenous full beta 2 receptor agonist. METHODS: Eight normal subjects were randomised to receive single d...

  4. Activity of beta-lactam beta-lactamase inhibitor combinations against extended spectrum beta-lactamase producing enterobacteriaceae in urinary isolates

    International Nuclear Information System (INIS)

    Objective: To determine the susceptibility pattern of beta-lactam beta-lactamase inhibitor combinations against extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae in urinary isolates. Study Design: Observational study. Place and Duration of Study: Ziauddin University Hospital, Karachi, from February to October 2008. Methodology: A total of 190 consecutive non-duplicate isolates of ESBL producing Enterobacteriaceae from urine samples of in-patients were included in the study. Urinary samples from out-patients, repeat samples and non-ESBL producing isolates were excluded. Detection of ESBL was carried out by double disk diffusion technique. Antimicrobial susceptibility testing was performed using modified Kirby Bauer's disk diffusion method according to CLSI guidelines. Statistical analysis was performed by SPSS version 10. Results: Of the 190 ESBL isolates tested, 88 cases (46.31%) were sensitive and 6 cases (3.15%) were resistant to all three combinations, the rest 96 cases (50.52%) were resistant to at least one of the combinations. Susceptibility pattern of cefoperazone/sulbactam, piperacillin/tazobactam, and amoxicillin/clavulanic acid was 95.26, 92.10, and 44.31 percent respectively. Conclusion: Cefoperazone/sulbactam exhibited the best activity against ESBL producing Enterobacteriaceae followed by piperacillin/tazobactam. Hospital antibiotic policies should be reviewed periodically to reduce the usage of extended spectrum cephalosporins and replace them with beta-lactam beta-lactamase inhibitor combinations agent for treating urinary tract infections. (author)

  5. Screening of some medicinal plants from cameroon for beta-lactamase inhibitory activity. (United States)

    Gangoué-Piéboji, Joseph; Baurin, Stéphane; Frère, Jean-Marie; Ngassam, Pierre; Ngameni, Bathelemy; Azebaze, Anatole; Pegnyemb, Dieudonné Emmanuel; Watchueng, Jean; Goffin, Colette; Galleni, Moreno


    In efforts to find new bioactive beta-lactamase inhibitors, this study investigated 16 Cameroonian plants belonging to 10 families which were evaluated for anti-beta-lactamase activity. The investigation showed that extracts 2, 6, 3 and 5 of the 16 plants investigated presented interesting in vitro beta-lactamase inhibition (over 90%), respectively, of the beta-lactamases TEM-1, OXA-10, IMP-1 and P99. These extracts were from Mammea africana (all beta-lactamases), Garcinia lucida, G. kola (OXA-10, IMP-1 and P99), Bridelia micrantha (OXA-10, P99), Ochna afzelii (OXA-10, P99), Prunus africana (IMP-1) and Adenia lobata (TEM-1). After elimination of tannins (according to the European Pharmacopoeia) the extracts from B. micrantha, G. lucida and M. africana were tested further for their anti-beta-lactamase activity. The extracts from B. micrantha and G. lucida exhibited potent inhibitory activity, respectively, of beta-lactamase OXA-10 (IC(50) = 0.02 mg/mL) and P99 (IC(50) = 0.01 mg/mL). The anti-beta-lactamase activity of M. africana extract was weak. The isolation and the structural elucidation of the active constituents of G. lucida and B. micrantha will provide useful leads in the development of beta-lactamase inhibitors.

  6. Comparison of Salivary Beta Glucuronidase Activity in Chronic Periodontitis Patients with and without Diabetes Mellitus


    Ramamurthy, Jaiganesh; ND, Jayakumar; Varghese, Sheeja


    Aim of the study: The aim of the study was to estimate the salivary beta glucuronidase (β) activity in patients with chronic periodontitis with and without diabetes mellitus and to evaluate the relationship between Beta Glucuronidase activity and Periodontal clinical parameters.

  7. Angiotensin administration stimulates renal 11 beta-hydroxysteroid dehydrogenase activity in healthy men

    NARCIS (Netherlands)

    Kerstens, MN; van der Kleij, FGH; Boonstra, AH; Sluiter, WJ; van der Molen, JC; Navis, G; Dullaart, RPF


    Background. We examined whether acute administration of angiotensin modulates the activity of 11beta-hydroxysteroid dehydrogenase (11betaHSD), the intracellular enzyme catalyzing the interconversion between the hormonally active cortisol and inactive cortisone. Methods. Twenty-one male healthy subje

  8. A 2-oxoglutarate-dependent dioxygenase from Ruta graveolens L. exhibits p-coumaroyl CoA 2'-hydroxylase activity (C2'H): a missing step in the synthesis of umbelliferone in plants. (United States)

    Vialart, Guilhem; Hehn, Alain; Olry, Alexandre; Ito, Kyoko; Krieger, Celia; Larbat, Romain; Paris, Cedric; Shimizu, Bun-Ichi; Sugimoto, Yukihiro; Mizutani, Masaharu; Bourgaud, Frederic


    Coumarins are important compounds that contribute to the adaptation of plants to biotic or abiotic stresses. Among coumarins, umbelliferone occupies a pivotal position in the plant phenylpropanoid network. Previous studies indicated that umbelliferone is derived from the ortho-hydroxylation of p-coumaric acid by an unknown biochemical step to yield 2,4-dihydroxycinnamic acid, which then undergoes spontaneous lactonization. Based on a recent report of a gene encoding a 2-oxoglutarate-dependent dioxygenase from Arabidopsis thaliana that exhibited feruloyl CoA 6'-hydroxylase activity (Bourgaud et al., 2006), we combined a bioinformatic approach and a cDNA library screen to identify an orthologous ORF (Genbank accession number JF799117) from Ruta graveolens L. This ORF shares 59% amino acid identity with feruloyl CoA 6'-hydroxylase, was functionally expressed in Escherichia coli, and converted feruloyl CoA into scopoletin and p-coumaroyl CoA into umbelliferone with equal activity. Its bi-functionality was further confirmed in planta: transient expression of JF799117 in Nicotiana benthamiana yielded plants with leaves containing high levels of umbelliferone and scopoletin when compared to control plants, which contained barely detectable traces of these compounds. The expression of JF799117 was also tightly correlated to the amount of umbelliferone that was found in UV-elicited R. graveolens leaves. Therefore, JF799117 encodes a p-coumaroyl CoA 2'-hydroxylase in R. graveolens, which represents a previously uncharacterized step in the synthesis of umbelliferone in plants. Psoralen, which is an important furanocoumarin in R. graveolens, was found to be a competitive inhibitor of the enzyme, and it may exert this effect through negative feedback on the enzyme at an upstream position in the pathway.

  9. Effect of gamma-irradiation on the whitening activity of {beta}-glucan

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jae Hun; Sung, Nak Yun; Jung, Pil Moon; Choi, Jong Il; Kim, Jin Kyu; Lee, Ju Woon [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of); Byun, Eui Hong [Chungnam Naitonal University, Daejeon (Korea, Republic of)


    This study evaluated the change in whitening activity of {beta}-glucan by gamma-irradiation. Tyrosinase inhibition was significantly increased in the samples with 30, 50, 10 kGy irradiated {beta}-glucan. Melanin synthesis of irradiated {beta}-glucan was measured from B16BL6 melanoma cell line treated with {alpha}-melanin stimulating hormone. Melanin synthesis was increased in the {alpha}-melanin stimulating hormone added group. However, it was decreased in the groups of 30, 50 and 100 kGy gamma-irradiated {beta}-glucan treated with {alpha}-melanin stimulating hormone. These results indicate that gamma irradiated {beta}-glucan may elevate the whitening activity. Therefore, gamma-irradiated {beta}-glucan could be used for nutraceutical foods in cosmetic industry.

  10. Platensimycin activity against mycobacterial beta-ketoacyl-ACP synthases.

    Directory of Open Access Journals (Sweden)

    Alistair K Brown

    Full Text Available BACKGROUND: There is an urgent need for the discovery and development of new drugs against Mycobacterium tuberculosis, the causative agent of tuberculosis, especially due to the recent emergence of multi-drug and extensively-drug resistant strains. Herein, we have examined the susceptibility of mycobacteria to the natural product platensimycin. METHODS AND FINDINGS: We have demonstrated that platensimycin has bacteriostatic activity against the fast growing Mycobacterium smegmatis (MIC = 14 microg/ml and against Mycobacterium tuberculosis (MIC = 12 microg/ml. Growth in the presence of paltensimycin specifically inhibited the biosynthesis of mycolic acids suggesting that the antibiotic targeted the components of the mycolate biosynthesis complex. Given the inhibitory activity of platensimycin against beta-ketoacyl-ACP synthases from Staphylococcus aureus, M. tuberculosis KasA, KasB or FabH were overexpressed in M. smegmatis to establish whether these mycobacterial KAS enzymes were targets of platensimycin. In M. smegmatis overexpression of kasA or kasB increased the MIC of the strains from 14 microg/ml, to 30 and 124 microg/ml respectively. However, overexpression of fabH on did not affect the MIC. Additionally, consistent with the overexpression data, in vitro assays using purified proteins demonstrated that platensimycin inhibited Mt-KasA and Mt-KasB, but not Mt-FabH. SIGNIFICANCE: Our results have shown that platensimycin is active against mycobacterial KasA and KasB and is thus an exciting lead compound against M. tuberculosis and the development of new synthetic analogues.

  11. Reduction of beta activity from depleted derbies, ingots and crucibles

    International Nuclear Information System (INIS)

    The reduction of beta radiation on uranium ingot and crucible surfaces was demonstrated in the production casting operation by adding a mixture of slag liner material (MgF2) and calcium fluoride to the remelt charge. The beta emitters (234Th and 234Pa) are largely discharged with the fluorides into drums during a remote crucible burnout operation; thereby, reducing operator exposure to beta radiation. A production test showed that very low beta radiation from uranium flat castings can be achieved by using derbies recently prepared by reduction. Plant tests with fluoride addition indicate that pickling of derbies may not be necessary for casting uranium flats from a plasma sprayed (ZrO2) crucible. Also, ingots produced with fluoride additions had less pipe as compared to standard production technique. 2 references, 5 tables

  12. Co-expression pattern of dopamine beta-hydroxylase (DβH) and neuropeptide Y (NPY) within sympathetic innervation of ovary and umbilical cord of the European bison (Bison bonasus L.). (United States)

    Skobowiat, Cezary; Panasiewicz, Grzegorz; Gizejewski, Zygmunt; Szafranska, Bozena


    Co-expression of dopamine β-hydroxylase (DβH) and neuropeptide Y (NPY) has never been examined in ovary (OV) and umbilical cord (UC) of the European bison (Eb), the endangered wild species. The OV and UC samples were harvested from seasonally eliminated Eb females (45-120 days post coitum). Frozen histological sections were examined by double fluorescent immunohistochemistry (dF-IHC), using the primary mouse anti-DβH monoclonals and rabbit anti-NPY polyclonals and then the immunocomplexes were visualized with FITC and CY3 fluorophores, respectively. Numerous DβH immunoreactive nerve fibers (DβH-IRs) and a little less frequent NPY-IRs were found in the bundle-like structures, innervating mainly perivascular regions of the OV. The NPY-IRs constantly co-expressed DβH, while some DβH-IRs did not express NPY. This specific pattern of innervation was observed both in the stromal and cortical regions of the OV. The simultaneous co-expression of DβH and NPY were also detected in the UC, in which specific single or bundle-like structures ran along the smooth muscles of blood vessels. The spatial-specific co-expression of DβH and NPY in OV and UC, may suggest that these markers are involved in the control of vascularization that regulates nourishing blood circulation required for proper pregnancy maintenance and efficient embryo/fetus development in the Eb.

  13. Tumor-produced, active Interleukin-1 {beta} regulates gene expression in carcinoma-associated fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Dudas, Jozsef, E-mail: [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Fullar, Alexandra, E-mail: [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); 1st Institute of Pathology and Experimental Cancer Research, Semmelweis University, Ulloei ut 26, H-1085 Budapest (Hungary); Bitsche, Mario, E-mail: [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Schartinger, Volker, E-mail: [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Kovalszky, Ilona, E-mail: [1st Institute of Pathology and Experimental Cancer Research, Semmelweis University, Ulloei ut 26, H-1085 Budapest (Hungary); Sprinzl, Georg Mathias, E-mail: [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Riechelmann, Herbert, E-mail: [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria)


    Recently we described a co-culture model of periodontal ligament (PDL) fibroblasts and SCC-25 lingual squamous carcinoma cells, which resulted in conversion of normal fibroblasts into carcinoma-associated fibroblasts (CAFs), and in epithelial-mesenchymal transition (EMT) of SCC-25 cells. We have found a constitutive high interleukin-1{beta} (IL1-{beta}) expression in SCC-25 cells in normal and in co-cultured conditions. In our hypothesis a constitutive IL1-{beta} expression in SCC-25 regulates gene expression in fibroblasts during co-culture. Co-cultures were performed between PDL fibroblasts and SCC-25 cells with and without dexamethasone (DEX) treatment; IL1-{beta} processing was investigated in SCC-25 cells, tumor cells and PDL fibroblasts were treated with IL1-{beta}. IL1-{beta} signaling was investigated by western blot and immunocytochemistry. IL1-{beta}-regulated genes were analyzed by real-time qPCR. SCC-25 cells produced 16 kD active IL1-{beta}, its receptor was upregulated in PDL fibroblasts during co-culture, which induced phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1), and nuclear translocalization of NF{kappa}B{alpha}. Several genes, including interferon regulatory factor 1 (IRF1) interleukin-6 (IL-6) and prostaglandin-endoperoxide synthase 2 (COX-2) were induced in CAFs during co-culture. The most enhanced induction was found for IL-6 and COX-2. Treatment of PDL fibroblasts with IL1-{beta} reproduced a time- and dose-dependent upregulation of IL1-receptor, IL-6 and COX-2. A further proof was achieved by DEX inhibition for IL1-{beta}-stimulated IL-6 and COX-2 gene expression. Constitutive expression of IL1-{beta} in the tumor cells leads to IL1-{beta}-stimulated gene expression changes in tumor-associated fibroblasts, which are involved in tumor progression. -- Graphical abstract: SCC-25 cells produce active, processed IL1-{beta}. PDL fibroblasts possess receptor for IL1-{beta}, and its expression is increased 4.56-times in the

  14. Enhanced Activity of Nanocrystalline Beta Zeolite for Acylation of Veratrole with Acetic Anhydride. (United States)

    Aisha Mahmood Abdulkareem, Al-Turkustani; Selvin, Rosilda


    Friedel-Craft acylation of veratrole using homogeneous acid catalysts such as AlCl3, FeCl3, ZnCl2, and HF etc. produces acetoveratrone, (3',4'-dimethoxyacetophenone), which is the intermediate for synthesis of papavarine alkaloids. The problems associated with these homogeneous catalysts can be overcome by using heterogeneous solid catalysts. Since acetoveratrone is a larger molecule, large pore Beta zeolites with smaller particle sizes are beneficial for the liquid-phase acylation of veratrole, for easy diffusion of reactants and products. The present study aims in the acylation of veratrole with acetic anhydride using nanocrystalline Beta Zeolite catalyst. A systematic investigation of the effects of various reaction parameters was done. The catalysts were characterized for their structural features by using XRD, TEM and DLS analyses. The catalytic activity of nanocrystalline Beta zeolite was compared with commercial Beta zeolite for the acylation and was found that nanocrystalline Beta zeolite possessed superior activity.

  15. Ablation of PGC-1beta results in defective mitochondrial activity, thermogenesis, hepatic function, and cardiac performance.

    Directory of Open Access Journals (Sweden)

    Christopher J Lelliott


    Full Text Available The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1beta (PGC-1beta has been implicated in important metabolic processes. A mouse lacking PGC-1beta (PGC1betaKO was generated and phenotyped using physiological, molecular, and bioinformatic approaches. PGC1betaKO mice are generally viable and metabolically healthy. Using systems biology, we identified a general defect in the expression of genes involved in mitochondrial function and, specifically, the electron transport chain. This defect correlated with reduced mitochondrial volume fraction in soleus muscle and heart, but not brown adipose tissue (BAT. Under ambient temperature conditions, PGC-1beta ablation was partially compensated by up-regulation of PGC-1alpha in BAT and white adipose tissue (WAT that lead to increased thermogenesis, reduced body weight, and reduced fat mass. Despite their decreased fat mass, PGC1betaKO mice had hypertrophic adipocytes in WAT. The thermogenic role of PGC-1beta was identified in thermoneutral and cold-adapted conditions by inadequate responses to norepinephrine injection. Furthermore, PGC1betaKO hearts showed a blunted chronotropic response to dobutamine stimulation, and isolated soleus muscle fibres from PGC1betaKO mice have impaired mitochondrial function. Lack of PGC-1beta also impaired hepatic lipid metabolism in response to acute high fat dietary loads, resulting in hepatic steatosis and reduced lipoprotein-associated triglyceride and cholesterol content. Altogether, our data suggest that PGC-1beta plays a general role in controlling basal mitochondrial function and also participates in tissue-specific adaptive responses during metabolic stress.

  16. Immune activation in multiple sclerosis and interferon-beta therapy

    DEFF Research Database (Denmark)

    Krakauer, Martin


    of inflammation or secondary lymphatic organs. Chemokine receptors are differentially expressed in T cells in blood and cerebrospinal fluid, indicating their role for in T-cell-recruitment to the CNS. Interferon (IFN)-beta is a first-line treatment for MS. The mechanism of action is unclear, but probably includes...

  17. Proinflammatory cytokines activate the intrinsic apoptotic pathway in beta-cells

    DEFF Research Database (Denmark)

    Grunnet, Lars G; Aikin, Reid; Tonnesen, Morten F;


    OBJECTIVE: Proinflammatory cytokines are cytotoxic to beta-cells and have been implicated in the pathogenesis of type 1 diabetes and islet graft failure. The importance of the intrinsic mitochondrial apoptotic pathway in cytokine-induced beta-cell death is unclear. Here, cytokine activation of the...... intrinsic apoptotic pathway and the role of the two proapoptotic Bcl-2 proteins, Bad and Bax, were examined in beta-cells. RESEARCH DESIGN AND METHODS: Human and rat islets and INS-1 cells were exposed to a combination of proinflammatory cytokines (interleukin-1beta, interferon-gamma, and/or tumor necrosis...... factor-alpha). Activation of Bad was determined by Ser136 dephosphorylation, mitochondrial stress by changes in mitochondrial metabolic activity and cytochrome c release, downstream apoptotic signaling by activation of caspase-9 and -3, and DNA fragmentation. The inhibitors FK506 and V5 were used to...

  18. A method for the determination of residual beta activity in drinking water samples

    Energy Technology Data Exchange (ETDEWEB)

    Idoeta, R. [Dpto. Ingenieria Nuclear y Mecanica de Fluidos, E. T. S. Ingenieria de Bilbao - Universidad del Pais Vasco (UPV/EHU), Alda. Urquijo s/n. 48013 Bilbao (Spain)], E-mail:; Herranz, M.; Abelairas, A.; Legarda, F. [Dpto. Ingenieria Nuclear y Mecanica de Fluidos, E. T. S. Ingenieria de Bilbao - Universidad del Pais Vasco (UPV/EHU), Alda. Urquijo s/n. 48013 Bilbao (Spain)


    The determination of residual beta activity in drinking water is usually needed in most monitoring programs. In this work a procedure for its determination is described and expressions for the calculations of detection limits and uncertainties are proposed.

  19. Gross alpha and gross beta activity in the products and by-product of amang tin tailings process

    International Nuclear Information System (INIS)

    Gross alpha and gross beta activities were determined for mineral samples collected from five amang tailing factories. The measured activity of alpha ranged from 31 to 220,000 Bq kg-1 with an average 9,154 Bq kg-1, whereas for beta activity ranged from 9 to 552,000 Bq kg-1 with an average 19,811 Bq kg-1. The higher gross alpha and gross beta observed of monazite while the lowest recorded of waste (sand) and pyrite for gross alpha and gross beta activity respectively. The minimum detectable activities were 180 and 40 Bq kg-1 for gross alpha and gross beta respectively. (author)

  20. Identification of thermostable beta-xylosidase activities produced by Aspergillus brasiliensis and Aspergillus niger

    DEFF Research Database (Denmark)

    Pedersen, Mads; Lauritzen, H.K.; Frisvad, Jens Christian;


    Twenty Aspergillus strains were evaluated for production of extracellular cellulolytic and xylanolytic activities. Aspergillus brasiliensis, A. niger and A. japonicus produced the highest xylanase activities with the A. brasiliensis and A. niger strains producing thermostable beta-xylosidases. Th...... is a well known enzyme producer, this is the first report of xylanase and thermostable beta-xylosidase production from the newly identified, non-ochratoxin-producing species A. brasiliensis....

  1. Identification and characterization of phenol hydroxylase from phenol-degrading Candida tropicalis strain JH8. (United States)

    Long, Yan; Yang, Sheng; Xie, Zhixiong; Cheng, Li


    The gene phhY encoding phenol hydroxylase from Candida tropicalis JH8 was cloned, sequenced, and expressed in Escherichia coli. The gene phhY contained an open reading frame of 2130 bp encoding a polypeptide of 709 amino acid residues. From its sequence analysis, it is a member of a family of flavin-containing aromatic hydroxylases and shares 41% amino acid identity with phenol hydroxylase from Trichosporon cutaneum. The recombinant phenol hydroxylase exists as a homotetramer structure with a native molecular mass of 320 kDa. Recombinant phenol hydroxylase was insensitive to pH treatment; its optimum pH was at 7.6. The optimum temperature for the enzyme was 30 °C, and its activity was rapidly lost at temperatures above 60 °C. Under the optimal conditions with phenol as substrate, the K(m) and V(max) of recombinant phenol hydroxylase were 0.21 mmol·L(-1) and 0.077 μmol·L(-1)·min(-1), respectively. This is the first paper presenting the cloning and expression in E. coli of the phenol hydroxylase gene from C. tropicalis and the characterization of the recombinant phenol hydroxylase.

  2. Cytochrome P3-450 cDNA encodes aflatoxin B1-4-hydroxylase. (United States)

    Faletto, M B; Koser, P L; Battula, N; Townsend, G K; Maccubbin, A E; Gelboin, H V; Gurtoo, H L


    Aflatoxin B1 (AFB1), a potent hepatocarcinogen and ubiquitous dietary contaminant in some countries, is detoxified to aflatoxin M1 (AFM1) via cytochrome P-450-mediated AFB1-4-hydroxylase. Genetic studies in mice have demonstrated that the expression of AFB1-4-hydroxylase is regulated by the aryl hydrocarbon locus and suggested that different cytochrome P-450 isozymes catalyze AFB1-4-hydroxylase and aryl hydrocarbon hydroxylase activities. We have now examined lysates from mammalian cells infected with recombinant vaccinia viruses containing expressible cytochrome P1-450 or P3-450 cDNAs for their ability to metabolize AFB1 to AFM1. Our results show that cytochrome P3-450 cDNA specifies AFB1-4-hydroxylase. This is the first direct assignment of a specific cytochrome P-450 to an AFB1 detoxification pathway. This finding may have relevance to the dietary modulation of AFB1 hepatocarcinogenesis.

  3. Peroxisomal beta-oxidation activities and gamma-decalactone production by the yeast Yarrowia lipolytica. (United States)

    Pagot, Y; Le Clainche, A; Nicaud, J M; Wache, Y; Belin, J M


    gamma-Decalactone is a peachy aroma compound resulting from the peroxisomal beta-oxidation of ricinoleic acid by yeasts. The expression levels of acyl-CoA oxidase (gene deletion) and 3-ketoacyl-CoA thiolase activities (gene amplification on replicative plasmids) were modified in the yeast Yarrowia lipolytica. The effects of these modifications on beta-oxidation were measured. Overexpression of thiolase activity did not have any effect on the overall beta-oxidation activity. The disruption of one of the acyl-CoA oxidase genes resulted in an enhanced activity. The enhancement led to an increase of overall beta-oxidation activity but reduced the gamma-decalactone production rates. This seemed to indicate a non-rate-limiting role for beta-oxidation in the biotransformation of ricinoleic acid to gamma-decalactone by the yeast Yarrowia lipolytica. All strains produced and then consumed gamma-decalactone. We checked the ability of the different strains to consume gamma-decalactone in a medium containing the lactone as sole carbon source. The consumption of the strain overexpressing acyl-CoA oxidase activity was higher than that of the wild-type strain. We concluded that peroxisomal beta-oxidation is certainly involved in gamma-decalactone catabolism by the yeast Y. lipolytica. The observed production rates probably depend on an equilibrium between production and consumption of the lactone. PMID:9581293

  4. Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine [beta]-synthase

    Energy Technology Data Exchange (ETDEWEB)

    Koutmos, Markos; Kabil, Omer; Smith, Janet L.; Banerjee, Ruma (Michigan-Med)


    The catalytic potential for H{sub 2}S biogenesis and homocysteine clearance converge at the active site of cystathionine {beta}-synthase (CBS), a pyridoxal phosphate-dependent enzyme. CBS catalyzes {beta}-replacement reactions of either serine or cysteine by homocysteine to give cystathionine and water or H{sub 2}S, respectively. In this study, high-resolution structures of the full-length enzyme from Drosophila in which a carbanion (1.70 {angstrom}) and an aminoacrylate intermediate (1.55 {angstrom}) have been captured are reported. Electrostatic stabilization of the zwitterionic carbanion intermediate is afforded by the close positioning of an active site lysine residue that is initially used for Schiff base formation in the internal aldimine and later as a general base. Additional stabilizing interactions between active site residues and the catalytic intermediates are observed. Furthermore, the structure of the regulatory 'energy-sensing' CBS domains, named after this protein, suggests a mechanism for allosteric activation by S-adenosylmethionine.

  5. Cholesterol enhances amyloid {beta} deposition in mouse retina by modulating the activities of A{beta}-regulating enzymes in retinal pigment epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jiying [Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan); Ohno-Matsui, Kyoko, E-mail: [Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan); Morita, Ikuo [Section of Cellular Physiological Chemistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan)


    Highlights: Black-Right-Pointing-Pointer Cholesterol-treated RPE produces more A{beta} than non-treated RPE. Black-Right-Pointing-Pointer Neprilysin expression and activity decreased in cholesterol-treated RPE. Black-Right-Pointing-Pointer {alpha}-Secretase expression and activity decreased in cholesterol-treated RPE. Black-Right-Pointing-Pointer Cholesterol-enriched diet induced subRPE deposits in aged mice. Black-Right-Pointing-Pointer A{beta} were present in cholesterol-enriched-diet-induced subRPE deposits in aged mice. -- Abstract: Subretinally-deposited amyloid {beta} (A{beta}) is a main contributor of developing age-related macular degeneration (AMD). However, the mechanism causing A{beta} deposition in AMD eyes is unknown. Hypercholesterolemia is a significant risk for developing AMD. Thus, we investigated the effects of cholesterol on A{beta} production in retinal pigment epithelial (RPE) cells in vitro and in the mouse retina in vivo. RPE cells isolated from senescent (12-month-old) C57BL/6 mice were treated with 10 {mu}g/ml cholesterol for 48 h. A{beta} amounts in culture supernatants were measured by ELISA. Activity and expression of enzymes and proteins that regulate A{beta} production were examined by activity assay and real time PCR. The retina of mice fed cholesterol-enriched diet was examined by transmission electron microscopy. Cholesterol significantly increased A{beta} production in cultured RPE cells. Activities of A{beta} degradation enzyme; neprilysin (NEP) and anti-amyloidogenic secretase; {alpha}-secretase were significantly decreased in cell lysates of cholesterol-treated RPE cells compared to non-treated cells, but there was no change in the activities of {beta}- or {gamma}-secretase. mRNA levels of NEP and {alpha}-secretase (ADAM10 and ADAM17) were significantly lower in cholesterol-treated RPE cells than non-treated cells. Senescent (12-month-old) mice fed cholesterol-enriched chow developed subRPE deposits containing A{beta}, whereas

  6. Lysyl hydroxylases:characterization of mouse lysyl hydroxylases and generation of genetically modified lysyl hydroxylase 3 mouse lines


    Ruotsalainen, H. (Heidi)


    Abstract Lysyl hydroxylase (EC, procollagen-lysine, 2-oxyglutarate, 5-dioxygenase, Plod) catalyzes the hydroxylation of certain lysine residues in collagens and in other proteins with collagenous domains. Three lysyl hydroxylase isoforms have been cloned from human and rat. The importance of lysyl hydroxylase 1 in collagen biosynthesis is demonstrated by the heritable disorder, Ehlers-Danlos syndrome type VI, which is characterized by joint laxity, progressive scoliosis, muscle h...

  7. Inhibition of AmpC beta-lactamase through a destabilizing interaction in the active site

    Energy Technology Data Exchange (ETDEWEB)

    Trehan, I.; Beadle, B.M.; Shoichet, B.K. (NWU)


    {beta}-Lactamases hydrolyze {beta}-lactam antibiotics, including penicillins and cephalosporins; these enzymes are the most widespread resistance mechanism to these drugs and pose a growing threat to public health. {beta}-Lactams that contain a bulky 6(7){alpha} substituent, such as imipenem and moxalactam, actually inhibit serine {beta}-lactamases and are widely used for this reason. Although mutant serine {beta}-lactamases have arisen that hydrolyze {beta}-lactamase resistant {beta}-lactams (e.g., ceftazidime) or avoid mechanism-based inhibitors (e.g., clavulanate), mutant serine {beta}-lactamases have not yet arisen in the clinic with imipenemase or moxalactamase activity. Structural and thermodynamic studies suggest that the 6(7){alpha} substituents of these inhibitors form destabilizing contacts within the covalent adduct with the conserved Asn152 in class C {beta}-lactamases (Asn132 in class A {beta}-lactamases). This unfavorable interaction may be crucial to inhibition. To test this destabilization hypothesis, we replaced Asn152 with Ala in the class C {beta}-lactamase AmpC from Escherichia coli and examined the mutant enzyme's thermodynamic stability in complex with imipenem and moxalactam. Consistent with the hypothesis, the Asn152 {yields} Ala substitution relieved 0.44 and 1.10 kcal/mol of strain introduced by imipenem and moxalactam, respectively, relative to the wild-type complexes. However, the kinetic efficiency of AmpC N152A was reduced by 6300-fold relative to that of the wild-type enzyme. To further investigate the inhibitor's interaction with the mutant enzyme, the X-ray crystal structure of moxalactam in complex with N152A was determined to a resolution of 1.83 {angstrom}. Moxalactam in the mutant complex is significantly displaced from its orientation in the wild-type complex; however, moxalactam does not adopt an orientation that would restore competence for hydrolysis. Although Asn152 forces {beta}-lactams with 6(7){alpha

  8. Retinal and choroidal TGF-beta in the tree shrew model of myopia: isoform expression, activation and effects on function. (United States)

    Jobling, Andrew Ian; Wan, Ran; Gentle, Alex; Bui, Bang Viet; McBrien, Neville Anthony


    A visually evoked signalling cascade, which begins in the retina, transverses the choroid, and mediates scleral remodelling, is considered to control eye growth. The ubiquitous cytokine TGF-beta has been associated with alterations in ocular growth, where alterations in scleral TGF-beta isoforms mediate the scleral remodelling that results in myopia. However, while the TGF-beta isoforms have been implicated in the scleral change during myopia development, it is unclear whether alterations in retinal and choroidal isoforms constitute part of the retinoscleral cascade. This study characterised the retinal and choroidal TGF-beta isoform profiles and TGF-beta2 activation during different stages of myopia development, as induced by form deprivation, in a mammalian model of eye growth. Using quantitative real-time PCR, the mRNA for all three mammalian isoforms of TGF-beta was detected in tree shrew retina and choroid. Distinct tissue-specific isoform profiles were observed for the retina (TGF-beta1:TGF-beta2:TGF-beta3=20:2085:1) and choroid (TGF-beta1:TGF-beta2:TGF-beta3=16:23:1), which remained constant over the development period under investigation. The active and latent pools of retinal TGF-beta2 were quantified using ELISA with the majority (>94%) of total TGF-beta2 found in the latent form. Unlike previous scleral data showing early and continuous decreases in TGF-beta isoform expression during myopia development, the levels of the three isoforms remained within normal ranges for retinal (TGF-beta1, -14 to +14%; TGF-beta2, -2 to +20%; TGF-beta3, -10 to +26%) and choroidal (TGF-beta1, -19 to +21%; TGF-beta2, -26 to +8%; TGF-beta3, -11 to +28%) tissues during myopia development (induction times of 3h, 7h, 11h, 24h, and 5 days). A 40% decrease in retinal TGF-beta2 activation was observed after 5 days of myopia development, however, there was no functional correlate of altered TGF-beta2 activity, as assessed by the retinal ERG response. Overall, these data highlight

  9. Histone acetylation influences the transcriptional activation of POX in Beta vulgaris L. and Beta maritima L. under salt stress. (United States)

    Yolcu, Seher; Ozdemir, Filiz; Güler, Aybüke; Bor, Melike


    Acetylation of histone proteins is a type of chromatin modification which facilitates the activation of genes. Recent studies brought up the importance of this reversible and rapid process for the regulation of gene expression especially in plant defense against a variety of environmental stresses. Deciphering the exact mechanisms of chromatin modifications under abiotic stress conditions is important for improving crop plants' performance and yield. In a previous study we compared the salt stress responses of Beta vulgaris (sugar beet) and Beta maritima (wild beet). In accordance with those results we suggested that chromatin remodeling can be an active process in the regulation of genes related to salt stress tolerance of these plants. Therefore we performed ChIP assay in control and salt stressed (250 and 500 mM NaCl) plants and compared the enrichment of acetylation in the associated chromatin sites. We found that the transcriptional activation of one peroxidase (POX) encoding gene was associated with the elevated levels of acetylation in H3K9 and H3K27 sites. The acetylation patterns were remarkably different between two species in which the highest acetylation levels were found at H3K9 and H3K27 in wild beet and sugar beet respectively.

  10. Histone acetylation influences the transcriptional activation of POX in Beta vulgaris L. and Beta maritima L. under salt stress. (United States)

    Yolcu, Seher; Ozdemir, Filiz; Güler, Aybüke; Bor, Melike


    Acetylation of histone proteins is a type of chromatin modification which facilitates the activation of genes. Recent studies brought up the importance of this reversible and rapid process for the regulation of gene expression especially in plant defense against a variety of environmental stresses. Deciphering the exact mechanisms of chromatin modifications under abiotic stress conditions is important for improving crop plants' performance and yield. In a previous study we compared the salt stress responses of Beta vulgaris (sugar beet) and Beta maritima (wild beet). In accordance with those results we suggested that chromatin remodeling can be an active process in the regulation of genes related to salt stress tolerance of these plants. Therefore we performed ChIP assay in control and salt stressed (250 and 500 mM NaCl) plants and compared the enrichment of acetylation in the associated chromatin sites. We found that the transcriptional activation of one peroxidase (POX) encoding gene was associated with the elevated levels of acetylation in H3K9 and H3K27 sites. The acetylation patterns were remarkably different between two species in which the highest acetylation levels were found at H3K9 and H3K27 in wild beet and sugar beet respectively. PMID:26773543

  11. Localization of beta-D-glucosidase activity and glucovanillin in vanilla bean (Vanilla planifolia Andrews). (United States)

    Odoux, E; Escoute, J; Verdeil, J-L; Brillouet, J-M


    The morphology, anatomy and histology of mature green vanilla beans were examined by light and transmission electron microscopy. Beans have a triangular cross-section with a central cavity containing seeds. Each angle is lined with tubular cells, or papillae, while the cavity sides consist of placental laminae. The epicarp and endocarp are formed by one or two layers of very small cells, while the mesocarp contains large, highly vacuolarized cells, the cytoplasm being restricted to a thin layer along the cell walls. The radial distributions of glucovanillin and beta-glucosidase activity, measured on p-nitrophenyl-beta-glucopyranoside and glucovanillin, are superimposable and show how beta-glucosidase activity increases from the epicarp towards the placental zone, whereas glucovanillin is exclusively located in the placentae and papillae. Subcellular localization of beta-glucosidase activity was achieved by incubating sections of vanilla beans in a buffer containing 5-bromo-4-chloro-3-indolyl-beta-d-glucopyranoside as a substrate. Activity was observed in the cytoplasm (and/or the periplasm) of mesocarp and endocarp cells, with a more diffuse pattern observed in the papillae. A possible mechanism for the hydrolysis of glucovanillin and release of the aromatic aglycon vanillin involves the decompartmentation of cytoplasmic (and/or periplasmic) beta-glucosidase and vacuolar glucovanillin.

  12. Preparation of high activity HTO using recovered tritium from expired beta light sources

    International Nuclear Information System (INIS)

    In this paper the technological procedures for treatment of expired beta light sources as radioactive wastes with tritium recovering and use in synthesis of high specific activity HTO were analyzed. Technological procedures for treatment of beta light sources consist in: envelope breaking into vacuumed enclosure, the radioactive gaseous mixture pumping and its storing onto metallic sodium. The obtained 3T2-3He mixture was used in the synthesis of HTO with high radioactivity concentration. (authors)

  13. In situ detection of TGF betas, TGF beta receptor Ⅱ mRNA and telomerase activity in rat cholangiocarcinogenesis

    Institute of Scientific and Technical Information of China (English)

    Jian-Ping Lu; Jian-Qun Mao; Ming-Sheng Li; Shi-Lun Lu; Xi-Qi Hu; Shi-Neng Zhu; Shintaro Nomura


    AIM: Initial report on the in situ examination of the mRNA expression of transforming growth factor betas (TGFβs),TGFβ type Ⅱ receptor (TβRII) and telomerase activity in the experimental rat liver tissue during cholangiocarcinogenesis.METHODS: Rat liver cholangiocarcinogenesis was induced by 3'-methyl 4-dimethylazobenzene (3'Me-DAB). In situhybridization was used to examine the TGFβs) and TGFβ type Ⅱ receptor (TβRⅡ) mRNA, in situ TRAP was used to check the telomerase activity in the tissue samples.RESULTS: There was no TGFβs, TβRⅡ mRNA expression or telomerase activity in the control rat cholangiocytes. The expression of TGFβ1, TβRⅡ was increased in regenerative,hyperplastic, dysplastic cholangiocytes and cholangiocarcinoma (CC) cells. The expression of TGFβ2 mRNA was observed in only a part of hyperplastic, dysplastic cholangiocytes. TGFβ3expression was very weak, only in hyperplastic lesion. There was positive telomerase activity in the regenerative,hyperplastic, dysplastic cholangiocytes, and CC cells. Stroma fibroblasts of these lesions also showed positive TGFβs, TβRⅡ mRNA expression and telomerase activity.CONCLUSION: There were TGFβs, TβRⅡ expression and telomerase activity in hyperplastic, dysplastic cholangiocytes,cholangiocarcinoma cells as well as in stroma fibroblasts during cholangiocarcinogenesis. Their expression or activity is important in cholangiocarcinogenesis andstroma formation.

  14. Radioactivity Measurement Method for Environmental Monitoring Gross Alpha/beta Activities in Drinking Water in Turkey. (United States)

    Kahraman, Gülten; Aslan, Nazife; Şahin, Mihriban; Yüksek, Simay


    The determination of gross alpha/beta activity concentrations of drinking water is the first step of the environmental monitoring studies and can provide a rapid evaluation of the radioactive content of a sample. In this study, a procedure using liquid scintillation spectrometry (LSS) for the simultaneously monitoring of gross alpha/beta activity concentration in drinking water was determined, verificated with proficiency test sample and applied to the real drinking water samples in Turkey. The results indicate that the method provides good accuracy and precision. LSS can be employed as a screening technique in high activity concentrations. PMID:26454594

  15. Evaluation of the Association of Menopausal Status with Delta and Beta EEG Activity during Sleep (United States)

    Campbell, Ian G.; Bromberger, Joyce T.; Buysse, Daniel J.; Hall, Martica H.; Hardin, Kimberly A.; Kravitz, Howard M.; Matthews, Karen A.; Rasor, Marianne O'Neill; Utts, Jessica; Gold, Ellen


    Study Objectives: Women report increasing sleep difficulties during menopause, but polysomnographic measures do not detect sleep disturbances. We examined whether two spectral analysis sleep measures, delta and beta power, were related to menopausal status. Design: The Study of Women's Health Across the Nation (SWAN) Sleep Study compared cross-sectionally spectral sleep measures in women in different stages of menopause. Setting: Sleep EEG was recorded in the participants' homes with ambulatory recorders. Participants: A multi-ethnic cohort of premenopausal and early perimenopausal (n = 189), late perimenopausal (n = 73), and postmenopausal (n = 59) women. Measurements: EEG power in the delta and beta frequency bands was calculated for all night NREM and all night REM sleep. Physical, medical, psychological, and socioeconomic data were collected from questionnaires and diaries. Results: Beta EEG power in NREM and REM sleep in late perimenopausal and postmenopausal women exceeded that in pre- and early perimenopausal women. Neither all night delta power nor the trend in delta power across the night differed by menopausal status. In a multivariate model that controlled for the physical, demographic, behavioral, psychological, and health-related changes that accompany menopause, beta power in both NREM and REM sleep EEG was significantly related to menopausal status. The frequency of hot flashes explained part but not all of the relation of beta power to menopausal status. Conclusions: Elevated beta EEG power in late perimenopausal and postmenopausal women provides an objective measure of disturbed sleep quality in these women. Elevated beta EEG activity suggests that arousal level during sleep is higher in these women. Citation: Campbell IG; Bromberger JT; Buysse DJ; Hall MH; Hardin KA; Kravitz HM; Matthews KA; Rasor MO; Utts J; Gold E. Evaluation of the association of menopausal status with delta and beta EEG activity during sleep. SLEEP 2011;34(11):1561-1568. PMID

  16. Interleukin-1beta induced vascular permeability is dependent on induction of endothelial tissue factor (TF) activity. (United States)

    Puhlmann, Markus; Weinreich, David M; Farma, Jeffrey M; Carroll, Nancy M; Turner, Ewa M; Alexander, H Richard


    IL-1beta is a pleotropic cytokine that may mediate increased procoagulant activity and permeability in endothelial tissue during inflammatory conditions. The procoagulant effects of IL-1beta are mediated through induction of tissue factor (TF) but its alterations on vascular permeability are not well characterized. We found that IL-1beta induced a rapid and dose-dependent increase in TF activity in human umbilical vein endothelial cells (ECs) under routine culture conditions. However, IL-1beta caused a rapid and marked increase in permeability across confluent EC monolayers using a two-compartment in vitro model only in the presence of factor VIII-deficient plasma that was completely abrogated by neutralizing anti-TF antibody pre-treatment. In vitro permeability was associated with loss of EC surface expression of VE-cadherin and contraction of F-actin cytoskeletal elements that resulted in EC intercellular gap formation. These data demonstrate that IL-1beta induces marked changes in permeability across activated endothelium via a TF dependent mechanism and suggest that modulation of TF activity may represent a strategy to treat various acute and chronic inflammatory conditions mediated by this cytokine.

  17. TLR2 is a primary receptor for Alzheimer's amyloid beta peptide to trigger neuroinflammatory activation.

    NARCIS (Netherlands)

    Liu, S.; Liu, Y.; Hao, W.; Wolf, L.; Kiliaan, A.J.; Penke, B.; Rube, C.E.; Walter, J.; Heneka, M.T.; Hartmann, T.; Menger, M.D.; Fassbender, K.


    Microglia activated by extracellularly deposited amyloid beta peptide (Abeta) act as a two-edged sword in Alzheimer's disease pathogenesis: on the one hand, they damage neurons by releasing neurotoxic proinflammatory mediators (M1 activation); on the other hand, they protect neurons by triggering an

  18. F-18-FEAnGA for PET of beta-Glucuronidase Activity in Neuroinflammation

    NARCIS (Netherlands)

    Antunes, Ines F.; Doorduin, Janine; Haisma, Hidde J.; Elsinga, Philip H.; van Waarde, Aren; Willemsen, Antoon T. M.; Dierckx, Rudi A.; de Vries, Erik F. J.


    Activation of microglia is a hallmark of inflammatory, infectious, and degenerative diseases of the central nervous system. Several studies have indicated that there is an increase in release of beta-glucuronidase by activated microglia into the extracellular space at the site of neuroinflammation.

  19. Simulation of the dose rate per activity of beta-emitting radionuclides

    International Nuclear Information System (INIS)

    The dose rate per activity was simulated for 10 beta-emitting radionuclides and for different activity distributions (point source, areal sources and a semi-infinite volume source). The results are given for 7 different distances from the source (from 0.01 to 2 m) for both contributions: the beta- and electron-emission, and the X- and gamma-emission. Data are provided for both operational quantities and organ doses: Hp(0.07), Hp(3), Hp(10), Hskin and Hlens. Finally, a software application to interpolate the dose rate per activity due to the beta-emission of arbitrary radionuclides is presented and a simple superposition of these data and of gamma-ray dose constants to calculate the total dose rate is described. (authors)

  20. MHD activity and energy loss during beta saturation and collapse at high beta poloidal in PBX

    International Nuclear Information System (INIS)

    High-β experiments, in medium to high-q tokamak plasmas, exhibit a temporal β saturation and collapse. This behavior has been attributed to ballooning, ideal kink, or tearing modes. In PBX, a unique diagnostic capability allowed studies of the relation between MHD and energy loss for neutral-beam-heated (<6 MW), mildly indented (10 to 15%), nearly steady I/sub p/ discharges that approached the Troyon-Gruber limit. Under these conditions, correlations between MHD activity and energy losses have shown that the latter can be almost fully accounted for by various long wavelength MHD instabilities and that there is no need to invoke high-n ballooning modes in PBX. 6 refs., 4 figs

  1. Long-term controlled GDNF over-expression reduces dopamine transporter activity without affecting tyrosine hydroxylase expression in the rat mesostriatal system. (United States)

    Barroso-Chinea, Pedro; Cruz-Muros, Ignacio; Afonso-Oramas, Domingo; Castro-Hernández, Javier; Salas-Hernández, Josmar; Chtarto, Abdelwahed; Luis-Ravelo, Diego; Humbert-Claude, Marie; Tenenbaum, Liliane; González-Hernández, Tomás


    The dopamine (DA) transporter (DAT) is a plasma membrane glycoprotein expressed in dopaminergic (DA-) cells that takes back DA into presynaptic neurons after its release. DAT dysfunction has been involved in different neuro-psychiatric disorders including Parkinson's disease (PD). On the other hand, numerous studies support that the glial cell line-derived neurotrophic factor (GDNF) has a protective effect on DA-cells. However, studies in rodents show that prolonged GDNF over-expression may cause a tyrosine hydroxylase (TH, the limiting enzyme in DA synthesis) decline. The evidence of TH down-regulation suggests that another player in DA handling, DAT, may also be regulated by prolonged GDNF over-expression, and the possibility that this effect is induced at GDNF expression levels lower than those inducing TH down-regulation. This issue was investigated here using intrastriatal injections of a tetracycline-inducible adeno-associated viral vector expressing human GDNF cDNA (AAV-tetON-GDNF) in rats, and doxycycline (DOX; 0.01, 0.03, 0.5 and 3mg/ml) in the drinking water during 5weeks. We found that 3mg/ml DOX promotes an increase in striatal GDNF expression of 12× basal GDNF levels and both DA uptake decrease and TH down-regulation in its native and Ser40 phosphorylated forms. However, 0.5mg/ml DOX promotes a GDNF expression increase of 3× basal GDNF levels with DA uptake decrease but not TH down-regulation. The use of western-blot under non-reducing conditions, co-immunoprecipitation and in situ proximity ligation assay revealed that the DA uptake decrease is associated with the formation of DAT dimers and an increase in DAT-α-synuclein interactions, without changes in total DAT levels or its compartmental distribution. In conclusion, at appropriate GDNF transduction levels, DA uptake is regulated through DAT protein-protein interactions without interfering with DA synthesis. PMID:26777664

  2. Elicitor and resistance-inducing activities of beta-1,4 cellodextrins in grapevine, comparison with beta-1,3 glucans and alpha-1,4 oligogalacturonides. (United States)

    Aziz, Aziz; Gauthier, Adrien; Bézier, Annie; Poinssot, Benoît; Joubert, Jean-Marie; Pugin, Alain; Heyraud, Alain; Baillieul, Fabienne


    Cellodextrins (CD), water-soluble derivatives of cellulose composed of beta-1,4 glucoside residues, have been shown to induce a variety of defence responses in grapevine (Vitis vinifera L.) cells. The larger oligomers of CD rapidly induced transient generation of H2O2 and elevation in free cytosolic calcium, followed by a differential expression of genes encoding key enzymes of the phenylpropanoid pathway and pathogenesis-related (PR) proteins as well as stimulation of chitinase and beta-1,3 glucanase activities. Most of these defence reactions were also induced by linear beta-1,3 glucans (betaGlu) and alpha-1,4 oligogalacturonides (OGA) of different degree of polymerization (DP), but the intensity of some reactions induced by CD was different when compared with betaGlu and OGA effects. Moreover, desensitization assays using H2O2 production showed that cells treated with CD remained fully responsive to a second application of OGA, suggesting a different mode of perception of these oligosaccharides by grape cells. None of CD, betaGlu, or OGA induced HSR gene expression nor did they induce cell death. In accordance with elicitor activity in grapevine cells, CD-incubated leaves challenged with Botrytis cinerea also resulted in a significant reduction of the disease. Data suggest that CD could operate via other distinct reaction pathways than betaGlu and OGA. They also highlight the requirement of a specific DP for each oligosaccharide to induce the defence response. PMID:17322548

  3. Antifungal activity of beta-asarone from rhizomes of Acorus gramineus. (United States)

    Lee, Jee Yeon; Lee, Jung Yeop; Yun, Bong-Sik; Hwang, Byung Kook


    An antifungal substance was isolated from the extract of Acorus gramineus using various chromatographic procedures. The antibiotic was identified as beta-asarone, cis-2,4,5-trimethoxy-1-propenylbenzene, on the basis of the high-resolution EI-mass, NMR, and UV spectral data. Beta-asarone completely inhibited mycelial growth of some plant pathogenic fungi, Cladosporium cucumerinum,Colletotrichum orbiculare, Magnaporthe grisea, and Pythium ultimum, in a range of 0.5-30 microg/mL. The growth of Bacillus subtilis, Erwinia carotovora subsp. carotovora, Ralstonia solanacearum, and Xanthomonas campestris pv. vesicatoria was slightly suppressed by beta-asarone. As the concentration of beta-asarone increased, M. grisea infection was drastically inhibited on rice leaves. Treatment with 500 microg/mL of beta-asarone also greatly suppressed lesion formation of Co. orbiculare on cucumber leaves. This is the first study to demonstrate in vitro and in vivo antifungal activity of beta-asarone against plant fungal pathogens M. grisea and C. orbiculare. PMID:14969530

  4. Age-related differences in EEG beta activity during an assessment of ankle proprioception. (United States)

    Toledo, Diana R; Barela, José A; Manzano, Gilberto M; Kohn, André F


    The aim of this work was to compare cortical beta oscillatory activity between young (YA) and older (OA) adults during the assessment of ankle proprioception. We analyzed the response time (RT) to kinesthetic perception and beta event-related desynchronization/synchronization (ERD/ERS) in response to passive ankle movement applied at a slow speed, 0.5°/s. The relationship between ERD/ERS and RT was investigated by classifying the signals into fast-, medium-, and slow-RT. The results showed a temporal relationship between beta oscillation changes and RT for both groups, i.e., earlier ERD and ERS were obtained for trials with faster response time. ERD was larger and delayed in OA compared to the YA, and beta ERS was present only for OA. These findings suggest that a less efficient proprioceptive signaling reaching the brain of OA requires a higher level of brain processing and hence the differences in ERD potentials between YA and OA. Furthermore, the occurrence of ERS in OA might represent a compensatory strategy of active cortical resetting for adequate sensorimotor behavior due to the age-related reduced peripheral input and neuromuscular impairments. Altered balance between excitatory and inhibitory intracortical activity in older adults presumably explains the changes in beta oscillations. PMID:27085535

  5. [Determination of beta-mannanase activity by viscosimetric and spectrophotometric methods]. (United States)

    Firantas, S G; Venozhinskene, Iu I; Pauliukonis, A B


    The activity of beta-mannanase from Bacillus subtilis was measured viscosimetrically and spectrophotometrically. As substrate galactomannane of Ceratonia siliqua was used. Relationships between the beta-mannanase activity and the substrate concentration as well as the enzyme content were investigated. The kinetic parameters of the enzymes obeying the Michaelis-Menten equation were calculated. It was found viscosimetrically that Vmax of the commercial enzyme preparation was 1.4 mucat/g (at pH 5.8 and 40 degrees) and Km was 0.6 mM. The viscosimetric method shows high sensitivity, whereas the spectrophotometric technique suits mass-scale analyses.

  6. Complete genomic structure of mouse lysyl hydroxylase 2 and lysyl hydroxylase 3/collagen glucosyltransferase. (United States)

    Ruotsalainen, H; Vanhatupa, S; Tampio, M; Sipilä, L; Valtavaara, M; Myllylä, R


    Lysyl hydroxylase is an enzyme involved in collagen biosynthesis, catalyzing the hydroxylation of lysyl residues as a post-translational event. Three isoforms have been characterized so far (LH1, LH2, LH3). Our recent findings indicate that LH3 possesses, not only lysyl hydroxylase activity, but also galactosylhydroxylysyl glucosyltransferase activity [Heikkinen et al., J. Biol. Chem. 275 (2000) 36158-36163]. We report here the characterization of mouse LH2 (Plod2) and LH3/glucosyltransferase (Plod3) genes. Plod2 spans approximately 50 kb of the genomic DNA, and is organized in 20 exons, one of the exons being alternatively spliced in the RNA processing. Plod3 spans approximately 10 kb of the genomic DNA, and contains 19 exons. Analysis of the 5' flanking region with many transcription start sites reveals the lack of a TATAA box in both genes. Sequence analysis indicated many retroposon-like elements within the Plod3 gene. A comparison was carried out among the LH1, LH2 and LH3 gene structures characterized so far from different species. PMID:11334715

  7. Rapid determination of gross alpha/beta activity in milk using liquid scintilation counter technique

    Directory of Open Access Journals (Sweden)

    Sas Daniel


    Full Text Available Rapid determination of gross alpha and beta emitters in milk by liquid scintillation counter is discussed. This method is based on direct addition of different types of milk into scintillation cocktail and therefore it is very promising for fast determination of alpha/beta activity due to direct alpha and beta separation, measurement in close 4p geometry and without sample treatment. The selected group of radionuclides was chosen with the respect to military significance, radio-toxicity, and possibility of potential misuse. As model radionuclides 241Am, 239Pu, and 90Sr were selected. The Liquid Scintilation Counter Hidex 300 SL equipped with triple-double-coincidence-ratio technique was used for sample measurement. The aim of the work was focused on comparison of different cocktails produced by Hidex and Perkin Elmer, choosing the best cocktail based on our measurement results and adjustment of its appropriate volume. Furthermore, the optimization of ratio between the volume of scintillation cocktail and the volume of urine was investigated with the respect to the model radionuclides. According to the obtained results, the efficiency for alpha emitters was greater than 85% and for beta, greater than 95%. The obtained results allowed this method to be used for rapid determination of gross alpha/beta activity in cases where time is an essence, such as first responders or mass-scale samples, where ordinary means suffer from lack of capacity or simply collapse under the onslaught.

  8. Prolyl 4 Hydroxylase: A Critical Target in the Pathophysiology of Diseases


    Kant, Ravi; Bali, Anjana; Singh, Nirmal; Jaggi, Amteshwar Singh


    Prolyl 4 hydroxylases (P4H) are iron- and 2-oxoglutamate-dependent dioxygenase enzymes and hypoxia-inducible transcription factor (HIF)-P4Hs play a critical role in the regulating oxygen homeostasis in the local tissues as well in the systemic circulation. Over a period of time, a number of prolyl hydroxylase inhibitors and activators have been developed. By employing the pharmacological tools and transgenic knock out animals, the critical role of these enzymes has been established in the pat...

  9. Direct Evidence for a Phenylalanine Site in the Regulatory Domain of Phenylalanine Hydroxylase


    Li, Jun; Ilangovan, Udayar; Daubner, S. Colette; Hinck, Andrew P.; Fitzpatrick, Paul F.


    The hydroxylation of phenylalanine to tyrosine by the liver enzyme phenylalanine hydroxylase is regulated by the level of phenylalanine. Whether there is a distinct allosteric binding site for phenylalanine outside of the active site has been unclear. The enzyme contains an N-terminal regulatory domain that extends through Thr117. The regulatory domain of rat phenylalanine hydroxylase was expressed in E. coli. The purified protein behaves as a dimer on a gel filtration column. In the presence...

  10. Effects of methoxychlor and its metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane on human and rat 17α-hydroxylase/17,20-lyase activity. (United States)

    Ye, Leping; Chen, Xiaomin; Li, Xiaoheng; Zhu, Qiqi; Yu, Lin; Guo, Jingjing; Chen, Bingbing; Akingbemi, Benson T; Ge, Ren-Shan; Li, Hui


    Exposure to methoxychlor, an agricultural pesticide, has been associated with reduced testicular androgen secretion. However, methoxychlor is converted to 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) in the liver, which then acts as its biologically active metabolite. Both methoxychlor and HPTE have been credited with estrogenic properties and have a weak anti-androgenic activity. However, the exact mechanisms of steroidogenic enzyme inhibition remain to be clarified. In the present study, human and rat testis microsomes were employed to investigate the inhibitory activities of methoxychlor and HPTE on 17α-hydroxylase/17,20-lyase (CYP17A1). The CYP17A1 enzyme is critical for androgen biosynthesis and catalyzes conversion of progesterone into androstenedione. The results demonstrated that HPTE directly inhibited human and rat CYP17A1 activities, while methoxychlor had no effects on this enzyme activity even at a concentration of 100 μM. The IC50 values of HPTE were 1.13±0.10 (human) and 6.87±0.13 μM (rat), respectively. When HPTE was incubated with rat immature Leydig cells, it also inhibited CYP17A1 activity with an IC50 value of 6.29±0.1 μM. Results of enzyme inhibition were supported by the observation that HPTE inhibited luteinizing hormone-stimulated 5α-androstane-3α,17β-diol and testosterone secretion by immature Leydig cells with IC50 values of 6.61±0.03 and 3.78±0.003 μM, respectively. The mode of action of HPTE on CYP17A1 activity was determined to be uncompetitive with the substrate progesterone. In conclusion, HPTE, the metabolite of MXC, directly inhibited human and rat testis CYP17A1 activities.

  11. JBP1 and JBP2 are two distinct thymidine hydroxylases involved in J biosynthesis in genomic DNA of African trypanosomes. (United States)

    Cliffe, Laura J; Kieft, Rudo; Southern, Timothy; Birkeland, Shanda R; Marshall, Marion; Sweeney, Kate; Sabatini, Robert


    Genomic DNA of African trypanosomes contains a hypermodified thymidine residue termed base J (beta-d-glucosyl-HOMedU). This modified base is localized primarily to repetitive DNA, namely the telomeres, and is implicated in the regulation of antigenic variation. The base is synthesized in a two-step pathway. Initially, a thymidine residue in DNA is hydroxylated by a thymidine hydroxylase (TH). This intermediate (HOMedU) is then glucosylated to form base J. Two proteins involved in J synthesis, JBP1 (J binding protein 1) and JBP2, contain a putative TH domain related to the family of Fe(2+)/2-oxoglutarate-dependent hydroxylases. We have previously shown that mutations in the TH domain of JBP1 kill its ability to stimulate J synthesis. Here we show that mutation of key residues in the TH domain of JBP2 ablate its ability to induce de novo J synthesis. While the individual JBP1 null and JBP2 null trypanosomes have reduced J levels, the deletion of both JBP1 and JBP2 generates a cell line that completely lacks base J but still contains glucosyl-transferase activity. Reintroduction of JBP2 in the J-null trypanosome stimulates HOMedU formation and site-specific synthesis of base J. We conclude that JBP2 and JBP1 are the TH enzymes involved in J biosynthesis. PMID:19136460

  12. Beta-ionone activates and bleaches visual pigment in salamander photoreceptors. (United States)

    Isayama, Tomoki; McCabe England, S L; Crouch, R K; Zimmerman, A L; Makino, C L


    Vision begins with photoisomerization of 11-cis retinal to the all-trans conformation within the chromophore-binding pocket of opsin, leading to activation of a biochemical cascade. Release of all-trans retinal from the binding pocket curtails but does not fully quench the ability of opsin to activate transducin. All-trans retinal and some other analogs, such as beta-ionone, enhance opsin's activity, presumably on binding the empty chromophore-binding pocket. By recording from isolated salamander photoreceptors and from patches of rod outer segment membrane, we now show that high concentrations of beta-ionone suppressed circulating current in dark-adapted green-sensitive rods by inhibiting the cyclic nucleotide-gated channels. There were also decreases in circulating current and flash sensitivity, and accelerated flash response kinetics in dark-adapted blue-sensitive (BS) rods and cones, and in ultraviolet-sensitive cones, at concentrations too low to inhibit the channels. These effects persisted in BS rods even after incubation with 9-cis retinal to ensure complete regeneration of their visual pigment. After long exposures to high concentrations of beta-ionone, recovery was incomplete unless 9-cis retinal was given, indicating that visual pigment had been bleached. Therefore, we propose that beta-ionone activates and bleaches some types of visual pigments, mimicking the effects of light.

  13. Receptor density is key to the alpha2/beta interferon differential activities. (United States)

    Moraga, Ignacio; Harari, Daniel; Schreiber, Gideon; Uzé, Gilles; Pellegrini, Sandra


    Multiple type I interferons (IFN-alpha/beta) elicit Jak/Stat activation, rapid gene induction, and pleiotropic effects, such as differentiation, antiviral protection, and blocks in proliferation, which are dependent on the IFN subtype and the cellular context. To date, ligand- and receptor-specific molecular determinants underlying IFN-alpha/beta differential activities or potencies have been well characterized. To analyze cellular determinants that impact subtype-specific potency, human fibrosarcoma U5A-derived clones, exhibiting a gradient of IFN sensitivity by virtue of increasing receptor levels, were monitored for Jak/Stat signaling, gene induction, cell cycle lengthening, and apoptosis. In cells with scarce receptors, IFN-beta was more potent than IFN-alpha2 in antiproliferative activity, while the two subtypes were equipotent in all other readouts. Conversely, in cells with abundant receptors, IFN-alpha2 matched or even surpassed IFN-beta in all readouts tested. Our results suggest that the differential activities of the IFN subtypes are dictated not only by the intrinsic ligand/receptor binding kinetics but also by the density of cell surface receptor components.

  14. Lipidated alpha-Peptide/beta-Peptoid Hybrids with Potent Antiinflammatory Activity

    DEFF Research Database (Denmark)

    Skovbakke, Sarah L.; Larsen, Camilla J.; Heegaard, Peter M. H.;


    , in contrast to polymyxin B, displayed only minor activity in the Limulus amebocyte lysate assay. Flow cytometry data showed specific interaction of a fluorophore-labeled lipidated a-peptide/beta-peptoid hybrid with monocytes and granulocytes indicating a cellular target expressed by these leukocyte subsets....

  15. Immunochemical characterization of brain and pineal tryptophan hydroxylase.


    Chung, Y. I.; Park, D. H.; Kim, M.; Baker, H; Joh, T H


    Recombinant mouse tryptophan hydroxylase (TPH) was expressed in Escherichia coli, using a bacterial expression vector and has been purified to homogeneity by sonication followed by Sepharose 4B column chromatography and native slab gel electrophoresis. This purified enzymatically active TPH protein was used for production of a specific antiserum. This antiserum identified the predicted TPH band (molecular weight, 54 kDa) on Western blot of crude extracts from the rat and mouse dorsal raphe, a...

  16. Discoidin domain receptor 1 is activated independently of beta(1) integrin

    DEFF Research Database (Denmark)

    Vogel, W; Brakebusch, C; Fässler, R;


    Various types of collagen have been identified as potential ligands for the two mammalian discoidin domain receptor (DDR) tyrosine kinases, DDR1 and DDR2. It is presently unclear whether collagen-induced DDR receptor activation, which occurs with very slow kinetics, involves additional proteins...... blocking antibodies for alpha(2)beta(1) integrin or in cells with a targeted deletion of the beta(1) integrin gene. Finally, we show that overexpression of dominant negative DDR1 in the myoblast cell line C2C12 blocks cellular differentiation and the formation of myofibers....

  17. A note on starch hydrolysis and beta-glucuronidase activity among flavobacteria. (United States)

    Petzel, J P; Hartman, P A


    Most flavobacteria tested with the fluorogenic substrate 4-methylumbelliferyl-beta-D-glucuronide possessed beta-glucuronidase (GUD), but when some of the same strains were tested with the API ZYM gallery, all were negative for GUD. Conflicting reports also appear in the literature about starch hydrolysis among flavobacteria. We observed that the results obtained can depend on the medium used and the length of incubation. Our results indicate that GUD activity and starch hydrolysis are more widely distributed in the genus Flavobacterium than previously reported. PMID:3804862

  18. Hormone-sensitive lipase, the rate-limiting enzyme in triglyceride hydrolysis, is expressed and active in beta-cells. (United States)

    Mulder, H; Holst, L S; Svensson, H; Degerman, E; Sundler, F; Ahrén, B; Rorsman, P; Holm, C


    Triglycerides in the beta-cell may be important for stimulus-secretion coupling, through provision of a lipid-derived signal, and for pathogenetic events in NIDDM, where lipids may adversely affect beta-cell function. In adipose tissues, hormone-sensitive lipase (HSL) is rate-limiting in triglyceride hydrolysis. Here, we investigated whether this enzyme is also expressed and active in beta-cells. Northern blot analysis and reverse transcription-polymerase chain reaction demonstrated that HSL is expressed in rat islets and in the clonal beta-cell lines INS-1, RINm5F, and HIT-T15. Western blot analysis identified HSL in mouse and rat islets and the clonal beta-cells. In mouse and rat, immunocytochemistry showed a predominant occurrence of HSL in beta-cells, with a presumed cytoplasmic localization. Lipase activity in homogenates of the rodent islets and clonal beta-cells constituted 2.1 +/- 0.6% of that in adipocytes; this activity was immunoinhibited by use of antibodies to HSL. The established HSL expression and activity in beta-cells offer a mechanism whereby lipids are mobilized from intracellular stores. Because HSL in adipocytes is activated by cAMP-dependent protein kinase (PKA), PKA-regulated triglyceride hydrolysis in beta-cells may participate in the regulation of insulin secretion, possibly by providing a lipid-derived signal, e.g., long-chain acyl-CoA and diacylglycerol.

  19. Assessing adrenocortical activity by determining levels of urinary free cortisol and urinary 6 beta-hydroxycortisol. (United States)

    Nakamura, J; Yakata, M


    A comparative study of urinary free cortisol and urinary 6 beta-hydroxycortisol levels as a diagnostic test for hypercortisolemic states was carried out by measuring the excretion in 24-h specimens from 289 apparently healthy subjects and 10 Cushing patients. The diurnal variations of both variables were examined in normal subjects and subjects with altered adrenal activities. Two of the 289 apparently normal subjects had high values of urinary free cortisol; one had a high, the other a normal 6 beta-hydroxycortisol level; they were later diagnosed as having Cushing's syndrome and infertility, respectively. Three other subjects had high values of the urinary variables, but during 5 years of follow-up did not show any clinical evidence of hypercortisolism. The two urinary variables gave no false-negative results in the Cushing patients. The diurnal variation revealed that levels of 6 beta-hydroxycortisol change in parallel with those of free cortisol in normal subjects and in subjects with altered adrenal activities. However, the ratio of 6 beta-hydroxycortisol to free cortisol during the diurnal variation varied from low values when free cortisol levels were high to high values when free cortisol levels were low. In normal subjects, 1 mg of dexamethasone taken orally at 23.00 h completely suppressed the levels of both variables on the following day. It is concluded that urinary 6 beta-hydroxycortisol is correlated to urinary free cortisol so that measurement of urinary 6 beta-hydroxycortisol levels can be used as a diagnostic test for hypercortisolism in a way comparable to the method using urinary free cortisol.

  20. Concurrent Transient Activation of Wnt/{beta}-Catenin Pathway Prevents Radiation Damage to Salivary Glands

    Energy Technology Data Exchange (ETDEWEB)

    Hai Bo; Yang Zhenhua; Shangguan Lei; Zhao Yanqiu [Institute for Regenerative Medicine, Scott and White Hospital, Molecular and Cellular Medicine Department, Texas A and M Health Science Center, Temple, Texas (United States); Boyer, Arthur [Department of Radiology, Scott and White Hospital, Temple, Texas (United States); Liu, Fei, E-mail: [Institute for Regenerative Medicine, Scott and White Hospital, Molecular and Cellular Medicine Department, Texas A and M Health Science Center, Temple, Texas (United States)


    Purpose: Many head and neck cancer survivors treated with radiotherapy suffer from permanent impairment of their salivary gland function, for which few effective prevention or treatment options are available. This study explored the potential of transient activation of Wnt/{beta}-catenin signaling in preventing radiation damage to salivary glands in a preclinical model. Methods and Materials: Wnt reporter transgenic mice were exposed to 15 Gy single-dose radiation in the head and neck area to evaluate the effects of radiation on Wnt activity in salivary glands. Transient Wnt1 overexpression in basal epithelia was induced in inducible Wnt1 transgenic mice before together with, after, or without local radiation, and then saliva flow rate, histology, apoptosis, proliferation, stem cell activity, and mRNA expression were evaluated. Results: Radiation damage did not significantly affect activity of Wnt/{beta}-catenin pathway as physical damage did. Transient expression of Wnt1 in basal epithelia significantly activated the Wnt/{beta}-catenin pathway in submandibular glands of male mice but not in those of females. Concurrent transient activation of the Wnt pathway prevented chronic salivary gland dysfunction following radiation by suppressing apoptosis and preserving functional salivary stem/progenitor cells. In contrast, Wnt activation 3 days before or after irradiation did not show significant beneficial effects, mainly due to failure to inhibit acute apoptosis after radiation. Excessive Wnt activation before radiation failed to inhibit apoptosis, likely due to extensive induction of mitosis and up-regulation of proapoptosis gene PUMA while that after radiation might miss the critical treatment window. Conclusion: These results suggest that concurrent transient activation of the Wnt/{beta}-catenin pathway could prevent radiation-induced salivary gland dysfunction.

  1. Bace1 activity impairs neuronal glucose metabolism: rescue by beta-hydroxybutyrate and lipoic acid

    Directory of Open Access Journals (Sweden)

    John A Findlay


    Full Text Available Glucose hypometabolism and impaired mitochondrial function in neurons have been suggested to play early and perhaps causative roles in Alzheimer’s disease (AD pathogenesis. Activity of the aspartic acid protease, beta-site amyloid precursor protein (APP cleaving enzyme 1 (BACE1, responsible for beta amyloid peptide generation, has recently been demonstrated to modify glucose metabolism. We therefore examined, using a human neuroblastoma (SH-SY5Y cell line, whether increased BACE1 activity is responsible for a reduction in cellular glucose metabolism. Overexpression of active BACE1, but not a protease-dead mutant BACE1, protein in SH-SY5Y cells reduced glucose oxidation and the basal oxygen consumption rate, which was associated with a compensatory increase in glycolysis. Increased BACE1 activity had no effect on the mitochondrial electron transfer process but was found to diminish substrate delivery to the mitochondria by inhibition of key mitochondrial decarboxylation reaction enzymes. This BACE1 activity-dependent deficit in glucose oxidation was alleviated by the presence of beta hydroxybutyrate or α-lipoic acid. Consequently our data indicate that raised cellular BACE1 activity drives reduced glucose oxidation in a human neuronal cell line through impairments in the activity of specific tricarboxylic acid cycle enzymes. Because this bioenergetic deficit is recoverable by neutraceutical compounds we suggest that such agents, perhaps in conjunction with BACE1 inhibitors, may be an effective therapeutic strategy in the early-stage management or treatment of AD.

  2. Extraction of single-trial cortical beta oscillatory activities in EEG signals using empirical mode decomposition

    Directory of Open Access Journals (Sweden)

    Wu Chi-Hsun


    Full Text Available Abstract Background Brain oscillatory activities are stochastic and non-linearly dynamic, due to their non-phase-locked nature and inter-trial variability. Non-phase-locked rhythmic signals can vary from trial-to-trial dependent upon variations in a subject's performance and state, which may be linked to fluctuations in expectation, attention, arousal, and task strategy. Therefore, a method that permits the extraction of the oscillatory signal on a single-trial basis is important for the study of subtle brain dynamics, which can be used as probes to study neurophysiology in normal brain and pathophysiology in the diseased. Methods This paper presents an empirical mode decomposition (EMD-based spatiotemporal approach to extract neural oscillatory activities from multi-channel electroencephalograph (EEG data. The efficacy of this approach manifests in extracting single-trial post-movement beta activities when performing a right index-finger lifting task. In each single trial, an EEG epoch recorded at the channel of interest (CI was first separated into a number of intrinsic mode functions (IMFs. Sensorimotor-related oscillatory activities were reconstructed from sensorimotor-related IMFs chosen by a spatial map matching process. Post-movement beta activities were acquired by band-pass filtering the sensorimotor-related oscillatory activities within a trial-specific beta band. Signal envelopes of post-movement beta activities were detected using amplitude modulation (AM method to obtain post-movement beta event-related synchronization (PM-bERS. The maximum amplitude in the PM-bERS within the post-movement period was subtracted by the mean amplitude of the reference period to find the single-trial beta rebound (BR. Results The results showed single-trial BRs computed by the current method were significantly higher than those obtained from conventional average method (P Conclusions The EMD-based method is effective for artefact removal and extracting

  3. Correlation between electrical activity and ACTH/beta-endorphin secretion in mouse pituitary tumor cells



    The electrical and secretory activities of mouse pituitary tumor cells (AtT-20/D-16v), which contain and release the ACTH/beta-endorphin family of peptides, were studied by means of intracellular recordings and radioimmunoassays. Injection of depolarizing current pulses evoked action potentials in all cells and the majority (82%) displayed spontaneous action potential activity. Action potentials were found to be calcium-dependent. Barium increased membrane resistance, action potential amplitu...

  4. HIF hydroxylase pathways in cardiovascular physiology and medicine. (United States)

    Bishop, Tammie; Ratcliffe, Peter J


    Hypoxia inducible factors (HIFs) are α/β heterodimeric transcription factors that direct multiple cellular and systemic responses in response to changes in oxygen availability. The oxygen sensitive signal is generated by a series of iron and 2-oxoglutarate-dependent dioxygenases that catalyze post-translational hydroxylation of specific prolyl and asparaginyl residues in HIFα subunits and thereby promote their destruction and inactivation in the presence of oxygen. In hypoxia, these processes are suppressed allowing HIF to activate a massive transcriptional cascade. Elucidation of these pathways has opened several new fields of cardiovascular research. Here, we review the role of HIF hydroxylase pathways in cardiac development and in cardiovascular control. We also consider the current status, opportunities, and challenges of therapeutic modulation of HIF hydroxylases in the therapy of cardiovascular disease.

  5. Suppression of natural killer cell activity by rabbit antibody to human beta 2-microglobulin (beta 2m) is an Fc-mediated phenomenon and is not beta 2m specific. (United States)

    Jones, R A; Richards, S J; Patel, D; Scott, C S


    Natural killer (NK) cell cytotoxicity constitutes an important component of the host immune defence system. The NK effector cell has been relatively well defined in terms of immunophenotypic characteristics, but in contrast to the functional T-cell receptor molecule associated with major histocompatibility complex (MHC)-restricted cytotoxic activity, the NK cell receptor has not to date been defined. However, several studies have suggested that the beta 2-microglobulin (beta 2m) molecule is functionally associated with NK cell activity. Using various heterospecific and monoclonal antibodies, this study has shown that intact rabbit IgG antibody bound either directly or indirectly to peripheral mononuclear cell (PMNC) effector populations significantly reduced their lytic activity against K562 targets. Substitution of F(ab)2 fragments for rabbit IgG antibodies, or the use of monoclonal antibodies alone, failed to reduce peripheral blood mononuclear cell (PMNC) lytic activity. Addition of non-NK cell components labelled with rabbit anti-beta 2m to purified NK-enriched effector cell populations also suppressed K562 lysis. In contrast, pre-treatment of a NK-enriched PMNC fraction with rabbit anti-beta 2m enhanced target lysis. These results strongly suggest that antibody-induced suppression of PMNC NK activity is mediated via rabbit Fc attached to co-existing non-NK cells in the mononuclear fraction, and are inconsistent with the previously suggested functional association between NK activity and membrane beta 2m.

  6. Synergistic activation of vascular TRPC6 channel by receptor and mechanical stimulation via phospholipase C/diacylglycerol and phospholipase A2/¿-hydroxylase/20-HETE pathways

    DEFF Research Database (Denmark)

    Inoue, Ryuji; Jensen, Lars Jørn; Jian, Zhong;


    TRPC6 is a non-voltage-gated Ca(2+) entry/depolarization channel associated with vascular tone regulation and remodeling. Expressed TRPC6 channel responds to both neurohormonal and mechanical stimuli, the mechanism for which remains controversial. In this study, we examined the possible interacti......TRPC6 is a non-voltage-gated Ca(2+) entry/depolarization channel associated with vascular tone regulation and remodeling. Expressed TRPC6 channel responds to both neurohormonal and mechanical stimuli, the mechanism for which remains controversial. In this study, we examined the possible...... or Arg8 vasopressin was greatly enhanced by mechanical stimuli via 20-HETE production. Furthermore, myogenic response of pressurized mesenteric artery was significantly enhanced by weak receptor stimulation dependently on 20-HETE production. These results collectively suggest that simultaneous operation...... of receptor and mechanical stimulations may synergistically amplify transmembrane Ca(2+) mobilization through TRPC6 activation, thereby enhancing the vascular tone via phospholipase C/diacylglycerol and phospholipase A(2)/omega-hydroxylase/20-HETE pathways....

  7. Enzymatic hydrolysis of water-soluble wheat arabinoxylan. 1. Synergy between alpha-L-arabinofuranosidases, endo-1,4-beta-xylanases, and beta-xylosidase activities

    DEFF Research Database (Denmark)

    Sørensen, H.R.; Meyer, Anne Boye Strunge; Pedersen, S.


    , but a synergistic interaction in xylose release was found between Ultraflo L and Celluclast 1.5 L. On the basis of high-performance anion exchange chromatography (HPAEC) analysis of the hydrolysates after enzymatic reaction, we propose that the observed synergism between Celluclast 1.5 L and Ultraflo L...... is the result of positive interaction between alpha-L-arabinofuranosidase and endo-1,4-beta-xylanase activities present in Ultraflo L that released arabinose, xylobiose and xylotriose, and beta-xylosidase activities in Celluclast 1.5 L, capable of catalyzing the hydrolysis of xylobiose and xylotriose to xylose....

  8. Retroviral transfer of a human tyrosine hydroxylase cDNA in various cell lines: regulated release of dopamine in mouse anterior pituitary AtT-20 cells.


    Horellou, P; Guibert, B; Leviel, V; Mallet, J


    Little is known about the molecular events mediating neurotransmitter release, a crucial step in synaptic transmission. In this paper, the biosynthesis and release of L-beta-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine were analyzed in three heterologous cell lines after retroviral-mediated gene transfer of tyrosine hydroxylase (EC, the rate-limiting enzyme in catecholamine synthesis. A recombinant retrovirus encoding human tyrosine hydroxylase type I as well as neomycin-resist...

  9. Catalytic activity of nuclear PLC-beta(1) is required for its signalling function during C2C12 differentiation. (United States)

    Ramazzotti, Giulia; Faenza, Irene; Gaboardi, Gian Carlo; Piazzi, Manuela; Bavelloni, Alberto; Fiume, Roberta; Manzoli, Lucia; Martelli, Alberto M; Cocco, Lucio


    Here we report that PLC-beta(1) catalytic activity plays a role in the increase of cyclin D3 levels and induces the differentiation of C2C12 skeletal muscle cells. PLC-beta(1) mutational analysis revealed the importance of His(331) and His(378) for the catalysis. The expression of PLC-beta(1) and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that PLC-beta(1) activates cyclin D3 promoter during the differentiation of myoblasts to myotubes, indicating that PLC-beta(1) is a crucial regulator of the mouse cyclin D3 gene. We show that after insulin treatment cyclin D3 mRNA levels are lower in cells overexpressing the PLC-beta(1) catalytically inactive form in comparison to wild type cells. We describe a novel signalling pathway elicited by PLC-beta(1) that modulates AP-1 activity. Gel mobility shift assay and supershift performed with specific antibodies indicate that the c-jun binding site is located in a cyclin D3 promoter region specifically regulated by PLC-beta(1) and that c-Jun binding activity is significantly increased by insulin and PLC-beta(1) overexpression. Mutation of AP-1 site decreased the basal cyclin D3 promoter activity and eliminated its induction by insulin and PLC-beta(1). These results hint at the fact that PLC-beta(1) catalytic activity signals a c-jun/AP-1 target gene, i.e. cyclin D3, during myogenic differentiation.

  10. Effect of clavulanic acid on activity of beta-lactam antibiotics in Serratia marcescens isolates producing both a TEM beta-lactamase and a chromosomal cephalosporinase. (United States)

    Bush, K; Flamm, R K; Ohringer, S; Singer, S B; Summerill, R; Bonner, D P


    An isolate of Serratia marcescens that produced both an inducible chromosomal and a plasmid-mediated TEM-1 beta-lactamase was resistant to ampicillin and amoxicillin and also demonstrated decreased susceptibility to extended-spectrum beta-lactam antibiotics (ESBAs). Clavulanic acid did not lower the MICs of the ESBAs, but it decreased the MICs of the penicillins. The TEM-1-producing plasmid was transferred to a more susceptible S. marcescens strain that produced a well-characterized inducible chromosomal beta-lactamase. The MICs of the ESBAs remained at a low level for the transconjugant. Ampicillin and amoxicillin which were good substrates for the plasmid-mediated enzyme, were not well hydrolyzed by the chromosomal enzymes; the ESBAs were hydrolyzed slowly by all the enzymes. When each of the S. marcescens strains was grown with these beta-lactam antibiotics, at least modest increases in chromosomal beta-lactamase activity were observed. When organisms were grown in the presence of clavulanic acid and an ESBA, no enhanced induction was observed. The increases in the MICs of the ESBAs observed for the initial clinical isolate may have been due to a combination of low inducibility, slow hydrolysis, and differences in permeability between the S. marcescens isolates. When clavulanic acid and a penicillin were added to strains that produced both a plasmid-mediated TEM and a chromosomal beta-lactamase, much higher levels of chromosomal beta-lactamase activity were present than were observed in cultures induced by the penicillin alone. This was due to the higher levels of penicillin that were available for induction as a result of inhibition of the TEM enzyme by clavulanate. Images PMID:1803992

  11. Characterization of nimesulide/{beta}-cyclodextrin composite obtained by solid state activation

    Energy Technology Data Exchange (ETDEWEB)

    Magarotto, L. [Eurand Int. S.p.A., Unit of Trieste (Italy); Bertini, S.; Cosentino, C.; Torri, G. [Ist. Scientifico di Chimica e Biochimica ' G. Ronzoni' , Milano (Italy)


    The mechano-chemical activation was used to obtain a new composite of a anti-inflammatory drug (Nimesulide) and a polysaccharide carrier ({beta}-cyclodextrin). The original industrial process for activation, patented by Eurand Int. S.p.A., permits an improvement of the physico-chemical properties of the drug, which reaches a higher thermodynamic state (activation state). This work wants to demonstrate that the interaction between the drug and the carrier as a composite causes the thermodynamic activation of the Nimesulide as a new physical compound. (orig.)

  12. Absolute measurement of {beta} activities and application to the determination of neutronic densities; Mesure absolue d'activites {beta} et application a la determination des densites neutronique

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, R. [Commissariat a l' Energie Atomique, Lab. du Fort de Chatillon, Fontenay-aux-Roses (France). Centre d' Etudes Nucleaires


    M. Berthelot, to my entrance to the ''Commissariat a l 'Energie Atomique'', proposed me to study the absolute measurement of neutron densities. Very quickly the problem of the absolute activity of {beta} sources became the central object of this work. In a first part, we will develop the methods of absolute determination for {beta} activities. The use of a 4{pi} counter permits to get the absolute activity of all beta radioactive source, susceptible to be put as thin leaf and of period superior than some minutes. The method is independent of the spectra of the measured radioelement. we will describe in the second part some applications which use neutron densities measurement, neutron sources intensities and ratio of cross sections of capture of thermal neutrons. (M.B.) [French] M. Berthelot, a mon entree au ''Commissariat a l 'Energie Atomique'', m'a propose d'etudier la mesure absolue des densites neutroniques. Tres rapidement le probleme de l'activite absolue des sources beta est devenu l'objet central de ce travail. Dans une premiere partie, on abordera les methodes de determination absolue des activites beta. L'utilisation d'un compteur 4{pi} permet d 'obtenir l'activite absolue de toute source radioactive beta, susceptible d'etre mise sous forme de feuille mince et de periode superieure a quelques minutes. La methode est independante du spectre du radioelement mesure. On decrira dans la seconde partie quelques applications a des mesures de densites neutroniques, d'intensites de sources de neutrons et de rapport de sections efficaces de capture de neutrons thermiques. (M.B.)

  13. In vitro synergistic activities of tobramycin and selected beta-lactams against 75 gram-negative clinical isolates.


    Owens, R C; Banevicius, M A; Nicolau, D. P.; Nightingale, C H; Quintiliani, R


    The microdilution checkerboard technique was utilized to distinguish synergistic activity between tobramycin and four beta-lactams: piperacillin-tazobactam, ticarcillin-clavulanate, ceftazidime, and ceftriaxone. Beta-lactam-aminoglycoside combinations were tested against 75 clinical isolates of Pseudomonas aeruginosa, Acinetobacter baumanii, Citrobacterfreundii, Serratia marcescens, and Enterobacter cloacae. Despite in vitro susceptibilities, all isolates demonstrated either synergism or indi...

  14. An advanced method of activity determination of large area beta emitting sources

    International Nuclear Information System (INIS)

    The presented advanced method of activity determination of large area beta emitting sources is based on a version of efficiency tracing method using a test foil placed between the source and a conventional large area detector. It is shown that the total efficiency of the measuring system may depend on a dimensionless parameter derived from the difference in count rates caused by inserting the test foil while other disturbing effects are mostly reduced or compensated. - Highlights: • Efficiency tracing transmission method of beta activity determination. • Efficiency determined by means of a parameter independent of initial absorption conditions. • Parameter is derived from two counting results obtained with using a test foil. • Particularly useful for calibration and measurement of radionuclide standard sources

  15. LXR/RXR ligand activation enhances basolateral efflux of beta-sitosterol in CaCo-2 cells. (United States)

    Field, F Jeffrey; Born, Ella; Mathur, Satya N


    To examine whether intestinal ABCA1 was responsible for the differences observed between cholesterol and beta-sitosterol absorption, ABCA1-facilitated beta-sitosterol efflux was investigated in CaCo-2 cells following liver X receptor/retinoid X receptor (LXR/RXR) activation. Both the LXR agonist T0901317 and the natural RXR/LXR agonists 22-hydroxycholesterol and 9-cis retinoic acid enhanced the basolateral efflux of beta-sitosterol without altering apical efflux. LXR-mediated enhanced beta-sitosterol efflux occurred between 6 h and 12 h after activation, suggesting that transcription, protein synthesis, and trafficking was likely necessary prior to facilitating efflux. The transcription inhibitor actinomycin D prevented the increase in beta-sitosterol efflux by T0901317. Glybenclamide, an inhibitor of ABCA1 activity, and arachidonic acid, a fatty acid that interferes with LXR activation, also prevented beta-sitosterol efflux in response to the LXR ligand activation. Influx of beta-sitosterol mass did not alter the basolateral or apical efflux of the plant sterol, nor did it alter ABCA1, ABCG1, ABCG5, or ABCG8 gene expression or ABCA1 mass. Similar to results observed with intestinal ABCA1-facilitated cholesterol efflux, LXR/RXR ligand activation enhanced the basolateral efflux of beta-sitosterol without affecting apical efflux. The results suggest that ABCA1 does not differentiate between cholesterol and beta-sitosterol and thus is not responsible for the selectivity of sterol absorption by the intestine. ABCA1, however, may play a role in beta-sitosterol absorption.

  16. Synthesis and activity of 2-oxoamides containing long chain beta-amino acids. (United States)

    Constantinou-Kokotou, Violetta; Peristeraki, Anna; Kokotos, Christoforos G; Six, David A; Dennis, Edward A


    2-Oxoamides based on long chain beta-amino acids were synthesized. 1-Benzyl substituted long chain amines, needed for such synthesis, were synthesized starting from Boc-phenylalaninol. The oxidative conversion of a phenyl group to a carboxyl group was used as the key transformation synthetic step. The compounds synthesized were studied for their activity against GIVA PLA(2), and were proven to be weak inhibitors. PMID:15635664

  17. Magnetic Field and Activity of the Single Late-type Giant Beta Ceti


    Tsvetkova, S.; Auriere, M.; Konstantinova-Antova, R.; Wade, G. A.; Bogdanovski, R. G.; Petit, P.


    We present the behavior of the magnetic field and activity indicators of the single late-type giant Beta Ceti in the period June 19, 2010 - December 14, 2010. We used spectropolarimetric data obtained with two telescopes - the NARVAL spectropolarimeter at Telescope Bernard Lyot, Pic du Midi, France and the ESPaDOnS spectropolarimeter at CFHT, Hawaii. The data were processed using the method of Least Square Deconvolution which enables to derive the mean photospheric profiles of Stokes I and V ...

  18. Antibacterial Activity of Some Plant Extracts Against Extended- Spectrum Beta-Lactamase Producing Escherichia coli Isolates


    Saeidi, Saeide; Amini Boroujeni, Negar; Ahmadi, Hassan; Hassanshahian, Mehdi


    Background: The extended-spectrum beta-lactamase (ESBL) -producing Escherichia coli isolates make many serious infections, especially urinary tract infections. Objectives: The purpose of this study was to determine the antibacterial activities of some natural plant extracts against ESBL-producing E. coli isolates, which harbor the TEM gene in urine samples of the patients who have urinary tract infections. Materials and Methods: Evaluation has to be exactly determined for both methods of disk...

  19. Endothelin-1 activates phospholipase D and thymidine incorporation in fibroblasts overexpressing protein kinase C beta 1.


    Pai, J K; Dobek, E A; Bishop, W R


    Endothelins (ETs) are a family of extremely potent vasoconstrictor peptides. In addition, ET-1 acts as a potent mitogen and activates phospholipase C in smooth muscle cells and fibroblasts. We examined the effects of ET-1 on phosphatidylcholine (PC) metabolism and thymidine incorporation in control Rat-6 fibroblasts and in cells that overexpress protein kinase C beta 1 (PKC). PC pools were labeled with [3H]myristic acid, and formation of phosphatidylethanol (PEt), an unambiguous marker of pho...

  20. Modulation of transmission in rat sympathetic ganglia by activation of presynaptic alpha- and beta-adrenoceptors.


    Medgett, I.C.


    1 Conditions under which transmission in rat isolated superior cervical ganglia may be affected by activation of presynaptic alpha- and beta-adrenoceptors have been investigated by means of an extracellular recording method. 2 Clonidine caused a small hyperpolarization of the ganglia (mean EC50 approximately 2 nM) in unstimulated preparations; with continuous preganglionic stimulation at 0.2 Hz, clonidine markedly decreased the height of the compound action potential (mean EC50 approximately ...

  1. Induction and characterization of a cytochrome P-450-dependent camphor hydroxylase in tissue cultures of common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

    Funk, C.; Croteau, R. (Washington State Univ., Pullman (United States))


    (+)-Camphor, a major monoterpene of the essential oil of common sage (Salvia officinalis), is catabolized in senescent tissue, and the pathway for the breakdown of this bicyclic ketone has been previously elucidated in sage cell-suspension cultures. In the initial step of catabolism, camphor is oxidized to 6-exo-hydroxycamphor, and the corresponding NADPH- and O[sub 2]-dependent hydroxylase activity was demonstrated in microsomal preparations of sage cells. Several well-established inhibitors of cytochrome P-450-dependent reactions, including cytochrome c, clotrimazole, and CO, inhibited the hydroxylation of camphor, and CO-dependent inhibition was partially reversed by blue light. Upon treatment of sage suspension cultures with 30 mM MnCl[sub 2], camphor-6-hydroxylase activity was induced up to 7-fold. A polypeptide with estimated molecular mass of 58 kD from sage microsomal membranes exhibited antigenic cross-reactivity in western blot experiments with two heterologous polyclonal antibodies raised against cytochrome P-450 camphor-5-exo-hydroxylase from Pseudomonas putida and cytochrome P-450 limonene-6S-hydroxylase from spearmint (Mentha spicata). Dot blotting indicated that the concentration of this polypeptide increased with camphor hydroxylase activity in microsomes of Mn[sup 2+]-induced sage cells. These results suggest that camphor-6-exo-hydroxylase from sage is a microsomal cytochrome P-450 monooxygenase that may share common properties and epitopes with bacterial and other plant monoterpene hydroxylases. 44 refs., 6 figs., 2 tabs.

  2. Phenylalanine hydroxylase deficiency: diagnosis and management guideline. (United States)

    Vockley, Jerry; Andersson, Hans C; Antshel, Kevin M; Braverman, Nancy E; Burton, Barbara K; Frazier, Dianne M; Mitchell, John; Smith, Wendy E; Thompson, Barry H; Berry, Susan A


    Phenylalanine hydroxylase deficiency, traditionally known as phenylketonuria, results in the accumulation of phenylalanine in the blood of affected individuals and was the first inborn error of metabolism to be identified through population screening. Early identification and treatment prevent the most dramatic clinical sequelae of the disorder, but new neurodevelopmental and psychological problems have emerged in individuals treated from birth. The additional unanticipated recognition of a toxic effect of elevated maternal phenylalanine on fetal development has added to a general call in the field for treatment for life. Two major conferences sponsored by the National Institutes of Health held >10 years apart reviewed the state of knowledge in the field of phenylalanine hydroxylase deficiency, but there are no generally accepted recommendations for therapy. The purpose of this guideline is to review the strength of the medical literature relative to the treatment of phenylalanine hydroxylase deficiency and to develop recommendations for diagnosis and therapy of this disorder. Evidence review from the original National Institutes of Health consensus conference and a recent update by the Agency for Healthcare Research and Quality was used to address key questions in the diagnosis and treatment of phenylalanine hydroxylase deficiency by a working group established by the American College of Medical Genetics and Genomics. The group met by phone and in person over the course of a year to review these reports, develop recommendations, and identify key gaps in our knowledge of this disorder. Above all, treatment of phenylalanine hydroxylase deficiency must be life long, with a goal of maintaining blood phenylalanine in the range of 120-360 µmol/l. Treatment has predominantly been dietary manipulation, and use of low protein and phenylalanine medical foods is likely to remain a major component of therapy for the immediate future. Pharmacotherapy for phenylalanine

  3. Phenylalanine hydroxylase deficiency: diagnosis and management guideline. (United States)

    Vockley, Jerry; Andersson, Hans C; Antshel, Kevin M; Braverman, Nancy E; Burton, Barbara K; Frazier, Dianne M; Mitchell, John; Smith, Wendy E; Thompson, Barry H; Berry, Susan A


    Phenylalanine hydroxylase deficiency, traditionally known as phenylketonuria, results in the accumulation of phenylalanine in the blood of affected individuals and was the first inborn error of metabolism to be identified through population screening. Early identification and treatment prevent the most dramatic clinical sequelae of the disorder, but new neurodevelopmental and psychological problems have emerged in individuals treated from birth. The additional unanticipated recognition of a toxic effect of elevated maternal phenylalanine on fetal development has added to a general call in the field for treatment for life. Two major conferences sponsored by the National Institutes of Health held >10 years apart reviewed the state of knowledge in the field of phenylalanine hydroxylase deficiency, but there are no generally accepted recommendations for therapy. The purpose of this guideline is to review the strength of the medical literature relative to the treatment of phenylalanine hydroxylase deficiency and to develop recommendations for diagnosis and therapy of this disorder. Evidence review from the original National Institutes of Health consensus conference and a recent update by the Agency for Healthcare Research and Quality was used to address key questions in the diagnosis and treatment of phenylalanine hydroxylase deficiency by a working group established by the American College of Medical Genetics and Genomics. The group met by phone and in person over the course of a year to review these reports, develop recommendations, and identify key gaps in our knowledge of this disorder. Above all, treatment of phenylalanine hydroxylase deficiency must be life long, with a goal of maintaining blood phenylalanine in the range of 120-360 µmol/l. Treatment has predominantly been dietary manipulation, and use of low protein and phenylalanine medical foods is likely to remain a major component of therapy for the immediate future. Pharmacotherapy for phenylalanine

  4. Latent transforming growth factor beta1 activation in situ: quantitative and functional evidence after low-dose gamma-irradiation (United States)

    Ehrhart, E. J.; Segarini, P.; Tsang, M. L.; Carroll, A. G.; Barcellos-Hoff, M. H.; Chatterjee, A. (Principal Investigator)


    The biological activity of transforming growth factor beta1 (TGF-beta) is controlled by its secretion as a latent complex in which it is noncovalently associated with latency-associated peptide (LAP). Activation is the extracellular process in which TGF-beta is released from LAP, and is considered to be a primary regulatory control. We recently reported rapid and persistent changes in TGF-beta immunoreactivity in conjunction with extracellular matrix remodeling in gamma-irradiated mouse mammary gland. Our hypothesis is that these specific changes in immunoreactivity are indicative of latent TGF-beta activation. In the present study, we determined the radiation dose response and tested whether a functional relationship exists between radiation-induced TGF-beta and collagen type III remodeling. After radiation exposures as low as 0.1 Gy, we detected increased TGF-beta immunoreactivity in the mammary epithelium concomitant with decreased LAP immunostaining, which are events consistent with activation. Quantitative image analysis demonstrated a significant (P=0.0005) response at 0.1 Gy without an apparent threshold and a linear dose response to 5 Gy. However, in the adipose stroma, loss of LAP demonstrated a qualitative threshold at 0.5 Gy. Loss of LAP paralleled induction of collagen III immunoreactivity in this tissue compartment. We tested whether TGF-beta mediates collagen III expression by treating animals with TGF-beta panspecific monoclonal antibody, 1D11.16, administered i.p. shortly before irradiation. Radiation-induced collagen III staining in the adipose stroma was blocked in an antibody dose-dependent manner, which persisted through 7 days postirradiation. RNase protection assay revealed that radiation-induced elevation of total gland collagen III mRNA was also blocked by neutralizing antibody treatment. These data provide functional confirmation of the hypothesis that radiation exposure leads to latent TGF-beta activation, support our interpretation of the

  5. AGN-2979, an inhibitor of tryptophan hydroxylase activation, does not affect serotonin synthesis in Flinders Sensitive Line rats, a rat model of depression, but produces a significant effect in Flinders Resistant Line rats. (United States)

    Kanemaru, Kazuya; Nishi, Kyoko; Diksic, Mirko


    The neurotransmitter, serotonin, is involved in several brain functions, including both normal, physiological functions, and pathophysiological functions. Alterations in any of the normal parameters of serotonergic neurotransmission can produce several different psychiatric disorders, including major depression. In many instances, brain neurochemical variables are not able to be studied properly in humans, thus making the use of good animal models extremely valuable. One of these animal models is the Flinders Sensitive Line (FSL) of rats, which has face, predictive and constructive validities in relation to human depression. The objective of this study was to quantify the effect of the tryptophan hydroxylase (TPH) activation inhibitor, AGN-2979, on the FSL rats (rats with depression-like behaviour), and compare it to the effect on the Flinders Resistant Line (FRL) of rats used as the control rats. The effect was evaluated by measuring changes in regional serotonin synthesis in the vehicle treated rats (FSL-VEH and FRL-VEH) relative to those measured in the AGN-2979 treated rats (FSL-AGN and FRL-AGN). Regional serotonin synthesis was measured autoradiographically in more than 30 brain regions. The measurements were performed using alpha-[(14)C]methyl-l-tryptophan as the tracer. The results indicate that AGN-2979 did not produce a significant reduction of TPH activity in the AGN-2979 group relative to the vehicle group (a reduction would have been observed if there had been an activation of TPH by the experimental setup) in the FSL rats. On the other hand, there was a highly significant reduction of synthesis in the FRL rats treated by AGN-2979, relative to the vehicle group. Together, the results demonstrate that in the FSL rats, AGN-2979 does not affect serotonin synthesis. This suggests that there was no activation of TPH in the FSL rats during the experimental procedure, but such activation did occur in the FRL rats. Because of this finding, it could be

  6. Liquid argon as active shielding and coolant for bare germanium detectors. A novel background suppression method for the GERDA 0{nu}{beta}{beta} experiment

    Energy Technology Data Exchange (ETDEWEB)

    Peiffer, J.P.


    Two of the most important open questions in particle physics are whether neutrinos are their own anti-particles (Majorana particles) as required by most extensions of the StandardModel and the absolute values of the neutrino masses. The neutrinoless double beta (0{nu}{beta}{beta}) decay, which can be investigated using {sup 76}Ge (a double beta isotope), is the most sensitive probe for these properties. There is a claim for an evidence for the 0{nu}{beta}{beta} decay in the Heidelberg-Moscow (HdM) {sup 76}Ge experiment by a part of the HdM collaboration. The new {sup 76}Ge experiment Gerda aims to check this claim within one year with 15 kg.y of statistics in Phase I at a background level of {<=}10{sup -2} events/(kg.keV.y) and to go to higher sensitivity with 100 kg.y of statistics in Phase II at a background level of {<=}10{sup -3} events/(kg.keV.y). In Gerda bare germanium semiconductor detectors (enriched in {sup 76}Ge) will be operated in liquid argon (LAr). LAr serves as cryogenic coolant and as high purity shielding against external background. To reach the background level for Phase II, new methods are required to suppress the cosmogenic background of the diodes. The background from cosmogenically produced {sup 60}Co is expected to be {proportional_to}2.5.10{sup -3} events/(kg.keV.y). LAr scintillates in UV ({lambda}=128 nm) and a novel concept is to use this scintillation light as anti-coincidence signal for background suppression. In this work the efficiency of such a LAr scintillation veto was investigated for the first time. In a setup with 19 kg active LAr mass a suppression of a factor 3 has been achieved for {sup 60}Co and a factor 17 for {sup 232}Th around Q{sub {beta}}{sub {beta}} = 2039 keV. This suppression will further increase for a one ton active volume (factor O(100) for {sup 232}Th and {sup 60}Co). LAr scintillation can also be used as a powerful tool for background diagnostics. For this purpose a new, very stable and robust wavelength

  7. 2-(2-Pyridyl) Benzimidazole Analogs and their beta-Glucuronidase Inhibitory Activity

    International Nuclear Information System (INIS)

    Synthesis of 2-(2-Pyridyl) benzimidazole analogs 1-11 have been carried out and evaluated for in vitro beta-glucuronidase inhibitory potential. The compounds 4 (IC/sub 50/ = 4.06 ± 0.34 meuM), 5 (IC/sub 50/ = 09.63 ± 0.81 meuM), 1 (IC/sub 50/ = 19.66 ± 0.44 meuM), 7 (IC/sub 50/ = 24.75 ± 0.25 meuM), 6 (IC/sub 50/ = 26.30 ± 1.37 meuM), and 3 (IC/sub 50/ = 32.11 ± 0.89 meuM), showed beta-glucuronidase inhibitory activity superior to the standard D-saccharic acid 1,4-lactone, with (IC/sub 50/ = 48.4 ± 1.25 meuM). Based on structure-activity relationship, we discover a new class of potent beta-glucuronidase inhibitors. (author)

  8. Reconstitution of Bacillus cereus 5/B/6 metallo-[beta]-lactamase activity with copper

    Energy Technology Data Exchange (ETDEWEB)

    Hilliard, N.P.; Shaw, R.W. (Texas Tech Univ., Lubbock (United States))


    Pathogenic bacteria become resistant to [beta]-lactam antibiotics such as penicillins and cephalosporins through the production of enzymes called [beta]-lactamases. The authors have successfully reconstituted the enzymatic activity of the metallo-[beta]-lactamase of Bacillus cereus 5/B/6 purified from an E. coli expression vector system by the addition of Cu(II) to the apoenzyme. This is the first report that copper supports catalytic activity in this enzyme. Maximal activity of the copper-reconstituted enzyme was achieved by a careful addition of a stoichiometric amount of CuSO[sub 4] to 200 [mu]M apoenzyme. Using either benzylpenicillin or cephalosporin C as the substrate, reconstitution of the activity by addition of copper to the apoenzyme resulted in the recovery of approximately 35% of the control activity of the native Zn(II) enzyme. In agreement with previous reports, in the presence of excess Cu(II), the preparation did not possess measurable catalytic activity. Electronic spectra of the copper-reconstituted enzyme displayed adsorption maxima at 394, 698 and 1,022 nm with extinction coefficients of 2,656, 55 and < 3 M[sup [minus]1]cm[sup [minus]1] respectively. Circular dichorism spectra in the ultraviolent region (UVCD) of the copper-reconstituted enzyme were identical with those of the native Zn(II) enzyme. Addition of excess cephalosporin C to the copper-reconstituted enzyme caused a decrease of about 50% of the absorbance of the 394 nm band and the formation of a new feature at 350 nm.

  9. The inhibition activity of selected beta-carboline alkaloids on enzymes of acetylcholinesterase and butyrylcholinesterase. (United States)

    Krsková, Zuzana; Martin, Jan; Dusek, Jaroslav


    This thesis deals with testing of inhibition activity beta-carboline alkaloids on activity of enzymes acetylcholinesterase (ACHE) and butyrylcholinesterase (BUCHE) using test "Fast Blue B salt" at TLC desk and Ellman's test using spectrophotometer. It was also investigated how dimethylsulfoxide used as a solvent in combination with water affects activity of enzymes and alkaloids. Results show harmine in form of base and salt in water and in mixture of DMSO and water has the hightest inhibition activity on ACHE using eserine as reference substance. Harmalol in form of salt in water and harmine in form of base and salt in mixture of DMSO and water has the hightest activity on BUCHE. It was find out that DMSO considerably affects activity of enzymes and alkaloids. PMID:21838142

  10. 24-Hydroxylase in Cancer: Impact on Vitamin D-based Anticancer Therapeutics


    Luo, Wei; Hershberger, Pamela A.; Trump, Donald L.; Johnson, Candace S.


    The active vitamin D hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in regulating calcium homeostasis and bone mineralization. 1,25(OH)2D3 also modulates cellular proliferation and differentiation in a variety of cell types. 24-hydroxylase, encoded by the CYP24A1 gene, is the key enzyme which converts 1,25(OH)2D3 to less active calcitroic acid. Nearly all cell types express 24-hydroxylase, the highest activity being observed in the kidney. There is increasing evidence linki...

  11. Chaperone-like activities of different molecular forms of beta-casein. Importance of polarity of N-terminal hydrophilic domain. (United States)

    Yousefi, Reza; Shchutskaya, Yulia Y; Zimny, Jaroslaw; Gaudin, Jean-Charles; Moosavi-Movahedi, Ali A; Muronetz, Vladimir I; Zuev, Yuriy F; Chobert, Jean-Marc; Haertlé, Thomas


    As a member of intrinsically unstructured protein family, beta-casein (beta-CN) contains relatively high amount of prolyl residues, adopts noncompact and flexible structure and exhibits chaperone-like activity in vitro. Like many chaperones, native beta-CN does not contain cysteinyl residues and exhibits strong tendencies for self-association. The chaperone-like activities of three recombinant beta-CNs wild type (WT) beta-CN, C4 beta-CN (with cysteinyl residue in position 4) and C208 beta-CN (with cysteinyl residue in position 208), expressed and purified from E. coli, which, consequently, lack the phosphorylated residues, were examined and compared with that of native beta-CN using insulin and alcohol dehydrogenase as target/substrate proteins. The dimers (beta-CND) of C4-beta-CN and C208 beta-CN were also studied and their chaperone-like activities were compared with those of their monomeric forms. Lacking phosphorylation, WT beta-CN, C208 beta-CN, C4 beta-CN and C4 beta-CND exhibited significantly lower chaperone-like activities than native beta-CN. Dimerization of C208 beta-CN with two distal hydrophilic domains considerably improved its chaperone-like activity in comparison with its monomeric form. The obtained results demonstrate the significant role played by the polar contributions of phosphorylated residues and N-terminal hydrophilic domain as important functional elements in enhancing the chaperone-like activity of native beta-CN. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 623-632, 2009.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at PMID:19322774

  12. Beta-Lactam-beta-lactamase-inhibitor combinations are active in experimental endocarditis caused by beta-lactamase-producing oxacillin-resistant staphylococci.


    Hirano, L; Bayer, A S


    Optimal therapeutic strategies for serious infections caused by borderline and heterotypic oxacillin-resistant Staphylococcus aureus (BORSA and ORSA) strains have not been fully characterized. Recent evidence suggests that the dominant penicillin-binding protein of ORSA strains (PBP 2a) shows good affinity for ampicillin and that these strains commonly produce beta-lactamase. Therefore, we compared the in vivo efficacy of the combination of ampicillin plus sulbactam with that of vancomycin ag...

  13. Effect of porin loss on the activity of tigecycline against Klebsiella pneumoniae producing extended-spectrum beta-lactamases or plasmid-mediated AmpC-type beta-lactamases. (United States)

    Conejo, M Carmen; Hernández, J Ramón; Pascual, Alvaro


    Tigecycline showed excellent in vitro activity against 50 clinical isolates of Klebsiella pneumoniae producing extended-spectrum beta-lactamases, plasmid-mediated AmpC-type beta-lactamases, or both. This activity was not affected by porin loss. Porin loss, however, did affect the activity of imipenem against strains that expressed both types of enzymes. PMID:18339509

  14. A liver X receptor (LXR)-{beta} alternative splicing variant (LXRBSV) acts as an RNA co-activator of LXR-{beta}

    Energy Technology Data Exchange (ETDEWEB)

    Hashimoto, Koshi, E-mail: [Department of Medicine and Molecular Science, Graduate School of Medicine, Gunma University, Maebashi, Gunma 371-8511 (Japan); Ishida, Emi; Matsumoto, Shunichi; Shibusawa, Nobuyuki; Okada, Shuichi [Department of Medicine and Molecular Science, Graduate School of Medicine, Gunma University, Maebashi, Gunma 371-8511 (Japan); Monden, Tsuyoshi [Department of Endocrinology and Metabolism, Dokkyo Medical College, Mibu, Tochigi (Japan); Satoh, Tetsurou; Yamada, Masanobu; Mori, Masatomo [Department of Medicine and Molecular Science, Graduate School of Medicine, Gunma University, Maebashi, Gunma 371-8511 (Japan)


    We report the isolation and functional characterization of a novel transcriptional co-activator, termed LXRBSV. LXRBSV is an alternative splicing variant of liver X receptor (LXR)-{beta} LXRBSV has an intronic sequence between exons 2 and 3 in the mouse LXR-{beta} gene. The LXRBSV gene is expressed in various tissues including the liver and brain. We sub-cloned LXRBSV into pSG5, a mammalian expression vector, and LXRBSV in pSG5 augmented human Sterol Response Element Binding Protein (SREBP)-1c promoter activity in HepG2 cells in a ligand (TO901317) dependent manner. The transactivation mediated by LXRBSV is selective for LXR-{beta}. The LXRBSV protein was deduced to be 64 amino acids in length; however, a GAL4-LXRBSV fusion protein was not able to induce transactivation. Serial deletion constructs of LXRBSV demonstrated that the intronic sequence inserted in LXRBSV is required for its transactivation activity. An ATG mutant of LXRBSV was able to induce transactivation as wild type. Furthermore, LXRBSV functions in the presence of cycloheximide. Taken together, we have concluded that LXRBSV acts as an RNA transcript not as a protein. In the current study, we have demonstrated for the first time that an alternative splicing variant of a nuclear receptor acts as an RNA co-activator.

  15. Detection and characterization of p-coumaric acid hydroxylase in mung bean, Vigna mungo, seedlings. (United States)

    Kojima, M; Takeuchi, W


    A new p-coumaric acid (4-hydroxycinnamic acid) hydroxylase was detected in mung bean seedlings treated with tentoxin, a fungal toxin, in which polyphenol oxidase that hydroxylates a wide variety of monophenols in vitro was completely eliminated. The enzyme required molecular oxygen and showed a pH optimum of 5.0. The enzyme acted only on p-coumaric acid (Km, 3.0 X 10(-5) M), while its specificity for the electron donor was rather broad. The Km value for NADPH (1.5 X 10(-4) M) was much lower than that for L-ascorbic acid (1.0 X 10(-2) M), although the Vmax value was almost the same with both electron donors. The enzyme was potently inhibited by beta-mercaptoethanol (Ki, 3.5 X 10(-6) M) and diethyldithiocarbamate (Ki, 2.3 X 10(-4) M), but was insensitive to p-chloromercuribenzoate. The enzyme was localized in the cell organelles which sedimented between mitochondria and endplasmic reticulum on sucrose density gradient centrifugation. The enzyme activity in the seedling was changed in response to induction by light in a manner suggesting its involvement in biosynthesis of phenolic compounds in mung bean seedlings.

  16. Synthesis of 16 alpha-3H androgen and estrogen substrates for 16 alpha-hydroxylase. (United States)

    Cantineau, R; Kremers, P; De Graeve, J; Cornelis, A; Laszlo, P; Gielen, J E; Lambotte, R


    The synthesis of 16 alpha-3H androgens and estrogens is described. 1-(3H)-Acetic acid in the presence of zinc dust reacts with 16 alpha-bromo-17-ketosteroids to produce 16 alpha-3H-17-ketosteroids. This chemical reaction was used to prepare 16 alpha-3H-dehydroepiandrosterone (I) and 16 alpha-3H-estrone acetate (XI) from 16 alpha-bromo-dehydroepiandrosterone (X) and from 16 alpha-bromo-estrone acetate (XII), respectively. Using appropriate microbiological techniques, it was possible to convert these radiolabelled substrates into 16 alpha-3H-androstenedione (II) and 16 alpha-3H-estradiol-17 beta (VII). 16 alpha-3H-Estrone (VI) was obtained by the chemical hydrolysis of 16 alpha-3H-estrone acetate. The label distribution as determined by microbiological 16 alpha-hydroxylations indicated a specific labelling of 77% for androgens and 65% for estrogens in the 16 alpha position. These substrates can be used for measuring the 16 alpha hydroxylase activity, an important step in the biosynthesis of estriol (VIII) and estetrol (IX). PMID:7013160

  17. L-653,328: an ocular hypotensive agent with modest beta receptor blocking activity

    Energy Technology Data Exchange (ETDEWEB)

    Sugrue, M.F.; Gautheron, P.; Grove, J.; Mallorga, P.; Viader, M.P.; Baldwin, J.J.; Ponticello, G.S.; Varga, S.L.


    L-653,328 is the acetate ester of L-652,698 ((S)-3-tert-butylamino-1-(4-(2(hydroxy)ethyl)phenoxy)2-propanol). The penetration of L-652,698 into the albino rabbit eye was enhanced when the compound was instilled as its prodrug acetate ester. The instillation (one drop of 50 microliter) of 0.01, 0.05 and 0.1% solutions of L-653,328 significantly decreased in a dose-dependent manner the elevated intraocular pressure (IOP) of alpha-chymotrypsinized rabbits by 3.2, 4.7 and 6.1 mm Hg, respectively. A 0.01% solution of L-652,698 failed to significantly lower IOP, whereas this dose of timolol (3.8 mm Hg) and betaxolol (3.3 mm Hg) was effective. L-652,698 was active at 0.05% and 0.1%. Extraocular beta-adrenoceptor blockade was quantified in ganglion-blocked, conscious rabbits by determining effects on heart rate and blood pressure changes to i.v. isoproterenol (0.5 microgram/kg). Doses of timolol blocking isoproterenol-induced hypotension and tachycardia by 50% were 0.0065% and 0.03%, respectively. The corresponding doses for betaxolol were greater than 3% (43% inhibition) and 0.3%. Heart rate and blood pressure changes to isoproterenol were blocked by 18 and 36%, respectively, after the instillation of a 3% solution of L-653,328. The reduced propensity of L-653,328 for extraocular beta-adrenoceptor blockade stems from the modest affinity of L-652,698, its active moiety, for beta-adrenoceptors. The Ki values of L-652,698 for displacement of 125I-iodocyanopindolol binding to beta 1-(left ventricle) and beta 2-binding sites (iris + ciliary body) in the rabbit were 5.7 microM and 7.3 microM, respectively. In marked contrast, the corresponding values for timolol were 12 nM and 1.8 nM.

  18. Impediments to enhancement of CPT-11 anticancer activity by E. coli directed beta-glucuronidase therapy.

    Directory of Open Access Journals (Sweden)

    Yuan-Ting Hsieh

    Full Text Available CPT-11 is a camptothecin analog used for the clinical treatment of colorectal adenocarcinoma. CPT-11 is converted into the therapeutic anti-cancer agent SN-38 by liver enzymes and can be further metabolized to a non-toxic glucuronide SN-38G, resulting in low SN-38 but high SN-38G concentrations in the circulation. We previously demonstrated that adenoviral expression of membrane-anchored beta-glucuronidase could promote conversion of SN-38G to SN-38 in tumors and increase the anticancer activity of CPT-11. Here, we identified impediments to effective tumor therapy with E. coli that were engineered to constitutively express highly active E. coli beta-glucuronidase intracellularly to enhance the anticancer activity of CPT-11. The engineered bacteria, E. coli (lux/βG, could hydrolyze SN-38G to SN-38, increased the sensitivity of cultured tumor cells to SN-38G by about 100 fold and selectively accumulated in tumors. However, E. coli (lux/βG did not more effectively increase CPT-11 anticancer activity in human tumor xenografts as compared to non-engineered E. coli. SN-38G conversion to SN-38 by E. coli (lux/βG appeared to be limited by slow uptake into bacteria as well as by segregation of E. coli in necrotic regions of tumors that may be relatively inaccessible to systemically-administered drug molecules. Studies using a fluorescent glucuronide probe showed that significantly greater glucuronide hydrolysis could be achieved in mice pretreated with E. coli (lux/βG by direct intratumoral injection of the glucuronide probe or by intratumoral lysis of bacteria to release intracellular beta-glucuronidase. Our study suggests that the distribution of beta-glucuronidase, and possibly other therapeutic proteins, in the tumor microenvironment might be an important barrier for effective bacterial-based tumor therapy. Expression of secreted therapeutic proteins or induction of therapeutic protein release from bacteria might therefore be a promising strategy

  19. Cinnamic acid 4-hydroxylase of sorghum [Sorghum biocolor (L.) Moench] gene SbC4H1 restricts lignin synthesis in Arabidopsis (United States)

    Cinnamic acid 4-hydroxylase (C4H) is the first hydroxylase enzyme of the phenylpropanoid pathway, and its content and activity affects the lignin synthesis. In this study, we isolated a C4H gene SbC4H1 from the suppression subtractive hybridization library of brown midrib (bmr) mutants of Sorghum b...

  20. Using total beta-activity measurements in milk to derive thyroid doses from Chernobyl fallout

    International Nuclear Information System (INIS)

    Following the Chernobyl accident, more than 200 childhood thyroid cancer cases have been observed in Brest Oblast of Belarus in territories slightly contaminated with 137Cs, but with suspected relatively high 131I fallout. The most helpful measurements available that can be used to estimate thyroid doses for the population of Brest Oblast are the total beta-activity measurements in cow's milk performed using DP-100 device within a few weeks after the accident. The 131I concentrations in milk were derived from the total beta-activity measurements on the basis of (1) a radioecological model used to estimate the variation with time of the radionuclide composition in milk and (2) the determination of the calibration factors of the DP-100 device for the most important radionuclides present in milk. As a result, 131I concentrations in milk were reconstructed for territories with different levels of 137Cs deposition. A non-linear dependence of the 131I concentration in milk on the 137Cs deposition density was obtained; it was used to estimate the thyroid doses from the consumption of 131I-contaminated cow's milk by the population of Brest Oblast. The average individual thyroid doses have been estimated to be 0.15, 0.18, 0.12, 0.06, 0.04 and 0.03 Gy for newborn, children aged 1, 5, 10 and 15 y and adults, respectively. The collective thyroid dose for the entire population of Brest Oblast is estimated to be 64,500 man Gy, the contribution from the adult population being about one half of the total. The methodology that is described could be applied in the framework of epidemiological studies of the relationship between radiation exposure to the thyroid gland and thyroid cancer in areas where numerous total beta-activity measurements in cow's milk were performed within a few weeks after the accident. (authors)

  1. Purification and Characterization of Tryptophan Hydroxylase

    DEFF Research Database (Denmark)

    Haahr, Lærke Tvedebrink

    This thesis deals with the purification and characterization of the iron-containing enzyme tryptophan hydroxylase (TPH). TPH exists in two isoforms, called TPH1 and TPH2. Each isoform consists of threestructural distinct domains: the regulatory, the catalytic and the tetramerization domain. TPH c...

  2. Expression and purification of the metal-containing monooxygenases tryptophan hydroxylase and dopamine β-hydroxylase

    DEFF Research Database (Denmark)

    Karlsen, Pernille Efferbach

    to abnormal levels of the neurotransmitters serotonin, dopamine and norepinephrine and the regulation of tryptophan hydroxylase and dopamine β-hydroxylase. These include depression, anxiety disorders, obsessive compulsive disorder (OCD), schizophrenia, Parkinson's disease and attention deficit...... spectrometry analysis. Tetrameric DβH was deglycosylated and separated from the deglycosylation enzyme in another purification step. 0.2 mg/l culture deglycosylated DβH was obtained after this step and it was used for screening of crystallization conditions....

  3. Highly Potent, Water Soluble Benzimidazole Antagonist for Activated (alpha)4(beta)1 Integrin

    Energy Technology Data Exchange (ETDEWEB)

    Carpenter, R D; Andrei, M; Lau, E Y; Lightstone, F C; Liu, R; Lam, K S; Kurth, M J


    The cell surface receptor {alpha}{sub 4}{beta}{sub 1} integrin, activated constitutively in lymphoma, can be targeted with the bisaryl urea peptidomimetic antagonist 1 (LLP2A). However, concerns on its preliminary pharmacokinetic (PK) profile provided an impetus to change the pharmacophore from a bisaryl urea to a 2-arylaminobenzimidazole moiety resulting in improved solubility while maintaining picomolar potency [5 (KLCA4); IC{sub 50} = 305 pM]. With exceptional solubility, this finding has potential for improving PK to help diagnose and treat lymphomas.

  4. Are striatal tyrosine hydroxylase interneurons dopaminergic? (United States)

    Xenias, Harry S; Ibáñez-Sandoval, Osvaldo; Koós, Tibor; Tepper, James M


    Striatal GABAergic interneurons that express the gene for tyrosine hydroxylase (TH) have been identified previously by several methods. Although generally assumed to be dopaminergic, possibly serving as a compensatory source of dopamine (DA) in Parkinson's disease, this assumption has never been tested directly. In TH-Cre mice whose nigrostriatal pathway had been eliminated unilaterally with 6-hydroxydopamine, we injected a Cre-dependent virus coding for channelrhodopsin-2 and enhanced yellow fluorescent protein unilaterally into the unlesioned midbrain or bilaterally into the striatum. Fast-scan cyclic voltammetry in striatal slices revealed that both optical and electrical stimulation readily elicited DA release in control striata but not from contralateral striata when nigrostriatal neurons were transduced. In contrast, neither optical nor electrical stimulation could elicit striatal DA release in either the control or lesioned striata when the virus was injected directly into the striatum transducing only striatal TH interneurons. This demonstrates that striatal TH interneurons do not release DA. Fluorescence immunocytochemistry in enhanced green fluorescent protein (EGFP)-TH mice revealed colocalization of DA, l-amino acid decarboxylase, the DA transporter, and vesicular monoamine transporter-2 with EGFP in midbrain dopaminergic neurons but not in any of the striatal EGFP-TH interneurons. Optogenetic activation of striatal EGFP-TH interneurons produced strong GABAergic inhibition in all spiny neurons tested. These results indicate that striatal TH interneurons are not dopaminergic but rather are a type of GABAergic interneuron that expresses TH but none of the other enzymes or transporters necessary to operate as dopaminergic neurons and exert widespread GABAergic inhibition onto direct and indirect spiny neurons.

  5. Magnetic Field and Activity of the Single Late-type Giant Beta Ceti

    CERN Document Server

    Tsvetkova, S; Konstantinova-Antova, R; Wade, G A; Bogdanovski, R G; Petit, P


    We present the behavior of the magnetic field and activity indicators of the single late-type giant Beta Ceti in the period June 19, 2010 - December 14, 2010. We used spectropolarimetric data obtained with two telescopes - the NARVAL spectropolarimeter at Telescope Bernard Lyot, Pic du Midi, France and the ESPaDOnS spectropolarimeter at CFHT, Hawaii. The data were processed using the method of Least Square Deconvolution which enables to derive the mean photospheric profiles of Stokes I and V parameters. We measured the surface-averaged longitudinal magnetic field Bl, which varies in the interval 0.1 - 8.2 G, the line activity indicators CaII K, H_alpha, CaII IR (854.2 nm) and radial velocity. By analyzing the Bl variations, was identified a possible rotational period P = 118 days. A single, large magnetic spot which dominates the field topology is a possible explanation for the Bl and activity indicator variations of Beta Ceti.

  6. Beta-Carotene, Vitamin E, MDA, Glutathione Reductase and Arylesterase Activity Levels in Patients with Active Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    N Aryaeian


    Full Text Available "nBackground: Many studies have investigated the possible role of reactive oxygen species in the etiology and patho­gene­sis of Rheumatoid Arthritis (RA. The aim of this study was to investigate the activities of some antioxidants in RA patients."nMethods: In this case-control study, 59 RA patients and 60 healthy sex and age-matched controls were selected. Vitamin E and Beta-carotene were determined using HPLC. Erythrocytes glutathione reductase (GR activity was meas­ured spec­trophotometrically, and malondialdehyde (MDA was determined by colorimetric method. Aryles­terase activity (AEA was measured by Phenylacetate. The clinical data were determined by a rheumatologist, medical history and filling the questionnaire by interview. Statistical analyses were carried out using the SPSS software."nResults: In patients with RA, serum MDA level was significantly higher and plasma concentration of vitamin E, Beta-carotene and GR activity, were significantly lower than healthy control (P<0.001. AEA activity differences between two groups were non-significant."nConclusions: Oxidative stress may play an important role in the inflammation and pathogenesis of RA.  

  7. The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase. (United States)

    Yu, Zhong; Genest, Paul-André; ter Riet, Bas; Sweeney, Kate; DiPaolo, Courtney; Kieft, Rudo; Christodoulou, Evangelos; Perrakis, Anastassis; Simmons, Jana M; Hausinger, Robert P; van Luenen, Henri G A M; Rigden, Daniel J; Sabatini, Robert; Borst, Piet


    Trypanosomatids contain an unusual DNA base J (beta-d-glucosylhydroxymethyluracil), which replaces a fraction of thymine in telomeric and other DNA repeats. To determine the function of base J, we have searched for enzymes that catalyze J biosynthesis. We present evidence that a protein that binds to J in DNA, the J-binding protein 1 (JBP1), may also catalyze the first step in J biosynthesis, the conversion of thymine in DNA into hydroxymethyluracil. We show that JBP1 belongs to the family of Fe(2+) and 2-oxoglutarate-dependent dioxygenases and that replacement of conserved residues putatively involved in Fe(2+) and 2-oxoglutarate-binding inactivates the ability of JBP1 to contribute to J synthesis without affecting its ability to bind to J-DNA. We propose that JBP1 is a thymidine hydroxylase responsible for the local amplification of J inserted by JBP2, another putative thymidine hydroxylase. PMID:17389644

  8. The biological activities of (1,3)-(1,6)-{beta}-d-glucan and porous electrospun PLGA membranes containing {beta}-glucan in human dermal fibroblasts and adipose tissue-derived stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Woo, Yeon I; Park, Bong Joo; Kim, Hye-Lee; Lee, Mi Hee; Kim, Jungsung; Park, Jong-Chul [Department of Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Yang, Young-Il [Department of Pathology, School of Medicine, Paik Institute for Clinical Research, Inje University, 633-165 Gae-dong, Busan-jin-gu, Busan 614-735 (Korea, Republic of); Kim, Jung Koo [Department of Biomedical Engineering, College of Biomedical Science and Engineering, Inje University, Kimhae 621-749 (Korea, Republic of); Tsubaki, Kazufumi [R and D division, Asahi Denka Co. Ltd, 7-2-35 Higashi-ogu, Arakawa-ku, Tokyo 116-8554 (Japan); Han, Dong-Wook, E-mail: parkjc@yuhs.a [Department of Nanomedical Engineering, College of Nanoscience and Nanotechnology, Pusan National University, geumjeong-gu, Busan 609-735 (Korea, Republic of)


    In this study, we investigated the possible roles of (1,3)-(1,6)-{beta}-d-glucan ({beta}-glucan) and porous electrospun poly-lactide-co-glycolide (PLGA) membranes containing {beta}-glucan for skin wound healing, especially their effect on adult human dermal fibroblast (aHDF) and adipose tissue-derived stem cell (ADSC) activation, proliferation, migration, collagen gel contraction and biological safety tests of the prepared membrane. This study demonstrated that {beta}-glucan and porous PLGA membranes containing {beta}-glucan have enhanced the cellular responses, proliferation and migration, of aHDFs and ADSCs and the result of a collagen gel contraction assay also revealed that collagen gels contract strongly after 4 h post-gelation incubation with {beta}-glucan. Furthermore, we confirmed that porous PLGA membranes containing {beta}-glucan are biologically safe for wound healing study. These results indicate that the porous PLGA membranes containing {beta}-glucan interacted favorably with the membrane and the topical administration of {beta}-glucan was useful in promoting wound healing. Therefore, our study suggests that {beta}-glucan and porous PLGA membranes containing {beta}-glucan may be useful as a material for enhancing wound healing.

  9. Simultaneous stimulation of GABA and beta adrenergic receptors stabilizes isotypes of activated adenylyl cyclase heterocomplex

    Directory of Open Access Journals (Sweden)

    Robichon Alain


    Full Text Available Abstract Background We investigated how the synthesis of cAMP, stimulated by isoproterenol acting through β-adrenoreceptors and Gs, is strongly amplified by simultaneous incubation with baclofen. Baclofen is an agonist of δ-aminobutyric acid type B receptors [GABAB], known to inhibit adenylyl cyclase via Gi. Because these agents have opposite effects on cAMP levels, the unexpected increase in cAMP synthesis when they are applied simultaneously has been intensively investigated. From previous reports, it appears that cyclase type II contributes most significantly to this phenomenon. Results We found that simultaneous application of isoproterenol and baclofen specifically influences the association/dissociation of molecules involved in the induction and termination of cyclase activity. Beta/gamma from [GABA]B receptor-coupled Gi has a higher affinity for adenylyl cyclase isoform(s when these isoforms are co-associated with Gs. Our data also suggest that, when beta/gamma and Gαs are associated with adenylyl cyclase isoform(s, beta/gamma from [GABA]B receptor-coupled Gi retards the GTPase activity of Gαs from adrenergic receptor. These reciprocal regulations of subunits of the adenylyl cyclase complex might be responsible for the drastic increase of cAMP synthesis in response to the simultaneous signals. Conclusions Simultaneous signals arriving at a particular synapse converge on molecular detectors of coincidence and trigger specific biochemical events. We hypothesize that this phenomenon comes from the complex molecular architectures involved, including scaffolding proteins that make reciprocal interactions between associated molecules possible. The biochemistry of simultaneous signaling is addressed as a key to synaptic function.

  10. Early peroxisome proliferator-activated receptor gamma regulated genes involved in expansion of pancreatic beta cell mass

    Directory of Open Access Journals (Sweden)

    Vivas Yurena


    Full Text Available Abstract Background The progression towards type 2 diabetes depends on the allostatic response of pancreatic beta cells to synthesise and secrete enough insulin to compensate for insulin resistance. The endocrine pancreas is a plastic tissue able to expand or regress in response to the requirements imposed by physiological and pathophysiological states associated to insulin resistance such as pregnancy, obesity or ageing, but the mechanisms mediating beta cell mass expansion in these scenarios are not well defined. We have recently shown that ob/ob mice with genetic ablation of PPARγ2, a mouse model known as the POKO mouse failed to expand its beta cell mass. This phenotype contrasted with the appropriate expansion of the beta cell mass observed in their obese littermate ob/ob mice. Thus, comparison of these models islets particularly at early ages could provide some new insights on early PPARγ dependent transcriptional responses involved in the process of beta cell mass expansion Results Here we have investigated PPARγ dependent transcriptional responses occurring during the early stages of beta cell adaptation to insulin resistance in wild type, ob/ob, PPARγ2 KO and POKO mice. We have identified genes known to regulate both the rate of proliferation and the survival signals of beta cells. Moreover we have also identified new pathways induced in ob/ob islets that remained unchanged in POKO islets, suggesting an important role for PPARγ in maintenance/activation of mechanisms essential for the continued function of the beta cell. Conclusions Our data suggest that the expansion of beta cell mass observed in ob/ob islets is associated with the activation of an immune response that fails to occur in POKO islets. We have also indentified other PPARγ dependent differentially regulated pathways including cholesterol biosynthesis, apoptosis through TGF-β signaling and decreased oxidative phosphorylation.

  11. Interaction of Plasminogen Activator Inhibitor-2 and Proteasome Subunit, Beta Type 1

    Institute of Scientific and Technical Information of China (English)

    JingFAN; Yu-QingZHANG; PingLI; MinHOU; LiTAN; XiaWANG; Yun-SongZHU


    The apoptosis protection by plasminogen activator inhibitor-2(PAI-2) is dependent on a 33 amino acid fragment between helix C and D of PAI-2 which is probably due to the interaction of PAI-2 with unknown intracellular proteins. In this study, we used the fragment between helix C and D of PAI-2 as bait to screen a HeLa cell cDNA library constructed during apoptosis in a yeast two-hybrid system and retrieved a clone encoding 241 amino acids of proteasome (prosome, macropain) subunit, beta type 1(PSMβ1) which plays important roles in NF-κB activation. GST-pulldown experiments confirmed the interaction between PAI-2 and PSMβ1 in vitro. These data suggest that the antiapoptosis activity of PAI-2 is probably related to its interation with PSMβ1.

  12. Histochemical demonstration of activity of acid phosphatase and beta-glucuronidase in bovine incisor tooth germs

    DEFF Research Database (Denmark)

    Kirkeby, S; Salling, E; Moe, D


    Activity of acid phosphatase and beta-glucuronidase was shown in bovine preodontoblasts and preameloblasts prior to the onset of secretion. In the preameloblasts the rather weak reaction consisted of small discrete granules dispersed in the cytoplasm apical, lateral, and proximal to the nucleus....... After initiation of enamel formation, a change in localization and intensity of the colored reaction product was observed in the ameloblasts. The activity appeared stronger and was restricted to a narrow zone just apical to the nucleus. It is proposed that the acid hydrolases in the tooth forming cells...... are located to the Golgi complex. The differences in activity of acid hydrolases between bone and tooth forming cells are expounded....

  13. Cyclic AMP-and beta-agonist-activated chloride conductance of a toad skin epithelium. (United States)

    Willumsen, N J; Vestergaard, L; Larsen, E H


    1. The control by intracellular cyclic AMP and beta-adrenergic stimulation of chloride conductance was studied in toad skin epithelium mounted in a chamber on the stage of an upright microscope. Impalement of identified principal cells from the serosal side with single-barrelled conventional or double-barrelled Cl(-)-sensitive microelectrodes was performed at x500 magnification. For blocking the active sodium current 50 microM-amiloride was present in the mucosal bath. 2. When clamped at transepithelial potential difference V = 0 mV, the preparations generated clamping currents of 0.9 +/- 1 microA/cm2 (mean +/- S.E.M.; number of observations n = 55). The intracellular potential of principal cells (Vb) was -96 +/- 2 mV with a fractional resistance of the basolateral membrane (fRb) of 0.016 +/- 0.003 (n = 54), and an intracellular Cl- activity of 40 +/- 2 mM (n = 24). 3. At V = 0 mV, serosal application of a cyclic AMP analogue, dibutyryl cyclic AMP (500 microM) or a beta-adrenergic agonist, isoprenaline (5 microM) resulted in a sixfold increase in transepithelial Cl- conductance identified by standard 36Cl- tracer technique. 4. The clamping current at V = 0 mV was unaffected by cyclic AMP (short-circuit current Isc = 0.1 +/- 0.3 microA/cm2, n = 16) indicating that subepidermal Cl(-)-secreting glands are not functioning in our preparations obtained by collagenase treatment. 5. Cyclic AMP- or isoprenaline-induced chloride conductance (Gcl) activation (V = 0 mV) was not reflected in membrane potential and intracellular Cl- activity in principal cells. Intracellular chloride activity was constant at approximately 40 mM at membrane potentials between -90 and -100 mV. Therefore, it can be concluded that the principal cells are not contributing to activated Cl- currents. 6. At V = -100 mV where the voltage-dependent chloride conductance of mitochondria-rich (MR) cells was already fully activated, GCl was unaffected by cyclic AMP or isoprenaline. The major effect of these

  14. 17-Beta-estradiol inhibits transforming growth factor-beta signaling and function in breast cancer cells via activation of extracellular signal-regulated kinase through the G protein-coupled receptor 30. (United States)

    Kleuser, Burkhard; Malek, Daniela; Gust, Ronald; Pertz, Heinz H; Potteck, Henrik


    Breast cancer development and breast cancer progression involves the deregulation of growth factors leading to uncontrolled cellular proliferation, invasion and metastasis. Transforming growth factor (TGF)-beta plays a crucial role in breast cancer because it has the potential to act as either a tumor suppressor or a pro-oncogenic chemokine. A cross-communication between the TGF-beta signaling network and estrogens has been postulated, which is important for breast tumorigenesis. Here, we provide evidence that inhibition of TGF-beta signaling is associated with a rapid estrogen-dependent nongenomic action. Moreover, we were able to demonstrate that estrogens disrupt the TGF-beta signaling network as well as TGF-beta functions in breast cancer cells via the G protein-coupled receptor 30 (GPR30). Silencing of GPR30 in MCF-7 cells completely reduced the ability of 17-beta-estradiol (E2) to inhibit the TGF-beta pathway. Likewise, in GPR30-deficient MDA-MB-231 breast cancer cells, E2 achieved the ability to suppress TGF-beta signaling only after transfection with GPR30-encoding plasmids. It is most interesting that the antiestrogen fulvestrant (ICI 182,780), which possesses agonistic activity at the GPR30, also diminished TGF-beta signaling. Further experiments attempted to characterize the molecular mechanism by which activated GPR30 inhibits the TGF-beta pathway. Our results indicate that GPR30 induces the stimulation of the mitogen-activated protein kinases (MAPKs), which interferes with the activation of Smad proteins. Inhibition of MAPK activity prevented the ability of E2 from suppressing TGF-beta signaling. These findings are of great clinical relevance, because down-regulation of TGF-beta signaling is associated with the development of breast cancer resistance in response to antiestrogens.

  15. Antimicrobial activity of beta-lactams against multiresistant micro-organisms from the family Enterobacteriaceae, and genus Pseudomonas. (United States)

    Niebla, A; González, I; Vallín, C


    The antimicrobial activity of twenty beta-lactams was determined against multiresistant micro-organisms from the Enterobacteriaceae family (450) and the genus Pseudomonas (90). The antimicrobial susceptibility was assessed by the disk diffusion method. The most effective antibiotics were cephalosporins of the second and third generation, and non-classical beta-lactams (imipenem and moxalactam). A pronounced resistance was found to carbenicillin, ampicillin, cephalotin and cefazolin. These resistance patterns corresponded to a high consumption of these antibiotics.

  16. Proteolytically modified human beta 2-microglobulin augments the specific cytotoxic activity in murine mixed lymphocyte culture

    DEFF Research Database (Denmark)

    Nissen, Mogens Holst; Claësson, M H


    (M-beta 2-m) bind to murine lymphocytes expressing H-2 class I antigens; M-beta 2-m, when added at day 0 and 1 of culture in nanomolar concentrations to a one-way murine allogeneic mixed lymphocyte culture (MLC) augments the generation of specific cytotoxic T lymphocytes; M-beta 2-m increases...... the endogenous production of interleukin 2 in the MLC culture; monoclonal antibody which reacts with both the native beta 2-m and M-beta 2-m molecule blocks the augmentation of cytotoxic T lymphocyte production induced by M-beta 2-m; murine as well as human MLC responder cells can proteolytically modify native...

  17. Beta activity in the premotor cortex is increased during stabilized as compared to normal walking

    Directory of Open Access Journals (Sweden)

    Sjoerd M. Bruijn


    Full Text Available Walking on two legs is inherently unstable. Still, we humans perform remarkable well at it, mostly without falling. To gain more understanding of the role of the brain in controlling gait stability we measured brain activity using electro-encephalography (EEG during stabilized and normal walking.Subjects walked on a treadmill in two conditions, each lasting 10 minutes; normal, and while being laterally stabilized by elastic cords. Kinematics of trunk and feet, electro-myography (EMG of neck muscles, as well as 64-channel EEG were recorded. To assess gait stability the local divergence exponent, step width, and trunk range of motion were calculated from the kinematic data. We used independent component analysis to remove movement, EMG, and eyeblink artifacts from the EEG, after which dynamic imaging of coherent sources beamformers were determined to identify cortical sources that showed a significant difference between conditions. Stabilized walking led to a significant increase in gait stability, i.e. lower local divergence exponents. Beamforming analysis of the beta band activity revealed significant sources in bilateral pre-motor cortices. Projection of sensor data on these sources showed a significant difference only in the left premotor area, with higher beta power during stabilized walking, specifically around push-off, although only significant around contralateral push-off. It appears that even during steady gait the cortex is involved in the control of stability.

  18. Phenylalanine binding is linked to dimerization of the regulatory domain of phenylalanine hydroxylase. (United States)

    Zhang, Shengnan; Roberts, Kenneth M; Fitzpatrick, Paul F


    Analytical ultracentrifugation has been used to analyze the oligomeric structure of the isolated regulatory domain of phenylalanine hydroxylase. The protein exhibits a monomer-dimer equilibrium with a dissociation constant of ~46 μM; this value is unaffected by the removal of the 24 N-terminal residues or by phosphorylation of Ser16. In contrast, phenylalanine binding (Kd = 8 μM) stabilizes the dimer. These results suggest that dimerization of the regulatory domain of phenylalanine hydroxylase is linked to allosteric activation of the enzyme.

  19. A mutation in human CMP-sialic acid hydroxylase occurred after the Homo-Pan divergence


    Chou, Hsun-Hua; Takematsu, Hiromu; Diaz, Sandra; Iber, Jane; Nickerson, Elizabeth; Wright, Kerry L.; Muchmore, Elaine A; Nelson, David L.; Warren, Stephen T.; Varki, Ajit


    Sialic acids are important cell-surface molecules of animals in the deuterostome lineage. Although humans do not express easily detectable amounts of N-glycolylneuraminic acid (Neu5Gc, a hydroxylated form of the common sialic acid N-acetylneuraminic acid, Neu5Ac), it is a major component in great ape tissues, except in the brain. This difference correlates with lack of the hydroxylase activity that converts CMP-Neu5Ac to CMP-Neu5Gc. Here we report cloning of human and chimpanzee hydroxylase c...

  20. The Effect of the Arg389Gly Beta-1 Adrenoceptor Polymorphism on Plasma Renin Activity and Heart Rate and the Genotype-Dependent Response to Metoprolol Treatment

    DEFF Research Database (Denmark)

    Petersen, Morten; Andersen, Jon T; Jimenez-Solem, Espen;


    A gene-drug interaction has been indicated between beta-1 selective beta-blockers and the Arg389Gly polymorphism (rs1801253) in the adrenergic beta-1 receptor gene (ADRB1). We studied the effect of the ADRB1 Arg389Gly polymorphism on plasma renin activity (PRA) and heart rate (HR) and the genotype...

  1. Activation of transmembrane bile acid receptor TGR5 stimulates insulin secretion in pancreatic {beta} cells

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Divya P.; Rajagopal, Senthilkumar; Mahavadi, Sunila [Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Mirshahi, Faridoddin [Division of Gastroenterology, Hepatology and Nutrition, Department of Internal Medicine, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Grider, John R. [Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Murthy, Karnam S., E-mail: [Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Sanyal, Arun J., E-mail: [Division of Gastroenterology, Hepatology and Nutrition, Department of Internal Medicine, Virginia Commonwealth University School of Medicine, Richmond, VA (United States)


    Highlights: Black-Right-Pointing-Pointer G protein coupled receptor TGR5 is expressed in mouse and human islets. Black-Right-Pointing-Pointer TGR5 is coupled to activation of Gs and Ca{sup 2+} release via cAMP/Epac/PLC-{epsilon} pathway. Black-Right-Pointing-Pointer Activation of TGR5 by bile salts and selective ligands causes insulin secretion. Black-Right-Pointing-Pointer TGR5 could be a potential therapeutic target to treat diabetes. -- Abstract: Bile acids act as signaling molecules and stimulate the G protein coupled receptor, TGR5, in addition to nuclear farnesoid X receptor to regulate lipid, glucose and energy metabolism. Bile acid induced activation of TGR5 in the enteroendocrine cells promotes glucagon like peptide-1 (GLP-1) release, which has insulinotropic effect in the pancreatic {beta} cells. In the present study, we have identified the expression of TGR5 in pancreatic {beta} cell line MIN6 and also in mouse and human pancreatic islets. TGR5 selective ligands, oleanolic acid (OA) and INT-777 selectively activated G{alpha}{sub s} and caused an increase in intracellular cAMP and Ca{sup 2+}. OA and INT-777 also increased phosphoinositide (PI) hydrolysis and the increase was blocked by NF449 (a selective G{alpha}{sub s} inhibitor) or (U73122) (PI hydrolysis inhibitor). OA, INT-777 and lithocholic acid increased insulin release in MIN6 and human islets and the increase was inhibited by treatment with NF449, (U73122) or BAPTA-AM (chelator of calcium), but not with myristoylated PKI (PKA inhibitor), suggesting that the release is dependent on G{sub s}/cAMP/Ca{sup 2+} pathway. 8-pCPT-2 Prime -O-Me-cAMP, a cAMP analog, which activates Epac, but not PKA also stimulated PI hydrolysis. In conclusion, our study demonstrates that the TGR5 expressed in the pancreatic {beta} cells regulates insulin secretion and highlights the importance of ongoing therapeutic strategies targeting TGR5 in the control of glucose homeostasis.

  2. Characterization of two carnation petal prolyl 4 hydroxylases. (United States)

    Vlad, Florina; Tiainen, Päivi; Owen, Carolyn; Spano, Thodhoraq; Daher, Firas Bou; Oualid, Fatiha; Senol, Namik Ozer; Vlad, Daniela; Myllyharju, Johanna; Kalaitzis, Panagiotis


    Prolyl 4-hydroxylases (P4Hs) catalyze the proline hydroxylation, a major post-translational modification, of hydroxyproline-rich glycoproteins. Two carnation petal P4H cDNAs, (Dianthus caryophyllus prolyl 4-hydroxylase) DcP4H1 and DcP4H2, were identified and characterized at the gene expression and biochemical level in order to investigate their role in flower senescence. Both mRNAs showed similar patterns of expression with stable transcript abundance during senescence progression and differential tissue-specific expression with DcP4H1 and DcP4H2 strongly expressed in ovaries and stems, respectively. Recombinant DcP4H1 and DcP4H2 proteins were produced and their catalytic properties were determined. Pyridine 2,4-dicarboxylate (PDCA) was identified as a potent inhibitor of the in vitro enzyme activity of both P4Hs and used to determine whether inhibition of proline hydroxylation in petals is involved in senescence progression of cut carnation flowers. PDCA suppressed the climacteric ethylene production indicating a strong correlation between the inhibition of DcP4H1 and DcP4H2 activity in vitro by PDCA and the suppression of climacteric ethylene production in cut carnation flowers.

  3. Beta glucosidase from Bacillus polymyxa is activated by glucose-6-phosphate. (United States)

    Weiss, Paulo H E; Álvares, Alice C M; Gomes, Anderson A; Miletti, Luiz C; Skoronski, Everton; da Silva, Gustavo F; de Freitas, Sonia M; Magalhães, Maria L B


    Optimization of cellulose enzymatic hydrolysis is crucial for cost effective bioethanol production from lignocellulosic biomass. Enzymes involved in cellulose hydrolysis are often inhibited by their end-products, cellobiose and glucose. Efforts have been made to produce more efficient enzyme variants that are highly tolerant to product accumulation; however, further improvements are still necessary. Based on an alternative approach we initially investigated whether recently formed glucose could be phosphorylated into glucose-6-phosphate to circumvent glucose accumulation and avoid inhibition of beta-glucosidase from Bacillus polymyxa (BGLA). The kinetic properties and structural analysis of BGLA in the presence of glucose-6-phosphate (G6P) were investigated. Kinetic studies demonstrated that enzyme was not inhibited by G6P. In contrast, the presence of G6P activated the enzyme, prevented beta glucosidase feedback inhibition by glucose accumulation and improved protein stability. G6P binding was investigated by fluorescence quenching experiments and the respective association constant indicated high affinity binding of G6P to BGLA. Data reported here are of great impact for future design strategies for second-generation bioethanol production. PMID:26116788

  4. [Activity of cefpodoxime and other oral beta-lactams against Haemophilus influenzae and Streptococcus pneumoniae with different susceptibilities to penicillin]. (United States)

    Fenoll, A; Robledo, O; Lerma, M; Giménez, M J; Cebrián, L; Casal, J; Aguilar, L; Gómez-Lus, M L


    This study explores the influence on the intrinsic activity of different oral beta-lactams of beta-lactamase production in Haemophilus influenzae and penicillin resistance in Streptococcus pneumoniae. Three substudies were performed: a) a general susceptibility study, analyzing 550 strains received by the Spanish Laboratorio de Referencia de Neumococos throughout February and March 2005; b) a study on the influence of penicillin resistance on the activity of beta-lactams, analyzing 251 penicillin-susceptible strains (MICor=2 mg/l) randomly chosen among those received by the Spanish Laboratorio de Referencia de Neumococos throughout 2005; and c) an H. influenzae susceptibility study analyzing 150 strains received by Instituto Valenciano de Microbiologia throughout 2005. A total of 71% of S. pneumoniae strains were susceptible to penicillin, 21% exhibited intermediate resistance and 8% strains presented full resistance. H. influenzae beta-lactamase production rate was 18.6%. Of the non-beta-lactamase-producing strains, 3% were not susceptible to ampicillin. Cefpodoxime and cefixime exhibited the highest intrinsic activity against H. influenzae, while amoxicillin and cefpodoxime were the most active compounds against S. pneumoniae. All H. influenzae strains were susceptible to oral cephalosporins and amoxicillin/clavulanic acid. The increase in penicillin resistance in S. pneumoniae influenced cefixime, cefaclor and cefuroxime to a higher degree than amoxicillin and cefpodoxime.

  5. Plasmin-induced migration requires signaling through protease-activated receptor 1 and integrin alpha(9)beta(1). (United States)

    Majumdar, Mousumi; Tarui, Takehiko; Shi, Biao; Akakura, Nobuaki; Ruf, Wolfram; Takada, Yoshikazu


    Plasmin is a major extracellular protease that elicits intracellular signals to mediate platelet aggregation, chemotaxis of peripheral blood monocytes, and release of arachidonate and leukotriene from several cell types in a G protein-dependent manner. Angiostatin, a fragment of plasmin(ogen), is a ligand and an antagonist for integrin alpha(9)beta(1). Here we report that plasmin specifically interacts with alpha(9)beta(1) and that plasmin induces of cells expressing migration recombinant alpha(9)beta(1) (alpha(9)-Chinese hamster ovary (CHO) cells). Migration was dependent on an interaction of the kringle domains of plasmin with alpha(9)beta(1) as well as the catalytic activity of plasmin. Angiostatin, representing the kringle domains of plasmin, alone did not induce the migration of alpha(9)-CHO cells, but simultaneous activation of the G protein-coupled protease-activated receptor (PAR)-1 with an agonist peptide induced the migration on angiostatin, whereas PAR-2 or PAR-4 agonist peptides were without effect. Furthermore, a small chemical inhibitor of PAR-1 (RWJ 58259) and a palmitoylated PAR-1-blocking peptide inhibited plasmin-induced migration of alpha(9)-CHO cells. These results suggest that plasmin induces migration by kringle-mediated binding to alpha(9)beta(1) and simultaneous proteolytic activation of PAR-1. PMID:15247268

  6. A series of beta-carboline derivatives inhibit the kinase activity of PLKs.

    Directory of Open Access Journals (Sweden)

    Xiaomin Han

    Full Text Available Polo-like kinases play an essential role in the ordered execution of mitotic events and 4 mammalian PLK family members have been identified. Accumulating evidence indicates that PLK1 is an attractive target for anticancer drugs. In this paper, a series of beta-carboline derivatives were synthesized and three compounds, DH281, DH285 and DH287, were identified as potent new PLK inhibitors. We employed various biochemical and cellular approaches to determine the effects of these compounds on the activity of PLK1 and other mitotic kinases and on cell cycle progression. We found that these three compounds could selectively inhibit the kinase activity of purified PLK1, PLK2 and PLK3 in vitro. They show strong antitumor activity against a number of cancer cell lines with relatively low micromolar IC(50s, but are relatively less toxic to non-cancer cells (MRC5. Moreover, these compounds could induce obvious accumulation of HeLa cells in G(2/M and S phases and trigger apoptosis. Although MRC5 cells show clear S-phase arrest after treatment with these compounds, the G2/M arrest and apoptosis are less insignificant, indicating the distinct sensitivity between normal and cancer cells. We also found that HeLa cells treated with these drugs exhibit monopolar spindles and increased Wee1 protein levels, the characteristics of cells treated with PLK1 inhibitors. Together, these results demonstrate that DH281, DH285 and DH287 beta-carboline compounds are new PLK inhibitors with potential for cancer treatment.

  7. Direct interaction of the kringle domain of urokinase-type plasminogen activator (uPA) and integrin alpha v beta 3 induces signal transduction and enhances plasminogen activation. (United States)

    Tarui, Takehiko; Akakura, Nobuaki; Majumdar, Mousumi; Andronicos, Nicholas; Takagi, Junichi; Mazar, Andrew P; Bdeir, Khalil; Kuo, Alice; Yarovoi, Serge V; Cines, Douglas B; Takada, Yoshikazu


    It has been questioned whether there are receptors for urokinase-type plasminogen activator (uPA) that facilitate plasminogen activation other than the high affinity uPA receptor (uPAR/CD87) since studies of uPAR knockout mice did not support a major role of uPAR in plasminogen activation. uPA also promotes cell adhesion, chemotaxis, and proliferation besides plasminogen activation. These uPA-induced signaling events are not mediated by uPAR, but mediated by unidentified, lower-affinity receptors for the uPA kringle. We found that uPA binds specifically to integrin alpha v beta 3 on CHO cells depleted of uPAR. The binding of uPA to alpha v beta 3 required the uPA kringle domain. The isolated uPA kringle domain binds specifically to purified, recombinant soluble, and cell surface alpha v beta 3, and other integrins (alpha 4 beta 1 and alpha 9 beta 1), and induced migration of CHO cells in an alpha v beta 3-dependent manner. The binding of the uPA kringle to alpha v beta 3 and uPA kringle-induced alpha v beta 3-dependent cell migration were blocked by homologous plasminogen kringles 1-3 or 1-4 (angiostatin), a known integrin antagonist. We studied whether the binding of uPA to integrin alpha v beta 3 through the kringle domain plays a role in plasminogen activation. On CHO cell depleted of uPAR, uPA enhanced plasminogen activation in a kringle and alpha v beta 3-dependent manner. Endothelial cells bound to and migrated on uPA and uPA kringle in an alpha v beta 3-dependent manner. These results suggest that uPA binding to integrins through the kringle domain plays an important role in both plasminogen activation and uPA-induced intracellular signaling. The uPA kringle-integrin interaction may represent a novel therapeutic target for cancer, inflammation, and vascular remodeling. PMID:16525582

  8. Measuring the Orientation of Taurine in the Active Site of the Non-Heme Fe (II)/α-Ketoglutarate Dependent Taurine Hydroxylase (TauD) using Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy


    Casey, Thomas M.; Grzyska, Piotr K.; Hausinger, Robert P.; McCracken, John


    The position and orientation of taurine near the non-heme Fe(II) center of the α-ketoglutarate (α-KG) dependent taurine hydroxylase (TauD) was measured using Electron Spin Echo Envelope Modulation (ESEEM) spectroscopy. TauD solutions containing Fe(II), α-KG, and natural abundance taurine or specifically deuterated taurine were prepared anaerobically and treated with nitric oxide (NO) to make an S=3/2 {FeNO}7 complex that is suitable for robust analysis with EPR spectroscopy. Using ratios of E...

  9. Design of a Quality Control Program for the Measurement of Gross Alpha and Gross Beta Activities (LMPR-CIEMAT)

    International Nuclear Information System (INIS)

    In accordance with international standards, general requirements for testing laboratories have to include a quality system for planning, implementing, and assessing the work performed by the organization and for carrying out required quality assurance and quality control. The purpose of internal laboratory quality control is to monitor performance, identify problems, and initiate corrective actions. This report describes the internal quality control to monitor the gross alpha and beta activities determination. Identification of specific performance indicators, the principles that govern their use and statistical means of evaluation are explained. Finally, calculation of alpha and beta specific activities, uncertainties and detection limits are performed. (Author) 10 refs.

  10. Penicillin-bound polyacrylate nanoparticles: restoring the activity of beta-lactam antibiotics against MRSA. (United States)

    Turos, Edward; Reddy, G Suresh Kumar; Greenhalgh, Kerriann; Ramaraju, Praveen; Abeylath, Sampath C; Jang, Seyoung; Dickey, Sonja; Lim, Daniel V


    This report describes the preparation of antibacterially active emulsified polyacrylate nanoparticles in which a penicillin antibiotic is covalently conjugated onto the polymeric framework. These nanoparticles were prepared in water by emulsion polymerization of an acrylated penicillin analogue pre-dissolved in a 7:3 (w:w) mixture of butyl acrylate and styrene in the presence of sodium dodecyl sulfate (surfactant) and potassium persulfate (radical initiator). Dynamic light scattering analysis and atomic force microscopy images show that the emulsions contain nanoparticles of approximately 40 nm in diameter. The nanoparticles have equipotent in vitro antibacterial properties against methicillin-susceptible and methicillin-resistant forms of Staphylococcus aureus and indefinite stability toward beta-lactamase. PMID:17420125

  11. Genomic organization of the mouse peroxisome proliferator-activated receptor beta/delta gene

    DEFF Research Database (Denmark)

    Larsen, Leif K; Amri, Ez-Zoubir; Mandrup, Susanne;


    Peroxisome proliferator-activated receptor (PPAR) beta/delta is ubiquitously expressed, but the level of expression differs markedly between different cell types. In order to determine the molecular mechanisms governing PPARbeta/delta gene expression, we have isolated and characterized the mouse...... gene encoding PPARbeta/delta. The gene spans approx. 41 kb and comprises 11 exons of which the six exons located in the 3'-end of the gene are included in all transcripts. Primer-extension and 5'-rapid amplification of cDNA ends experiments revealed the presence of multiple transcription start points...... and splice variants, originating from the use of at least four different promoters. One of these transcription start points was found to be used predominantly in all tissues examined. Initiation from this major transcription start point gives rise to a transcript with a 548 nt 5'-untranslated leader...

  12. Expression of hypoxia-inducible factor-1α and aspartyl-(asparaginyl) beta-hydroxylase in missed abortion patients' villi%HIF-1α和AAH在稽留流产患者绒毛中的表达研究

    Institute of Scientific and Technical Information of China (English)

    米春梅; 周昌菊; 薛敏; 卢翌


    Objective To investigate the expression of hypoxia-inducoble factor-1α (HIF-1α) and aspartyl-(asparaginyl) beta-hydroxylase (AAH) in missed abortion patients' villi, and explore the relationship between these two materials and missed abortion. Methods 50 patients with missed abortion were collected and categorized into two groups (test group 1 and test group 2). Patients in test group 1 (n=20) were of confirmed etiologies while those in test group 2 (n=30) showed no obviously etiological clues. In addition, 20 early pregnant women with artificial abortion surgery were used as control group, of whom the embryos were confirmed alive before surgery. According to the time that the embryo stayed in uterus after it died, the 50 patients of missed abortion were subdivided into the group of ≤4 weeks or the group of >4 weeks. Immunohistochemical technique and computer image analysis system were used to detect the expression loci and level of HIF-1α and AAH in chorionie villi. Results HIF-1α expression was detected in both missed abortion villi (test group) and normal early pregnancy villi (control group), and its loci were mainly located in endochylema and nucleus of trephocytes. The expression levels of HIF-1α in test groups were significantly lower than that in control group (P 0.05). The expression level of HIF-1α in villi from missed abortion women showed no significant difference between the groups of ≤4 or >4 weeks of arrested embryos. The AAH expression was found to have the similar result as HIF-1α's. Conclusions The expression level of HIF-1α and AAH in villi of missed abortion patients is much lower than that of normal early pregnant women. HIF-1α and AAH have a function of supporting normal pregnancy, so their low expression may be an important cause of missed abortion.%目的 研究缺氧诱导因子-1α(HIF-1α)及天冬氨酰(天冬酰胺酰)-β-羟化酶(AAH)在稽留流产患者绒毛中的表达情况,探讨其与稽留

  13. 醛固酮合酶基因和11-β羟化酶基因多态性及其单体型与醛固酮瘤发病风险的关系%Association of aldosterone synthase and 11-beta hydroxylase genes polymorphism and haplotype with susceptibility of aldosterone producing adenoma

    Institute of Scientific and Technical Information of China (English)

    王保军; 陈路遥; 李新涛; 马鑫; 杨素霞; 欧阳金芝; 张旭


    目的 探讨醛固酮合酶基因(CYP11B2)和11-β羟化酶基因(CYP11B1)多态性及其单体型与醛固酮瘤发病风险的关系.方法 提取81例醛固酮瘤患者和103例对照人群外周血DNA,应用2个独立聚合酶链反应(PCR)和TaqMan探针技术对CYP11B2和CYP11B1基因的7个多态性位点(rs6387、rs6410、rs3097、rs4539、intron2转位、rs1799998、rs 13268025)进行基因分型,采用Logistic回归模型分析不同等位基因、基因型和单体型与醛固酮瘤发病风险的相关性.结果 intron2转位多态性位点C等位基因在病例组中的分布频率(25.3%)高于对照组(15.0%),携带C等位基因的个体较携带W等位基因的个体发生醛固酮瘤的风险增加了1.04倍(P<0.01).rs13268025位点中携带C等位基因的个体较携带T等位基因的个体发生醛固酮瘤的风险增加了0.91倍(P<0.05).7个多态性位点间存在着不同程度的连锁不平衡.单体型分析示AGGAWTT为最常见的单体型,单体型GAGAWTT是AGGAWTT发病风险的4.43倍(P<0.01).结论 CYP11B2和CYP11B1基因多态性与醛固酮瘤发病风险明显相关,intron2转位多态性位点的C等位基因、rs13268025位点的C等位基因、单体型GAGAWTT是醛固酮瘤发病的危险因素.%Objective To evaluate the effects of aldosterone synthase (CYP1 1B2) and 11-beta hydroxylase (CYP1 1 B1) genes polymorphism and haplotype on the susceptibility of aldosterone producing adenoma.Methods Peripheral blood DNA was extracted from 81 aldosterone producing adenoma patients and 103 control subjects.Real-time TaqMan probes technique and two seperate PCRs were used for genotyping seven polymorphism sites of the CYP1 1B2 and CYP11B1 genes (rs6387,rs6410,rs3097,rs4539,intron 2 conversion,rs1799998,rs13268025).Logistic regression model was performed to analyze the relationship of different genotypes or haplotype and the susceptibility of aldosterone producing adenoma.Results The frequency for allele C at site intron 2

  14. 醛固酮合酶和11-β羟化酶基因多态性与原发性醛固酮增多症发病风险的相关性研究%Association of polymorphisms in aldosterone synthase and 11 beta-hydroxylase genes with the risk of primary aldosteronism

    Institute of Scientific and Technical Information of China (English)

    张国玺; 李宏召; 吴准; 刘双林; 张旭; 欧阳金芝; 王保军; 邓西元; 王超; 史涛坪; 居正华; 徐华; 马鑫


    Objective To determine the association of mutations in aldosterone synthase (CYPllB2)and 11 beta-hydroxylase(CYP11B1)genes with primary aldosteronism(PA).Methods Five mutations of CYP11B2 and CYP11B1 genes were analyzed in patients with PA and normal population.Among them,intron 2 was detected by 2 independent PCR reactions,and the others were analyzed using Taqman probes.The Haploview 4.0,SNPassoc 1.5-3 and Haplo.stats 1.3.8 were used to analyse the association between polymorphisms and PA.Results All the selected mutations were successfully genetyped.Only rs64lO allelic frequencies in patients with aldosterone-producing adenoma (APA)and idiopathic hyperaldosteronism(IHA)were significantly different with those in controls (P<0.05).There was a relative excess of AA homozygotes and AG heterozygotes of rs6410 allele in APA group compared with control group(P<0.01).There were significantly different genotypes AA and AG of rs6410 allele between patients with IHA and controls only after adjusted for age,gender,eeptible haplotype AAAWT was identified to be significantly associated with APA(OR=1.44,95%CI 1.19-1.76).Three susceptible haplotypes AAAWT,AGGWT and AGAWC were identified to be significantly associated with IHA(OR=1.55,95%CI 1.23-1.96;OR=1.49,95%CI 1.17-1.89;OR=1.40,95%CI 1.04-1.88).In contrast,1 protective haplotype GGAWT showed significant difference between patients with APA and controls(OR=0.73,95%CI 0.55-0.97).Conclusion There is a significant association between genetic variations in CYP11B2 and CYP11B1 genes and genetie predisposition to PA.%目的 研究醛固酮合酶基因(CYP11B2)和11-β羟化酶基因(CYP11B1)多态性与原发性醛同酮增多症(PA)发病风险之间的相关性.方法 对醛固酮瘤(APA)和特发性醛崮酮增多症(IHA)患者(APA 134例,IHA 45例)及正常人群(118名)中CYP11B2和CYP1181基因的5个多态性位点进行检测.其中,intron 2采用2个独立的PCR反应,其余位

  15. Mechanisms of transcriptional activation of the mouse claudin-5 promoter by estrogen receptor alpha and beta. (United States)

    Burek, Malgorzata; Steinberg, Katrin; Förster, Carola Y


    Claudin-5 is an integral membrane protein and a critical component of endothelial tight junctions that control paracellular permeability. Claudin-5 is expressed at high levels in the brain vascular endothelium. Estrogens have multiple effects on vascular physiology and function. The biological actions of estrogens are mediated by two different estrogen receptor (ER) subtypes, ER alpha and ER beta. Estrogens have beneficial effects in several vascular disorders. Recently we have cloned and characterized a murine claudin-5 promoter and demonstrated 17beta-estradiol (E2)-mediated regulation of claudin-5 in brain and heart microvascular endothelium on promoter, mRNA and protein level. Sequence analysis revealed a putative estrogen response element (ERE) and a putative Sp1 transcription factor binding site in the claudin-5 promoter. The aim of the present study was to further characterize the estrogen-responsive elements of claudin-5 promoter. First, we introduced point mutations in ERE or Sp1 site in -500/+111 or in Sp1 site of -268/+111 claudin-5 promoter construct, respectively. Basal and E2-mediated transcriptional activation of mutated constructs was abrogated in the luciferase reporter gene assay. Next, we examined whether estrogen receptor subtypes bind to the claudin-5 promoter region. For this purpose we performed chromatin immunoprecipitation assays using anti-estrogen receptor antibodies and cellular lysates of E2-treated endothelial cells followed by quantitative PCR analysis. We show enrichment of claudin-5 promoter fragments containing the ERE- and Sp1-binding site in immunoprecipitates after E2 treatment. Finally, in a gel mobility shift assay, we demonstrated DNA-protein interaction of both ER subtypes at ERE. In summary, this study provides evidence that both a non-consensus ERE and a Sp1 site in the claudin-5 promoter are functional and necessary for the basal and E2-mediated activation of the promoter.

  16. Code Betal to calculation Alpha/Beta activities in environmental samples; Programa de ordenador Betal para el calculo de la actividad Beta/Alfa de muestras ambientales

    Energy Technology Data Exchange (ETDEWEB)

    Romero, L.; Travesi, A.


    A codes, BETAL, was developed, written in FORTRAN IV, to automatize calculations and presentations of the result of the total alpha-beta activities measurements in environmental samples. This code performs the necessary calculations for transformation the activities measured in total counts, to pCi/1., bearing in mind the efficiency of the detector used and the other necessary parameters. Further more, it appraise the standard deviation of the result, and calculus the Lower limit of detection for each measurement. This code is written in iterative way by screen-operator dialogue, and asking the necessary data to perform the calculation of the activity in each case by a screen label. The code could be executed through any screen and keyboard terminal, (whose computer accepts Fortran IV) with a printer connected to the said computer. (Author) 5 refs.

  17. Effect of trimethyllead chloride on slowly activating (SV) channels in red beet (Beta vulgaris L.) taproots. (United States)

    Trela, Zenon; Burdach, Zbigniew; Przestalski, Stanisław; Karcz, Waldemar


    The patch-clamp technique was used to examine the effect of trimethyllead chloride (Met(3)PbCl) on SV channel activity in red beet (Beta vulgaris L.) taproot vacuoles. It was found that in the control bath the macroscopic currents showed the typical slow activation and a strong outward rectification of the steady-state currents. An addition of Met(3)PbCl to the bath solution blocked, in a concentration-dependent manner, SV currents in red beet vacuoles. The time constant τ increased several times in the presence of 100 μM trimethyllead chloride at all voltages tested. When single channel properties were analyzed, only little channel activity could be recorded in the presence of 100 μM Met(3)PbCl. Trimethyllead chloride decreased significantly (by about one order of magnitude) the open probability of single channels. The recordings of single channel activity obtained in the presence and absence of Met(3)PbCl showed that organolead only slightly (by ca. 10%) decreased the unitary conductance of single channels. It was also found that Met(3)PbCl diminished significantly the number of SV channel openings, whereas it did not change the opening times of the channels. Taken together, these results suggest that Met(3)PbCl binding site is located outside the channel selectivity filter. PMID:23312295

  18. Primary structure, tissue distribution, and chromosomal localization of a novel isoform of lysyl hydroxylase (lysyl hydroxylase 3) (United States)

    Valtavaara, M; Szpirer, C; Szpirer, J; Myllylä, R


    We report characterization of a novel isoform of lysyl hydroxylase (lysyl hydroxylase 3, LH3). The cDNA clones encode a polypeptide of 738 amino acids, including a signal peptide. The amino acid sequence has a high overall identity with LH1 and LH2, the isoforms characterized earlier. Conserved regions are present in the carboxyl-terminal portion of the isoforms and also in the central part of the molecules. Histidine and asparagine residues, which are conserved in the other isoforms and are known to be required for enzymatic activity, are also conserved in the novel isoform. The gene for LH3 (PLOD3) has been assigned to human chromosome 7q36 and rat chromosome 12. Gene expression of LH3 is highly regulated in adult human tissues. A strong hybridization signal, corresponding to an mRNA 2.75 kilobases in size, is obtained in heart, placenta and pancreas on multiple tissue RNA blots. Expression of the cDNA in vitro results in the synthesis of a protein that hydroxylates lysyl residues in collagenous sequences in a non-triple helical conformation. PMID:9582318

  19. Thymosin beta-10 is aberrantly expressed in pancreatic cancer and induces JNK activation. (United States)

    Li, Min; Zhang, Yuqing; Zhai, Qihui; Feurino, Louis W; Fisher, William E; Chen, Changyi; Yao, Qizhi


    Thymosin beta-10 (T beta 10) has been shown to be associated with several cancers; however, its role in pancreatic cancer is not understood. The expression of T beta 10 was determined by immunohistochemistry and real-time polymerase chain reaction. The phosphorylation of JNK and the cytokine secretion was determined by using the Bio-Plex phosphoprotein and cytokines assays. Pancreatic cancer tissues and cells expressed higher amounts of T beta 10 than normal surrounding tissues and human pancreatic duct epithelial cells. Exogenous T beta 10 caused the phosphorylation of JNK and increased the secretion of cytokines interleukin (IL)-7 and IL-8 in BxPC-3 cells. T beta 10 might be a promising marker and a novel therapeutic target for pancreatic cancer. PMID:19194824

  20. Inter-trial analysis of post-movement Beta activities in EEG signals using multivariate empirical mode decomposition. (United States)

    Chang, Hsiang-Chih; Lee, Po-Lei; Lo, Men-Tzung; Wu, Yu-Te; Wang, Kuo-Wei; Lan, Gong-Yau


    Event-related desynchronization/synchronization (ERD/ERS) is a technique to quantify subject's nonphase-locked neural activities underlying specific frequency bands, reactive to external/internal stimulus. However, conventional ERD/ERS studies usually utilize fixed frequency band determined from one or few channels to filter whole-head EEG/MEG data, which may inevitably include task-unrelated signals and result in underestimation of reactive oscillatory activities in multichannel studies. In this study, we adopted multivariate empirical mode decomposition (MEMD) to extract beta-related oscillatory activities in performing self-paced right and left index-finger lifting tasks. The MEMD extracts common modes from all channels in same-index intrinsic mode functions (IMFs) which allows the temporal-frequency features among different channels can be compared in each subband. The beta-band oscillatory activities were further bandpass filtered within trial-specific beta bands determined from sensorimotor-related channels (C3 and C4), and then rectified using amplitude modulation method to detect trial-by-trial beta rebound (BR) values in ERS time courses. The validity of the MEMD approach in BR values extraction has been demonstrated in multichannel EEG study which showed larger BR values than conventional ERS technique. The MEMD-based method enables the trial-by-trial extraction of sensorimotor oscillatory activities which might allow the exploration of subtle brain dynamics in future studies. PMID:23661320

  1. Antiviral activity of monoterpenes beta-pinene and limonene against herpes simplex virus in vitro.

    Directory of Open Access Journals (Sweden)

    Akram Astani


    Full Text Available Essential oils are complex mixtures containing compounds of several different functional- group classes. Depending on the structure, we can distinguish monoterpenes, phenylpropanes, and other components. Here in this study two monoterpene compounds of essential oils, i.e. β-pinene and limonene were examined for their antiviral activity against herpes simplex virus type 1 (HSV-1 in vitro.All antiviral assays were performed using RC-37 cells. Cytotoxicity was determined in a neutral red assay, antiviral assays were performed with HSV-1 strain KOS. The mode of antiviral action was evaluated at different periods during the viral replication cycle. Acyclovir was used as positive antiviral control.Beta-pinenene and limonenen reduced viral infectivity by 100 %. The mode of antiviral action has been determined, only moderate antiviral effects were revealed by monoterpenes when these drugs were added to host cells prior infection or after entry of HSV into cells. However, both monoterpenes exhibited high anti-HSV-1 activity by direct interaction with free virus particles. Both tested drugs interacted with HSV-1 in a dose-dependent manner thereby inactivating viral infection.These results suggest that monoterpenes in essential oils exhibit antiherpetic activity in the early phase of viral multiplication and might be used as potential antiviral agents.

  2. Elevation in sphingomyelin synthase activity is associated with increases in amyloid-beta peptide generation.

    Directory of Open Access Journals (Sweden)

    Jen-Hsiang T Hsiao

    Full Text Available A pathological hallmark of Alzheimer's disease (AD is the presence of amyloid-beta peptide (Aβ plaques in the brain. Aβ is derived from a sequential proteolysis of the transmenbrane amyloid precursor protein (APP, a process which is dependent on the distribution of lipids present in the plasma membrane. Sphingomyelin is a major membrane lipid, however its role in APP processing is unclear. Here, we assessed the expression of sphingomyelin synthase (SGMS1; the gene responsible for sphingomyelin synthesis in human brain and found that it was significantly elevated in the hippocampus of AD brains, but not in the cerebellum. Secondly, we assessed the impact of altering SGMS activity on Aβ generation. Inhibition of SGMS activity significantly reduced the level of Aβ in a dose- and time dependent manner. The decrease in Aβ level occurred without changes in APP expression or cell viability. These results when put together indicate that SGMS activity impacts on APP processing to produce Aβ and it could be a contributing factor in Aβ pathology associated with AD.

  3. Activation of band 3 mediates group A Streptococcus streptolysin S-based beta-haemolysis. (United States)

    Higashi, Dustin L; Biais, Nicolas; Donahue, Deborah L; Mayfield, Jeffrey A; Tessier, Charles R; Rodriguez, Kevin; Ashfeld, Brandon L; Luchetti, Jeffrey; Ploplis, Victoria A; Castellino, Francis J; Lee, Shaun W


    Streptococcus pyogenes, or group A Streptococcus (GAS), is a human bacterial pathogen that can manifest as a range of diseases from pharyngitis and impetigo to severe outcomes such as necrotizing fasciitis and toxic shock syndrome. GAS disease remains a global health burden with cases estimated at over 700 million annually and over half a million deaths due to severe infections(1). For over 100 years, a clinical hallmark of diagnosis has been the appearance of complete (beta) haemolysis when grown in the presence of blood. This activity is due to the production of a small peptide toxin by GAS known as streptolysin S. Although it has been widely held that streptolysin S exerts its lytic activity through membrane disruption, its exact mode of action has remained unknown. Here, we show, using high-resolution live cell imaging, that streptolysin S induces a dramatic osmotic change in red blood cells, leading to cell lysis. This osmotic change was characterized by the rapid influx of Cl(-) ions into the red blood cells through SLS-mediated disruption of the major erythrocyte anion exchange protein, band 3. Chemical inhibition of band 3 function significantly reduced the haemolytic activity of streptolysin S, and dramatically reduced the pathology in an in vivo skin model of GAS infection. These results provide key insights into the mechanism of streptolysin S-mediated haemolysis and have implications for the development of treatments against GAS. PMID:27571972

  4. Activities of UDP-glucuronyltransferase, beta-glucuronidase and deiodinase types I and II in hyper- and hypothyroid rats

    NARCIS (Netherlands)

    Heide, S.M. van der; Joosten, B.H.G.M.; Everts, M.E.; Klaren, P.H.M.


    We have investigated the hypothesis that uridine 5'-diphosphate (UDP)-glucuronyltransferases (UGTs) and beta-glucuronidase are jointly involved in a mechanism for the storage and mobilization of iodothyronine metabolites in liver, kidney, heart and brain. Specifically, we predicted UGT activities to


    NARCIS (Netherlands)



    Activation of the retinoic acid receptor (RAR) beta2 promoter is known to be mediated by a RA response element located in the proximity of the TATA-box. By deletion studies in P19 embryonal carcinoma cells we have analyzed the RARbeta2 promoter for the presence of additional regulatory elements. We

  6. Glucose-induced repression of PPARalpha gene expression in pancreatic beta-cells involves PP2A activation and AMPK inactivation

    DEFF Research Database (Denmark)

    Ravnskjaer, Kim; Boergesen, Michael; Dalgaard, Louise T;


    Tight regulation of fatty acid metabolism in pancreatic beta-cells is important for beta-cell viability and function. Chronic exposure to elevated concentrations of fatty acid is associated with beta-cell lipotoxicity. Glucose is known to repress fatty acid oxidation and hence to augment the toxi......Tight regulation of fatty acid metabolism in pancreatic beta-cells is important for beta-cell viability and function. Chronic exposure to elevated concentrations of fatty acid is associated with beta-cell lipotoxicity. Glucose is known to repress fatty acid oxidation and hence to augment...... but not AMPKalpha1 using RNAi suppressed PPARalpha expression, thereby mimicking the effect of glucose. These results indicate that activation of protein phosphatase 2A and subsequent inactivation of AMPK is necessary for glucose repression of PPARalpha expression in pancreatic beta-cells....

  7. Expression of mouse beta defensin 2 in Escherichia coli and its broad-spectrum antimicrobial activity

    Directory of Open Access Journals (Sweden)

    Tianxiang Gong


    Full Text Available Mature mouse beta defensin 2 (mBD2 is a small cationic peptide with antimicrobial activity. Here we established a prokaryotic expression vector containing the cDNA of mature mBD2 fused with thioredoxin (TrxA, pET32a-mBD2. The vector was transformed into Escherichia Coli (E. coli Rosseta-gami (2 for expression fusion protein. Under the optimization of fermentation parameters: induce with 0.6 mM isopropylthiogalactoside (IPTG at 34ºC in 2×YT medium and harvest at 6 h postinduction, fusion protein TrxA-mBD2 was high expressed in the soluble fraction (>95%. After cleaved fusion protein by enterokinase, soluble mature mBD2 was achieved 6 mg/L with a volumetric productivity. Purified recombinant mBD2 demonstrated clear broad-spectrum antimicrobial activity for fungi, bacteria and virus. The MIC of antibacterial activity of against Staphylococcus aureus was 50 µg/ml. The MIC of against Candida albicans (C. albicans and Cryptococcus neoformans (C. neoformans was 12.5µg/ml and 25µg/ml, respectively. Also, the antimicrobial activity of mBD2 was effected by NaCl concentration. Additionally, mBD2 showed antiviral activity against influenza A virus (IAV, the protective rate for Madin-Darby canine kidney cells (MDCK was 93.86% at the mBD2 concentration of 100 µg/ml. These works might provide a foundation for the following research on the mBD2 as therapeutic agent for medical microbes.

  8. CRFR1 activation protects against cytokine-induced beta cell death

    DEFF Research Database (Denmark)

    Blaabjerg, Lykke; Christensen, Gitte Lund; Matsumoto, Masahito;


    During diabetes development beta cells are exposed to elevated concentrations of proinflammatory cytokines, TNFα and IL-1β which in vitro, induce beta cell death. The class B G-protein-coupled receptors (GPCRs): Corticotropin releasing factor receptor 1 (CRFR1) and CRFR2 are expressed in pancreat...

  9. Hydroxylase inhibition attenuates colonic epithelial secretory function and ameliorates experimental diarrhea.

    LENUS (Irish Health Repository)

    Ward, Joseph B J


    Hydroxylases are oxygen-sensing enzymes that regulate cellular responses to hypoxia. Transepithelial Cl(-) secretion, the driving force for fluid secretion, is dependent on O(2) availability for generation of cellular energy. Here, we investigated the role of hydroxylases in regulating epithelial secretion and the potential for targeting these enzymes in treatment of diarrheal disorders. Ion transport was measured as short-circuit current changes across voltage-clamped monolayers of T(84) cells and mouse colon. The antidiarrheal efficacy of dimethyloxallyl glycine (DMOG) was tested in a mouse model of allergic disease. Hydroxylase inhibition with DMOG attenuated Ca(2+)- and cAMP-dependent secretory responses in voltage-clamped T(84) cells to 20.2 +\\/- 2.6 and 38.8 +\\/- 6.7% (n=16; P<\\/=0.001) of those in control cells, respectively. Antisecretory actions of DMOG were time and concentration dependent, being maximal after 18 h of DMOG (1 mM) treatment. DMOG specifically inhibited Na(+)\\/K(+)-ATPase pump activity without altering its expression or membrane localization. In mice, DMOG inhibited agonist-induced secretory responses ex vivo and prevented allergic diarrhea in vivo. In conclusion, hydroxylases are important regulators of epithelial Cl(-) and fluid secretion and present a promising target for development of new drugs to treat transport disorders.

  10. Hydroxylase inhibition attenuates colonic epithelial secretory function and ameliorates experimental diarrhea.

    LENUS (Irish Health Repository)

    Ward, Joseph B J


    Hydroxylases are oxygen-sensing enzymes that regulate cellular responses to hypoxia. Transepithelial Cl(-) secretion, the driving force for fluid secretion, is dependent on O(2) availability for generation of cellular energy. Here, we investigated the role of hydroxylases in regulating epithelial secretion and the potential for targeting these enzymes in treatment of diarrheal disorders. Ion transport was measured as short-circuit current changes across voltage-clamped monolayers of T(84) cells and mouse colon. The antidiarrheal efficacy of dimethyloxallyl glycine (DMOG) was tested in a mouse model of allergic disease. Hydroxylase inhibition with DMOG attenuated Ca(2+)- and cAMP-dependent secretory responses in voltage-clamped T(84) cells to 20.2 ± 2.6 and 38.8 ± 6.7% (n=16; P≤0.001) of those in control cells, respectively. Antisecretory actions of DMOG were time and concentration dependent, being maximal after 18 h of DMOG (1 mM) treatment. DMOG specifically inhibited Na(+)\\/K(+)-ATPase pump activity without altering its expression or membrane localization. In mice, DMOG inhibited agonist-induced secretory responses ex vivo and prevented allergic diarrhea in vivo. In conclusion, hydroxylases are important regulators of epithelial Cl(-) and fluid secretion and present a promising target for development of new drugs to treat transport disorders.

  11. Tumors initiated by constitutive Cdk2 activation exhibit transforming growth factor beta resistance and acquire paracrine mitogenic stimulation during progression

    DEFF Research Database (Denmark)

    Corsino, P.; Davis, B.; Law, M.;


    mediate some of the transforming effects that result from cyclin D1 overexpression in human breast cancers. MMTV-DIK2 cancer cells express the hepatocyte growth factor (HGF) receptor, c-Met. MMTV-D1K2 cancer cells also secrete transforming growth factor beta (TGF beta), but are relatively resistant to TGF......Cyclin D1/cyclin-dependent kinase 2 (Cdk2) complexes are present at high frequency in human breast cancer cell lines, but the significance of this observation is unknown. This report shows that expression of a cyclin D1-Cdk2 fusion protein under the control of the mouse mammary tumor virus (MMITV...... beta antiproliferative effects. Fibroblasts derived from MMTV-DIK2 tumors secrete factors that stimulate the proliferation of MMTV-D1K2 cancer cells, stimulate c-Met tyrosine phosphorylation, and stimulate the phosphorylation of the downstream signaling intermediates p70(s6k) and Akt on activating...

  12. In vitro activity of fosfomycin tromethamine against extended spectrum beta-lactamase producing urinary tract bacteria

    International Nuclear Information System (INIS)

    To determine the in vitro activity of Fosfomycin tromethamine against extended spectrum beta-lactamase producing uropathogens. Study Design: Experimental study. Place and Duration of Study: Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, from October 2011 to October 2012. Methodology: A total of 381 culture positive ESBL producing isolates from 2400 urine samples submitted over a period of one year were included in this study. Identification of isolates was done by standard biochemical profile of the organisms. The antimicrobial susceptibility of culture positive isolates was performed by disk diffusion method as recommended by Clinical Laboratory Standard Institute guidelines (CLSI). Results: The antimicrobial activity of Fosfomycin to various isolates revealed that 93% of E. coli, 64% Klebsiella spp. 50% Proteus spp. 75% Enterobacter cloacae, 100% Citrobacter freundii, 100% Burkholderia spp. 100% Serratia spp. and 50% Stenotrophomonas maltophilia were susceptible to this chemical compound. Conclusion: Fosfomycin showed excellent effectiveness to most of the common ESBL producing bacteria such as E. coli, Klebsiella and Proteus spp. (author)

  13. High-beta operation and magnetohydrodynamic activity on the TFTR tokamak

    Energy Technology Data Exchange (ETDEWEB)

    McGuire, K.; Arunasalam, V.; Barnes, C. W.; Bell, M. G.; Bitter, M.; Boivin, R.; Bretz, N. L.; Budny, R.; Bush, C. E.; Cavallo, A.; Chu, T. K.; Cohen, S. A.; Colestock, P.; Davis, S. L.; Dimock, D. L.; Dylla, H. F.; Efthimion, P. C.; Ehrhrardt, A. B.; Fonck, R. J.; Fredrickson, E.; Furth, H. P.; Gammel, G.; Goldston, R. J.; Greene, G.; Grek, B.; Grisham, L. R.; Hammett, G.; Hawryluk, R. J.; Hendel, H. W.; Hill, K. W.; Hinnov, E.; Hoffman, D. J.; Hosea, J.; Howell, R. B.; Hsuan, H.; Hulse, R. A.; Janos, A. C.; Jassby, D.; Jobes, F.; Johnson, D. W.; Johnson, L. C.; Kaita, R.; Kieras-Phillips, C.; Kilpatrick, S. J.; LaMarche, P. H.; LeBlanc, B.; Manos, D. M.; Mansfield, D. K.; Mazzucato, E.; McCarthy, M. P.; McCune, M. C.; McNeill, D. H.; Meade, D. M.; Medley, S. S.; Mikkelsen, D. R.; Monticello, D.; Motley, R.; Mueller, D.; Murphy, J. A.; Nagayama, Y.; Nazakian, D. R.; Neischmidt, E. B.; Owens, D. K.; Park, H.; Park, W.; Pitcher, S.; Ramsey, A. T.; Redi, M. H.; Roquemore, A. L.; Rutherford, P. H.; Schilling, G.; Schivell, J.; Schmidt, G. L.; Scott, S. D.; Sinnis, J. C.; Stevens, J.; Stratton, B. C.; Stodiek, W.; Synakowski, E. J.; Tang, W. M.; Taylor, G.; Timberlake, J. R.; Towner, H. H.; Ulrickson, M.; von Goeler, S.; Wieland, R.; Williams, M.; Wilson, J. R.; Wong, K.-L.; Yamada, M.; Yoshikawa, S.; Young, K. M.; Zarnstorff, M. C.; Zweben, S. J.


    Magnetohydrodynamic (MHD) activity within three zones (core, half-radius, and edge) of TFTR ( Plasma Physics and Controlled Nuclear Fusion Research 1986 (IAEA, Vienna, 1987), Vol. 1, p. 51) tokamak plasmas are discussed. Near the core of the plasma column, sawteeth are often observed. Two types of sawteeth are studied in detail; one with complete, and the other with incomplete, magnetic reconnection. Their characteristics are determined by the shape of the q profile. Near the half-radius the m/n =3/2 and 2/1 resistive ballooning modes are found to correlate with a beta collapse. The pressure and the pressure gradient at the mode rational surface are found to play an important role in stability. MHD activity is also studied at the plasma edge during limiter H modes. The edge localized modes (ELM's) are found to have a precursor mode with a frequency between 50--500 kHz and a mode number m/n=1/0. The mode does not show a ballooning structure. While these instabilities have been studied on many other machines, on TFTR the studies have been extended to high pressure (plasma pressure greater than 4 x 10⁵ Pa) and low collisionality ( vi*(a/2) < 0.002, ve* ( a/2)< 0.01).

  14. AMP-activated protein kinase (AMPK mediates nutrient regulation of thioredoxin-interacting protein (TXNIP in pancreatic beta-cells.

    Directory of Open Access Journals (Sweden)

    Maayan Shaked

    Full Text Available Thioredoxin-interacting protein (TXNIP regulates critical biological processes including inflammation, stress and apoptosis. TXNIP is upregulated by glucose and is a critical mediator of hyperglycemia-induced beta-cell apoptosis in diabetes. In contrast, the saturated long-chain fatty acid palmitate, although toxic to the beta-cell, inhibits TXNIP expression. The mechanisms involved in the opposing effects of glucose and fatty acids on TXNIP expression are unknown. We found that both palmitate and oleate inhibited TXNIP in a rat beta-cell line and islets. Palmitate inhibition of TXNIP was independent of fatty acid beta-oxidation or esterification. AMP-activated protein kinase (AMPK has an important role in cellular energy sensing and control of metabolic homeostasis; therefore we investigated its involvement in nutrient regulation of TXNIP. As expected, glucose inhibited whereas palmitate stimulated AMPK. Pharmacologic activators of AMPK mimicked fatty acids by inhibiting TXNIP. AMPK knockdown increased TXNIP expression in presence of high glucose with and without palmitate, indicating that nutrient (glucose and fatty acids effects on TXNIP are mediated in part via modulation of AMPK activity. TXNIP is transcriptionally regulated by carbohydrate response element-binding protein (ChREBP. Palmitate inhibited glucose-stimulated ChREBP nuclear entry and recruitment to the Txnip promoter, thereby inhibiting Txnip transcription. We conclude that AMPK is an important regulator of Txnip transcription via modulation of ChREBP activity. The divergent effects of glucose and fatty acids on TXNIP expression result in part from their opposing effects on AMPK activity. In light of the important role of TXNIP in beta-cell apoptosis, its inhibition by fatty acids can be regarded as an adaptive/protective response to glucolipotoxicity. The finding that AMPK mediates nutrient regulation of TXNIP may have important implications for the pathophysiology and treatment

  15. Determination of beta-glucosidase activity in soils with a bioanalytical sensor modified with multiwalled carbon nanotubes. (United States)

    Stege, Patricia W; Messina, Germán A; Bianchi, Guillermo; Olsina, Roberto A; Raba, Julio


    Soil microorganisms and enzymes are the primary mediators of soil biological processes, including organic matter degradation, mineralization, and nutrient recycling. They play an important role in maintaining soil ecosystem quality and functional diversity. Moreover, enzyme activities can provide an indication of quantitative changes in soil organic matter. Beta-glucosidase (beta-Glu) activity has been found to be sensitive to soil management and has been proposed as a soil quality indicator because it provides an early indication of changes in organic matter status and its turnover. The aims of the present study were to test and use a simple and convenient procedure for the assay of beta-Glu activity in agricultural soil. The method described here is based on the enzymatic degradation of cellobiose by beta-Glu present in the soil sample and the subsequent determination of glucose produced by the enzymatic reaction using screen-printed carbon electrodes modified with multiwalled carbon nanotubes (SPCE-CNT) equipped with coimmobilized glucose oxidase and horseradish peroxidase enzymes. The potential applied to the SPCE-CNT detection was -0.15 V versus a Ag/AgCl pseudo-reference electrode. A linear calibration curve was obtained in the range 2.7-11.3 mM with a correlation coefficient. In the present study, an easy and effective SPCE-CNT-modified electrode allowed an improved amperometric response to be achieved and this is attributed to the increased surface area upon electrode modification. PMID:20349226

  16. Maintained activity of glycogen synthase kinase-3{beta} despite of its phosphorylation at serine-9 in okadaic acid-induced neurodegenerative model

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Yong-Whan [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Yoon, Seung-Yong, E-mail: [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Choi, Jung-Eun [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Kim, Sang-Min [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Lee, Hui-Sun; Choe, Han [Department of Physiology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Lee, Seung-Chul [CrystalGenomics, Seoul (Korea, Republic of); Kim, Dong-Hou, E-mail: [Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul (Korea, Republic of)


    Glycogen synthase kinase-3{beta} (GSK3{beta}) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3{beta}. However, the inactive form of GSK3{beta} which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3{beta} substrates, such as {beta}-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly, OA also induces phosphorylation of GSK3{beta} at serine-9 and other substrates including tau, {beta}-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3{beta} inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3{beta} may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3{beta} inhibitors could be a valuable drug candidate in AD.

  17. Prolonged exposure of human beta cells to elevated glucose levels results in sustained cellular activation leading to a loss of glucose regulation.


    Z. Ling; Pipeleers, D G


    Human beta cells can be maintained in serum-free culture at 6 mmol/liter glucose, with 80% cell recovery and preserved glucose-inducible functions after 1 wk. Between 0 and 10 mmol/liter, glucose dose-dependently increases the number of beta cells in active protein synthesis (15% at 0 mmol/liter glucose, 60% at 5 mmol/liter, and 82% at 10 mmol/liter), while lacking such an effect in islet non-beta cells (> 75% activated irrespective of glucose concentrations). As in rat beta cells, this inter...

  18. Betacyanin accumulation and guaiacol peroxidase activity in Beta vulgaris L. leaves following copper stress

    Directory of Open Access Journals (Sweden)

    Janet M. León Morales


    Full Text Available The effect of copper stress on betacyanin accumulation and guaiacol peroxidase (GPOD activity in leaves of different age was evaluated in red beet (Beta vulgaris L. var. Crosby Egyptian plants. In hydroponic culture, plants were treated with 0.3 μM (control, 50 μM, 100 μM, and 250 μM of CuSO4 for 6 days. Copper was taken up and accumulated in old roots but was not translocated to leaves. However in young leaves, the increase of lipid peroxidation and reduction of growth were evident from day 3 of copper exposure; whereas in old leaves, the lipid peroxidation and growth were the same from either copper-treated or control plants. In response to copper exposure, the betacyanin accumulation was evident in young leaves by day 3, and continued to increase until day 6. Betacyanin only were accumulated in old leaves until day 6, but the contents were from 4 to 5 times lower than those observed in young leaves at the same copper concentrations. GPOD activity increased 3.3- and 1.4-fold in young and old leaves from day 3 of copper treatment respectively, but only in the young leaves was sustained at the same level until day 6. Old roots shown betacyanin in the control plants, but the betacyanin level and growth were reduced with the copper exposure. In contrast, young roots emerged by copper effect also accumulated copper and showed the highest betacyanin content of all plant parts assayed. These results indicate that betacyanin accumulation and GPOD activity are defense responses to copper stress in actively growing organs.

  19. Structure of human phytanoyl-CoA 2-hydroxylase identifies molecular mechanisms of Refsum disease. (United States)

    McDonough, Michael A; Kavanagh, Kathryn L; Butler, Danica; Searls, Timothy; Oppermann, Udo; Schofield, Christopher J


    Refsum disease (RD), a neurological syndrome characterized by adult onset retinitis pigmentosa, anosmia, sensory neuropathy, and phytanic acidaemia, is caused by elevated levels of phytanic acid. Many cases of RD are associated with mutations in phytanoyl-CoA 2-hydroxylase (PAHX), an Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the initial alpha-oxidation step in the degradation of phytenic acid in peroxisomes. We describe the x-ray crystallographic structure of PAHX to 2.5 A resolution complexed with Fe(II) and 2OG and predict the molecular consequences of mutations causing RD. Like other 2OG oxygenases, PAHX possesses a double-stranded beta-helix core, which supports three iron binding ligands (His(175), Asp(177), and His(264)); the 2-oxoacid group of 2OG binds to the Fe(II) in a bidentate manner. The manner in which PAHX binds to Fe(II) and 2OG together with the presence of a cysteine residue (Cys(191)) 6.7 A from the Fe(II) and two further histidine residues (His(155) and His(281)) at its active site distinguishes it from that of the other human 2OG oxygenase for which structures are available, factor inhibiting hypoxia-inducible factor. Of the 15 PAHX residues observed to be mutated in RD patients, 11 cluster in two distinct groups around the Fe(II) (Pro(173), His(175), Gln(176), Asp(177), and His(220)) and 2OG binding sites (Trp(193), Glu(197), Ile(199), Gly(204), Asn(269), and Arg(275)). PAHX may be the first of a new subfamily of coenzyme A-binding 2OG oxygenases.

  20. Cholesterol and bile acids regulate cholesterol 7 alpha-hydroxylase expression at the transcriptional level in culture and in transgenic mice.


    Ramirez, M.I.; Karaoglu, D; Haro, D; Barillas, C; Bashirzadeh, R; Gil, G.


    Cholesterol 7 alpha-hydroxylase (7 alpha-hydroxylase) is the rate-limiting enzyme in bile acid biosynthesis. It is subject to a feedback control, whereby high levels of bile acids suppress its activity, and cholesterol exerts a positive control. It has been suggested that posttranscriptional control plays a major part in that regulation. We have studied the mechanisms by which cholesterol and bile acids regulate expression of the 7 alpha-hydroxylase gene and found it to be solely at the trans...

  1. Cholesterol and bile acids regulate cholesterol 7 alpha-hydroxylase expression at the transcriptional level in culture and in transgenic mice. (United States)

    Ramirez, M I; Karaoglu, D; Haro, D; Barillas, C; Bashirzadeh, R; Gil, G


    Cholesterol 7 alpha-hydroxylase (7 alpha-hydroxylase) is the rate-limiting enzyme in bile acid biosynthesis. It is subject to a feedback control, whereby high levels of bile acids suppress its activity, and cholesterol exerts a positive control. It has been suggested that posttranscriptional control plays a major part in that regulation. We have studied the mechanisms by which cholesterol and bile acids regulate expression of the 7 alpha-hydroxylase gene and found it to be solely at the transcriptional level by using two different approaches. First, using a tissue culture system, we localized a liver-specific enhancer located 7 kb upstream of the transcriptional initiation site. We also showed that low-density lipoprotein mediates transcriptional activation of chimeric genes, containing either the 7 alpha-hydroxylase or the albumin enhancer in front of the 7 alpha-hydroxylase proximal promoter, to the same extent as the in vivo cholesterol-mediated regulation of 7 alpha-hydroxylase mRNA. In a second approach, using transgenic mice, we have found that expression of an albumin enhancer-7 alpha-hydroxylase-lacZ fusion gene is restricted to the liver and is regulated by cholesterol and bile acids in a manner quantitatively similar to that of the endogenous gene. We also found, that a liver-specific enhancer is necessary for expression of the rat 7 alpha-hydroxylase gene, in agreement with the tissue culture experiments. Together, these results demonstrate that cholesterol and bile acids regulate the expression of the 7 alpha-hydroxylase gene solely at the transcriptional level. PMID:8139578

  2. Intestinal lactase (beta-galactosidase) and other glycosidase activities in suckling and adult tammar wallabies (Macropus eugenii). (United States)

    Walcott, P J; Messer, M


    The activities of various glycosidases in homogenates of the small intestinal mucosa of two adult and 18 suckling tammar wallabies (M. eugenii) aged from 6 to 50 weeks were investigated. Lactase (beta-D-galactosidase), beta-N-acetylglucosaminidase, alpha-L-fucosidase and neuraminidase activities were high during the first 34 weeks post partum and then declined to very low levels. Maltase, isomaltase, sucrase and trehalase activities were very low or absent during the first 34 weeks, and then increased. The lactase activity was unusual in being greater in the distal than the middle or proximal thirds of the intestine, and in its low pH optimum (pH 4.6), inhibition by p-chloromercuribenzene sulfonate but not by Tris, and lack of cellobiase activity. These properties are those of a lysosomal acid beta-galactosidase rather than of a brush border neutral lactase. The maltase activity had the characteristics of a lysosomal acid alpha-glucosidase early in lactation and of a brush border neutral maltase in adult animals. The significance of these findings is discussed in relation to changes in dietary carbohydrates during weaning and to the mode of digestion of milk carbohydrates by the pouch young. PMID:6783021

  3. Association of the interleukin 1 beta gene and brain spontaneous activity in amnestic mild cognitive impairment

    Directory of Open Access Journals (Sweden)

    Zhuang Liying


    Full Text Available Abstract Purpose The inflammatory response has been associated with the pathogenesis of Alzheimer’s disease (AD. The purpose of this study is to determine whether the rs1143627 polymorphism of the interleukin-1 beta (IL-1β gene moderates functional magnetic resonance imaging (fMRI-measured brain regional activity in amnestic mild cognitive impairment (aMCI. Methods Eighty older participants (47 with aMCI and 33 healthy controls were recruited for this study. All of the participants were genotyped for variant rs1143627 in the IL1B gene and were scanned using resting-state fMRI. Brain activity was assessed by amplitude of low-frequency fluctuation (ALFF. Results aMCI patients had abnormal ALFF in many brain regions, including decreases in the inferior frontal gyrus, the superior temporal lobe and the middle temporal lobe, and increases in the occipital cortex (calcarine, parietal cortex (Pcu and cerebellar cortex. The regions associated with an interaction of group X genotypes of rs1143627 C/T were the parietal cortex (left Pcu, frontal cortex (left superior, middle, and medial gyrus, right anterior cingulum, occipital cortex (left middle lobe, left cuneus and the bilateral posterior lobes of the cerebellum. Regarding the behavioral significance, there were significant correlations between ALFF in different regions of the brain and with the cognitive scores of each genotype group. Conclusions The present study provided evidence that aMCI patients had abnormal ALFF in many brain regions. Specifically, the rs1143627 C/T polymorphism of the IL1B gene may modulate regional spontaneous brain activity in aMCI patients.

  4. Beat-induced fluctuations in auditory cortical beta-band activity: Using EEG to measure age-related changes

    Directory of Open Access Journals (Sweden)

    Laura K Cirelli


    Full Text Available People readily extract regularity in rhythmic auditory patterns, enabling prediction of the onset of the next beat. Recent magnetoencephalography (MEG research suggests that such prediction is reflected by the entrainment of oscillatory networks in the brain to the tempo of the sequence. In particular, induced beta-band oscillatory activity from auditory cortex decreases after each beat onset and rebounds prior to the onset of the next beat across tempi in a predictive manner. The objective of the present study was to examine the development of such oscillatory activity by comparing electroencephalography (EEG measures of beta-band fluctuations in 7-year-old children to adults. EEG was recorded while participants listened passively to isochronous tone sequences at three tempi (390, 585, and 780ms for onset-to-onset interval. In adults, induced power in the high beta-band (20-25 Hz decreased after each tone onset and rebounded prior to the onset of the next tone across tempo conditions, consistent with MEG findings. In children, a similar pattern was measured in the two slower tempo conditions, but was weaker in the fastest condition. The results indicate that the beta-band timing network works similarly in children, although there are age-related changes in consistency and the tempo range over which it operates.

  5. Beta vulgaris subspecies cicla var. flavescens (Swiss chard): flavonoids, hepatoprotective and hypolipidemic activities. (United States)

    Hashem, A N; Soliman, M S; Hamed, M A; Swilam, N F; Lindequist, U; Nawwar, M A


    The novel flavonoids, 2",2"'-di-O-α-rhamnopyranosyl-vicenin II, a di-C-glycosyl flavone, and herbacetin 3-O-β-xylopyranosyl- (1"' --> 2")-O-β-glucopyranoside, were isolated from the leaves of Beta vulgaris subspecies cicla L. var. flavescens, an edible plant which is consumed in the Mediterranean areas, additional to the known flavonoids, 6-C-glucosyl isoscutellarein, vitexin-(1"' --> 2")-O-β-xylopyranosyl, vitexin-(1'" --> 2")-O-α-rhamnopyranosyI and vitexin. All metabolites were established by conventional methods of analysis and their structures were confirmed by spectroscopic analysis, including 1 D and 2D-NMR and by HR-ESIMS, as well. The extract of the plant leaves shows hepatoprotective effects in rats intoxicated by administration of acetaminophen and exhibits hypolipidemic activity in rats with high-fat-diet induced hypercholesterolemia. The evaluation was done through measuring the liver function enzymes (aspartate and alanine aminotransferases and alkaline phosphatase, the lipid profile (total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol and triglycerides) and histopathological analysis of liver slides. PMID:27209705

  6. Beta-adrenergic receptor sensitivity, autonomic balance and serotonergic activity in practitioners of Transcendental Meditation

    Energy Technology Data Exchange (ETDEWEB)

    Hill, D.A.


    The aim of this thesis was to investigate the acute autonomic effects of the Transcendental Meditation Program (TM) and resolve the conflict arising from discrepant neurochemical and psychophysiological data. Three experimental investigations were performed. The first examined beta{sub 2}-adrenergic receptors (AR's) on peripheral blood lymphocytes, via (I{sup 125})iodocyanopindolol binding, in 10 male mediating and 10 age matched non-meditating control subjects, to test the hypothesis that the long-term practice of TM and the TM Sidhi Program (TMSP) reduces end organ sensitivity to adrenergic agonists. The second investigated respiratory sinus arrhythmia (an indirect measure of cardiac Parasympathetic Nervous System tone), and skin resistance (a measure of Sympathetic Nervous System tone) during periods of spontaneous respiratory apneusis, a phenomenon occurring during TM that is known to mark the subjective experience of transcending. The third was within subject investigation of the acute effects of the TMSP on 5-hydroxytryptamine (5-HT) activity. Platelet 5-HT was assayed by high pressure liquid chromatography with electrochemical detection, plasma prolactin (PL) and lutenizing hormone (LH) by radioimmunoassay, tryptophan by spectrofluorimetry, and alpha-1-acid glycoprotein (AGP, a modulator of 5-HT uptake) by radial immunodiffusion assay.

  7. Beta-receptor activation increases sodium current in guinea pig heart

    Institute of Scientific and Technical Information of China (English)

    Hong-wei WANG; Zhi-fang YANG; Yin ZHANG; Jian-min YANG; Yuan-mou LIU; Ci-zhen LI


    Aim: To study the influence of β-receptor activation on sodium channel current and the physiological significance of increased sodium current with regard to the increased cardiac output caused by sympathetic excitation.Methods: Multiple experimental approaches, including ECG, action potential recording with conventional microelectrodes, whole-cell current measurements, single-channel recordings, and pumping-force measurements, were applied to guinea pig hearts and isolated ventricular myocytes.Results: Isoprenaline was found to dose-dependently shorten QRS waves, increase the amplitude and the Vmaxof action potentials, aug-ment the fast sodium current, and increase the occurrence frequencies and open time constants of the long-open and burst modes of the sodium channel. Increased levels of membrane-permeable cAMP have similar effects. In the presence of a calcium channel blocker, TTX reversed the increased pumping force produced by isoprenaline.Conclusion: Beta-adrenergic modulation increases the inward sodium current and accelerates the conduction velocity within the ventri-cles by changing the sodium channel modes, which might both be conducive to the synchronous contraction of the heart and enhance its pumping function.

  8. TGF-beta 1 Gene-Activated Matrices Regulated the Osteogenic Differentiation of BMSCs

    Institute of Scientific and Technical Information of China (English)


    Poly (lactic acid/glycolic acid/asparagic acid-co-polyethylene glycol)(PLGA-[ASP-PEG]) scaffold materials were linked with a novel nonviral vector (K)16GRGDSPC through cross linker Sulfo-LC-SPDP to construct a new type of nonviral gene transfer system. Eukaryotic expressing vector containing transforming growth factor beta 1 (pcDNA3-TGFβ1) was encapsulated by the system. Bone marrow stromal cells (BMSCs) obtained from rabbit were cultured on PLGA-[ASP-PEG] modified by (K)16GRGDSPC and TGF-β1 gene and PLGA-[ASP-PEG] modified by (K)16GRGDSPC and empty vector pcDNA3 as control.The expressions of osteogenic makers of the BMSCs cultured on the TGF-β1 gene-activated scaffold materials were found significantly higher than those of the control group (P<0.05). A brand-new way was provided for regulating seed cells to directionally differentiate into osteoblasts for bone defect restoration in bone tissue engineering.

  9. Mechanistic studies of cancer cell mitochondria- and NQO1-mediated redox activation of beta-lapachone, a potentially novel anticancer agent

    International Nuclear Information System (INIS)

    Beta-lapachone (beta-Lp) derived from the Lapacho tree is a potentially novel anticancer agent currently under clinical trials. Previous studies suggested that redox activation of beta-Lp catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1) accounted for its killing of cancer cells. However, the exact mechanisms of this effect remain largely unknown. Using chemiluminescence and electron paramagnetic resonance (EPR) spin-trapping techniques, this study for the first time demonstrated the real-time formation of ROS in the redox activation of beta-lapachone from cancer cells mediated by mitochondria and NQO1 in melanoma B16–F10 and hepatocellular carcinoma HepG2 cancer cells. ES936, a highly selective NQO1 inhibitor, and rotenone, a selective inhibitor of mitochondrial electron transport chain (METC) complex I were found to significantly block beta-Lp meditated redox activation in B16–F10 cells. In HepG2 cells ES936 inhibited beta-Lp-mediated oxygen radical formation by ∼ 80% while rotenone exerted no significant effect. These results revealed the differential contribution of METC and NQO1 to beta-lapachone-induced ROS formation and cancer cell killing. In melanoma B16–F10 cells that do not express high NQO1 activity, both NOQ1 and METC play a critical role in beta-Lp redox activation. In contrast, in hepatocellular carcinoma HepG2 cells expressing extremely high NQO1 activity, redox activation of beta-Lp is primarily mediated by NQO1 (METC plays a minor role). These findings will contribute to our understanding of how cancer cells are selectively killed by beta-lapachone and increase our ability to devise strategies to enhance the anticancer efficacy of this potentially novel drug while minimizing its possible adverse effects on normal cells. - Highlights: • Both isolated mitochondria and purified NQO1 are able to generate ROS by beta-Lp. • The differential roles of mitochondria and NQO1 in mediating redox activation of beta-Lp • In cancer cells with

  10. Mechanistic studies of cancer cell mitochondria- and NQO1-mediated redox activation of beta-lapachone, a potentially novel anticancer agent

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jason Z. [Virginia Tech CRC, Blacksburg, VA (United States); Ke, Yuebin [Shenzhen Center for Disease Control and Prevention, Shenzhen 518055 (China); Misra, Hara P. [Virginia Tech CRC, Blacksburg, VA (United States); Trush, Michael A. [Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD (United States); Li, Y. Robert [Campbell University School of Osteopathic Medicine, Buies Creek, NC (United States); Virginia Tech-Wake Forest University SBES, Blacksburg, VA (United States); Department of Biology, University of North Carolina at Greensboro, NC (United States); Zhu, Hong, E-mail: [Campbell University School of Osteopathic Medicine, Buies Creek, NC (United States); Jia, Zhenquan, E-mail: [Department of Biology, University of North Carolina at Greensboro, NC (United States)


    Beta-lapachone (beta-Lp) derived from the Lapacho tree is a potentially novel anticancer agent currently under clinical trials. Previous studies suggested that redox activation of beta-Lp catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1) accounted for its killing of cancer cells. However, the exact mechanisms of this effect remain largely unknown. Using chemiluminescence and electron paramagnetic resonance (EPR) spin-trapping techniques, this study for the first time demonstrated the real-time formation of ROS in the redox activation of beta-lapachone from cancer cells mediated by mitochondria and NQO1 in melanoma B16–F10 and hepatocellular carcinoma HepG2 cancer cells. ES936, a highly selective NQO1 inhibitor, and rotenone, a selective inhibitor of mitochondrial electron transport chain (METC) complex I were found to significantly block beta-Lp meditated redox activation in B16–F10 cells. In HepG2 cells ES936 inhibited beta-Lp-mediated oxygen radical formation by ∼ 80% while rotenone exerted no significant effect. These results revealed the differential contribution of METC and NQO1 to beta-lapachone-induced ROS formation and cancer cell killing. In melanoma B16–F10 cells that do not express high NQO1 activity, both NOQ1 and METC play a critical role in beta-Lp redox activation. In contrast, in hepatocellular carcinoma HepG2 cells expressing extremely high NQO1 activity, redox activation of beta-Lp is primarily mediated by NQO1 (METC plays a minor role). These findings will contribute to our understanding of how cancer cells are selectively killed by beta-lapachone and increase our ability to devise strategies to enhance the anticancer efficacy of this potentially novel drug while minimizing its possible adverse effects on normal cells. - Highlights: • Both isolated mitochondria and purified NQO1 are able to generate ROS by beta-Lp. • The differential roles of mitochondria and NQO1 in mediating redox activation of beta-Lp • In cancer cells with

  11. Modeling clustered activity increase in amyloid-beta positron emission tomographic images with statistical descriptors

    Directory of Open Access Journals (Sweden)

    Shokouhi S


    Full Text Available Sepideh Shokouhi,1 Baxter P Rogers,1 Hakmook Kang,2 Zhaohua Ding,1 Daniel O Claassen,3 John W Mckay,1 William R Riddle1On behalf of the Alzheimer’s Disease Neuroimaging Initiative1Department of Radiology and Radiological Sciences, Vanderbilt University Institute of Imaging Science, 2Department of Biostatistics, 3Department of Neurology, Vanderbilt University, Nashville, TN, USABackground: Amyloid-beta (Aβ imaging with positron emission tomography (PET holds promise for detecting the presence of Aβ plaques in the cortical gray matter. Many image analyses focus on regional average measurements of tracer activity distribution; however, considerable additional information is available in the images. Metrics that describe the statistical properties of images, such as the two-point correlation function (S2, have found wide applications in astronomy and materials science. S2 provides a detailed characterization of spatial patterns in images typically referred to as clustering or flocculence. The objective of this study was to translate the two-point correlation method into Aβ-PET of the human brain using 11C-Pittsburgh compound B (11C-PiB to characterize longitudinal changes in the tracer distribution that may reflect changes in Aβ plaque accumulation.Methods: We modified the conventional S2 metric, which is primarily used for binary images and formulated a weighted two-point correlation function (wS2 to describe nonbinary, real-valued PET images with a single statistical function. Using serial 11C-PiB scans, we calculated wS2 functions from two-dimensional PET images of different cortical regions as well as three-dimensional data from the whole brain. The area under the wS2 functions was calculated and compared with the mean/median of the standardized uptake value ratio (SUVR. For three-dimensional data, we compared the area under the wS2 curves with the subjects’ cerebrospinal fluid measures.Results: Overall, the longitudinal changes in wS2

  12. Rhodocytin (aggretin) activates platelets lacking alpha(2)beta(1) integrin, glycoprotein VI, and the ligand-binding domain of glycoprotein Ibalpha

    DEFF Research Database (Denmark)

    Bergmeier, W; Bouvard, D; Eble, J A;


    Although alpha(2)beta(1) integrin (glycoprotein Ia/IIa) has been established as a platelet collagen receptor, its role in collagen-induced platelet activation has been controversial. Recently, it has been demonstrated that rhodocytin (also termed aggretin), a snake venom toxin purified from...... that collagen may activate platelets by a similar mechanism. In contrast to these findings, we provided evidence that rhodocytin does not bind to alpha(2)beta(1) integrin. Here we show that the Cre/loxP-mediated loss of beta(1) integrin on mouse platelets has no effect on rhodocytin-induced platelet activation......, excluding an essential role of alpha(2)beta(1) integrin in this process. Furthermore, proteolytic cleavage of the 45-kDa N-terminal domain of glycoprotein (GP) Ibalpha either on normal or on beta(1)-null platelets had no significant effect on rhodocytin-induced platelet activation. Moreover, mouse platelets...

  13. Synthesis of 16 alpha-/sub 3/H androgen and estrogen substrates for 16 alpha-hydroxylase

    Energy Technology Data Exchange (ETDEWEB)

    Cantineau, R.; Kremers, P.; De Graeve, J.; Cornelis, A.; Laszlo, P.; Gielen, J.E.; Lambotte, R.


    The synthesis of 16 alpha-/sup 3/H androgens and estrogens is described. 1-(/sup 3/H)-Acetic acid in the presence of zinc dust reacts with 16 alpha-bromo-17-ketosteroids to produce 16 alpha-/sup 3/H-17-ketosteroids. This chemical reaction was used to prepare 16 alpha-/sup 3/H-dehydroepiandrosterone (I) and 16 alpha-/sub 4/H-estrone acetate (XI) from 16 alpha-bromo-dehydroepiandrosterone (X) and from 16 alpha-bromo-estrone acetate (XII), respectively. Using appropriate microbiological techniques, it was possible to convert these radiolabelled substrates into 16 alpha-/sup 3/H-androstenedione (II) and 16 alpha-/sup 3/H-estradiol-17 beta (VII). 16 alpha-/sup 3/H-Estrone (VI) was obtained by the chemical hydrolysis of 16 alpha-/sup 3/H-estrone acetate. The label distribution as determined by microbiological 16 alpha-hydroxylations indicated a specific labelling of 77% for androgens and 65% for estrogens in the 16 alpha position. These substrates can be used for measuring the 16 alpha hydroxylase activity, an important step in the biosynthesis of estriol (VIII) and estetrol (IX).

  14. Sequential changes in chromatin structure during transcriptional activation in the beta globin LCR and its target gene. (United States)

    Kim, Kihoon; Kim, AeRi


    Chromatin structure is modulated during transcriptional activation. The changes include the association of transcriptional activators, formation of hypersensitive sites and covalent modifications of histones. To understand the order of the various changes accompanying transcriptional activation, we analyzed the mouse beta globin gene, which is transcriptionally inducible in erythroid MEL cells over a time course of HMBA treatment. Transcription of the globin genes requires the locus control region (LCR) consisting of several hypersensitive sites (HSs). Erythroid specific transcriptional activators such as NF-E2, GATA-1, TAL1 and EKLF were associated with the LCR in the uninduced state before transcriptional activation. The HSs of the LCR were formed in this state as revealed by high sensitivity to DNase I and MNase attack. However the binding of transcriptional activators and the depletion of histones were observed in the promoter of the beta globin gene only after transcriptional activation. In addition, various covalent histone modifications were sequentially detected in lysine residues of histone H3 during the activation. Acetylation of K9, K36 and K27 was notable in both LCR HSs and gene after induction but before transcriptional initiation. Inactive histone marks such as K9me2, K36me2 and K27me2 were removed coincident with transcriptional initiation in the gene region. Taken together, these results indicate that LCR has a substantially active structure in the uninduced state while transcriptional activation serially adds active marks, including histone modifications, and removes inactive marks in the target gene of the LCR.

  15. Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition (United States)

    Chang, Ching Ching; Few, Ling Ling; Konrad, Manfred; See Too, Wei Cun


    Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the ckβ gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme’s activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKβ by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis. PMID:27149373

  16. A novel dimeric thymosin beta 4 with enhanced activities accelerates the rate of wound healing

    Directory of Open Access Journals (Sweden)

    Xu TJ


    Full Text Available Tian-Jiao Xu,1,2,* Qi Wang,1,* Xiao-Wen Ma,1 Zhen Zhang,3 Wei Zhang,1 Xiao-Chang Xue,1 Cun Zhang,1 Qiang Hao,1 Wei-Na Li,1 Ying-Qi Zhang,1 Meng Li11State Key Laboratory of Cancer Biology, Biotechnology Center, School of Pharmacy, Fourth Military Medical University, Xi’an, People’s Republic of China; 2The Institute of Medicine, Qiqihar Medical University, Qiqihar, People’s Republic of China; 3Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, USA*These authors contributed equally to this workObjective: Thymosin beta 4 (Tβ4 is a peptide with 43 amino acids that is critical for repair and remodeling tissues on the skin, eye, heart, and neural system following injury. To fully realize its utility as a treatment for disease caused by injury, the authors constructed a cost-effective novel Tβ4 dimer and demonstrated that it was better able to accelerate tissue repair than native Tβ4.Methods: A prokaryotic vector harboring two complete Tβ4 genes with a short linker was constructed and expressed in Escherichia coli. A pilot-scale fermentation (10 L was performed to produce engineered bacteria and the Tβ4 dimer was purified by one-step hydrophobic interaction chromatography. The activities of the Tβ4 dimer to promote endothelial cell proliferation, migration, and sprouting were assessed by tetramethylbenzidine (methylthiazol tetrazolium, trans-well, scratch, and tube formation assays. The ability to accelerate dermal healing was assessed on rats.Results: After fermentation, the Tβ4 dimer accounted for about 30% of all the bacteria proteins. The purity of the Tβ4 dimer reached 98% after hydrophobic interaction chromatography purification. An average of 562.4 mg/L Tβ4 dimer was acquired using a 10 L fermenter. In each assay, the dimeric Tβ4 exhibited enhanced activities compared with native Tβ4. Notably, the ability of the dimeric Tβ4 to promote cell migration was almost two times higher

  17. Stimulation of Na{sup +}/K{sup +} ATPase activity and Na{sup +} coupled glucose transport by {beta}-catenin

    Energy Technology Data Exchange (ETDEWEB)

    Sopjani, Mentor [Department of Physiology, University of Tuebingen (Germany); Department of Chemistry, University of Prishtina, Kosovo (Country Unknown); Alesutan, Ioana; Wilmes, Jan [Department of Physiology, University of Tuebingen (Germany); Dermaku-Sopjani, Miribane [Department of Physiology, University of Tuebingen (Germany); Faculty of Medicine, University of Prishtina, Kosovo (Country Unknown); Lam, Rebecca S. [Department of Physiology, University of Tuebingen (Germany); Department of Molecular Neurogenetics, Max Planck Institute of Biophysics, Frankfurt/Main (Germany); Koutsouki, Evgenia [Department of Physiology, University of Tuebingen (Germany); Jakupi, Muharrem [Faculty of Medicine, University of Prishtina, Kosovo (Country Unknown); Foeller, Michael [Department of Physiology, University of Tuebingen (Germany); Lang, Florian, E-mail: [Department of Physiology, University of Tuebingen (Germany)


    Research highlights: {yields} The oncogenic transcription factor {beta}-catenin stimulates the Na{sup +}/K{sup +}-ATPase. {yields} {beta}-Catenin stimulates SGLT1 dependent Na{sup +}, glucose cotransport. {yields} The effects are independent of transcription. {yields} {beta}-Catenin sensitive transport may contribute to properties of proliferating cells. -- Abstract: {beta}-Catenin is a multifunctional protein stimulating as oncogenic transcription factor several genes important for cell proliferation. {beta}-Catenin-regulated genes include the serum- and glucocorticoid-inducible kinase SGK1, which is known to stimulate a variety of transport systems. The present study explored the possibility that {beta}-catenin influences membrane transport. To this end, {beta}-catenin was expressed in Xenopus oocytes with or without SGLT1 and electrogenic transport determined by dual electrode voltage clamp. As a result, expression of {beta}-catenin significantly enhanced the ouabain-sensitive current of the endogeneous Na{sup +}/K{sup +}-ATPase. Inhibition of vesicle trafficking by brefeldin A revealed that the stimulatory effect of {beta}-catenin on the endogenous Na{sup +}/K{sup +}-ATPase was not due to enhanced stability of the pump protein in the cell membrane. Expression of {beta}-catenin further enhanced glucose-induced current (Ig) in SGLT1-expressing oocytes. In the absence of SGLT1 Ig was negligible irrespective of {beta}-catenin expression. The stimulating effect of {beta}-catenin on both Na{sup +}/K{sup +} ATPase and SGLT1 activity was observed even in the presence of actinomycin D, an inhibitor of transcription. The experiments disclose a completely novel function of {beta}-catenin, i.e. the regulation of transport.

  18. BETA-S, Multi-Group Beta-Ray Spectra

    International Nuclear Information System (INIS)

    1 - Description of program or function: BETA-S calculates beta-decay source terms and energy spectra in multigroup format for time-dependent radionuclide inventories of actinides, fission products, and activation products. Multigroup spectra may be calculated in any arbitrary energy-group structure. The code also calculates the total beta energy release rate from the sum of the average beta-ray energies as determined from the spectral distributions. BETA-S also provides users with an option to determine principal beta-decaying radionuclides contributing to each energy group. The CCC-545/SCALE 4.3 (or SCALE4.2) code system must be installed on the computer before installing BETA-S, which requires the SCALE subroutine library and nuclide-inventory generation from the ORIGEN-S code. 2 - Methods:Well-established models for beta-energy distributions are used to explicitly represent allowed, and 1., 2. - and 3. -forbidden transition types. Forbidden non-unique transitions are assumed to have a spectral shape of allowed transitions. The multigroup energy spectra are calculated by numerically integrating the energy distribution functions using an adaptive Simpson's Rule algorithm. Nuclide inventories are obtained from a binary interface produced by the ORIGEN-S code. BETA-S calculates the spectra for all isotopes on the binary interface that have associated beta-decay transition data in the ENSDF-95 library, developed for the BETA-S code. This library was generated from ENSDF data and contains 715 materials, representing approximately 8500 individual beta transition branches. 3 - Restrictions on the complexity of the problem: The algorithms do not treat positron decay transitions or internal conversion electrons. The neglect of positron transitions in inconsequential for most applications involving aggregate fission products, since most of the decay modes are via electrons. The neglect of internal conversion electrons may impact on the accuracy of the spectrum in the low

  19. Chemoprotective activity of boldine: modulation of drug-metabolizing enzymes. (United States)

    Kubínová, R; Machala, M; Minksová, K; Neca, J; Suchý, V


    Possible chemoprotective effects of the naturally occurring alkaloid boldine, a major alkaloid of boldo (Peumus boldus Mol.) leaves and bark, including in vitro modulations of drug-metabolizing enzymes in mouse hepatoma Hepa-1 cell line and mouse hepatic microsomes, were investigated. Boldine manifested inhibition activity on hepatic microsomal CYP1A-dependent 7-ethoxyresorufin O-deethylase and CYP3A-dependent testosterone 6 beta-hydroxylase activities and stimulated glutathione S-transferase activity in Hepa-1 cells. In addition to the known antioxidant activity, boldine could decrease the metabolic activation of other xenobiotics including chemical mutagens. PMID:11265593

  20. A connective tissue disorder caused by mutations of the lysyl hydroxylase 3 gene. (United States)

    Salo, Antti M; Cox, Helen; Farndon, Peter; Moss, Celia; Grindulis, Helen; Risteli, Maija; Robins, Simon P; Myllylä, Raili


    Lysyl hydroxylase 3 (LH3, encoded by PLOD3) is a multifunctional enzyme capable of catalyzing hydroxylation of lysyl residues and O-glycosylation of hydroxylysyl residues producing either monosaccharide (Gal) or disaccharide (Glc-Gal) derivatives, reactions that form part of the many posttranslational modifications required during collagen biosynthesis. Animal studies have confirmed the importance of LH3, particularly in biosynthesis of the highly glycosylated type IV and VI collagens, but to date, the functional significance in vivo of this enzyme in man is predominantly unknown. We report here a human disorder of LH3 presenting as a compound heterozygote with recessive inheritance. One mutation dramatically reduced the sugar-transfer activity of LH3, whereas another abrogated lysyl hydroxylase activity; these changes were accompanied by reduced LH3 protein levels in cells. The disorder has a unique phenotype causing severe morbidity as a result of features that overlap with a number of known collagen disorders. PMID:18834968

  1. Molecular cloning and expression of gene fragments from corynebacteriophage beta encoding enzymatically active peptides of diphtheria toxin.


    Tweten, R K; Collier, R J


    Two restriction fragments from corynebacteriophage beta vir tox+ that encode peptides similar to diphtheria toxin fragment A and the chain termination fragment, CRM45, have been cloned into Escherichia coli in plasmid pBR322. Clones containing the recombinant plasmids produced gene products that were active in catalyzing the ADP ribosylation of elongation factor 2 and were reactive with diphtheria toxin antiserum. Toxin-related peptides were found primarily in the periplasmic compartment and ...

  2. Transcription factor activating protein 2 beta (TFAP2B) mediates noradrenergic neuronal differentiation in neuroblastoma. (United States)

    Ikram, Fakhera; Ackermann, Sandra; Kahlert, Yvonne; Volland, Ruth; Roels, Frederik; Engesser, Anne; Hertwig, Falk; Kocak, Hayriye; Hero, Barbara; Dreidax, Daniel; Henrich, Kai-Oliver; Berthold, Frank; Nürnberg, Peter; Westermann, Frank; Fischer, Matthias


    Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2'-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma. PMID:26598443

  3. Magneto-Hydrodynamic Activity and Energetic Particles - Application to Beta Alfven Eigenmodes

    International Nuclear Information System (INIS)

    The goal of magnetic fusion research is to extract the power released by fusion reactions and carried by the product of these reactions, liberated at energies of the order of a few MeV. The feasibility of fusion energy production relies on our ability to confine these energetic particles, while keeping the thermonuclear plasma in safe operating conditions. For that purpose, it is necessary to understand and find ways to control the interaction between energetic particles and the thermonuclear plasma. Reaching these two goals is the general motivation for this work. More specifically, our focus is on one type of instability, the Beta Alfven Eigenmode (BAE), which can be driven by energetic particles and impact on the confinement of both energetic and thermal particles. In this work, we study the characteristics of BAEs analytically and derive its dispersion relation and structure. Next, we analyze the linear stability of the mode in the presence of energetic particles. First, a purely linear description is used, which makes possible to get an analytical linear criterion for BAE destabilization in the presence of energetic particles. This criterion is compared with experiments conducted in the Tore-Supra tokamak. Secondly, because the linear analysis reveals some features of the BAE stability which are subject to a strong nonlinear modification, the question is raised of the possibility of a sub-critical activity of the mode. We propose a simple scenario which makes possible the existence of meta-stable modes, verified analytically and numerically. Such a scenario is found to be relevant to the physics and scales characterizing BAEs. (author)

  4. Inhibition of phosphorylated tyrosine hydroxylase attenuates ethanol-induced hyperactivity in adult zebrafish (Danio rerio). (United States)

    Nowicki, Magda; Tran, Steven; Chatterjee, Diptendu; Gerlai, Robert


    Zebrafish have been successfully employed in the study of the behavioural and biological effects of ethanol. Like in mammals, low to moderate doses of ethanol induce motor hyperactivity in zebrafish, an effect that has been attributed to the activation of the dopaminergic system. Acute ethanol exposure increases dopamine (DA) in the zebrafish brain, and it has been suggested that tyrosine hydroxylase, the rate-limiting enzyme of DA synthesis, may be activated in response to ethanol via phosphorylation. The current study employed tetrahydropapaveroline (THP), a selective inhibitor of phosphorylated tyrosine hydroxylase, for the first time, in zebrafish. We treated zebrafish with a THP dose that did not alter baseline motor responses to examine whether it can attenuate or abolish the effects of acute exposure to alcohol (ethanol) on motor activity, on levels of DA, and on levels of dopamine's metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). We found that 60-minute exposure to 1% alcohol induced motor hyperactivity and an increase in brain DA. Both of these effects were attenuated by pre-treatment with THP. However, no differences in DOPAC levels were found among the treatment groups. These findings suggest that tyrosine hydroxylase is activated via phosphorylation to increase DA synthesis during alcohol exposure in zebrafish, and this partially mediates alcohol's locomotor stimulant effects. Future studies will investigate other potential candidates in the molecular pathway to further decipher the neurobiological mechanism that underlies the stimulatory properties of this popular psychoactive drug.

  5. 24-Hydroxylase in Cancer: Impact on Vitamin D-based Anticancer Therapeutics (United States)

    Luo, Wei; Hershberger, Pamela A.; Trump, Donald L.; Johnson, Candace S.


    The active vitamin D hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in regulating calcium homeostasis and bone mineralization. 1,25(OH)2D3 also modulates cellular proliferation and differentiation in a variety of cell types. 24-hydroxylase, encoded by the CYP24A1 gene, is the key enzyme which converts 1,25(OH)2D3 to less active calcitroic acid. Nearly all cell types express 24-hydroxylase, the highest activity being observed in the kidney. There is increasing evidence linking the incidence and prognosis of certain cancers to low serum 25 (OH)D3 levels and high expression of vitamin D 24-hydroxylase supporting the idea that elevated CYP24A1 expression may stimulate degradation of vitamin D metabolites including 25-(OH)D3 and 1,25(OH)2D3. The over expression of CYP24A1 in cancer cells may be a factor affecting 1,25(OH)2D3 bioavailability and anti-proliferative activity pre-clinically and clinically. The combination of 1,25(OH)2D3 with CYP24A1 inhibitors enhances 1,25(OH)2D3 mediated signaling and anti-proliferative effects and may be useful in overcoming effects of aberrant CYP24 expression. PMID:23059474

  6. Lysyl Hydroxylase 3 Glucosylates Galactosylhydroxylysine Residues in Type I Collagen in Osteoblast Culture*


    Sricholpech, Marnisa; Perdivara, Irina; Nagaoka, Hideaki; Yokoyama, Megumi; Tomer, Kenneth B.; Yamauchi, Mitsuo


    Lysyl hydroxylase 3 (LH3), encoded by Plod3, is the multifunctional collagen-modifying enzyme possessing LH, hydroxylysine galactosyltransferase (GT), and galactosylhydroxylysine-glucosyltransferase (GGT) activities. Although an alteration in type I collagen glycosylation has been implicated in several osteogenic disorders, the role of LH3 in bone physiology has never been investigated. To elucidate the function of LH3 in bone type I collagen modifications, we used a short hairpin RNA technol...

  7. Inducibility of aryl hydrocarbon hydroxylase in BALB/c/ki mice exposed to urban air pollution. (United States)

    Mostardi, R A; Ely, D L; Liebelt, A; Grossman, S; Fu, M M


    In two separate experiments BALB/c/ki mice were exposed to urban air pollution. Mice exposed to clean air served as controls. In both experiments there were no obvious quantitative or qualitative differences in lung or liver tissue examined by light microscopy. In both experiments higher aryl hydrocarbon hydroxylase activities and higher trace metal concentrations were observed in the mice exposed to polluted urban air. These data are interpreted in terms of health hazards of urban air pollutants. PMID:7265310

  8. Inducibility of aryl hydrocarbon hydroxylase in BALB/c/Ki mice exposed to urban air pollution

    Energy Technology Data Exchange (ETDEWEB)

    Mostardi, R.A. (Univ. of Akron, OH); Ely, D.L.; Liebelt, A.; Grossman, S.; Fu, M.M.


    In two separate experiments BALB/c/Kl mice were exposed to urban air pollution. Mice exposed to clean air served as controls. In both experiments there were no obvious quantitative or qualitative differences in lung or liver tissue examined by light microscopy. In both experiments higher aryl hydrocarbon hydroxylase activities and higher trace metal concentrations were observed in the mice exposed to polluted urban air. These data are interpreted in terms of health hazards of urban air pollutants.

  9. Synthesis and antitumor activity of 2-. beta. -D-ribofuranosylselenazole-4-carboxamide and related derivatives

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, P.C.; Robins, R.K.


    Treatment of 2,3,5-tri-O-benzoyl-..beta..-D-ribofuranosyl-1-carbonitrile with hydrogen selenide provided 2,5-anhydro-3,4,6-tri-O-benzoyl-D-allonselenoamide (3). Compound 3 was treated with ethyl bromopyruvate to provide ethyl 2-(2,3,5-tri-O-benzoyl-D-ribofuranosyl)selenazole-4-carboxylates, which after ammonolysis were converted to 2-..beta..-D-ribofuranosylselenazole-4-carboxamide (6) and its ..cap alpha..-analogue 7, respectively. Acetylation of nucleoside 6 provided 2-(2,3,5-tri-O-acetyl-..beta..-D-ribofuranosyl)selenazole-4-carboxamide, and phosphorylation of 6 provided the corresponding 5'-phosphate 9. Compounds 6 and 9 were found to be cytotoxic toward P388 and L1210 cells in culture and effective against Lewis lung carcinoma in mice.

  10. Glucose amplifies fatty acid-induced endoplasmic reticulum stress in pancreatic beta-cells via activation of mTORC1.

    Directory of Open Access Journals (Sweden)

    Etti Bachar

    Full Text Available BACKGROUND: Palmitate is a potent inducer of endoplasmic reticulum (ER stress in beta-cells. In type 2 diabetes, glucose amplifies fatty-acid toxicity for pancreatic beta-cells, leading to beta-cell dysfunction and death. Why glucose exacerbates beta-cell lipotoxicity is largely unknown. Glucose stimulates mTORC1, an important nutrient sensor involved in the regulation of cellular stress. Our study tested the hypothesis that glucose augments lipotoxicity by stimulating mTORC1 leading to increased beta-cell ER stress. PRINCIPAL FINDINGS: We found that glucose amplifies palmitate-induced ER stress by increasing IRE1alpha protein levels and activating the JNK pathway, leading to increased beta-cell apoptosis. Moreover, glucose increased mTORC1 activity and its inhibition by rapamycin decreased beta-cell apoptosis under conditions of glucolipotoxicity. Inhibition of mTORC1 by rapamycin did not affect proinsulin and total protein synthesis in beta-cells incubated at high glucose with palmitate. However, it decreased IRE1alpha expression and signaling and inhibited JNK pathway activation. In TSC2-deficient mouse embryonic fibroblasts, in which mTORC1 is constitutively active, mTORC1 regulated the stimulation of JNK by ER stressors, but not in response to anisomycin, which activates JNK independent of ER stress. Finally, we found that JNK inhibition decreased beta-cell apoptosis under conditions of glucolipotoxicity. CONCLUSIONS/SIGNIFICANCE: Collectively, our findings suggest that mTORC1 mediates glucose amplification of lipotoxicity, acting through activation of ER stress and JNK. Thus, mTORC1 is an important transducer of ER stress in beta-cell glucolipotoxicity. Moreover, in stressed beta-cells mTORC1 inhibition decreases IRE1alpha protein expression and JNK activity without affecting ER protein load, suggesting that mTORC1 regulates the beta-cell stress response to glucose and fatty acids by modulating the synthesis and activity of specific

  11. Calcium has a permissive role in interleukin-1beta-induced c-jun N-terminal kinase activation in insulin-secreting cells

    DEFF Research Database (Denmark)

    Størling, Joachim; Zaitsev, Sergei V; Kapelioukh, Iouri L;


    -acetoxymethyl], an inhibitor of calmodulin (W7), and inhibitors of Ca(2+)/calmodulin-dependent kinase (KN62 and KN93) partially reduced IL-1beta-stimulated c-jun phosphorylation in INS-1 or betaTC3 cells. Our data suggest that Ca(2+) plays a permissive role in IL-1beta activation of the JNK signaling pathway in insulin......-cells are largely unknown. In this study, we investigated whether Ca(2+) plays a role for IL-1beta-induced JNK activation. In insulin-secreting rat INS-1 cells cultured in the presence of 11 mm glucose, combined pharmacological blockade of L- and T-type Ca(2+) channels suppressed IL-1beta-induced in vitro...

  12. Event-Related Beta EEG Changes During Active, Passive Movement and Functional Electrical Stimulation of the Lower Limb. (United States)

    Qiu, Shuang; Yi, Weibo; Xu, Jiapeng; Qi, Hongzhi; Du, Jingang; Wang, Chunfang; He, Feng; Ming, Dong


    A number of electroencephalographic (EEG) studies have reported on event-related desynchronization/synchronization (ERD/ERS) during active movements, passive movements, and the movements induced by functional electrical stimulation (FES). However, the quantitative differences in ERD values and affected frequency bands associated with the lower limb have not been discussed. The goal of this paper was to quantitatively compare the ERD patterns during active movement, passive movement and FES-induced movement of the lower limb. 64-channel EEG signals were recorded to investigate the brain oscillatory patterns during active movement, passive movement and FES-induced movement of the lower limb in twelve healthy subjects. And passive movement and FES-induced movement were also performed in a hemiplegic stroke patient. For healthy subjects, FES-induced movement presented significantly higher characteristic frequency of central beta ERD while there was no significant difference in ERD values compared with active or passive movement. Meanwhile, beta ERD values of FES-induced movement were significantly correlated with those of active movement, and spatial distribution of beta ERD pattern for FES-induced movement was more correlated with that for active movement. In addition, the stroke patient presented central ERD patterns during FES-induced movement, while no ERD with similar frequencies could be found during passive movement. This work implies that the EEG oscillatory pattern under FES-induced movement tends more towards active movement instead of passive movement. The quantification of ERD patterns could be expected as a potential technique to evaluate the brain response during FES-induced movement. PMID:26441422

  13. Purification, characterization, and directed evolution study of a vitamin D3 hydroxylase from Pseudonocardia autotrophica

    International Nuclear Information System (INIS)

    Vitamin D3 (VD3) is a fat-soluble prohormone that plays a crucial role in bone metabolism, immunity, and control of cell proliferation and cell differentiation in mammals. The actinomycete Pseudonocardia autotrophica is capable of bioconversion of VD3 into its physiologically active forms, namely, 25(OH)VD3 or 1α,25(OH)2VD3. In this study, we isolated and characterized Vdh (vitamin D3 hydroxylase), which hydroxylates VD3 from P. autotrophica NBRC 12743. The vdh gene encodes a protein containing 403 amino acids with a molecular weight of 44,368 Da. This hydroxylase was found to be homologous with the P450 belonging to CYP107 family. Vdh had the same ratio of the Vmax values for VD3 25-hydroxylation and 25(OH)VD3 1α-hydroxylation, while other enzymes showed preferential regio-specific hydroxylation on VD3. We characterized a collection of Vdh mutants obtained by random mutagenesis and obtained a Vdh-K1 mutant by the combination of four amino acid substitutions. Vdh-K1 showed one-order higher VD3 25-hydroxylase activity than the wild-type enzyme. Biotransformation of VD3 into 25(OH)VD3 was successfully accomplished with a Vdh-expressed recombinant strain of actinobacterium Rhodococcus erythropolis. Vdh may be a useful enzyme for the production of physiologically active forms of VD3 by a single cytochrome P450.

  14. Phagocytosis of haemozoin (malarial pigment enhances metalloproteinase-9 activity in human adherent monocytes: Role of IL-1beta and 15-HETE

    Directory of Open Access Journals (Sweden)

    Giribaldi Giuliana


    Full Text Available Abstract Background It has been shown previously that human monocytes fed with haemozoin (HZ or trophozoite-parasitized RBCs displayed increased matrix metalloproteinase-9 (MMP-9 enzyme activity and protein/mRNA expression and increased TNF production, and showed higher matrix invasion ability. The present study utilized the same experimental model to analyse the effect of phagocytosis of: HZ, delipidized HZ, beta-haematin (lipid-free synthetic HZ and trophozoites on production of IL-1beta and MMP-9 activity and expression. The second aim was to find out which component of HZ was responsible for the effects. Methods Native HZ freshly isolated from Plasmodium falciparum (Palo Alto strain, Mycoplasma-free, delipidized HZ, beta-haematin (lipid-free synthetic HZ, trophozoites and control meals such as opsonized non-parasitized RBCs and inert latex particles, were fed to human monocytes. The production of IL-1beta by differently fed monocytes, in presence or absence of specific MMP-9 inhibitor or anti-hIL-1beta antibodies, was quantified in supernatants by ELISA. Expression of IL-1beta was analysed by quantitative real-time RT-PCR. MMP-9 activity and protein expression were quantified by gelatin zymography and Western blotting. Results Monocytes fed with HZ or trophozoite-parasitized RBCs generated increased amounts of IL-1beta and enhanced enzyme activity (in cell supernatants and protein/mRNA expression (in cell lysates of monocyte MMP-9. The latter appears to be causally related to enhanced IL-1beta production, as enhancement of both expression and enzyme activity were abrogated by anti-hIL-1beta Abs. Upregulation of IL-1beta and MMP-9 were absent in monocytes fed with beta-haematin or delipidized HZ, indicating a role for HZ-attached or HZ-generated lipid components. 15-HETE (15(S,R-hydroxy-6,8,11,13-eicosatetraenoic acid a potent lipoperoxidation derivative generated by HZ from arachidonic acid via haem-catalysis was identified as one mediator

  15. Measuring phospholipase D activity in insulin-secreting pancreatic beta-cells and insulin-responsive muscle cells and adipocytes. (United States)

    Cazzolli, Rosanna; Huang, Ping; Teng, Shuzhi; Hughes, William E


    Phospholipase D (PLD) is an enzyme producing phosphatidic acid and choline through hydrolysis of phosphatidylcholine. The enzyme has been identified as a member of a variety of signal transduction cascades and as a key regulator of numerous intracellular vesicle trafficking processes. A role for PLD in regulating glucose homeostasis is emerging as the enzyme has recently been identified in events regulating exocytosis of insulin from pancreatic beta-cells and also in insulin-stimulated glucose uptake through controlling GLUT4 vesicle exocytosis in muscle and adipose tissue. We present methodologies for assessing cellular PLD activity in secretagogue-stimulated insulin-secreting pancreatic beta-cells and also insulin-stimulated adipocyte and muscle cells, two of the principal insulin-responsive cell types controlling blood glucose levels. PMID:19160674

  16. Measuring phospholipase D activity in insulin-secreting pancreatic beta-cells and insulin-responsive muscle cells and adipocytes. (United States)

    Cazzolli, Rosanna; Huang, Ping; Teng, Shuzhi; Hughes, William E


    Phospholipase D (PLD) is an enzyme producing phosphatidic acid and choline through hydrolysis of phosphatidylcholine. The enzyme has been identified as a member of a variety of signal transduction cascades and as a key regulator of numerous intracellular vesicle trafficking processes. A role for PLD in regulating glucose homeostasis is emerging as the enzyme has recently been identified in events regulating exocytosis of insulin from pancreatic beta-cells and also in insulin-stimulated glucose uptake through controlling GLUT4 vesicle exocytosis in muscle and adipose tissue. We present methodologies for assessing cellular PLD activity in secretagogue-stimulated insulin-secreting pancreatic beta-cells and also insulin-stimulated adipocyte and muscle cells, two of the principal insulin-responsive cell types controlling blood glucose levels.

  17. Conclusion on the peer review of the pesticide risk assessment of the active substance beta-cypermethrin

    Directory of Open Access Journals (Sweden)

    European Food Safety Authority


    Full Text Available The conclusions of the European Food Safety Authority (EFSA following the peer review of the initial risk assessments carried out by the competent authority of the rapporteur Member State the United Kingdom, for the pesticide active substance beta-cypermethrin are reported. The context of the peer review was that required by Commission Regulation (EU No 188/2011. The conclusions were reached on the basis of the evaluation of the representative uses of beta-cypermethrin as an insecticide on oilseed rape, wheat and maize. The reliable endpoints concluded as being appropriate for use in regulatory risk assessment, derived from the available studies and literature in the dossier peer reviewed, are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are identified as regards the risk to aquatic organisms, bees and non-target arthropods.

  18. Shedding of Clostridium difficile, fecal beta-lactamase activity, and gastrointestinal symptoms in 51 volunteers treated with oral cefixime. (United States)

    Chachaty, E; Bourneix, C; Renard, S; Bonnay, M; Andremont, A


    Microbial changes including the shedding of Clostridium difficile, fecal beta-lactamase activity, and gastrointestinal symptoms were assessed in 51 healthy volunteers given 200 mg of cefixime twice daily for 8 days. The number of organisms of the family Enterobacteriaceae (means +/- standard deviations) dropped from 6.9 +/- 1.1 to 3.9 +/- 1.8 log CFU/g of feces (P < 0.01), whereas counts of enterococci rose from 7.0 +/- 1.5 to 9.0 +/- 1.0 log CFU/g of feces (P < 0.01). Both counts returned to their initial levels 50 days after the cessation of treatment. Cefixime did not significantly modify the frequency of fecal excretion of Pseudomonas aeruginosa, Staphylococcus spp., yeasts, or members of the Enterobacteriaceae resistant to ceftazidime or ampicillin. The proportion of subjects shedding C. difficile rose from 6% before treatment to 57% (P < 0.01) at the end of treatment but returned to 8% 50 days thereafter. No case of pseudomembranous colitis was observed. Stool changes occurred in 13 volunteers during treatment (25%) and in 2 others more than 10 days after the end of treatment (4%). These changes were not significantly associated with the shedding of toxigenic strains of C. difficile or with the presence of toxin A in feces. By contrast, during treatment, stool changes occurred in 8 of the 18 volunteers (44%) who had antibiotic activity in their feces but in only 5 of the 33 (15%) for whom no such activity was found (P < 0.05). The absence of antibiotic activity in the feces was itself linked with the presence of beta-lactamase activity in the feces. Since we had found earlier that fecal beta-lactamase activity afforded protection against alteration in stool consistency during treatments with oral cephalosporins, the present study confirmed our previous preliminary results in this respect. PMID:8363371

  19. Segmented Ge detector rejection of internal beta activity produced by neutron irradiation (United States)

    Varnell, L. S.; Callas, J. L.; Mahoney, W. A.; Pehl, R. H.; Landis, D. A.


    Future Ge spectrometers flown in space to observe cosmic gamma-ray sources will incorporate segmented detectors to reduce the background from radioactivity produced by energetic particle reactions. To demonstrate the effectiveness of a segmented Ge detector in rejecting background events due to the beta decay of internal radioactivity, a laboratory experiment has been carried out in which radioactivity was produced in the detector by neutron irradiation. A Cf-252 source of neutrons was used to produce, by neutron capture on Ge-74 (36.5 percent of natural Ge) in the detector itself, Ge-75 (t sub 1/2 = 82.78 min), which decays by beta emission with a maximum electron kinetic energy of 1188 keV. By requiring that an ionizing event deposit energy in two or more of the five segments of the detector, each about 1-cm thick, the beta particles, which have a range of about 1-mm, are rejected, while most external gamma rays incident on the detector are counted. Analysis of this experiment indicates that over 85 percent of the beta events from the decay of Ge-75 are rejected, which is in good agreement with Monte Carlo calculations.

  20. Identification of bacteria with beta-galactosidase activity in faeces from lactase non-persistent subjects

    NARCIS (Netherlands)

    Tao, H; Priebe, MG; Vonk, RJ; Welling, GW


    Previous studies suggest that, besides the maldigestion of lactose in the small intestine, the colonic processing of lactose might play a role in lactose intolerance. beta-Galactosidase is the bacterial enzyme which catalyzes the first step of lactose fermentation in the colon. We propose a practica

  1. Design of a Quality Control Program for the Measurement of Gross Alpha and Gross Beta Activities (LMPR-CIEMAT); Diseno del Control de Calidad de las Medidas de Actividad Alfa-Beta Total (LMPR-CIEMAT)

    Energy Technology Data Exchange (ETDEWEB)

    Alvarez, A.; Yague, L.; Gasco, C.; Navarro, N.; Higueras, E.; Noguerales, C.


    In accordance with international standards, general requirements for testing laboratories have to include a quality system for planning, implementing, and assessing the work performed by the organization and for carrying out required quality assurance and quality control. The purpose of internal laboratory quality control is to monitor performance, identify problems, and initiate corrective actions. This report describes the internal quality control to monitor the gross alpha and beta activities determination. Identification of specific performance indicators, the principles that govern their use and statistical means of evaluation are explained. Finally, calculation of alpha and beta specific activities, uncertainties and detection limits are performed. (Author) 10 refs.

  2. A tyrosine hydroxylase assay in microwells using coupled nonenzymatic decarboxylation of dopa

    Energy Technology Data Exchange (ETDEWEB)

    Bostwick, J.R.; Le, W.D. (Department of Neurology, Baylor College of Medicine, Houston, Texas (USA))


    A radiometric assay for tyrosine hydroxylase employing a coupled nonenzymatic decarboxylation of L-{sup 14}CDopa formed from L-{sup 14}Ctyrosine has been adapted for performance in a 96 microwell culture plate. The method uses an easily manufactured plate holder to compress blotting paper impregnated with methylbenzethonium hydroxide against the top rim of each well. This forms isolated, airtight compartments in which 14CO2 is evolved and quantitatively absorbed into the blotting paper. The method is sensitive enough to detect the production of less than 5 pmol of 14CO2. A major advantage of this system is that cells can be grown in tissue culture and subsequently assayed for tyrosine hydroxylase activity in the same well. The method is more facile than previously devised procedures, allowing for the simultaneous assay of up to 96 samples totally contained in a single, compact, portable unit.

  3. Enzymic synthesis of alpha- and beta-D-glucosides of 1-deoxynojirimycin and their glycosidase inhibitory activities. (United States)

    Asano, N; Oseki, K; Kaneko, E; Matsui, K


    1-Deoxynojirimycin (1) is a potent inhibitor of mammalian and rice alpha-glucosidase. Several glucosides of 1 were synthesized by use of the native and immobilized enzyme and their effect on various enzymes was investigated. Transglucosylation reactions using rice alpha-glucosidase, yeast alpha- and beta-glucosidases purified from Rhodotorula lactosa were performed with maltose or cellobiose as a glucose donor and N-(benzyloxycarbonyl)-1-deoxynojirimycin (2) as an acceptor. The transglucosylation reaction using native rice alpha-glucosidase afforded 3-O-alpha-D-glucopyranosyl-N-(benzyloxycarbonyl)-1-deoxynojirimycin (4), 4-O-alpha-D-glucopyranosyl-N-(benzyloxycarbonyl)-1-deoxynojirimycin (5), and 2-O-alpha-D-glucopyranosyl-N-(benzyloxycarbonyl)-1-deoxynojirimycin (3) in yields of 40, 13, and 2%, respectively, after 30 min. The transglucosylation reaction using immobilized rice alpha-glucosidase was similar to that using the native enzyme. In the system using native yeast alpha-glucosidase, 3, 5, and 4 were formed in yields of 34, 13, and 6%, respectively, after 15 h. The immobilization of yeast alpha-glucosidase caused a significant decrease in transglucosylation activity. Yeast beta-glucosidase showed a high transglucosylation activity and incubation with the reaction system afforded 2-O-beta-D-glucopyranosyl-N-(benzyloxycarbonyl)-1-deoxynojirimycin (6) and 4-O-beta-D-glucopyranosyl-N-(benzyloxycarbonyl)-1-deoxynojirimycin (7) in yields of 69 and 3%, respectively, after 3 h. The transglucosylation reaction using immobilized yeast beta-glucosidase preferentially afforded 6 in a yield of 73% after 3 h. After removal of N-benzyloxycarbonyl group from the product glucosides, their glycosidase inhibitory activities were measured. 3-O-alpha-D-Glucopyranosyl-1-deoxynojirimycin (9) retained the potent inhibition of 1 against rat intestinal sucrase activity and was more effective than 1 against rice alpha-glucosidase. 4-O-alpha-D-Glucopyranosyl-1-deoxynojirimycin (10

  4. Activated endothelial interleukin-1beta, -6, and -8 concentrations and intercellular adhesion molecule-1 expression are attenuated by lidocaine.

    LENUS (Irish Health Repository)

    Lan, Wei


    Endothelial cells play a key role in ischemia reperfusion injury. We investigated the effects of lidocaine on activated human umbilical vein endothelial cell (HUVEC) interleukin (IL)-1beta, IL-6, and IL-8 concentrations and intercellular adhesion molecule-1 (ICAM-1) expression. HUVECs were pretreated with different concentrations of lidocaine (0 to 0.5 mg\\/mL) for 60 min, thereafter tumor necrosis factor-alpha was added at a concentration of 2.5 ng\\/mL and the cells incubated for 4 h. Supernatants were harvested, and cytokine concentrations were analyzed by enzyme-linked immunosorbent assay. Endothelial ICAM-1 expression was analyzed by using flow cytometry. Differences were assessed using analysis of variance and post hoc unpaired Student\\'s t-test where appropriate. Lidocaine (0.5 mg\\/mL) decreased IL-1beta (1.89 +\\/- 0.11 versus 4.16 +\\/- 1.27 pg\\/mL; P = 0.009), IL-6 (65.5 +\\/- 5.14 versus 162 +\\/- 11.5 pg\\/mL; P < 0.001), and IL-8 (3869 +\\/- 785 versus 14,961 +\\/- 406 pg\\/mL; P < 0.001) concentrations compared with the control. IL-1beta, IL-6, and IL-8 concentrations in HUVECs treated with clinically relevant plasma concentrations of lidocaine (0.005 mg\\/mL) were similar to control. ICAM-1 expression on lidocaine-treated (0.05 mg\\/mL) HUVECs was less than on controls (198 +\\/- 52.7 versus 298 +\\/- 50.3; Mean Channel Fluorescence; P < 0.001). Activated endothelial IL-1beta, IL-6, and IL-8 concentrations and ICAM-1 expression are attenuated only by lidocaine at concentrations larger than clinically relevant concentrations.

  5. Structural, Functional and Evolutionary Studies on Prolyl-Hydroxylases


    Scotti, John Salvatore; Christopher J. Schofield


    This thesis studies the prolyl-hydroxylase family of 2-oxoglutarate dependent oxygenases from structural, functional and evolutionary perspectives. The role of prolyl-hydroxylation was first identified in collagen, wherein hydroxyproline was found to stabilise the collagen triple helix. In the 1960s, the presence of hydroxyproline in collagen was found to be a result of enzyme catalysed protein modification. An enzyme, now known as collagen prolyl-4-hydroxylase (CP4H), was found to be com...

  6. Development of Rapid Radiochemical Method for Gross Alpha and Gross Beta Activity Concentration in Flowback and Produced Waters from Hydraulic Fracturing Operations (United States)

    This report summarizes the development and validation of an improved method for the Determination of Gross Alpha and Gross Beta Activity in Flowback and Produced Waters from Hydraulic Fracturing Operations (FPWHFO). Flowback and produced waters are characterized by high concentra...

  7. Measuring the orientation of taurine in the active site of the non-heme Fe(II)/α-ketoglutarate-dependent taurine hydroxylase (TauD) using electron spin echo envelope modulation (ESEEM) spectroscopy. (United States)

    Casey, Thomas M; Grzyska, Piotr K; Hausinger, Robert P; McCracken, John


    The position and orientation of taurine near the non-heme Fe(II) center of the α-ketoglutarate (α-KG)-dependent taurine hydroxylase (TauD) was measured using Electron Spin Echo Envelope Modulation (ESEEM) spectroscopy. TauD solutions containing Fe(II), α-KG, and natural abundance taurine or specifically deuterated taurine were prepared anaerobically and treated with nitric oxide (NO) to make an S = 3/2 {FeNO}(7) complex that is suitable for robust analysis with EPR spectroscopy. Using ratios of ESEEM spectra collected for TauD samples having natural abundance taurine or deuterated taurine, (1)H and (14)N modulations were filtered out of the spectra and interactions with specific deuterons on taurine could be studied separately. The Hamiltonian parameters used to calculate the amplitudes and line shapes of frequency spectra containing isolated deuterium ESEEM were obtained with global optimization algorithms. Additional statistical analysis was performed to validate the interpretation of the optimized parameters. The strongest (2)H hyperfine coupling was to a deuteron on the C1 position of taurine and was characterized by an effective dipolar distance of 3.90 ± 0.25 Å from the {FeNO}(7) paramagnetic center. The principal axes of this C1-(2)H hyperfine coupling and nuclear quadrupole interaction tensors were found to make angles of 26 ± 5 and 52 ± 17°, respectively, with the principal axis of the {FeNO}(7) zero-field splitting tensor. These results are discussed within the context of the orientation of substrate taurine prior to the initiation of hydrogen abstraction. PMID:23937570

  8. Measuring the orientation of taurine in the active site of the non-heme Fe(II)/α-ketoglutarate-dependent taurine hydroxylase (TauD) using electron spin echo envelope modulation (ESEEM) spectroscopy. (United States)

    Casey, Thomas M; Grzyska, Piotr K; Hausinger, Robert P; McCracken, John


    The position and orientation of taurine near the non-heme Fe(II) center of the α-ketoglutarate (α-KG)-dependent taurine hydroxylase (TauD) was measured using Electron Spin Echo Envelope Modulation (ESEEM) spectroscopy. TauD solutions containing Fe(II), α-KG, and natural abundance taurine or specifically deuterated taurine were prepared anaerobically and treated with nitric oxide (NO) to make an S = 3/2 {FeNO}(7) complex that is suitable for robust analysis with EPR spectroscopy. Using ratios of ESEEM spectra collected for TauD samples having natural abundance taurine or deuterated taurine, (1)H and (14)N modulations were filtered out of the spectra and interactions with specific deuterons on taurine could be studied separately. The Hamiltonian parameters used to calculate the amplitudes and line shapes of frequency spectra containing isolated deuterium ESEEM were obtained with global optimization algorithms. Additional statistical analysis was performed to validate the interpretation of the optimized parameters. The strongest (2)H hyperfine coupling was to a deuteron on the C1 position of taurine and was characterized by an effective dipolar distance of 3.90 ± 0.25 Å from the {FeNO}(7) paramagnetic center. The principal axes of this C1-(2)H hyperfine coupling and nuclear quadrupole interaction tensors were found to make angles of 26 ± 5 and 52 ± 17°, respectively, with the principal axis of the {FeNO}(7) zero-field splitting tensor. These results are discussed within the context of the orientation of substrate taurine prior to the initiation of hydrogen abstraction.

  9. Active Stability Control of a High-Beta Self-Organized Compact Torus

    International Nuclear Information System (INIS)

    Full text: A magnetized coaxial plasma gun (MCPG) has been proposed as an effective device for control of a high-beta self-organized compact torus of field-reversed configuration (FRC). The initial results demonstrate that the application of an MCPG suppresses the most prominent FRC instability of the centrifugally-driven interchange mode with toroidal mode number n = 2. This observation was made on the Nihon University Compact Torus Experiment (NUCTE), a flexible theta-pinch-based FRC facility. In the series of experiments, a MCPG generates a spheromak-like plasmoid which can then travel axially to merge with a pre-existing FRC. Since the MCPG is mounted on-axis and generates a significant helicity, it provides the FRC-relevant version of coaxial helicity injection (CHI) that has been applied in both spheromaks and spherical tokamaks. When CHI is applied, the onset of elliptical deformation of FRC cross-section is delayed until 45 - 50 μs from FRC formation compared to the onset time of 25 μs in the case without CHI. Besides delaying instability, MCPG application reduces the toroidal rotation frequency from 67 kHz to 41 kHz. Moreover, the flux decay time is extended from 57 to 67 μs. These changes have been made despite the quite modest flux content of the plasmoid: ∼0.05 mWb of poloidal and 0.01 mWb of toroidal flux, compared with the 0.4 mWb of poloidal flux in the pre-formed FRC. The observed global stabilization and confinement improvements suggest that the MCPG can actively control the rotational instability. This global instability can also be suppressed by externally applied static multipole fields. However, it has been known that nonaxisymmetric multipole fields have adverse effects on confinement. This indicates an advantage of MCPG in that it shows both improved confinement and stability. The conventional technique does not slow the toroidal rotation down. Therefore, MCPG introduces a different stabilization mechanism that may be the same as that

  10. Antibacterial Activity of Some Plant Extracts Against Extended- Spectrum Beta-Lactamase Producing Escherichia coli Isolates (United States)

    Saeidi, Saeide; Amini Boroujeni, Negar; Ahmadi, Hassan; Hassanshahian, Mehdi


    Background: The extended-spectrum beta-lactamase (ESBL) -producing Escherichia coli isolates make many serious infections, especially urinary tract infections. Objectives: The purpose of this study was to determine the antibacterial activities of some natural plant extracts against ESBL-producing E. coli isolates, which harbor the TEM gene in urine samples of the patients who have urinary tract infections. Materials and Methods: Evaluation has to be exactly determined for both methods of disk diffusion test and polymerase chain reaction (PCR), separately. We evaluated 120 strains of E. coli isolates from the urine culture of the patients in Boo-Ali Hospital (Zahedan, south-eastern Iran) who were suffering from urinary tract infections. The ESBL-producing E. coli isolates were evaluated by disk diffusion test and PCR through TEM gene detection. The minimal inhibitory concentration (MIC) of commonly used antibiotics including ceftazidime, ceftriaxon, amikacin, gentamicin and ciprofloxacin along with the MIC of the alcoholic extract of different natural plants including Myrtus communis L (Myrtaceae), Amaranthus retraflexus (Amaranthaceae), Cyminum cuminum L (Apiaceae), Marrubium vulgare (Laminaceae) and Peganum. harmala (Zygrophyllaceae) against the ESBL-producing E. coli isolates, which harbor the TEM genes, were determined using the microdulition method. Results: Results of this study showed that in disk diffusion method, 80 samples of E. coli produced ESBLs. In PCR method, the TEM gene distribution in the isolated ESBL-producing organisms was 50 (41.6%). Amikacin was the most effective anti-bacterial agent and ciprofloxacin was the least effective against E. coli isolates. All the natural plant extracts mentioned above, especially P. harmala, were effective against the selected isolates of ESBL-producing E. coli. The most frequent ESBL rate producing E. coli isolates (32 out of 50) had MIC of 2.5 mg/mL in ethanol extract of P. harmala. Conclusions: The alcoholic

  11. Tyrosine Hydroxylase as a Target for Deltamethrin in the Nigrostriatal Dopaminergic Pathway

    Institute of Scientific and Technical Information of China (English)


    Objective To study the effects of deltamethrin on tyrosine hydroxylase in nigrostriatum of male rats. Methods Sprague-Dawley rats were daily treated with deltamethrin at 6.25 or 12.5 mg/kg body weight by gavage for 10 days. Then HPLC-fluorescence detection was used to analyze the contents of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homoranillic acid (HVA) in substantial nigra and striatum. The activities of tyrosine hydroxylase (TH) were also detected by HPLC-fluorescence detection. TH mRNA or TH protein levels were measured by RT-PCR and immunohistochemistry method. Results The content of DA in striatum was significantly decreased by the treatments, suggesting an inhibition of DA synthesis by deltamethrin. The contents of DA metabolites DOPAC and HVA increased, indicating increased dopamine turnover. Furthermore, deltamethrin significantly decreased the activity, as well as the mRNA and protein levels of TH.Conclusions These findings reveal a novel aspect of deltamethrin neurotoxicity and suggest tyrosine hydroxylase as a molecular target of deltamethin on dopamine metabolism in the nigrostriatal pathway.

  12. A revised model for AMP-activated protein kinase structure: The alpha-subunit binds to both the beta- and gamma-subunits although there is no direct binding between the beta- and gamma-subunits. (United States)

    Wong, Kelly A; Lodish, Harvey F


    The 5'-AMP-activated protein kinase (AMPK) is a master sensor for cellular metabolic energy state. It is activated by a high AMP/ATP ratio and leads to metabolic changes that conserve energy and utilize alternative cellular fuel sources. The kinase is composed of a heterotrimeric protein complex containing a catalytic alpha-subunit, an AMP-binding gamma-subunit, and a scaffolding beta-subunit thought to bind directly both the alpha- and gamma-subunits. Here, we use coimmunoprecipitation of proteins in transiently transfected cells to show that the alpha2-subunit binds directly not only to the beta-subunit, confirming previous work, but also to the gamma1-subunit. Deletion analysis of the alpha2-subunit reveals that the C-terminal 386-552 residues are sufficient to bind to the beta-subunit. The gamma1-subunit binds directly to the alpha2-subunit at two interaction sites, one within the catalytic domain consisting of alpha2 amino acids 1-312 and a second within residues 386-552. Binding of the alpha2 and the gamma1-subunits was not affected by 400 mum AMP or ATP. Furthermore, we show that the beta-subunit C terminus is essential for binding to the alpha2-subunit but, in contrast to previous work, the beta-subunit does not bind directly to the gamma1-subunit. Taken together, this study presents a new model for AMPK heterotrimer structure where through its C terminus the beta-subunit binds to the alpha-subunit that, in turn, binds to the gamma-subunit. There is no direct interaction between the beta- and gamma-subunits.

  13. An application of LSC method for the measurement of gross alpha and beta activities in spiked water and drinking water samples

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    Çakal Gaye Özgür


    Full Text Available In this study, after the pulse shape calibration of a liquid scintillation counting (LSC spectrometer (Quantulus 1220, the effi ciency was determined depending on sample quenching parameters. Then, gross alpha and beta activities in two spiked water samples obtained from International Atomic Energy Agency (IAEA were used for the validation of the ASTM D7283-06 method, which is a standard test method for alpha and beta activity in water by LSC. Later, the drinking water samples (35 tap water and 9 bottled water obtained from different districts of Ankara, Turkey, were measured. The maximum gross alpha activities are measured to be 0.08 Bq/L for tap waters and 0.13 Bq/L for bottled waters, whereas the maximum gross beta activities are found to be 0.18 Bq/L for tap waters and 0.16 Bq/L for bottled waters. These results indicate that these drinking water samples are below the required limits, which are 0.1 Bq/L for alpha emitting radionuclides and 1 Bq/L for beta emitting radionuclides. As a result, gross alpha and beta activities in drinking water of Ankara were determined accurately by this validated LSC method. It is also worth noting that LSC is a rapid and accurate method for the determination of gross alpha and beta activities without requiring a tedious sample preparation.

  14. E6 and E7 oncoproteins from human papillomavirus type 16 induce activation of human transforming growth factor beta1 promoter throughout Sp1 recognition sequence. (United States)

    Peralta-Zaragoza, Oscar; Bermúdez-Morales, Víctor; Gutiérrez-Xicotencatl, Lourdes; Alcocer-González, Juan; Recillas-Targa, Félix; Madrid-Marina, Vicente


    Human Papillomavirus (HPV) infection is the main etiologic agent of cervical cancer and HPV E6 and E7 oncogenes trans-regulate many cellular genes. An association between TGF-beta1 gene expression and cervical cancer development has been suggested; however, the mechanisms by which HPV influences TGF-beta1 expression remain unclear. In the present study we analyzed the mechanism through which HPV-16 E6 and E7 oncoproteins regulate the TGF-beta1 promoter in cervical tumor cells. Our results showed that E6 and E7 increased TGF-beta1 promoter activity. Furthermore, we identified a specific DNA sequence motif in the TGF-beta1 core promoter that is responsible for trans-activation and that corresponds to the Sp1e-binding site associated with HPV-16 E6 and E7 oncoproteins. Mutational analysis showed that the Sp1e recognition site abolished the trans-activation caused by E6 and E7. These results suggest a physical interaction and functional cooperation between viral oncoproteins and cellular regulatory elements of the TGF-beta1 promoter, and may explain the contribution of HPV-16 to TGF-beta1 gene expression in cervical cancer. PMID:16987065

  15. Xalpha-DVM investigation of double water molecule interactions with active sites of alpha- and beta-subunits of hemoglobin (United States)

    Yuryeva, Elmira I.

    In this work, the results of Xalpha-discrete variation method calculations of the electronic structure and interatomic parameters of chemical bonding between iron (II) and oxygen molecule with and without extra electrons and protons in active site (AS) of alpha- and beta-subunits of oxyhemoglobin are presented. The Skulachev model of O2 molecule existing in respiration medium in the 2H2O form was used. The introduction of extra electrons does not change considerably the interaction of the iron atom with the O2 oxygen molecule, but strengthens the repulsion in the Fe bond N bonds. In this case, the estimated effective charge of the iron atom is +1.8/1.5e for AS of alpha-/beta-subunits of oxyhemoglobin, and the magnetic moment of iron atoms becomes zero. The deoxygenation effect of the AS of the alpha- and beta-subunits of oxyhemoglobin is due to the ability of extra protons to break down covalent attraction between the iron atom and the nearest oxygen atom and also to weakening of the repulsive component of the covalent Fe bond N interactions.

  16. The role of 14-3-3{beta} in transcriptional activation of estrogen receptor {alpha} and its involvement in proliferation of breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yoonseo; Kim, Hyungjin; Jang, Sung-Wuk [School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of); Ko, Jesang, E-mail: [School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of)


    Highlights: {yields} 14-3-3{beta} interacts with ER{alpha} and the interaction is Akt-dependent. {yields} 14-3-3{beta} regulates the transcriptional activity of ER{alpha} in a ligand-dependent manner. {yields} 14-3-3{beta} increases expressions of ER{alpha} target genes. {yields} 14-3-3{beta} increases breast cancer cell proliferation. -- Abstract: The estrogen receptor (ER) functions as a transcription factor that mediates the effects of estrogen. ER{alpha}, which plays a crucial role in the development and progression of breast cancer, is activated by estrogen binding, leading to receptor phosphorylation, dimerization, and recruitment of co-activators and chaperons to the estrogen-bound receptor complex. The 14-3-3 proteins bind to target proteins via phosphorylation and influence many cellular events by altering their subcellular localization or acting as a chaperone. However, regulation of ER{alpha} expression and transactivation by the 14-3-3 proteins has not been reported. We demonstrate that 14-3-3{beta} functions as a positive regulator of ER{alpha} through a direct protein-protein interaction in an estrogen-dependent manner. Ectopic expression of 14-3-3{beta} stimulated ER{alpha}-mediated transcriptional activity in MCF-7 breast cancer cells. Enhanced ER{alpha} transcriptional activity due to 14-3-3{beta} increased the expressions of the endogenous ER{alpha} target genes, leading to proliferation of breast cancer cells. We suggest that 14-3-3{beta} has oncogenic potential in breast cancer via binding to ER{alpha} and activation of the transcriptional activity of ER{alpha}.

  17. Cellular Oxygen Sensing: Crystal Structure of Hypoxia-Inducible Factor Prolyl Hydroxylase (PHD2)

    Energy Technology Data Exchange (ETDEWEB)

    McDonough,M.; Li, V.; Flashman, E.; Chowdhury, R.; Mohr, C.; Lienard, B.; Zondlo, J.; Oldham, N.; Clifton, I.; et al.


    Cellular and physiological responses to changes in dioxygen levels in metazoans are mediated via the posttranslational oxidation of hypoxia-inducible transcription factor (HIF). Hydroxylation of conserved prolyl residues in the HIF-{alpha} subunit, catalyzed by HIF prolyl-hydroxylases (PHDs), signals for its proteasomal degradation. The requirement of the PHDs for dioxygen links changes in dioxygen levels with the transcriptional regulation of the gene array that enables the cellular response to chronic hypoxia; the PHDs thus act as an oxygen-sensing component of the HIF system, and their inhibition mimics the hypoxic response. We describe crystal structures of the catalytic domain of human PHD2, an important prolyl-4-hydroxylase in the human hypoxic response in normal cells, in complex with Fe(II) and an inhibitor to 1.7 Angstroms resolution. PHD2 crystallizes as a homotrimer and contains a double-stranded {beta}-helix core fold common to the Fe(II) and 2-oxoglutarate-dependant dioxygenase family, the residues of which are well conserved in the three human PHD enzymes (PHD 1-3). The structure provides insights into the hypoxic response, helps to rationalize a clinically observed mutation leading to familial erythrocytosis, and will aid in the design of PHD selective inhibitors for the treatment of anemia and ischemic disease.

  18. Phenylalanine hydroxylase: function, structure, and regulation. (United States)

    Flydal, Marte I; Martinez, Aurora


    Mammalian phenylalanine hydroxylase (PAH) catalyzes the rate-limiting step in the phenylalanine catabolism, consuming about 75% of the phenylalanine input from the diet and protein catabolism under physiological conditions. In humans, mutations in the PAH gene lead to phenylketonuria (PKU), and most mutations are mainly associated with PAH misfolding and instability. The established treatment for PKU is a phenylalanine-restricted diet and, recently, supplementation with preparations of the natural tetrahydrobiopterin cofactor also shows effectiveness for some patients. Since 1997 there has been a significant increase in the understanding of the structure, catalytic mechanism, and regulation of PAH by its substrate and cofactor, in addition to improved correlations between genotype and phenotype in PKU. Importantly, there has also been an increased number of studies on the structure and function of PAH from bacteria and lower eukaryote organisms, revealing an additional anabolic role of the enzyme in the synthesis of melanin-like pigments. In this review, we discuss these recent studies, which contribute to define the evolutionary adaptation of the PAH structure and function leading to sophisticated regulation for effective catabolic processing of phenylalanine in mammalian organisms.

  19. Cardiovascular response to beta-adrenergic blockade or activation in 23 inbred mouse strains.

    Directory of Open Access Journals (Sweden)

    Corinne Berthonneche

    Full Text Available We report the characterisation of 27 cardiovascular-related traits in 23 inbred mouse strains. Mice were phenotyped either in response to chronic administration of a single dose of the beta-adrenergic receptor blocker atenolol or under a low and a high dose of the beta-agonist isoproterenol and compared to baseline condition. The robustness of our data is supported by high trait heritabilities (typically H(2>0.7 and significant correlations of trait values measured in baseline condition with independent multistrain datasets of the Mouse Phenome Database. We then focused on the drug-, dose-, and strain-specific responses to beta-stimulation and beta-blockade of a selection of traits including heart rate, systolic blood pressure, cardiac weight indices, ECG parameters and body weight. Because of the wealth of data accumulated, we applied integrative analyses such as comprehensive bi-clustering to investigate the structure of the response across the different phenotypes, strains and experimental conditions. Information extracted from these analyses is discussed in terms of novelty and biological implications. For example, we observe that traits related to ventricular weight in most strains respond only to the high dose of isoproterenol, while heart rate and atrial weight are already affected by the low dose. Finally, we observe little concordance between strain similarity based on the phenotypes and genotypic relatedness computed from genomic SNP profiles. This indicates that cardiovascular phenotypes are unlikely to segregate according to global phylogeny, but rather be governed by smaller, local differences in the genetic architecture of the various strains.

  20. Capillary electrophoretic behaviors of pharmacologically active xanthones from Securidaca inappendiculata with beta-cyclodextrin as a buffer additive. (United States)

    Bo, Tao; Huang, Yongfa; Yang, Xuedong; Li, Ke An; Liu, Huwei; Xu, Lizhen


    The capillary electrophoretic (CE) behaviors of ten xanthones in the presence of beta-cyclodextrin (CD) are investigated, and apparent analyte-selector binding constants between beta-CD and the xanthones in the CE running buffer are calculated to elucidate the migration order. Also, the separation selectivity with beta-CD additive is compared with that of sulfated beta-CD additive. It is indicated that beta-CD can greatly change the separation selectivity of xanthones, and the electrophoretic behaviors of xanthones are rather different when using beta-CD from that when using sulfated beta-CD as an additive. PMID:12803804

  1. Structural and Functional Analyses of a Glycoside Hydrolase Family 5 Enzyme with an Unexpected [beta]-Fucosidase Activity

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Shosuke; Park, David S.; Bae, Brian; Mackie, Roderick; Cann, Isaac K.O.; Nair, Satish K. (UIUC)


    We present characterization of PbFucA, a family 5 glycoside hydrolase (GH5) from Prevotella bryantii B{sub 1}4. While GH5 members typically are xylanases, PbFucA shows no activity toward xylan polysaccharides. A screen against a panel of p-nitrophenol coupled sugars identifies PbFucA as a {beta}-D-fucosidase. We also present the 2.2 {angstrom} resolution structure of PbFucA and use structure-based mutational analysis to confirm the role of catalytically essential residues. A comparison of the active sites of PbFucA with those of family 5 and 51 glycosidases reveals that while the essential catalytic framework is identical between these enzymes, the steric contours of the respective active site clefts are distinct and likely account for substrate discrimination. Our results show that members of this cluster of orthologous group (COG) 5520 have {beta}-D-fucosidase activities, despite showing an overall sequence and structural similarity to GH-5 xylanases.

  2. Mineralocorticoid replacement during infancy for salt wasting congenital adrenal hyperplasia due to 21-hydroxylase deficiency

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    Larissa G. Gomes


    Full Text Available OBJECTIVE: The protocols for glucocorticoid replacement in children with salt wasting 21-hydroxylase deficiency are well established; however, the current recommendation for mineralocorticoid replacement is general and suggests individualized dose adjustments. This study aims to retrospectively review the 9-∝-fludrocortisone dose regimen in salt wasting 21-hydroxylase deficient children who have been adequately treated during infancy. METHODS: Twenty-three salt wasting 21-hydroxylase deficient patients with good anthropometric and hormonal control were followed in our center since diagnosis. The assessments of cortisone acetate and 9-∝-fludrocortisone doses, anthropometric parameters, and biochemical and hormonal levels were rigorously evaluated in pre-determined intervals from diagnosis to two years of age. RESULTS: The 9-∝-fludrocortisone doses decreased over time during the first and second years of life; the median fludrocortisone doses were 200 µg at 0-6 months, 150 µg at 7-18 months and 125 µg at 19-24 months. The cortisone acetate dose per square meter was stable during follow-up (median = 16.8 mg/m²/day. The serum sodium, potassium and plasma rennin activity levels during treatment were normal, except in the first month of life, when periodic 9-∝-fludrocortisone dose adjustments were made. CONCLUSIONS: The mineralocorticoid needs of salt wasting 21-hydroxylase deficient patients are greater during early infancy and progressively decrease during the first two years of life, which confirms that a partial aldosterone resistance exists during this time. Our study proposes a safety regiment for mineralocorticoid replacement during this critical developmental period.

  3. Sesamin Modulates Tyrosine Hydroxylase, Superoxide Dismutase, Catalase, Inducible No Synthase and Interleukin-6 Expression in Dopaminergic Cells Under Mpp+-Induced Oxidative Stress

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    Vicky Lahaie-Collins


    Full Text Available Oxidative stress is regarded as a mediator of nerve cell death in several neurodegenerative disorders, such as Parkinson's disease. Sesamin, a lignan mainly found in sesame oil, is currently under study for its anti-oxidative and possible neuroprotective properties. We used 1-methyl-4-phenyl-pyridine (MPP+ ion, the active metabolite of the potent parkinsonism-causing toxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine, to produce oxidative stress and neurodegeneration in neuronal PC12 cells, which express dopamine, as well as neurofilaments. Our results show that picomolar doses of sesamin protected neuronal PC12 cells from MPP+-induced cellular death, as revealed by colorimetric measurements and production of reactive oxygen species. We also demonstrated that sesamin acted by rescuing tyrosine hydroxylase levels from MPP+-induced depletion. Sesamin, however, did not modulate dopamine transporter levels, and estrogen receptor-alpha and -beta protein expression. By examining several parameters of cell distress, we found that sesamin also elicited a strong increase in superoxide dismutase activity as well as protein expression and decreased catalase activity and the MPP+ stimulated inducible nitric oxide synthase protein expression, in neuronal PC12 cells. Finally, sesamin possessed significant anti-inflammatory properties, as disclosed by its potential to reduce MPP+-induced interleukin-6 mRNA levels in microglia. From these studies, we determined the importance of the lignan sesamin as a neuroprotective molecule and its possible role in complementary and/or preventive therapies of neurodegenerative diseases.

  4. CYP17A1 intron mutation causing cryptic splicing in 17α-hydroxylase deficiency.

    Directory of Open Access Journals (Sweden)

    Daw-Yang Hwang

    Full Text Available 17α-Hydroxylase/17, 20-lyase deficiency (17OHD is an autosomal recessive disease causing congenital adrenal hyperplasia and a rare cause of hypertension with hypokalemia. The CYP17A1 gene mutation leads to 17OHD and its clinical features. We described an 18 y/o female with clinical features of 17α-hydroxylase/17, 20-lyase deficiency and characterized the functional consequences of an intronic CYP17A1 mutation. The coding regions and flanking intronic bases of the CYP17A1 gene were amplified by PCR and sequenced. The patient is a compound heterozygote for the previously described p.R358X and IVS1 +2T>C mutations. A first intron splice donor site mutation was re-created in minigene and full-length expression vectors. Pre-mRNA splicing of the variant CYP17A1 intron was studied in transfected cells and in a transformed lymphoblastoid cell line. When the full-length CYP17A1 gene and minigene containing the intronic mutation was expressed in transfected cells, the majority (>90% of mRNA transcripts were incorrectly spliced. Only the p.R358X transcript was detected in the EBV-transformed lymphoblastoid cell line. The IVS1 +2T>C mutation abolished most 17α-hydroxylase/17, 20-lyase enzyme activity by aberrant mRNA splicing to an intronic pseudo-exon, causing a frame shift and early termination.

  5. The Effect of Glutaraldehyde Cross-Linking on the Enzyme Activity of Immobilized &beta-Galactosidase on Chitosan Bead

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    He Chen


    Full Text Available The effect of glutaraldehyde solution concentration, cross-linking time, cross-linking pH and cross-linking temperature on the enzyme activity of the immobilized &beta-galactosidase on Chitosan beads were studied. The enzyme activity was measured after immobilized by detecting the absorbance value at 420 nm.The results were as follows: the immobilized enzyme activity reached the maximum when the concentration of glutaraldehyde solution was 0.3%.The immobilized enzyme had optimal cross-linking time of 3h, optimal cross-linking pH of 6.5, optimal cross-linking temperature of 25°C, under these conditions, the immobilized enzyme activity reached 101, 96, 90 U/g, respectively.

  6. The tissue plasminogen activator-plasminogen proteolytic cascade accelerates amyloid-beta (Abeta) degradation and inhibits Abeta-induced neurodegeneration. (United States)

    Melchor, Jerry P; Pawlak, Robert; Strickland, Sidney


    Accumulation of the amyloid-beta (Abeta) peptide depends on both its generation and clearance. To better define clearance pathways, we have evaluated the role of the tissue plasminogen activator (tPA)-plasmin system in Abeta degradation in vivo. In two different mouse models of Alzheimer's disease, chronically elevated Abeta peptide in the brain correlates with the upregulation of plasminogen activator inhibitor-1 (PAI-1) and inhibition of the tPA-plasmin system. In addition, Abeta injected into the hippocampus of mice lacking either tPA or plasminogen persists, inducing PAI-1 expression and causing activation of microglial cells and neuronal damage. Conversely, Abeta injected into wild-type mice is rapidly cleared and does not cause neuronal degeneration. Thus, the tPA-plasmin proteolytic cascade aids in the clearance of Abeta, and reduced activity of this system may contribute to the progression of Alzheimer's disease.

  7. Cosmogenic-neutron activation of TeO2 and implications for neutrinoless double-beta decay experiments

    CERN Document Server

    Wang, Barbara S; Scielzo, Nicholas D; Smith, Alan R; Thomas, Keenan J; Wender, Stephen A


    Flux-averaged cross sections for cosmogenic-neutron activation of natural tellurium were measured using a neutron beam containing neutrons of kinetic energies up to $\\sim$800 MeV, and having an energy spectrum similar to that of cosmic-ray neutrons at sea-level. Analysis of the radioisotopes produced reveals that 110mAg will be a dominant contributor to the cosmogenic-activation background in experiments searching for neutrinoless double-beta decay of 130Te, such as CUORE and SNO+. An estimate of the cosmogenic-activation background in the CUORE experiment has been obtained using the results of this measurement and cross-section measurements of proton activation of tellurium. Additionally, the measured cross sections in this work are also compared with results from semi-empirical cross-section calculations.

  8. Genetic diversity of tyrosine hydroxylase (TH and dopamine b-hydroxylase (DBH genes in cattle breeds

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    Diana Lelidett Lourenco-Jaramillo


    Full Text Available DNA from four cattle breeds was used to re-sequence all of the exons and 56% of the introns of the bovine tyrosine hydroxylase (TH gene and 97% and 13% of the bovine dopamine b-hydroxylase (DBH coding and non-coding sequences, respectively. Two novel single nucleotide polymorphisms (SNPs and a microsatellite motif were found in the TH sequences. The DBH sequences contained 62 nucleotide changes, including eight non-synonymous SNPs (nsSNPs that are of particular interest because they may alter protein function and therefore affect the phenotype. These DBH nsSNPs resulted in amino acid substitutions that were predicted to destabilize the protein structure. Six SNPs (one from TH and five from DBH non-synonymous SNPs were genotyped in 140 animals; all of them were polymorphic and had a minor allele frequency of > 9%. There were significant differences in the intra- and inter-population haplotype distributions. The haplotype differences between Brahman cattle and the three B. t. taurus breeds (Charolais, Holstein and Lidia were interesting from a behavioural point of view because of the differences in temperament between these breeds.

  9. Genetic diversity of tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) genes in cattle breeds. (United States)

    Lourenco-Jaramillo, Diana Lelidett; Sifuentes-Rincón, Ana María; Parra-Bracamonte, Gaspar Manuel; de la Rosa-Reyna, Xochitl Fabiola; Segura-Cabrera, Aldo; Arellano-Vera, Williams


    DNA from four cattle breeds was used to re-sequence all of the exons and 56% of the introns of the bovine tyrosine hydroxylase (TH) gene and 97% and 13% of the bovine dopamine β-hydroxylase (DBH) coding and non-coding sequences, respectively. Two novel single nucleotide polymorphisms (SNPs) and a microsatellite motif were found in the TH sequences. The DBH sequences contained 62 nucleotide changes, including eight non-synonymous SNPs (nsSNPs) that are of particular interest because they may alter protein function and therefore affect the phenotype. These DBH nsSNPs resulted in amino acid substitutions that were predicted to destabilize the protein structure. Six SNPs (one from TH and five from DBH non-synonymous SNPs) were genotyped in 140 animals; all of them were polymorphic and had a minor allele frequency of > 9%. There were significant differences in the intra- and inter-population haplotype distributions. The haplotype differences between Brahman cattle and the three B. t. taurus breeds (Charolais, Holstein and Lidia) were interesting from a behavioural point of view because of the differences in temperament between these breeds. PMID:22888292

  10. Safrole oxide induces neuronal apoptosis through inhibition of integrin beta4/SOD activity and elevation of ROS/NADPH oxidase activity. (United States)

    Su, Le; Zhao, BaoXiang; Lv, Xin; Wang, Nan; Zhao, Jing; Zhang, ShangLi; Miao, JunYing


    Neuronal apoptosis is a very important event in the development of the central nervous system (CNS), but the underlying mechanisms remain to be elucidated. We have previously shown that safrole oxide, a small molecule, induces integrin beta4 expression and promotes apoptosis in vascular endothelial cells. In this study, the effects of safrole oxide on cell growth and apoptosis have been examined in primary cultures of mouse neurons. Safrole oxide was found to significantly inhibit neuronal cell growth and to induce apoptosis. The inhibitory and apoptotic activities of safrole oxide followed a dose- and time-dependent manner. Interestingly, the expression of integrin beta4 was significantly inhibited with safrole oxide treatment. Furthermore, safrole oxide dramatically increases the level of intracellular reactive oxygen species (ROS) and the activity of NADPH oxidase. Moreover, manganese-dependent superoxide dismutase (MnSOD) activity was decreased significantly with safrole oxide treatment. Our study thus demonstrates that safrole oxide induces neuronal apoptosis through integrin beta4, ROS, NADPH, and MnSOD.

  11. Acetylcholine release in mouse hippocampal CA1 preferentially activates inhibitory-selective interneurons via alpha4 beta2* nicotinic receptor activation

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    L. Andrew Bell


    Full Text Available Acetylcholine (ACh release onto nicotinic receptors directly activates subsets of inhibitory interneurons in hippocampal CA1. However, the specific interneurons activated and their effect on the hippocampal network is not completely understood. Therefore, we investigated subsets of hippocampal CA1 interneurons that respond to ACh release through the activation of nicotinic receptors and the potential downstream effects this may have on hippocampal CA1 network function. ACh was optogenetically released in mouse hippocampal slices by expressing the excitatory optogenetic protein oChIEF-tdTomato in medial septum/diagonal band of Broca cholinergic neurons using Cre recombinase-dependent adeno-associated viral mediated transfection. The actions of optogenetically released ACh were assessed on both pyramidal neurons and different interneuron subtypes via whole cell patch clamp methods. Vasoactive intestinal peptide (VIP-expressing interneurons that selectively innervate other interneurons (VIP/IS were excited by ACh through the activation of nicotinic receptors containing alpah4 and beta2 subunits (alpha4 beta2*. ACh release onto VIP/IS was presynaptically inhibited by M2 muscarinic autoreceptors. ACh release produced spontaneous inhibitory postsynaptic current (sIPSC barrages blocked by dihydro-beta-erythroidine in interneurons but not pyramidal neurons. Optogenetic suppression of VIP interneurons did not inhibit these sIPSC barrages suggesting other interneuron-selective interneurons were also excited by 42* nicotinic receptor activation. In contrast, interneurons that innervate pyramidal neuron perisomatic regions were not activated by ACh release onto nicotinic receptors. Therefore, we propose ACh release in CA1 facilitates disinhibition through activation of 42* nicotinic receptors on interneuron-selective interneurons whereas interneurons that innervate pyramidal neurons are less affected by nicotinic receptor activation.

  12. Shedding of Clostridium difficile, fecal beta-lactamase activity, and gastrointestinal symptoms in 51 volunteers treated with oral cefixime.


    Chachaty, E; Bourneix, C; Renard, S; Bonnay, M.; Andremont, A


    Microbial changes including the shedding of Clostridium difficile, fecal beta-lactamase activity, and gastrointestinal symptoms were assessed in 51 healthy volunteers given 200 mg of cefixime twice daily for 8 days. The number of organisms of the family Enterobacteriaceae (means +/- standard deviations) dropped from 6.9 +/- 1.1 to 3.9 +/- 1.8 log CFU/g of feces (P < 0.01), whereas counts of enterococci rose from 7.0 +/- 1.5 to 9.0 +/- 1.0 log CFU/g of feces (P < 0.01). Both counts returned to...

  13. Arkadia enhances Nodal/TGF-beta signaling by coupling phospho-Smad2/3 activity and turnover.

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    Konstantinos J Mavrakis


    Full Text Available Regulation of transforming growth factor-beta (TGF-beta signaling is critical in vertebrate development, as several members of the TGF-beta family have been shown to act as morphogens, controlling a variety of cell fate decisions depending on concentration. Little is known about the role of intracellular regulation of the TGF-beta pathway in development. E3 ubiquitin ligases target specific protein substrates for proteasome-mediated degradation, and several are implicated in signaling. We have shown that Arkadia, a nuclear RING-domain E3 ubiquitin ligase, is essential for a subset of Nodal functions in the embryo, but the molecular mechanism of its action in embryonic cells had not been addressed. Here, we find that Arkadia facilitates Nodal signaling broadly in the embryo, and that it is indispensable for cell fates that depend on maximum signaling. Loss of Arkadia in embryonic cells causes nuclear accumulation of phospho-Smad2/3 (P-Smad2/3, the effectors of Nodal signaling; however, these must be repressed or hypoactive as the expression of their direct target genes is reduced or lost. Molecular and functional analysis shows that Arkadia interacts with and ubiquitinates P-Smad2/3 causing their degradation, and that this is via the same domains required for enhancing their activity. Consistent with this dual function, introduction of Arkadia in homozygous null (-/- embryonic stem cells activates the accumulated and hypoactive P-Smad2/3 at the expense of their abundance. Arkadia-/- cells, like Smad2-/- cells, cannot form foregut and prechordal plate in chimeras, confirming this functional interaction in vivo. As Arkadia overexpression never represses, and in some cells enhances signaling, the degradation of P-Smad2/3 by Arkadia cannot occur prior to their activation in the nucleus. Therefore, Arkadia provides a mechanism for signaling termination at the end of the cascade by coupling degradation of P-Smad2/3 with the activation of target gene

  14. Biphasic effect of IL-1beta on the activity of argininosuccinate synthetase in Caco-2 cells. Involvement of nitric oxide production. (United States)

    Brasse-Lagnel, Carole; Lavoinne, Alain; Fairand, Alain; Vavasseur, Karine; Deniel, Nicolas; Husson, Annie


    The expression of the argininosuccinate synthetase gene (ASS), the limiting enzyme of arginine synthesis, was previously shown to be rapidly induced by a short-term (4 h) exposure to IL-1beta in Caco-2 cells [Biochimie, 2005, 403-409]. The present report shows that, by contrast, a long-term (24 h) exposure to IL-1beta inhibited the ASS activity despite an increase in both specific mRNA level and protein amount, demonstrating a post-translational effect. Concerning the mechanism involved, we demonstrate that the inhibiting effect is linked to the production of nitric oxide (NO) induced by IL-1beta. Indeed, the inhibiting effect of IL-1beta was totally blocked in the presence of l-NMMA, an inhibitor of the inducible nitric oxide synthase, or by culturing the cells in an arginine-deprived medium. Moreover, a decrease in the ASS activity was induced by culturing the cells in the presence of SNAP, a NO donor. Conversely, blocking the action of NO by antioxidant agents, the stimulatory effect of IL-1beta on ASS activity was restored, as measured at 24 h. Finally, such an inhibiting effect of NO on ASS activity may be related, at least in part, to S-nitrosylation of the protein. The physiological relevance of the antagonistic effects of IL-1beta and NO on ASS is discussed.

  15. Anabolic function of phenylalanine hydroxylase in Caenorhabditis elegans. (United States)

    Calvo, Ana C; Pey, Angel L; Ying, Ming; Loer, Curtis M; Martinez, Aurora


    In humans, liver phenylalanine hydroxylase (PAH) has an established catabolic function, and mutations in PAH cause phenylketonuria, a genetic disease characterized by neurological damage, if not treated. To obtain novel evolutionary insights and information on molecular mechanisms operating in phenylketonuria, we investigated PAH in the nematode Caenorhabditis elegans (cePAH), where the enzyme is coded by the pah-1 gene, expressed in the hypodermis. CePAH presents similar molecular and kinetic properties to human PAH [S(0.5)(L-Phe) approximately 150 microM; K(m) for tetrahydrobiopterin (BH(4)) approximately 35 microM and comparable V(max)], but cePAH is devoid of positive cooperativity for L-Phe, an important regulatory mechanism of mammalian PAH that protects the nervous system from excess L-Phe. Pah-1 knockout worms show no obvious neurological defects, but in combination with a second cuticle synthesis mutation, they display serious cuticle abnormalities. We found that pah-1 knockouts lack a yellow-orange pigment in the cuticle, identified as melanin by spectroscopic techniques, and which is detected in C. elegans for the first time. Pah-1 mutants show stimulation of superoxide dismutase activity, suggesting that cuticle melanin functions as oxygen radical scavenger. Our results uncover both an important anabolic function of PAH and the change in regulation of the enzyme along evolution. PMID:18460651

  16. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein. (United States)

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun


    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders.

  17. Adrenaline activation of prejunctional beta-adrenoceptors in guinea-pig atria.


    Majewski, H.; McCulloch, M. W.; Rand, M J; Story, D. F.


    1. Adrenaline in a concentration of 1.0 microM depressed the stimulation-induced efflux of tritium from the guinea-pig atria incubated with [3H]-noradrenaline, whereas adrenaline in a concentration of 0.5 nM significantly enhanced the stimulation-induced efflux of tritium. This enhancement was blocked by metoprolol (0.1 microM) and thus appears to be mediated by beta-adrenoceptors. 2. In guinea-pig atria incubated with unlabelled adrenaline and then with [3H]-noradrenaline, both catecholamine...

  18. Lactate-modulated induction of THBS-1 activates transforming growth factor (TGF-beta2 and migration of glioma cells in vitro.

    Directory of Open Access Journals (Sweden)

    Corinna Seliger

    Full Text Available BACKGROUND: An important phenomenon observed in glioma metabolism is increased aerobic glycolysis in tumor cells, which is generally referred to as the Warburg effect. Transforming growth factor (TGF-beta2, which we previously showed to be induced by lactic acid, is a key pathophysiological factor in glioblastoma, leading to increased invasion and severe local immunosuppression after proteolytic cleavage from its latency associated peptide. In this study we tested the hypothesis, that lactate regulates TGF-beta2 expression and glioma cell migration via induction of Thrombospondin-1 (THBS-1, a TGF-beta activating protein. METHODS: Lactate levels were reduced by knockdown of LDH-A using specific small interfering RNA (siRNA and competitive inhibition of LDH-A by sodium oxamate. Knockdown of THBS-1 was performed using specific siRNA. Western Blot, qRT-PCR, and ELISA were used to investigate expression levels of LDH-A, LDH-B, TGF-beta2 and THBS-1. Migration of cells was examined by Spheroid, Scratch and Boyden Chamber assays. RESULTS: Knockdown of LDH-A with subsequent decrease of lactate concentration leads to reduced levels of THBS-1 and TGF-beta2 in glioma cells. Lactate addition increases THBS-1 protein, leading to increased activation of TGF-beta2. Inhibition of THBS-1 reduces TGF-beta2 protein and migration of glioma cells. Addition of synthetic THBS-1 can rescue reduced TGF-beta2 protein levels and glioma cell migration in siLDH-A treated cells. CONCLUSION: We define a regulatory cascade between lactate, THBS-1 and TGF-beta2, leading to enhanced migration of glioma cells. Our results demonstrate a specific interaction between tumor metabolism and migration and provide a better understanding of the mechanisms underlying glioma cell invasion.

  19. Bacillus anthracis capsule activates caspase-1 and induces interleukin-1beta release from differentiated THP-1 and human monocyte-derived dendritic cells. (United States)

    Cho, Min-Hee; Ahn, Hae-Jeong; Ha, Hyun-Joon; Park, Jungchan; Chun, Jeong-Hoon; Kim, Bong-Su; Oh, Hee-Bok; Rhie, Gi-Eun


    The poly-gamma-d-glutamic acid (PGA) capsule is one of the major virulence factors of Bacillus anthracis, which causes a highly lethal infection. The antiphagocytic PGA capsule disguises the bacilli from immune surveillance and allows unimpeded growth of bacilli in the host. Recently, efforts have been made to include PGA as a component of anthrax vaccine; however, the innate immune response of PGA itself has been poorly investigated. In this study, we characterized the innate immune response elicited by PGA in the human monocytic cell line THP-1, which was differentiated into macrophages with phorbol 12-myristate 13-acetate (PMA) and human monocyte-derived dendritic cells (hMoDCs). PGA capsules were isolated from the culture supernatant of either the pXO1-cured strain of B. anthracis H9401 or B. licheniformis ATCC 9945a. PGA treatment of differentiated THP-1 cells and hMoDCs led to the specific extracellular release of interleukin-1beta (IL-1beta) in a dose-dependent manner. Evaluation of IL-1beta processing by Western blotting revealed that cleaved IL-1beta increased in THP-1 cells and hMoDCs after PGA treatment. Enhanced processing of IL-1beta directly correlated with increased activation of its upstream regulator, caspase-1, also known as IL-1beta-converting enzyme (ICE). The extracellular release of IL-1beta in response to PGA was ICE dependent, since the administration of an ICE inhibitor prior to PGA treatment blocked induction of IL-1beta. These results demonstrate that B. anthracis PGA elicits IL-1beta production through activation of ICE in PMA-differentiated THP-1 cells and hMoDCs, suggesting the potential for PGA as a therapeutic target for anthrax. PMID:19737897

  20. Brain activation by short-term nicotine exposure in anesthetized wild-type and beta2-nicotinic receptors knockout mice: a BOLD fMRI study

    Energy Technology Data Exchange (ETDEWEB)

    Suarez, S.V.; Changeux, J.P.; Granon, S. [Unite de Neurobiologie Integrative du Systeme Cholinergique, URA CNRS 2182, Institut Pasteur, Departement de Neuroscience, 25 rue du Dr Roux, 75015 Paris (France); Amadon, A.; Giacomini, E.; Le Bihan, D. [Service Hospitalier Frederic Joliot, 4 place du general Leclerc, 91400 Orsay (France); Wiklund, A. [Section of Anaesthesiology and Intensive Care Medicine, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm (Sweden)


    Rationale: The behavioral effects of nicotine and the role of the beta2-containing nicotinic receptors in these behaviors are well documented. However, the behaviors altered by nicotine rely on the functioning on multiple brain circuits where the high-affinity {beta}2-containing nicotinic receptors ({beta}2*nAChRs) are located. Objectives We intend to see which brain circuits are activated when nicotine is given in animals naive for nicotine and whether the {beta}2*nAChRs are needed for its activation of the blood oxygen level dependent (BOLD) signal in all brain areas. Materials and methods: We used functional magnetic resonance imaging (fMRI) to measure the brain activation evoked by nicotine (1 mg/kg delivered at a slow rate for 45 min) in anesthetized C57BL/6J mice and {beta}2 knockout (KO) mice. Results: Acute nicotine injection results in a significant increased activation in anterior frontal, motor, and somatosensory cortices and in the ventral tegmental area and the substantia nigra. Anesthetized mice receiving no nicotine injection exhibited a major decreased activation in all cortical and subcortical structures, likely due to prolonged anesthesia. At a global level, {beta}2 KO mice were not rescued from the globally declining BOLD signal. However, nicotine still activated regions of a meso-cortico-limbic circuit likely via {alpha}7 nicotinic receptors. Conclusions: Acute nicotine exposure compensates for the drop in brain activation due to anesthesia through the meso-cortico-limbic network via the action of nicotine on {beta}2*nAChRs. The developed fMRI method is suitable for comparing responses in wild-type and mutant mice. (authors)

  1. Beta-arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors.


    Azzi, Mounia; Pascale G. Charest; Angers, Stéphane; Rousseau, Guy; Kohout, Trudy; Bouvier, Michel; Piñeyro, Graciela


    It is becoming increasingly clear that signaling via G protein-coupled receptors is a diverse phenomenon involving receptor interaction with a variety of signaling partners. Despite this diversity, receptor ligands are commonly classified only according to their ability to modify G protein-dependent signaling. Here we show that beta2AR ligands like ICI118551 and propranolol, which are inverse agonists for Gs-stimulated adenylyl cyclase, induce partial agonist responses for the mitogen-activat...

  2. Gross alpha and beta activity analyses in urine-a routine laboratory method for internal human radioactivity detection. (United States)

    Chen, Xiaowen; Zhao, Luqian; Qin, Hongran; Zhao, Meijia; Zhou, Yirui; Yang, Shuqiang; Su, Xu; Xu, Xiaohua


    The aim of this work was to develop a method to provide rapid results for humans with internal radioactive contamination. The authors hypothesized that valuable information could be obtained from gas proportional counter techniques by screening urine samples from potentially exposed individuals rapidly. Recommended gross alpha and beta activity screening methods generally employ gas proportional counting techniques. Based on International Standards Organization (ISO) methods, improvements were made in the evaporation process to develop a method to provide rapid results, adequate sensitivity, and minimum sample preparation and operator intervention for humans with internal radioactive contamination. The method described by an American National Standards Institute publication was used to calibrate the gas proportional counter, and urine samples from patients with or without radionuclide treatment were measured to validate the method. By improving the evaporation process, the time required to perform the assay was reduced dramatically. Compared with the reference data, the results of the validation samples were very satisfactory with respect to gross-alpha and gross-beta activities. The gas flow proportional counting method described here has the potential for radioactivity monitoring in the body. This method was easy, efficient, and fast, and its application is of great utility in determining whether a sample should be analyzed by a more complicated method, for example radiochemical and/or γ-spectroscopy. In the future, it may be used commonly in medical examination and nuclear emergency treatment.Health Phys. 106(5):000-000; 2014.

  3. Increased Specific Labeling of INS-1 Pancreatic Beta-Cell by Using RIP-Driven Cre Mutants with Reduced Activity.

    Directory of Open Access Journals (Sweden)

    Gen-cheng Gong

    Full Text Available Ectopically expressed Cre recombinase in extrapancreatic tissues in RIP-Cre mice has been well documented. The objective of this study was to find a simple solution that allows for improved beta-cell specific targeting. To this end, the RIP-Cre and reporter CMV-loxP-DsRed-loxP-EGFP expression cassettes were configurated into a one-plasmid and two-plasmid systems, which labeled approximately 80% insulin-positive INS-1 cells after 48 h transfection. However, off-target labeling was robustly found in more than 15% insulin-negative Ad293 cells. When an IRES element was inserted in front of Cre to reduce the translation efficiency, the ratio of recombination between INS-1 and Ad293 cells increased 3-4-fold. Further, a series of Cre mutants were generated by site-directed mutagenesis. When one of the mutants, Cre(H289P in both configurations, was used in the experiment, the percentage of recombination dropped to background levels in a number of insulin-negative cell lines, but decreased only slightly in INS-1 cells. Consistently, DNA substrate digestion assay showed that the enzymatic activity of Cre(H289P was reduced by 30-fold as compared to that of wild-type. In this study, we reported the generation of constructs containing RIP and Cre mutants, which enabled enhanced beta-cell specific labeling in vitro. These tools could be invaluable for beta-cell targeting and to the study of islet development.

  4. Naltrexone normalizes the suppression but not the surge of delta 5-3 beta-hydroxysteroid dehydrogenase activity in Leydig cells of stressed rat fetuses. (United States)

    Ward, I L; Ward, O B; Hayden, T; Weisz, J; Orth, J M


    Rat fetuses from mothers stressed chronically by immobilization and high intensity illumination beginning on day 14 of gestation have higher than normal levels of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity in Leydig cells on day 17 of gestation and lower than normal levels on days 18 and 19. Plasma testosterone titers in normal and stressed male fetuses closely parallel the activity of 3 beta HSD in fetal Leydig cells. In the present study quantitative cytochemistry was used to determine whether the stress-induced alterations in 3 beta HSD activity could be prevented by treating the mother with naltrexone, an opioid receptor blocker, before each stress session. Naltrexone normalized 3 beta HSD activity on days 18 and 19 of gestation, suggesting that the stress-induced suppression involves the endogenous opioid system. In contrast, naltrexone did not prevent the elevation in enzyme activity seen on day 17 in stressed fetuses. The persistence of a stress-induced surge on day 17, in spite of naltrexone therapy, suggests that some nonopioid mechanism is operational at that time. PMID:2361487

  5. A review on medicinal importance, pharmacological activity and bioanalytical aspects of beta-carboline alkaloid ‘‘Harmine’’

    Institute of Scientific and Technical Information of China (English)

    K Patel; M Gadewar; R Tripathi; SK Prasad; Dinesh Kumar Patel


    Harmine, a beta-carboline alkaloid, is widely distributed in the plants, marine creatures, insects, mammalians as well as in human tissues and body fluids. Harmine was originally isolated from seeds of Peganum harmal in 1847 having a core indole structure and a pyridine ring. Harmine has various types of pharmacological activities such as antimicrobial, antifungal, antitumor, cytotoxic, antiplasmodial, antioxidaant, antimutagenic, antigenotoxic and hallucinogenic properties. It acts on gamma-aminobutyric acid type A and monoamine oxidase A or B receptor, enhances insulin sensitivity and also produces vasorelaxant effect. Harmine prevents bone loss by suppressing osteoclastogenesis. The current review gives an overview on pharmacological activity and analytical techniques of harmine, which may be useful for researcheres to explore the hidden potential of harmine and and will also help in developing new drugs for the treatment of various diseases.

  6. Evaluating practicability of an LSC method for routine monitoring gross alpha and beta activities in water samples in Taiwan

    International Nuclear Information System (INIS)

    A procedure using liquid scintillation counting for the monitoring of gross alpha and beta activities in environmental water was implemented to improve the conventional procedure using GFPC adopted in Taiwan. The new procedure was acquired through calibration and validation, and then was applied to the monitoring of surface water in Taiwan. This procedure can improve 2–4 times of detection efficiencies and takes only 70–80% of analysis time with reliable accuracy. With these features, the newly developed procedure is favorable during emergency situations. - Highlights: ► An improvement of the monitoring of gross α/β activities in water in Taiwan. ► Improving 2–4 times of detection efficiencies and taking 70–80% of analysis time. ► Twenty three sites of surface water were monitored after the Fukushima accident.

  7. A review on medicinal importance, pharmacological activity and bioanalytical aspects of beta-carboline alkaloid “Harmine”

    Directory of Open Access Journals (Sweden)

    K Patel


    Full Text Available Harmine, a beta-carboline alkaloid, is widely distributed in the plants, marine creatures, insects, mammalians as well as in human tissues and body fluids. Harmine was originally isolated from seeds of Peganum harmal in 1847 having a core indole structure and a pyridine ring. Harmine has various types of pharmacological activities such as antimicrobial, antifungal, antitumor, cytotoxic, antiplasmodial, antioxidaant, antimutagenic, antigenotoxic and hallucinogenic properties. It acts on gamma–aminobutyric acid type A and monoamine oxidase A or B receptor, enhances insulin sensitivity and also produces vasorelaxant effect. Harmine prevents bone loss by suppressing osteoclastogenesis. The current review gives an overview on pharmacological activity and analytical techniques of harmine, which may be useful for researcheres to explore the hidden potential of harmine and and will also help in developing new drugs for the treatment of various diseases.

  8. Effect of ayurvedic medicines on beta-glucuronidase activity of Brunner's glands during recovery from cysteamine induced duodenal ulcers in rats. (United States)

    Nadar, T S; Pillai, M M


    Biochemical and histochemical studies revealed decreased beta-glucuronidase activity in the Brunner's glands of duodenal ulcerated rats. The enzyme activity showed gradual increase during recovery. Rats treated with a mixture of Ayurvedic medicines (Glycyrrhiza glabra, Terminalia chebula, Piper longum and Shanka Bhasma) recovered faster with concomitant increase in beta-glucuronidase activity in the Brunner's glands. It can be concluded that Ayurvedic medicines used do not act as antacid but improve the secretory status of Brunner's glands involved in the protection against duodenal ulcer. PMID:2620935

  9. Incorportion of oxygen-18 into the 25-position of cholecalciferol by hepatic cholecalciferol 25-hydroxylase. (United States)

    Madhok, T C; Schnoes, H K; DeLuca, H F


    The oxygen enzymically inserted as a hydroxy function by rat liver post-mitochondrial fraction into the 25-position of cholecalciferol to giver 25-hydroxycholecaliferol is derived exclusively from molecular O2. Therefore like the other two cholecalciferol hydroxylases, i.e. 25-hydroxycholecalciferol 1alpha-hydroxylase and 25-hydroxycholecalciferol 24-hydroxylase, the cholecalciferol 25-hydroxylase is also a mono-oxygenase ('mixed-function oxidase'). PMID:217341

  10. Transforming growth factor beta isoforms regulation of Akt activity and XIAP levels in rat endometrium during estrous cycle, in a model of pseudopregnancy and in cultured decidual cells

    Directory of Open Access Journals (Sweden)

    Asselin Eric


    during pseudopregnancy. In cultured stromal cells, we found that TGF-beta3 isoform induced Smad2 phosphorylation, indicating that the Smad pathway is activated by TGF-beta3 in these cells. Furthermore, TGF-beta2 and TGF-beta3 induced a dose-dependant increase of apoptosis in cultured stromal cells, as demonstrated by Hoechst nuclear staining. Noteworthy, TGF-beta2 and TGF-beta3 reduced the level of the anti-apoptotic XIAP protein, as well as the level of phosphorylated/active Akt, a well known survival protein, in a dose-dependent manner. Conclusion Those results suggest that TGF-beta might play an important role in the remodelling endometrium during the estrous cycle and in the regulation of apoptosis in rat decidual cells, in which inhibition of Akt survival pathway might be an important mechanism involved in the regulation of apoptosis.

  11. Endotoxin/lipopolysaccharide activates NF-kappa B and enhances tumor cell adhesion and invasion through a beta 1 integrin-dependent mechanism.

    LENUS (Irish Health Repository)

    Wang, Jiang Huai


    Beta(1) integrins play a crucial role in supporting tumor cell attachment to and invasion into the extracellular matrix. Endotoxin\\/LPS introduced by surgery has been shown to enhance tumor metastasis in a murine model. Here we show the direct effect of LPS on tumor cell adhesion and invasion in extracellular matrix proteins through a beta(1) integrin-dependent pathway. The human colorectal tumor cell lines SW480 and SW620 constitutively expressed high levels of the beta(1) subunit, whereas various low levels of alpha(1), alpha(2), alpha(4), and alpha(6) expression were detected. SW480 and SW620 did not express membrane-bound CD14; however, LPS in the presence of soluble CD14 (sCD14) significantly up-regulated beta(1) integrin expression; enhanced tumor cell attachment to fibronectin, collagen I, and laminin; and strongly promoted tumor cell invasion through the Matrigel. Anti-beta(1) blocking mAbs (4B4 and 6S6) abrogated LPS- plus sCD14-induced tumor cell adhesion and invasion. Furthermore, LPS, when combined with sCD14, resulted in NF-kappaB activation in both SW480 and SW620 cells. Inhibition of the NF-kappaB pathway significantly attenuated LPS-induced up-regulation of beta(1) integrin expression and prevented tumor cell adhesion and invasion. These results provide direct evidence that although SW480 and SW620 cells do not express membrane-bound CD14, LPS in the presence of sCD14 can activate NF-kappaB, up-regulate beta(1) integrin expression, and subsequently promote tumor cell adhesion and invasion. Moreover, LPS-induced tumor cell attachment to and invasion through extracellular matrix proteins is beta(1) subunit-dependent.

  12. Beta-defensins activate macrophages and synergize in pro-inflammatory cytokine expression induced by TLR ligands. (United States)

    Barabas, Nicola; Röhrl, Johann; Holler, Ernst; Hehlgans, Thomas


    Our previous studies indicated that mouse beta defensin 14 (mBD14, Defb14), a newly identified member of the beta-defensin super family, interacts with the chemokine receptors CCR2 and CCR6. In this study we report that pre-stimulation of primary mouse macrophages with mBD14 results in a synergistic, enhanced expression of pro-inflammatory cytokines and chemokines induced by TLR ligand re-stimulation. Experiments using specific inhibitors of G(i)-protein-coupled receptor signaling provide evidence that this effect seems to be mediated by a G(i)-protein-coupled receptor expressed on bone marrow derived macrophages. However, using primary macrophages derived from CCR6- and CCR2-deficient mice clearly demonstrated that the enhanced pro-inflammatory cytokine and chemokine expression is independent of the chemokine receptors CCR6 and CCR2. Additionally, signaling pathway analysis indicated that mBD14 is capable of inducing MAPK ERK1/2 phosphorylation and the induction of CD86 and F4/80 expression in bone marrow-derived macrophages after mBD14 stimulation. Collectively, our data indicate that β-defensins activate primary macrophages and enhance pro-inflammatory responses by using G(i)PCRs in order to support inflammatory reactions induced by TLR ligands. PMID:23332217

  13. Estrogen-2-hydroxylase in the brain of the male African catfish, Clarias gariepinus

    International Nuclear Information System (INIS)

    Estrogen-2-hydroxylase activity, involved in the biosynthesis of catecholestrogens, was localized in the brain of the male African catfish, Clarias gariepinus, by means of a radiometric assay using [2-3H]estradiol as substrate. Fore- and midbrain were divided in 18, 500-microns thick, transverse sections from which small defined areas were punched out and assayed. The estrogen-2-hydroxylase activity was calculated from the release of tritium during hydroxylation, and expressed in femtomole catecholestradiol.milligram-1 tissue.hour-1. The enzyme could be demonstrated throughout the brain. A high activity (greater than 350 fmol) was observed in the telencephalon, in particularly the rostral part and the area ventralis pars dorsalis; in the diencephalon in the preoptic region, including the magnocellular part of the preoptic nucleus and the rostral part of the anterior periventricular nucleus; and in the area tuberalis, including the nucleus lateralis tuberis, the rostral part of the nucleus anterior tuberis, the caudal part of the nucleus posterior periventricularis, and in the nucleus recessus posterioris. Also a high activity was detected in the mesencephalic tectum opticum and the dorsolateral part of the torus semicircularis. The ventral mesencephalon showed a moderate (200-350 fmol) to low (less than 200 fmol) activity, whereas the lowest activity was found in the hindbrain (118 fmol). The significance of the biosynthesis of catecholestrogens in the brain is discussed in light of the negative feedback mechanism of gonadal steroids on gonadotropin release

  14. Inducible Conditional Vascular-Specific Overexpression of Peroxisome Proliferator-Activated Receptor Beta/Delta Leads to Rapid Cardiac Hypertrophy (United States)

    Wagner, Kay-Dietrich; Vukolic, Ana; Baudouy, Delphine; Michiels, Jean-François


    Peroxisome proliferator-activated receptors are nuclear receptors which function as ligand-activated transcription factors. Among them, peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) is highly expressed in the heart and thought to have cardioprotective functions due to its beneficial effects in metabolic syndrome. As we already showed that PPARβ/δ activation resulted in an enhanced cardiac angiogenesis and growth without impairment of heart function, we were interested to determine the effects of a specific activation of PPARβ/δ in the vasculature on cardiac performance under normal and in chronic ischemic heart disease conditions. We analyzed the effects of a specific PPARβ/δ overexpression in endothelial cells on the heart using an inducible conditional vascular-specific mouse model. We demonstrate that vessel-specific overexpression of PPARβ/δ induces rapid cardiac angiogenesis and growth with an increase in cardiomyocyte size. Upon myocardial infarction, vascular overexpression of PPARβ/δ, despite the enhanced cardiac vessel formation, does not protect against chronic ischemic injury. Our results suggest that the proper balance of PPARβ/δ activation in the different cardiac cell types is required to obtain beneficial effects on the outcome in chronic ischemic heart disease. PMID:27057154



    Cheng, Shu-Yuan; Serova, Lidia I.; Glazkova, Dina; Sabban, Esther L.


    Egr1, a transcription factor rapidly induced by various stimuli including stress, can elevate transcription of genes for the catecholamine biosynthetic enzymes TH and PNMT. To examine if Egr1 also regulates dopamine β-hydroxylase (DBH) gene expression, PC12 cells were transfected with expression vector for full length or truncated inactive Egr1 and various DBH promoter-driven luciferase constructs. While Egr1 elevated TH promoter activity, DBH promoter activity was reduced. The reduction occu...

  16. Interleukin-1-mediated febrile responses in mice and interleukin-1 beta activation of NFkappaB in mouse primary astrocytes, involves the interleukin-1 receptor accessory protein. (United States)

    Zetterström, M; Lundkvist, J; Malinowsky, D; Eriksson, G; Bartfai, T


    The endogenous pyrogen interleukin-1 (IL-1) is considered as one of the key molecules in orchestrating the host response of injury and inflammation. IL-1 exerts its effects upon binding to the type I IL-1 receptor (IL-1RI). The IL-1-IL-1RI complex is further thought to associate with the IL-1 receptor accessory protein (IL-1RAcP), which is suggested to be important for most IL-1 signal transduction pathways. With the aim of investigating the importance of the IL-1RAcP in IL-1 signalling, IL-1alpha and IL-1beta induced febrile responses and IL-1beta-mediated activation of NFkappaB in primary astrocyte cultures were examined using IL-1RAcP-deficient (IL-1RAcP KO) and wild type mice, respectively. It was shown that neither recombinant rat IL-1alpha (rrIL-1alpha, 25 microg/kg), recombinant rat IL-1beta (rrIL-1beta, 40 microg/kg) nor recombinant human IL-1beta (rhIL-1beta, 50 microg/kg) injected i.p. could elicit febrile responses in the IL-1RAcP-deficient mice, while the same doses of rrIL-1alpha/beta or rhIL-1beta injected into wild type mice caused normal fever responses. A febrile response could be induced in the IL-1RAcP-deficient mice by i.p. administration of E. coli lipopolysaccharide (LPS, 50 microg/kg) and this response was similar to that obtained in wild type mice. Furthermore, it was shown that rhIL-1beta activated, in a concentration-dependent manner, nuclear translocation of the transcriptional nuclear factor kappa B (NFkappaB) in primary astrocyte cultures prepared from wild type mice, whereas no IL-1beta-induced translocation of NFkappaB could be detected in cultures prepared from IL-1RAcP-deficient mice, as revealed by electrophoretic mobility shift assay (EMSA). The rhIL-1beta-induced NFkappaB complexes were shown to contain p50 but no, or very little, p65 and cRel immunoreactive proteins.

  17. Activation of the GLP-1 Receptor Signalling Pathway: A Relevant Strategy to Repair a Deficient Beta-Cell Mass

    Directory of Open Access Journals (Sweden)

    Bernard Portha


    Full Text Available Recent preclinical studies in rodent models of diabetes suggest that exogenous GLP-1R agonists and DPP-4 inhibitors have the ability to increase islet mass and preserve beta-cell function, by immediate reactivation of beta-cell glucose competence, as well as enhanced beta-cell proliferation and neogenesis and promotion of beta-cell survival. These effects have tremendous implication in the treatment of T2D because they directly address one of the basic defects in T2D, that is, beta-cell failure. In human diabetes, however, evidence that the GLP-1-based drugs alter the course of beta-cell function remains to be found. Several questions surrounding the risks and benefits of GLP-1-based therapy for the diabetic beta-cell mass are discussed in this review and require further investigation.

  18. All-trans retinoic acid modulates mitogen-activated protein kinase pathway activation in human scleral fibroblasts through retinoic acid receptor beta (United States)

    Huo, Lijun; Cui, Dongmei; Yang, Xiao; Gao, Zhenya; Trier, Klaus


    Purpose All-trans retinoic acid (ATRA) is known to inhibit the proliferation of human scleral fibroblasts (HSFs) and to modulate the scleral intercellular matrix composition, and may therefore serve as a mediator for controlling eye growth. Cell proliferation is regulated by the mitogen-activated protein kinase (MAPK) pathway. The aim of the current study was to investigate whether changed activation of the MAPK pathway could be involved in the response of HSFs exposed to ATRA. Methods HSFs were cultured in Dulbecco Modified Eagle's Medium/F12 (DMEM/F12) and exposed to 1 μmol/l ATRA for 10 min, 30 min, 1 h, 8 h, or 24 h. The activation of extracellular signal-regulated kinase (ERK 1/2), p38, and c-Jun N-terminal kinase (JNK) in HSFs was assessed with western blot analysis and immunocytofluorescence. Results After exposure to ATRA for 24 h, the HSFs appeared shrunken and thinner than the control cells. The intercellular spaces were wider, and the HSFs appeared less numerous than in the control culture. Western blot showed decreased activation of ERK 1/2 in the HSFs from 30 min (p=0.01) to 24 h (p<0.01) after the start of exposure to ATRA, and increased activation of the JNK protein from 10 to 30 min (p<0.01) after the start of exposure to ATRA. Indirect immunofluorescence confirmed changes in activation of ERK 1/2 and JNK in HSFs exposed to ATRA. No change in activation of p38 in HSFs was observed after exposure to ATRA. Pretreatment of the HSFs with LE135, an antagonist of retinoic acid receptor beta (RARβ), abolished the ATRA-induced changes inactivation of ERK 1/2 and JNK. Conclusions ATRA inhibits HSF proliferation by a mechanism associated with modulation of ERK 1/2 and JNK activation and depends on stimulation of retinoic acid receptor beta. PMID:23946634

  19. Atividade antioxidante do beta-caroteno e da vitamina A. Estudo comparativo com antioxidante sintético beta-carotene and vitamin A antioxidant activity. Comparative study with synthetic antioxidant

    Directory of Open Access Journals (Sweden)

    José Afonso PASSOTTO


    Full Text Available Foi avaliada a atividade antioxidante da vitamina A na forma de acetato de retinol e de seu principal precursor, o beta-caroteno, adicionados a um sistema de óleo de soja previamente sensibilizado à oxidação. Os parâmetros utilizados como grau de atividade oxidativa foram: índice de peróxidos, teores de malonaldeído durante os intervalos de 24 a 72 horas, e perfil dos ácidos linoléico e linolênico após 144 horas de oxidação. Pelos resultados pode-se verificar que o retinol apresentou atividade antioxidante superior ao beta-caroteno. As determinações das atividades antioxidantes foram comparadas à do butilhidroxitolueno (BHT. A eficiência antioxidante da vitamina A e do beta-caroteno foram proporcionais às suas resistências à decomposição no sistema oxidativo. O acetato de retinol, a exemplo do BHT, mostrou uma rápida interação com os radicais ativos, pois já no início de sua adição ao óleo de soja, reduziu o nível da oxidação em relação ao respectivo controle.In soybean oil suceptible to oxidation the authors studied the antioxidant activity of the vitamin A as retinol acetate and the beta-carotene was studied. The oxidation index of the system was determined by peroxide and malonaldehyde values during the intervals from 24 to 72 hours and profile of the linoleic and linolenic acids after 144 hours of oxidation. It was observed that the retinol acetate had an antioxidant activity greater than beta-carotene. The antioxidant activity of retinol acetate and beta-carotene were compared to the butyl hidroxi toluene (BHT and was observed that the antioxidant efficiency was directly proportional to degradation resistance of them in the oxidative system. The retinol acetate, as such BHT, showed a fast interaction with actives radicals, in the beginning of the addition to the soybean oil, reducing the oxidation level when compared to the control.

  20. Therapeutic treatment with a novel hypoxia-inducible factor hydroxylase inhibitor (TRC160334 ameliorates murine colitis

    Directory of Open Access Journals (Sweden)

    Gupta R


    Full Text Available Ram Gupta,1 Anita R Chaudhary,2 Binita N Shah,1 Avinash V Jadhav,3 Shitalkumar P Zambad,1 Ramesh Chandra Gupta,4 Shailesh Deshpande,4 Vijay Chauthaiwale,4 Chaitanya Dutt4 1Department of Pharmacology, 2Cellular and Molecular Biology, 3Preclinical Safety Evaluation, 4Discovery, Torrent Research Centre, Torrent Pharmaceuticals Ltd, Gandhinagar, Gujarat, India Background and aim: Mucosal healing in inflammatory bowel disease (IBD can be achieved by improvement of intestinal barrier protection. Activation of hypoxia-inducible factor (HIF has been identified as a critical factor for barrier protection during mucosal insult and is linked with improvement in symptoms of colitis. Although prophylactic efficacy of HIF hydroxylase inhibitors in murine colitis have been established, its therapeutic efficacy in clinically relevant therapeutic settings have not been established. In the present study we aim to establish therapeutic efficacy of TRC160334, a novel HIF hydroxylase inhibitor, in animal models of colitis. Methods: The efficacy of TRC160334 was evaluated in two different mouse models of colitis by oral route. A prophylactic efficacy study was performed in a 2,4,6-trinitrobenzene sulfonic acid-induced mouse model of colitis representing human Crohn's disease pathology. Additionally, a therapeutic efficacy study was performed in a dextran sulfate sodium-induced mouse model of colitis, a model simulating human ulcerative colitis. Results: TRC160334 treatment resulted in significant improvement in disease end points in both models of colitis. TRC160334 treatment resulted into cytoprotective heatshock protein 70 induction in inflamed colon. TRC160334 successfully attenuated the rate of fall in body weight, disease activity index, and macroscopic and microscopic scores of colonic damage leading to overall improvement in study outcome. Conclusion: Our findings are the first to demonstrate that therapeutic intervention with a HIF hydroxylase inhibitor

  1. Validation the quantification of beta emitters activity in urine by scintillation spectrometry in the liquid phase; Validacion de la cuantificacion de actividad de emisores beta en orina mediante espectrometria de centelleo en fase liquida

    Energy Technology Data Exchange (ETDEWEB)

    Sierra, I.; Hernandez, C.; Benito, P.; Lopez, C.


    In this paper the methodology used in the validation of the technique for quantifying activity of some beta emitters in urine ({sup 3}H, {sup 1}4C, {sup 3}5S, {sup 3}2P and {sup 9}0Sr) by scintillation spectrometry Liquid Phase (Liquid Scintillation Counting, LSC) is described in bio elimination Laboratory Service CIEMAT Radiation Dosimetry accredited since last year for carrying out assays measure radiation dose based on ISO forth above. (Author)

  2. Quality control on the accuracy of the total Beta activity index in different sample matrices water; Control de calidad en la precision del indice de actividad beta total en diferentes matrices de muestras de aguas

    Energy Technology Data Exchange (ETDEWEB)

    Pujol, L.; Pablo, M. A. de; Payeras, J.


    The standard ISO/IEC 17025:2005 of general requirements for the technical competence of testing and calibration laboratories, provides that a laboratory shall have quality control procedures for monitoring the validity of tests and calibrations ago. In this paper, the experience of Isotopic Applications Laboratory (CEDEX) in controlling the accuracy rate of total beta activity in samples of drinking water, inland waters and marine waters is presented. (Author)

  3. Inhibition of transforming growth factor-beta-induced liver fibrosis by a retinoic acid derivative via the suppression of Col 1A2 promoter activity. (United States)

    Yang, Kun-Lin; Chang, Wen-Teng; Hung, Kuo-Chen; Li, Eric I C; Chuang, Chia-Chang


    Transforming growth factor-beta1 (TGF-beta1) mediates expression of collagen 1A2 (Col 1A2) gene via a synergistic cooperation between Smad2/Smad3 and Sp1, both act on the Col 1A2 gene promoter. In our previous study, we reported that a retinoic acid derivative obtained from Phellinus linteus (designated PL) antagonizes TGF-beta-induced liver fibrosis through regulation of ROS and calcium influx. In this continuing study we seek further the effect of PL on the Smad signaling pathway. We used a Col 1A2 promoter-luciferase construct to study the action of PL on Smad through TGF-beta. We found that PL decreases the promoter activity of Col 1A2, hinders the translocalization of phosphorylated Smad2/3-Smad 4 complex from cytosol into nucleus and inhibits Sp1 binding activity. These results suggest that PL inhibits TGF-beta1-induced Col 1A2 promoter activity through blocking ROS and calcium influx as well as impeding Sp1 binding and translocalization of pSmad 2/3-Smad4 complex into nucleus.

  4. Upregulating of Fas, integrin beta4 and P53 and depressing of PC-PLC activity and ROS level in VEC apoptosis by safrole oxide. (United States)

    Zhao, Jing; Miao, Junying; Zhao, Baoxiang; Zhang, Shangli


    Previously, we found that safrole oxide could trigger vascular endothelial cell (VEC) apoptosis. In this study, to investigate its mechanism to induce apoptosis in VECs, the activities of nitric oxide synthetase and phosphatidylcholine specific phospholipase C, the level of reactive oxygen species and the expressions of Fas, integrin beta4 and P53 were analyzed. The data showed that safrole oxide induced apoptosis by increasing the expressions of Fas, integrin beta4 and P53, and depressing the activity of Ca(2+)-independent phosphatidylcholine-specific phospholipase C and intracellular reactive oxygen species levels in VECs.

  5. NMDA-receptor activation but not ion flux is required for amyloid-beta induced synaptic depression.

    Directory of Open Access Journals (Sweden)

    Albert Tamburri

    Full Text Available Alzheimer disease is characterized by a gradual decrease of synaptic function and, ultimately, by neuronal loss. There is considerable evidence supporting the involvement of oligomeric amyloid-beta (Aβ in the etiology of Alzheimer's disease. Historically, AD research has mainly focused on the long-term changes caused by Aβ rather than analyzing its immediate effects. Here we show that acute perfusion of hippocampal slice cultures with oligomeric Aβ depresses synaptic transmission within 20 minutes. This depression is dependent on synaptic stimulation and the activation of NMDA-receptors, but not on NMDA-receptor mediated ion flux. It, therefore, appears that Aβ dependent synaptic depression is mediated through a use-dependent metabotropic-like mechanism of the NMDA-receptor, but does not involve NMDA-receptor mediated synaptic transmission, i.e. it is independent of calcium flux through the NMDA-receptor.

  6. Recommendations for the nutrition management of phenylalanine hydroxylase deficiency. (United States)

    Singh, Rani H; Rohr, Fran; Frazier, Dianne; Cunningham, Amy; Mofidi, Shideh; Ogata, Beth; Splett, Patricia L; Moseley, Kathryn; Huntington, Kathleen; Acosta, Phyllis B; Vockley, Jerry; Van Calcar, Sandra C


    The effectiveness of a phenylalanine-restricted diet to improve the outcome of individuals with phenylalanine hydroxylase deficiency (OMIM no. 261600) has been recognized since the first patients were treated 60 years ago. However, the treatment regime is complex, costly, and often difficult to maintain for the long term. Improvements and refinements in the diet for phenylalanine hydroxylase deficiency have been made over the years, and adjunctive therapies have proven to be successful for certain patients. Yet evidence-based guidelines for managing phenylalanine hydroxylase deficiency, optimizing outcomes, and addressing all available therapies are lacking. Thus, recommendations for nutrition management were developed using evidence from peer-reviewed publications, gray literature, and consensus surveys. The areas investigated included choice of appropriate medical foods, integration of adjunctive therapies, treatment during pregnancy, monitoring of nutritional and clinical markers, prevention of nutrient deficiencies, providing of access to care, and compliance strategies. This process has not only provided assessment and refinement of current nutrition management and monitoring recommendations but also charted a direction for future studies. This document serves as a companion to the concurrently published American College of Medical Genetics and Genomics guideline for the medical treatment of phenylalanine hydroxylase deficiency. PMID:24385075

  7. Chemical modification of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

    NARCIS (Netherlands)

    Wijnands, R.A.


    Chemical modification was used to examine the role of some amino acid residues in the binding of the substrates to the enzyme p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. Ionic strength dependent binding studies were used to investigate the role of the protein as a whole in the comple

  8. Association between Tryptophan Hydroxylase 2 Gene Polymorphism and Completed Suicide (United States)

    Fudalej, Sylwia; Ilgen, Mark; Fudalej, Marcin; Kostrzewa, Grazyna; Barry, Kristen; Wojnar, Marcin; Krajewski, Pawel; Blow, Frederic; Ploski, Rafal


    The association between suicide and a single nucleotide polymorphism (rs1386483) was examined in the recently identified tryptophan hydroxylase 2 (TPH2) gene. Blood samples of 143 suicide victims and 162 age- and sex-matched controls were examined. The frequency of the TT genotype in the TPH2 polymorphism was higher in suicide victims than in…

  9. Signal transducer and activator of transcription 5 activation is sufficient to drive transcriptional induction of cyclin D2 gene and proliferation of rat pancreatic beta-cells

    DEFF Research Database (Denmark)

    Friedrichsen, Birgitte N; Richter, Henrijette E; Hansen, Johnny A;


    cells transiently transfected with a cyclin D2 promoter-reporter construct revealed a 3- to 5-fold increase of transcriptional activity in response to hGH stimulation. Furthermore, coexpression of a constitutive active STAT5 mutant (either CA-STAT5a or CA-STAT5b) was sufficient to drive transactivation......-STAT5b stimulated transcriptional activation of the cyclin D2 promoter and induced hGH-independent proliferation in these cells. In primary beta-cells, adenovirus-mediated expression of CA-STAT5b profoundly stimulated DNA-synthesis (5.3-fold over control) in the absence of hGH. Our studies indicate...

  10. Promoter-distal RNA polymerase II binding discriminates active from inactive CCAAT/ enhancer-binding protein beta binding sites (United States)

    Savic, Daniel; Roberts, Brian S.; Carleton, Julia B.; Partridge, E. Christopher; White, Michael A.; Cohen, Barak A.; Cooper, Gregory M.; Gertz, Jason; Myers, Richard M.


    Transcription factors (TFs) bind to thousands of DNA sequences in mammalian genomes, but most of these binding events appear to have no direct effect on gene expression. It is unclear why only a subset of TF bound sites are actively involved in transcriptional regulation. Moreover, the key genomic features that accurately discriminate between active and inactive TF binding events remain ambiguous. Recent studies have identified promoter-distal RNA polymerase II (RNAP2) binding at enhancer elements, suggesting that these interactions may serve as a marker for active regulatory sequences. Despite these correlative analyses, a thorough functional validation of these genomic co-occupancies is still lacking. To characterize the gene regulatory activity of DNA sequences underlying promoter-distal TF binding events that co-occur with RNAP2 and TF sites devoid of RNAP2 occupancy using a functional reporter assay, we performed cis-regulatory element sequencing (CRE-seq). We tested more than 1000 promoter-distal CCAAT/enhancer-binding protein beta (CEBPB)-bound sites in HepG2 and K562 cells, and found that CEBPB-bound sites co-occurring with RNAP2 were more likely to exhibit enhancer activity. CEBPB-bound sites further maintained substantial cell-type specificity, indicating that local DNA sequence can accurately convey cell-type–specific regulatory information. By comparing our CRE-seq results to a comprehensive set of genome annotations, we identified a variety of genomic features that are strong predictors of regulatory element activity and cell-type–specific activity. Collectively, our functional assay results indicate that RNAP2 occupancy can be used as a key genomic marker that can distinguish active from inactive TF bound sites. PMID:26486725

  11. Cinnamate-4-hydroxylase expression in arabidopsis. Regulation in response to development and the environment

    Energy Technology Data Exchange (ETDEWEB)

    Bell-Lelong, D.A.; Cusumano, J.C.; Meyer, K.; Chapple, C. [Purdue Univ., West Lafayette, IN (United States)


    Cinnamate-r-hydroxylase (C4H) is the first Cyt P450-dependent monooxygenase of the phenylpropanoid pathway. To study the expression of this gene in Arabidopsis thaliana, a C4H cDNA clone from the Arabidopsis expressed sequence tag database was identified and used to isolate its corresponding genomic clone. The entire C4H coding sequence plus 2.9 kb of its promoter were isolated on a 5.4-kb HindIII fragment of this cosmid. Inspection of the promoter sequence revealed the presence of a number of putative regulatory motifs previously identified in the promoters of other phenylpropanoid pathway genes. The expression of C4H was analyzed by RNA blot hybridization analysis and in transgenic Arabidopsis carrying a C4H-{beta}-glucuronidase transcriptional fusion. C4H message accumulation was light-dependent, but was detectable even in dark-grown seedlings. Consistent with these data, C4H mRNA was accumulated to light-grown levels in etiolated det1-1 mutant seedlings. C4H is widely expressed in various Arabidopsis tissues, particularly in roots and cells undergoing lignification. The C4H-driven {beta}-glucuronidase expression accurately reflected the tissue-specificity and wound-inducibility of the C4H promoter indicated by RNA blot hybridization analysis. A modest increase in C4H expression was observed in the tt8 mutant of Arabidopsis. 77 refs., 5 figs.

  12. Threonine 79 is a hinge residue that governs the fidelity of DNA polymerase beta by helping to position the DNA within the active site. (United States)

    Maitra, Mausumi; Gudzelak, Andrew; Li, Shu-Xia; Matsumoto, Yoshihiro; Eckert, Kristin A; Jager, Joachim; Sweasy, Joann B


    DNA polymerase beta (pol beta) is an ideal system for studying the role of its different amino acid residues in the fidelity of DNA synthesis. In this study, the T79S variant of pol beta was identified using an in vivo genetic screen. T79S is located in the N-terminal 8-kDa domain of pol beta and has no contact with either the DNA template or the incoming dNTP substrate. The T79S protein produced 8-fold more multiple mutations in the herpes simplex virus type 1-thymidine kinase assay than wild-type pol beta. Surprisingly, T79S is a misincorporation mutator only when using a 3'-recessed primer-template. In the presence of a single nucleotide-gapped DNA substrate, T79S displays an antimutator phenotype when catalyzing DNA synthesis opposite template C and has similar fidelity as wild type opposite templates A, G, or T. Threonine 79 is located directly between two helix-hairpin-helix motifs located within the 8-kDa and thumb domains of pol beta. As the pol beta enzyme closes into its active form, the helix-hairpin-helix motifs appear to assist in the production and stabilization of a 90 degrees bend of the DNA. The function of the bent DNA is to present the templating base to the incoming nucleotide substrate. We propose that Thr-79 is part of a hydrogen bonding network within the helix-hairpin-helix motifs that is important for positioning the DNA within the active site. We suggest that alteration of Thr-79 to Ser disrupts this hydrogen bonding network and results in an enzyme that is unable to bend the DNA into the proper geometry for accurate DNA synthesis.

  13. Induction by phenobarbital of aniline-p-hydroxylase in mouse liver under the influence of X-irradiation and 2,4,6-triethyleneimino-1,3,5-triazine

    International Nuclear Information System (INIS)

    The phenobarbital-induced activity of aniline-p-hydroxylase in livers of mice was enhanced additionally when the animals were X-irradiated 4-16 hours before the administration of the inducer. The same effect could be demonstrated after repeated irradiation with low doses. 2,4,6-triethyleneimino-1,3,5-triazine (tretamine) inhibited the induction of aniline-p-hydroxylase only when administered in extremely high doses. Lower doses resulted in 'superinduciton'. (orig.)

  14. Synthesis and structure-activity relationship study of cytotoxic germanicane- and lupane-type 3beta-O-monodesmosidic saponins starting from betulin. (United States)

    Thibeault, Dominic; Gauthier, Charles; Legault, Jean; Bouchard, Jimmy; Dufour, Philippe; Pichette, André


    Germanicane-type triterpenes allobetulin (3) and 28-oxoallobetulin (4) can be obtained by the Wagner-Meerwein rearrangement of the more available lupane-type triterpenes betulin (1) and betulinic acid (2), respectively. The medical uses of betulinic acid (2) and its derivatives are limited because of their poor hydrosolubility and pharmacokinetics properties. In order to overcome this major problem, we synthesized and studied the in vitro anticancer activity of a series of 3beta-O-monodesmosidic saponins derived from betulin (14-16), betulinic acid (20-22), allobetulin (23-28) and 28-oxoallobetulin (29-34) based on six different natural sugar residues (d-glucose, l-rhamnose, d-arabinose, d-galactose, d-mannose and d-xylose). This structure-activity relationship study confirmed that betulinic acid saponins are generally better in vitro anticancer agents than those derived from betulin with the exception of betulin 3beta-O-alpha-d-mannopyranoside (15) which exerted a potent cytotoxic activity against lung carcinoma (A-549) and colorectal adenocarcinoma (DLD-1) human cell lines with IC(50) ranging from 7.3 to 10.1mumol/L. Furthermore, although the synthesis of novel germanicane-type saponins was carried out with success, the bioactivity measured for these glycosides was not as high as we anticipated since only the 3beta-O-beta-d-glucopyranoside and 3beta-O-beta-d-galactopyranoside of allobetulin (23,24) showed moderate anticancer activity (IC(50) 30-40 micromol/L). PMID:17614290

  15. Overexpression of EB1 in human esophageal squamous cell carcinoma (ESCC) may promote cellular growth by activating beta-catenin/TCF pathway. (United States)

    Wang, Yihua; Zhou, Xiaobo; Zhu, Hongxia; Liu, Shuang; Zhou, Cuiqi; Zhang, Guo; Xue, Liyan; Lu, Ning; Quan, Lanping; Bai, Jinfeng; Zhan, Qimin; Xu, Ningzhi


    Esophageal squamous cell carcinoma (ESCC) has a multifactorial etiology involving environmental and/or genetic factors. End-binding protein 1 (EB1), which was cloned as an interacting partner of the adenomatous polyposis coli (APC) tumor suppressor protein, was previously found overexpressed in ESCC. However, the precise role of EB1 in the development of this malignancy has not yet been elucidated. In this study, we analysed freshly resected ESCC specimens and demonstrated that EB1 was overexpressed in approximately 63% of tumor samples compared to matched normal tissue. We report that overexpression of EB1 in the ESCC line EC9706 significantly promotes cell growth, whereas suppression of EB1 protein level by RNA interference significantly inhibited growth of esophageal tumor cells. In addition, EB1 overexpression induced nuclear accumulation of beta-catenin and promoted the transcriptional activity of beta-catenin/T-cell factor (TCF). These effects were partially or completely abolished by coexpression of APC or DeltaN TCF4, respectively. Also, we found that EB1 affected the interaction between beta-catenin and APC. Furthermore, EB1 overexpression was correlated with cytoplasmic/nuclear accumulation of beta-catenin in primary human ESCC. Taken together, these results support the novel hypothesis that EB1 overexpression may play a role in the development of ESCC by affecting APC function and activating the beta-catenin/TCF pathway.

  16. The role of endo-beta-Mannanase activity in tomato seed germination.

    NARCIS (Netherlands)

    Toorop, P.E.


    The role of endo-β-mannanase activity in tomato seed germination was studied using the osmotic agent PEG 6000 and the plant hormones abscisic acid (ABA) and gibberellic acid. Endo-β-mannanase is known to degrade galactomannans in cell walls, and its activity was found in the lateral endosperm upon r

  17. beta-carotene does not change markers of enzymatic and nonenzymatic antioxidant activity in human blood

    DEFF Research Database (Denmark)

    Castenmiller, J.J.M.; Lauridsen, Søren T.; Dragsted, Lars O.;


    In vitamin A-replete populations, increased concentrations of serum carotenoids have been associated with a decreased risk of degenerative diseases. The mechanism of action of carotenoids in determining antioxidant activity is largely unknown. The aim of the study was to examine the effect...... of carotenoid supplementation and spinach intake on erythrocyte enzyme antioxidant activities, serum or plasma nonenzymatic antioxidant concentrations, and concentrations of oxidatively damaged amino acids in plasma; Subjects received for 3 wk a basic diet (n = 10), a basic diet with a carotenoid supplement (n...... and erythrocyte enzyme activities were assessed, and differences among experimental groups were tested. Consumption of spinach resulted in greater (P activity and lower (P activity and serum alpha-tocopherol concentration compared...

  18. Synthesis and biological activity of some heterocyclic compounds containing benzimidazole and beta-lactam moiety

    Indian Academy of Sciences (India)

    K F Ansari; C Lal


    A number of 1-substituted-2-methyl benzimidazole derivatives have been synthesized and tested for their antibacterial activities. The chemical structures of the newly synthesized compounds were verified on the basis of spectral and elemental methods of analyses. Investigation of antimicrobial activity of the compounds was done by disc diffusion method using Gram-positive (S. aureus, S. mutans and B. subtilis), Gram-negative (E. coli, S. typhi and P. aeruginosa) bacteria and fungi (C. albicans, A. flavus and A. niger). Among the compounds tested 5a, 5b, 5d, 5i, 5j and 5k exhibited good antibacterial activities against Gram positive bacteria, while 5d and 5i also showed notable antifungal activity. Specially compounds 5a and 5b exhibited appreciable activity against S. aureus and B. subtilis comparable to reference drugs.

  19. Improving of understanding of beta-hexachlorocyclohexane (HCH) adsorption on activated carbons by temperature-programmed desorption studies. (United States)

    Passé-Coutrin, Nady; Maisonneuve, Laetitia; Durimel, Axelle; Dentzer, Joseph; Gadiou, Roger; Gaspard, Sarra


    In order to understand the interactions between beta-hexachlorocyclohexane (HCH) and chemical groups at activated carbon (AC) surface, the solid samples were hydrogenated aiming to decrease the amounts of oxygenated groups. Two AC samples designated by BagH2O and BagP1.5 were prepared by water vapor activation and phosphoric acid activation, respectively, of sugarcane bagasse used as an AC precursor. A more simple molecule 1,2,3-trichloropropane (TCP) is used as a model of chlorinated compound. The AC were characterized by infrared, X-ray photoelectron spectroscopy (XPS), Raman resonance spectroscopies, as well as temperature-programmed desorption coupled with mass spectrometry (TPD-MS). BagP1.5 and BagH2O AC surface contained oxygenated groups. Upon hydrogenation, a decrease of most of these group amxounts was observed for both samples, while hydroxyl groups increased. On the basis of temperature-programmed desorption data obtained for AC samples contaminated with TCP or HCH, it was possible to determine the type of hydrogen bond formed between each AC and HCH. PMID:26018287

  20. Beta-Glucosidase Activity in Paddy Soils of the Taihu Lake Region, China

    Institute of Scientific and Technical Information of China (English)

    WANG Xiao-Chang; LU Qin


    The profile distribution ofβ-gulcosidase activity in twelve typical paddy soil profiles with high productivity in the Taihu Lake region of China were investigated. Activities ofβ-gulcosidase in the plow layers were in the range of 52.68-137.02μg PNP g-1 soil h-1 with a mean of 89.22μg PNP g-1 soil h-1. However, most plow layers ranged from 70 to 110μg PNP g-1 soil h-1. The profile distribution of β-gulcosidase activity in the 12 soil profiles decreased rapidly with soil depth, with activity at the 60 cm depth only about 10% of that in the surface layers (0-15 cm or 0-20 cm). In these soil profiles, β-gulcosidase activity was significantly positively correlated with soil organic carbon and arylsulphatase activity.Meanwhile, a significantly negative correlation was shown betweenβ-gulcosidase activity and soil pH.

  1. The Relationship Between Beta Endorphins and Emotional State in Physically Active Individuals Aged 45-55 (A Report on a Pilot Study)


    Kundziņa Ieva; Grants Juris


    Introduction. This sports-science-related article heavily relies on studies that have reported an increase in beta-endorphin (â-EP) concentration in plasma in response to physical activity. It examines the psychological and physiological effects of physical activity and exercise and reports on a research-experiment-based, endorphin-hypotheses-related pilot study aimed at exploring mood-related â-EP effects occurring in physically active male and female individuals aged 45-55 in response to ph...

  2. Deposition of atmospheric 210Pb and total beta activity in Finland

    International Nuclear Information System (INIS)

    The seasonal and regional variation of the atmospheric 210Pb deposition in Finland was studied. The 210Pb activity concentration in precipitation shows a decreasing trend from southeastern Finland north-westwards. An average deposition of 40 Bq/m2 during a 12 months period was observed. The deposition of 210Pb shows a seasonal variation with minimum in spring and maximum in autumn and winter. The specific activity of 210Pb (activity of 210Pb per unit mass of stable lead) in the atmosphere has returned to the level prior to World War II owing to the reduced lead emissions into the atmosphere. (author)

  3. Effectiveness of a Prudhoe Bay crude oil and its aliphatic, aromatic and heterocyclic fractions in inducing mortality and aryl hydrocarbon hydroxylase in chick embryo in ovo

    Energy Technology Data Exchange (ETDEWEB)

    Walters, P.; Khan, S.; O' Brien, P.J.O.; Rahimtula, A.T.; Payne, J.F.


    Prudhoe Bay crude oil (PBCO) and its aliphatic, aromatic and heterocyclic fractions were tested on the developing chick embryo for (i) embryotoxicity (ii) their ability to induce hepatic and renal cytochrome P450 levels as well as hepatic, renal and pulmonary aryl hydrocarbon hydroxylase activities. On the basis of its concentration in PBCO, the aromatic fraction was responsible for most of the embryotoxicity as well as for the enzyme inducing ability. The NOS fraction constituted less than 7% (w/v) of PbCO but, on a weight equivalent basis, was roughly as potent as the aromatic fraction in causing embryotoxicity and in inducing cytochrome P450 levels and aryl hydrocarbon hydroxylase. The aliphatic fraction was found to be essentially inactive. The results are consistent with the concept that elevation of aryl hydrocarbon hydroxylase levels by certain components of PBCO may lead to increased embroyotoxicity.

  4. Effect of treadmill exercise on social interaction and tyrosine hydroxylase expression in the attention-deficit/ hyperactivity disorder rats


    Baek, Dae-Jung; Lee, Chae-Bin; Baek, Seung-Soo


    Attention-deficit/hyperactivity disorder (ADHD) involves clinically heterogeneous dysfunctions of sustained attention, with behavioral hyper-activity and impulsivity. The exact underlying mechanisms of ADHD are not known, however, impairment of dopaminergic system in the nigrostriatal pathway was suggested as the one of the possible mechanisms of ADHD. Tyrosine hydroxylase (TH) is the rate-limiting enzyme that is involved in the synthesis of dopamine. Spontaneous hypertensive rats have been u...

  5. Receptor Density Is Key to the Alpha2/Beta Interferon Differential Activities


    Moraga, I.; Harari, D.; Schreiber, G.; Uze, G.; Pellegrini, S.


    Multiple type I interferons (IFN-α/β) elicit Jak/Stat activation, rapid gene induction, and pleiotropic effects, such as differentiation, antiviral protection, and blocks in proliferation, which are dependent on the IFN subtype and the cellular context. To date, ligand- and receptor-specific molecular determinants underlying IFN-α/β differential activities or potencies have been well characterized. To analyze cellular determinants that impact subtype-specific potency, human fibrosarcoma U5A-d...

  6. Crystal Structure of Full-length Mycobacterium tuberculosis H37Rv Glycogen Branching Enzyme; Insights of N-Terminal [beta]-Sandwich in Sustrate Specifity and Enzymatic Activity

    Energy Technology Data Exchange (ETDEWEB)

    Pal, Kuntal; Kumar, Shiva; Sharma, Shikha; Garg, Saurabh Kumar; Alam, Mohammad Suhail; Xu, H. Eric; Agrawal, Pushpa; Swaminathan, Kunchithapadam (NU Sinapore); (Van Andel); (IMT-India)


    The open reading frame Rv1326c of Mycobacterium tuberculosis (Mtb) H37Rv encodes for an {alpha}-1,4-glucan branching enzyme (MtbGlgB, EC, Uniprot entry Q10625). This enzyme belongs to glycoside hydrolase (GH) family 13 and catalyzes the branching of a linear glucose chain during glycogenesis by cleaving a 1 {yields} 4 bond and making a new 1 {yields} 6 bond. Here, we show the crystal structure of full-length MtbGlgB (MtbGlgBWT) at 2.33-{angstrom} resolution. MtbGlgBWT contains four domains: N1 {beta}-sandwich, N2 {beta}-sandwich, a central ({beta}/{alpha}){sub 8} domain that houses the catalytic site, and a C-terminal {beta}-sandwich. We have assayed the amylase activity with amylose and starch as substrates and the glycogen branching activity using amylose as a substrate for MtbGlgBWT and the N1 domain-deleted (the first 108 residues deleted) Mtb{Delta}108GlgB protein. The N1 {beta}-sandwich, which is formed by the first 105 amino acids and superimposes well with the N2 {beta}-sandwich, is shown to have an influence in substrate binding in the amylase assay. Also, we have checked and shown that several GH13 family inhibitors are ineffective against MtbGlgBWT and Mtb{Delta}108GlgB. We propose a two-step reaction mechanism, for the amylase activity (1 {yields} 4 bond breakage) and isomerization (1 {yields} 6 bond formation), which occurs in the same catalytic pocket. The structural and functional properties of MtbGlgB and Mtb{Delta}108GlgB are compared with those of the N-terminal 112-amino acid-deleted Escherichia coli GlgB (EC{Delta}112GlgB).

  7. Regulation of Gene Expression of Catecholamine Biosynthetic Enzymes in Dopamine-β-Hydroxylase- and CRH-Knockout Mice Exposed to Stress


    Richard, Kvetnansky; Olga, Krizanova; Andrej, Tillinger; Sabban Esther, L.; Thomas Steven, A; Lucia, Kubovcakova


    Norepinephrine-deficient mice harbor a disruption of the gene for dopamine-β-hydroxylase (DBH-KO). Corticotropin-releasing hormone knockout mice (CRH-KO) have markedly reduced HPA activity. The aim of the present work was to study how deficiency of DBH and CRH would affect tyrosine hydroxylase (TH), DBH, and phenylethanolamine N-methyltransferase (PNMT) gene expression and protein levels in the adrenal medulla (AM) and stellate ganglia (SG) of control and stressed mice. Both in AM and SG, sin...

  8. Elaboration of Co-60 sources on gilding vyns, with beta efficiencies 80 % or better and its activity measure by the coincidences method

    International Nuclear Information System (INIS)

    The measuring technique of radioactive activity by the coincidences method 4 π β- γ it requires of the elaboration of radioactive sources on thin supports in order to flows the biggest percentage of beta particles, those of the order of the 80 % or but. In this work a procedure for the elaboration of this type of sources, with gilding Vyns is reported. (Author)

  9. The nicotinic alpha7 acetylcholine receptor agonist ssr180711 is unable to activate limbic neurons in mice overexpressing human amyloid-beta1-42

    DEFF Research Database (Denmark)

    Soderman, A.; Spang-Thomsen, Mogens; Hansen, H.;


    Recent studies have demonstrated that amyloid-beta1-42 (Abeta1-42) binds to the nicotinergic alpha7 acetylcholine receptor (alpha7 nAChR) and that the application of Abeta1-42 to cells inhibits the function of the alpha7 nAChR. The in vivo consequences of the pharmacological activation of the alpha...

  10. Efficacy of attractive toxic sugar baits (ATSB) against Aedes albopictus with garlic oil encapsulated in beta-Cyclodextrin as the active ingredient (United States)

    We tested the efficacy of attractive toxic sugar bait (ATSB) with garlic oil microencapsulated in beta-cyclodextrin as active ingredient against Aedes albopictus in suburban Haifa, Israel. Two three-acre gardens with high numbers of Ae. albopictus were chosen for perimeter spray treatment with ATSB ...

  11. Glucocorticoids Inhibit Basal and Hormone-Induced Serotonin Synthesis in Pancreatic Beta Cells (United States)

    Hasni Ebou, Moina; Singh-Estivalet, Amrit; Launay, Jean-Marie; Callebert, Jacques; Tronche, François; Ferré, Pascal; Gautier, Jean-François; Guillemain, Ghislaine; Bréant, Bernadette


    Diabetes is a major complication of chronic Glucocorticoids (GCs) treatment. GCs induce insulin resistance and also inhibit insulin secretion from pancreatic beta cells. Yet, a full understanding of this negative regulation remains to be deciphered. In the present study, we investigated whether GCs could inhibit serotonin synthesis in beta cell since this neurotransmitter has been shown to be involved in the regulation of insulin secretion. To this aim, serotonin synthesis was evaluated in vitro after treatment with GCs of either islets from CD1 mice or MIN6 cells, a beta-cell line. We also explored the effect of GCs on the stimulation of serotonin synthesis by several hormones such as prolactin and GLP 1. We finally studied this regulation in islet in two in vivo models: mice treated with GCs and with liraglutide, a GLP1 analog, and mice deleted for the glucocorticoid receptor in the pancreas. We showed in isolated islets and MIN6 cells that GCs decreased expression and activity of the two key enzymes of serotonin synthesis, Tryptophan Hydroxylase 1 (Tph1) and 2 (Tph2), leading to reduced serotonin contents. GCs also blocked the induction of serotonin synthesis by prolactin or by a previously unknown serotonin activator, the GLP-1 analog exendin-4. In vivo, activation of the Glucagon-like-Peptide-1 receptor with liraglutide during 4 weeks increased islet serotonin contents and GCs treatment prevented this increase. Finally, islets from mice deleted for the GR in the pancreas displayed an increased expression of Tph1 and Tph2 and a strong increased serotonin content per islet. In conclusion, our results demonstrate an original inhibition of serotonin synthesis by GCs, both in basal condition and after stimulation by prolactin or activators of the GLP-1 receptor. This regulation may contribute to the deleterious effects of GCs on beta cells. PMID:26901633

  12. Glucocorticoids Inhibit Basal and Hormone-Induced Serotonin Synthesis in Pancreatic Beta Cells.

    Directory of Open Access Journals (Sweden)

    Moina Hasni Ebou

    Full Text Available Diabetes is a major complication of chronic Glucocorticoids (GCs treatment. GCs induce insulin resistance and also inhibit insulin secretion from pancreatic beta cells. Yet, a full understanding of this negative regulation remains to be deciphered. In the present study, we investigated whether GCs could inhibit serotonin synthesis in beta cell since this neurotransmitter has been shown to be involved in the regulation of insulin secretion. To this aim, serotonin synthesis was evaluated in vitro after treatment with GCs of either islets from CD1 mice or MIN6 cells, a beta-cell line. We also explored the effect of GCs on the stimulation of serotonin synthesis by several hormones such as prolactin and GLP 1. We finally studied this regulation in islet in two in vivo models: mice treated with GCs and with liraglutide, a GLP1 analog, and mice deleted for the glucocorticoid receptor in the pancreas. We showed in isolated islets and MIN6 cells that GCs decreased expression and activity of the two key enzymes of serotonin synthesis, Tryptophan Hydroxylase 1 (Tph1 and 2 (Tph2, leading to reduced serotonin contents. GCs also blocked the induction of serotonin synthesis by prolactin or by a previously unknown serotonin activator, the GLP-1 analog exendin-4. In vivo, activation of the Glucagon-like-Peptide-1 receptor with liraglutide during 4 weeks increased islet serotonin contents and GCs treatment prevented this increase. Finally, islets from mice deleted for the GR in the pancreas displayed an increased expression of Tph1 and Tph2 and a strong increased serotonin content per islet. In conclusion, our results demonstrate an original inhibition of serotonin synthesis by GCs, both in basal condition and after stimulation by prolactin or activators of the GLP-1 receptor. This regulation may contribute to the deleterious effects of GCs on beta cells.

  13. Activation of the Wnt/{beta}-catenin signaling pathway is associated with glial proliferation in the adult spinal cord of ALS transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yanchun [Department of Histology and Embryology, Weifang Medical University, Weifang, Shandong (China); Department of Histology and Embryology, Shandong University School of Medicine, Jinan, Shandong (China); Guan, Yingjun, E-mail: [Department of Histology and Embryology, Weifang Medical University, Weifang, Shandong (China); Department of Histology and Embryology, Shandong University School of Medicine, Jinan, Shandong (China); Liu, Huancai [Department of Orthopedic, Affiliated Hospital, Weifang Medical University, Weifang, Shandong (China); Wu, Xin; Yu, Li; Wang, Shanshan; Zhao, Chunyan; Du, Hongmei [Department of Histology and Embryology, Weifang Medical University, Weifang, Shandong (China); Wang, Xin, E-mail: [Department of Neurosurgery, Brigham and Women' s Hospital, Harvard Medical School, Boston, MA (United States)


    Highlights: Black-Right-Pointing-Pointer Wnt3a and Cyclin D1 were upregulated in the spinal cord of the ALS mice. Black-Right-Pointing-Pointer {beta}-catenin translocated from the cell membrane to the nucleus in the ALS mice. Black-Right-Pointing-Pointer Wnt3a, {beta}-catenin and Cyclin D1 co-localized for astrocytes were all increased. Black-Right-Pointing-Pointer BrdU/Cyclin D1 double-positive cells were increased in the spinal cord of ALS mice. Black-Right-Pointing-Pointer BrdU/Cyclin D1/GFAP triple-positive cells were detected in the ALS mice. -- Abstract: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive and fatal loss of motor neurons. In ALS, there is a significant cell proliferation in response to neurodegeneration; however, the exact molecular mechanisms of cell proliferation and differentiation are unclear. The Wnt signaling pathway has been shown to be involved in neurodegenerative processes. Wnt3a, {beta}-catenin, and Cyclin D1 are three key signaling molecules of the Wnt/{beta}-catenin signaling pathway. We determined the expression of Wnt3a, {beta}-catenin, and Cyclin D1 in the adult spinal cord of SOD1{sup G93A} ALS transgenic mice at different stages by RT-PCR, Western blot, and immunofluorescence labeling techniques. We found that the mRNA and protein of Wnt3a and Cyclin D1 in the spinal cord of the ALS mice were upregulated compared to those in wild-type mice. In addition, {beta}-catenin translocated from the cell membrane to the nucleus and subsequently activated transcription of the target gene, Cyclin D1. BrdU and Cyclin D1 double-positive cells were increased in the spinal cord of these mice. Moreover, Wnt3a, {beta}-catenin, and Cyclin D1 were also expressed in both neurons and astrocytes. The expression of Wnt3a, {beta}-catenin or Cyclin D1 in mature GFAP{sup +} astrocytes increased. Moreover, BrdU/Cyclin D1/GFAP triple-positive cells were detected in the ALS mice. Our findings suggest that

  14. The autophosphorylation and p34cdc2 phosphorylation sites of casein kinase-2 beta-subunit are not essential for reconstituting the fully-active heterotetrameric holoenzyme

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Issinger, O G;


    Two mutants of human casein kinase-2 beta-subunit with short deletions at either their amino (delta 1-4) or carboxy (delta 209-215) terminal side have been created that have lost the capability to undergo autophosphorylation and p34cdc2 mediated phosphorylation, respectively. Both mutants give rise...... to reconstituted CK2 holoenzymes displaying basal catalytic activity, thermostability and responsiveness to polylysine, identical to those of wild-type holoenzyme, whose reconstitution, moreover, is not affected by previous phosphorylation of the beta-subunit at either its N-terminal or C-terminal sites. Unlike...

  15. Real-time monitoring of beta-d-glucuronidase activity in sediment laden streams: A comparison of prototypes. (United States)

    Stadler, Philipp; Blöschl, Günter; Vogl, Wolfgang; Koschelnik, Juri; Epp, Markus; Lackner, Maximilian; Oismüller, Markus; Kumpan, Monika; Nemeth, Lukas; Strauss, Peter; Sommer, Regina; Ryzinska-Paier, Gabriela; Farnleitner, Andreas H; Zessner, Matthias


    Detection of enzymatic activities has been proposed as a rapid surrogate for the culture-based microbiological pollution monitoring of water resources. This paper presents the results of tests on four fully automated prototype instruments for the on-site monitoring of beta-d-glucuronidase (GLUC) activity. The tests were performed on sediment-laden stream water in the Hydrological Open Air Laboratory (HOAL) during the period of March 2014 to March 2015. The dominant source of faecal pollution in the stream was swine manure applied to the fields within the catchment. The experiments indicated that instrument pairs with the same construction design yielded highly consistent results (R(2) = 0.96 and R(2) = 0.94), whereas the results between different designs were less consistent (R(2) = 0.71). Correlations between the GLUC activity measured on-site and culture-based Escherichia coli analyses over the entire study period yielded R(2) = 0.52 and R(2) = 0.47 for the two designs, respectively. The correlations tended to be higher at the event scale. The GLUC activity was less correlated with suspended sediment concentrations than with E. coli, which is interpreted in terms of indicator applicability and the time since manure application. The study shows that this rapid assay can yield consistent results over a long period of on-site operation in technically challenging habitats. Although the use of GLUC activity as a proxy for culture-based assays could not be proven for the observed habitat, the study results suggest that this biochemical indicator has high potential for implementation in early warning systems. PMID:27262553

  16. Mutagenic activity of a fluorinated analog of the beta-adrenoceptor ligand carazolol in the Ames test

    NARCIS (Netherlands)

    Doze, P; Elsinga, PH; de Vries, EFJ; Van Waarde, A; Vaalburg, W


    S-1'[F-18]-Fluorocarazolol (FCAR) is a fluorinated analog of the nonmutagenic beta-blocker carazolol (CAR). Tn former studies FCAR proved to be suitable for quantification of beta-adrenoceptors in vivo with positron emission tomography (PET). We report here that FCAR displays no acute toxicity in ei

  17. Synthesis and Evaluation of [F-18]-FEAnGA as a PET Tracer for beta-Glucuronidase Activity

    NARCIS (Netherlands)

    Antunes, Ines F.; Haisma, Hidde J.; Elsinga, Philip H.; Dierckx, Ruch A.; de Vries, Erik F. J.


    To increase the therapeutic index of chemotherapeutic drugs, prodrugs have been investigated as anticancer agents, as they may present fewer eytotoxic side effects than conventional cytotoxic drugs, while therapeutic efficacy is maintained or even increased. Extracellular beta-glucuronidase (beta-GU

  18. [High beta tokamak research

    International Nuclear Information System (INIS)

    Our activities on High Beta Tokamak Research during the past 20 months of the present grant period can be divided into six areas: reconstruction and modeling of high beta equilibria in HBT; measurement and analysis of MHD instabilities observed in HBT; measurements of impurity transport; diagnostic development on HBT; numerical parameterization of the second stability regime; and conceptual design and assembly of HBT-EP. Each of these is described in some detail in the sections of this progress report

  19. Lithium chloride ameliorates learning and memory ability and inhibits glycogen synthase kinase-3 beta activity in a mouse model of fragile X syndrome

    Institute of Scientific and Technical Information of China (English)

    Shengqiang Chen; Xuegang Luo; Quan Yang; Weiwen Sun; Kaiyi Cao; Xi Chen; Yueling Huang; Lijun Dai; Yonghong Yi


    In the present study, Fmr1 knockout mice (KO mice) were used as the model for fragile X syndrome. The results of step-through and step-down tests demonstrated that Fmr1 KO mice had shorter latencies and more error counts, indicating a learning and memory disorder. After treatment with 30, 60, 90, 120, or 200 mg/kg lithium chloride, the learning and memory abilities of the Fmr1 KO mice were significantly ameliorated, in particular, the 200 mg/kg lithium chloride treatment had the most significant effect. Western blot analysis showed that lithium chloride significantly enhanced the expression of phosphorylated glycogen synthase kinase 3 beta, an inactive form of glycogen synthase kinase 3 beta, in the cerebral cortex and hippocampus of the Fmr1 KO mice. These results indicated that lithium chloride improved learning and memory in the Fmr1 KO mice, possibly by inhibiting glycogen synthase kinase 3 beta activity.

  20. A role for Peroxisome Proliferator-Activated Receptor Beta in T cell development (United States)

    Mothe-Satney, Isabelle; Murdaca, Joseph; Sibille, Brigitte; Rousseau, Anne-Sophie; Squillace, Raphaëlle; Le Menn, Gwenaëlle; Rekima, Akila; Larbret, Frederic; Pelé, Juline; Verhasselt, Valérie; Grimaldi, Paul A.; Neels, Jaap G.


    Metabolism plays an important role in T cell biology and changes in metabolism drive T cell differentiation and fate. Most research on the role of metabolism in T lymphocytes focuses on mature T cells while only few studies have investigated the role of metabolism in T cell development. In this study, we report that activation or overexpression of the transcription factor Peroxisome Proliferator-Activated Receptor β (PPARβ) increases fatty acid oxidation in T cells. Furthermore, using both in vivo and in vitro models, we demonstrate that PPARβ activation/overexpression inhibits thymic T cell development by decreasing proliferation of CD4−CD8− double-negative stage 4 (DN4) thymocytes. These results support a model where PPARβ activation/overexpression favours fatty acid- instead of glucose-oxidation in developing T cells, thereby hampering the proliferative burst normally occurring at the DN4 stage of T cell development. As a consequence, the αβ T cells that are derived from DN4 thymocytes are dramatically decreased in peripheral lymphoid tissues, while the γδ T cell population remains untouched. This is the first report of a direct role for a member of the PPAR family of nuclear receptors in the development of T cells. PMID:27680392

  1. Thymosin beta4 targeting impairs tumorigenic activity of colon cancer stem cells. (United States)

    Ricci-Vitiani, Lucia; Mollinari, Cristiana; di Martino, Simona; Biffoni, Mauro; Pilozzi, Emanuela; Pagliuca, Alfredo; de Stefano, Maria Chiara; Circo, Rita; Merlo, Daniela; De Maria, Ruggero; Garaci, Enrico


    Thymosin β4 (Tβ4) is an actin-binding peptide overexpressed in several tumors, including colon carcinomas. The aim of this study was to investigate the role of Tβ4 in promoting the tumorigenic properties of colorectal cancer stem cells (CR-CSCs), which are responsible for tumor initiation and growth. We first found that CR-CSCs from different patients have higher Tβ4 levels than normal epithelial cells. Then, we used a lentiviral strategy to down-regulate Tβ4 expression in CR-CSCs and analyzed the effects of such modulation on proliferation, survival, and tumorigenic activity of CR-CSCs. Empty vector-transduced CR-CSCs were used as a control. Targeting of the Tβ4 produced CR-CSCs with a lower capacity to grow and migrate in culture and, interestingly, reduced tumor size and aggressiveness of CR-CSC-based xenografts in mice. Moreover, such loss in tumorigenic activity was accompanied by a significant increase of phosphatase and tensin homologue (PTEN) and a concomitant reduction of the integrin-linked kinase (ILK) expression, which resulted in a decreased activation of protein kinase B (Akt). Accordingly, exogenous expression of an active form of Akt rescued all the protumoral features lost after Tβ4 targeting in CR-CSCs. In conclusion, Tβ4 may have important implications for therapeutic intervention for treatment of human colon carcinoma. PMID:20566622

  2. Unlike PPAR{gamma}, PPAR{alpha} or PPAR{beta}/{delta} activation does not promote human monocyte differentiation toward alternative macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Bouhlel, Mohamed Amine [Univ Lille Nord de France, F-59000 Lille (France); Inserm U545, F-59000 Lille (France); UDSL, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France); Brozek, John [Genfit, Loos (France); Derudas, Bruno [Univ Lille Nord de France, F-59000 Lille (France); Inserm U545, F-59000 Lille (France); UDSL, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France); Zawadzki, Christophe; Jude, Brigitte [Inserm ERI-9 and Equipe d' Accueil 2693, IFR114, Universite de Lille, Lille (France); Staels, Bart, E-mail: [Univ Lille Nord de France, F-59000 Lille (France); Inserm U545, F-59000 Lille (France); UDSL, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France); Chinetti-Gbaguidi, Giulia [Univ Lille Nord de France, F-59000 Lille (France); Inserm U545, F-59000 Lille (France); UDSL, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France)


    Macrophages adapt their response to micro-environmental signals. While Th1 cytokines promote pro-inflammatory M1 macrophages, Th2 cytokines promote an 'alternative' anti-inflammatory M2 macrophage phenotype. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors expressed in macrophages where they control the inflammatory response. It has been shown that PPAR{gamma} promotes the differentiation of monocytes into anti-inflammatory M2 macrophages in humans and mice, while a role for PPAR{beta}/{delta} in this process has been reported only in mice and no data are available for PPAR{alpha}. Here, we show that in contrast to PPAR{gamma}, expression of PPAR{alpha} and PPAR{beta}/{delta} overall does not correlate with the expression of M2 markers in human atherosclerotic lesions, whereas a positive correlation with genes of lipid metabolism exists. Moreover, unlike PPAR{gamma}, PPAR{alpha} or PPAR{beta}/{delta} activation does not influence human monocyte differentiation into M2 macrophages in vitro. Thus, PPAR{alpha} and PPAR{beta}/{delta} do not appear to modulate the alternative differentiation of human macrophages.

  3. FHL2 mediates dexamethasone-induced mesenchymal cell differentiation into osteoblasts by activating Wnt/beta-catenin signaling-dependent Runx2 expression. (United States)

    Hamidouche, Zahia; Haÿ, Eric; Vaudin, Pascal; Charbord, Pierre; Schüle, Roland; Marie, Pierre J; Fromigué, Olivia


    The differentiation of bone marrow mesenchymal stem cells (MSCs) into osteoblasts is a crucial step in bone formation. However, the mechanisms involved in the early stages of osteogenic differentiation are not well understood. In this study, we identified FHL2, a member of the LIM-only subclass of the LIM protein superfamily, that is up-regulated during early osteoblast differentiation induced by dexamethasone in murine and human MSCs. Gain-of-function studies showed that FHL2 promotes the expression of the osteoblast transcription factor Runx2, alkaline phosphatase, type I collagen, as well as in vitro extracellular matrix mineralization in murine and human mesenchymal cells. Knocking down FHL2 using sh-RNA reduces basal and dexamethasone-induced osteoblast marker gene expression in MSCs. We demonstrate that FHL2 interacts with beta-catenin, a key player involved in bone formation induced by Wnt signaling. FHL2-beta-catenin interaction potentiates beta-catenin nuclear translocation and TCF/LEF transcription, resulting in increased Runx2 and alkaline phosphatase expression, which was inhibited by the Wnt inhibitor DKK1. Reduction of Runx2 transcriptional activity using a mutant Runx2 results in inhibition of FHL2-induced alkaline phosphatase expression in MSCs. These findings reveal that FHL2 acts as an endogenous activator of mesenchymal cell differentiation into osteoblasts and mediates osteogenic differentiation induced by dexamethasone in MSCs through activation of Wnt/beta-catenin signaling- dependent Runx2 expression. PMID:18653765

  4. Adrenergic receptors and gastric secretion in dogs. Is a "tonic balance" relationship between vagal and beta 2-adrenergic activity a possibility?

    DEFF Research Database (Denmark)

    Gottrup, F; Hovendal, C; Bech, K;


    The relative influence of adrenergic receptors on gastric acid secretion in the dog stomach with different vagal activity or "tone" is almost unknown. beta-adrenoceptors seem to be most important for the direct effect of adrenergic stimulation on acid secretion. In this study the effects of vagot...... that a counterbalance between beta 2-adrenergic and cholinergic vagal tone exists. A "tonic balance theory" is suggested and is probably involved in the resulting acid secretion after vagotomy.......The relative influence of adrenergic receptors on gastric acid secretion in the dog stomach with different vagal activity or "tone" is almost unknown. beta-adrenoceptors seem to be most important for the direct effect of adrenergic stimulation on acid secretion. In this study the effects...... of vagotomy and beta 2-adrenoceptor activity were studied in conscious gastric fistula dogs. Pentagastrin stimulated acid output was increased slightly in non-vagotomized dogs and to its prevagotomy level in vagotomized dogs after propranolol infusion. Practolol showed no such effect. Histamine stimulated...

  5. Effects of osmotic stress on the activity of MAPKs and PDGFR-beta-mediated signal transduction in NIH-3T3 fibroblasts

    DEFF Research Database (Denmark)

    Nielsen, M-B; Christensen, Søren Tvorup; Hoffmann, E K


    in a serum- and nutrient-free inorganic medium (300 mosM) to analyze the effects of osmotic stress on MAPK activity and PDGF receptor (PDGFR)-beta-mediated signal transduction. We found that hypoosmolarity (cell swelling at 211 mosM) induced the phosphorylation and nuclear translocation of ERK1/2, most...... likely via a pathway independent of PDGFR-beta and MEK1/2. Conversely, hyperosmolarity (cell shrinkage at 582 mosM) moved nuclear and phosphorylated ERK1/2 to the cytoplasm and induced the phosphorylation and nuclear translocation of p38 and phosphorylation of JNK1/2. In a series of parallel experiments......, hypoosmolarity did not affect PDGF-BB-induced activation of PDGFR-beta, whereas hyperosmolarity strongly inhibited ligand-dependent PDGFR-beta activation as well as downstream mitogenic signal components of the receptor, including Akt and the MEK1/2-ERK1/2 pathway. Based on these results, we conclude that ligand...

  6. Anti-acetylcholinesterase and Antioxidant Activities of Inhaled Juniper Oil on Amyloid Beta (1-42)-Induced Oxidative Stress in the Rat Hippocampus. (United States)

    Cioanca, Oana; Hancianu, Monica; Mihasan, Marius; Hritcu, Lucian


    Juniper volatile oil is extracted from Juniperus communis L., of the Cupressaceae family, also known as common juniper. Also, in aromatherapy the juniper volatile oil is used against anxiety, nervous tension and stress-related conditions. In the present study, we identified the effects of the juniper volatile oil on amyloid beta (1-42)-induced oxidative stress in the rat hippocampus. Rats received a single intracerebroventricular injection of amyloid beta (1-42) (400 pmol/rat) and then were exposed to juniper volatile oil (200 μl, either 1 or 3 %) for controlled 60 min period, daily, for 21 continuous days. Also, the antioxidant activity in the hippocampus was assessed using superoxide dismutase, glutathione peroxidase and catalase specific activities, the total content of the reduced glutathione, protein carbonyl and malondialdehyde levels. Additionally, the acetylcholinesterase activity in the hippocampus was assessed. The amyloid beta (1-42)-treated rats exhibited the following: increase of the acetylcholinesterase, superoxide dismutase and catalase specific activities, decrease of glutathione peroxidase specific activity and the total content of the reduced glutathione along with an elevation of malondialdehyde and protein carbonyl levels. Inhalation of the juniper volatile oil significantly decreases the acetylcholinesterase activity and exhibited antioxidant potential. These findings suggest that the juniper volatile oil may be a potential candidate for the development of therapeutic agents to manage oxidative stress associated with Alzheimer's disease through decreasing the activity of acetylcholinesterase and anti-oxidative mechanism. PMID:25743585

  7. Live Borrelia burgdorferi preferentially activate interleukin-1 beta gene expression and protein synthesis over the interleukin-1 receptor antagonist. (United States)

    Miller, L C; Isa, S; Vannier, E; Georgilis, K; Steere, A C; Dinarello, C A


    Lyme arthritis is one of the few forms of chronic arthritis in which the cause is known with certainty. Because cytokines are thought to contribute to the pathogenesis of chronic arthritis, we investigated the effect of the Lyme disease spirochete, Borrelia burgdorferi, on the gene expression and synthesis of IL-1 beta and the IL-1 receptor antagonist (IL-1ra) in human peripheral blood mononuclear cells. Live B. burgdorferi induced fivefold more IL-1 beta than IL-1 alpha and sevenfold more IL-1 beta than IL-1ra; LPS or sonicated B. burgdorferi induced similar amounts of all three cytokines. This preferential induction of IL-1 beta was most dramatic in response to a low passage, virulent preparation of B. burgdorferi vs. three high passage avirulent strains. No difference in induction of IL-1ra was seen between these strains. The marked induction of IL-1 beta was partially diminished by heat-treatment and abrogated by sonication; IL-1ra was not affected. This suggested that a membrane component(s) accounted for the preferential induction of IL-1 beta. However, recombinant outer surface protein beta induced little IL-1 beta. By 4 h after stimulation, B. burgdorferi induced sixfold more IL-1 beta protein than LPS. In contrast to LPS-induced IL-1 beta mRNA which reached maximal accumulation after 3 h, B. burgdorferi-induced IL-1 beta mRNA showed biphasic elevations at 3 and 18 h. B. burgdorferi-induced IL-1ra mRNA peaked at 12 h, whereas LPS-induced IL-1ra mRNA peaked at 9 h. IL-1 beta synthesis increased in response to increasing numbers of spirochetes, whereas IL-1ra synthesis did not. The preferential induction by B. burgdorferi of IL-1 beta over IL-1ra is an example of excess agonist over antagonist synthesis induced by a microbial pathogen, and may contribute to the destructive lesion of Lyme arthritis. Images PMID:1387885

  8. Biotransformation of Cholesterol and 16α,17α-Epoxypregnenolone and Isolation of Hydroxylase in Burkholderia cepacia SE-1 (United States)

    Zhu, XiangDong; Pang, CuiPing; Cao, Yuting; Fan, Dan


    The metabolism of cholesterol is critical in eukaryotes as a precursor for vitamins, steroid hormones, and bile acids. Some steroid compounds can be transformed into precursors of steroid medicine by some microorganisms. In this study, the biotransformation products of cholesterol and 16α,17α-epoxypregnenolone produced by Burkholderia cepacia SE-1 were investigated, and a correlative enzyme, hydroxylase, was also studied. The biotransformation products, 7β-hydroxycholesterol, 7-oxocholesterol, and 20-droxyl-16α,17α-epoxypregn-1,4-dien-3-one, were purified by silica gel and Sephadex LH-20 column chromatography and identified by nuclear magnetic resonance and mass spectroscopy. The hydroxylase was isolated from the bacterium and the partial sequences of the hydroxylase, which belong to the catalases/peroxidase family, were analyzed using MS/MS analyses. The enzyme showed activity toward cholesterol and had a specific activity of 37.2 U/mg of protein at 30°C and pH 7.0. PMID:27340662

  9. The dopamine β-hydroxylase -1021C/T polymorphism is associated with the risk of Alzheimer's disease in the Epistasis Project

    NARCIS (Netherlands)

    O. Combarros (Onofre); D.R. Warden (Donald); N. Hammond (Naomi); M. Cortina-Borja (Mario); O. Belbin (Olivia); M.G. Lehmann (Michael); G.K. Wilcock (Gordon); K. Brown (Kristelle); P.G. Kehoe (Patrick); R. Barber (Rachel); E. Coto (Eliecer); V. Alvarez (Victoria); P. Deloukas (Panagiotis); R. Gwilliam (Rhian); R. Heun (Reinhard); H. Kölsch (Heike); I. Mateo (Ignacio); A. Oulhaj (Abderrahim); A. Arias-Vásquez (Alejandro); M. Schuur (Maaike); Y.S. Aulchenko (Yurii); M.A. Ikram (Arfan); M.M.B. Breteler (Monique); C.M. van Duijn (Cock); K. Morgan (Kevin); A.D. Smith (David)


    textabstractBackground: The loss of noradrenergic neurones of the locus coeruleus is a major feature of Alzheimer's disease (AD). Dopamine β-hydroxylase (DBH) catalyses the conversion of dopamine to noradrenaline. Interactions have been reported between the low-activity -1021T allele (rs1611115) of

  10. Structure of GES-1 at Atomic Resolution: Insights Into the Evolution of Carbapenamase Activity in the Class a Extended-Spectrum Beta-Lactamases

    Energy Technology Data Exchange (ETDEWEB)

    Smith, C.A.; /SLAC, SSRL; Caccamo, M.; /Notre Dame U.; Kantardjieff, K.A.; /Cal State, Fullerton; Vakulenko, S.; /Notre Dame U.


    The structure of the class A extended-spectrum {beta}-lactamase GES-1 from Klebsiella pneumoniae has been determined to 1.1 Angstrom resolution. GES-1 has the characteristic active-site disulfide bond of the carbapenemase family of {beta}-lactamases and has a structure that is very similar to those of other known carbapenemases, including NMC-A, SME-1 and KPC-2. Most residues implicated in the catalytic mechanism of this class of enzyme are present in the GES-1 active site, including Ser70, which forms a covalent bond with the carbonyl C atom of the {beta}-lactam ring of the substrate during the formation of an acyl-enzyme intermediate, Glu166, which is implicated as both the acylation and deacylation base, and Lys73, which is also implicated as the acylation base. A water molecule crucial to catalysis is observed in an identical location as in other class A {beta}-lactamases, interacting with the side chains of Ser70 and Glu166. One important residue, Asn170, also normally a ligand for the hydrolytic water, is missing from the GES-1 active site. This residue is a glycine in GES-1 and the enzyme is unable to hydrolyze imipenem. This points to this residue as being critically important in the hydrolysis of this class of {beta}-lactam substrate. This is further supported by flexible-docking studies of imipenem with in silico-generated Gly170Asn and Gly170Ser mutant GES-1 enzymes designed to mimic the active sites of imipenem-hydrolyzing point mutants GES-2 and GES-5.

  11. Simulation of TGF-Beta Activation by Low-Dose HZE Radiation in a Cell Culture (United States)

    Plante, Ianik; Cucinotta, Francis A.


    High charge (Z) and energy (E) (HZE) nuclei comprised in the galactic cosmic rays are main contributors to space radiation risk. They induce many lesions in living matter such as non-specific oxidative damage and the double-strand breaks (DSBs), which are considered key precursors of early and late effects of radiation. There is increasing evidence that cells respond collectively rather than individually to radiation, suggesting the importance of cell signaling1. The transforming growth factor (TGF ) is a signaling peptide that is expressed in nearly all cell type and regulates a large array of cellular processes2. TGF have been shown to mediate cellular response to DNA damage3 and to induce apoptosis in non-irradiated cells cocultured with irradiated cells4. TFG molecules are secreted by cells in an inactive complex known as the latency-associated peptide (LAP). TGF is released from the LAP by a conformational change triggered by proteases, thrombospondin-1, integrins, acidic conditions and .OH radical5. TGF then binds to cells receptors and activates a cascade of events mediated by Smad proteins6, which might interfere with the repair of DNA. Meanwhile, increasingly sophisticated Brownian Dynamics (BD) algorithms have appeared recently in the literature7 and can be applied to study the interaction of molecules with receptors. These BD computer models have contributed to the elucidation of signal transduction, ligand accumulation and autocrine loops in the epidermal growth factor (EGF) and its receptor (EFGR) system8. To investigate the possible roles of TGF in an irradiated cell culture, our Monte-Carlo simulation codes of the radiation track structure9 will be used to calculate the activation of TFG triggered by .OH produced by low doses of HZE ions. The TGF molecules will then be followed by a BD algorithm in a medium representative of a cell culture to estimate the number of activated receptors.

  12. Renal sodium retention in cirrhotic rats depends on glucocorticoid-mediated activation of mineralocorticoid receptor due to decreased renal 11beta-HSD-2 activity

    DEFF Research Database (Denmark)

    Thiesson, Helle; Jensen, Boye L; Bistrup, Claus;


    rats with decompensated liver cirrhosis and ascites 7 wk after bile duct ligation (BDL). Renal 11beta-HSD-2 mRNA, protein, and activity were significantly decreased in decompensated rats. The urinary Na(+)/K(+) ratio was reduced by 40%. Renal epithelial sodium channel (ENaC) mRNA and immunostaining......, and reduced ascites formation to the same degree as direct inhibition of MR with K-canrenoate. Total potassium balance was negative in the BDL rats, whereas renal potassium excretion was unchanged. In the distal colon, expression of ENaC was increased in BDL rats. Fecal potassium excretion was increased...... by endogenous glucocorticoids. In addition, the overall potassium loss in the BDL model is due to increased fecal potassium excretion, which is associated with upregulation of ENaC in distal colon....

  13. In-beam PET measurement of $^{7}Li^{3+}$ irradiation induced $\\beta^+}$-activity

    CERN Document Server

    Priegnitz, M; Parodi, K; Sommerer, F; Fiedler, F; Enghardt, W


    At present positron emission tomography (PET) is the only feasible method of an in situ and non-invasive monitoring of patient irradiation with ions. At the experimental carbon ion treatment facility of the Gesellschaft für Schwerionenforschung (GSI) Darmstadt an in-beam PET scanner has been integrated into the treatment site and lead to a considerable quality improvement of the therapy. Since ions other than carbon are expected to come into operation in future patient treatment facilities, it is highly desirable to extend in-beam PET also to other therapeutic relevant ions, e.g. 7Li. Therefore, by means of the in-beam PET scanner at GSI the β+-activity induced by 7Li3+ ions has been investigated for the first time. Targets of PMMA, water, graphite and polyethylene were irradiated with monoenergetic, pencil-like beams of 7Li3+ with energies between 129.1 A MeV and 205.3 A MeV and intensities ranging from 3.0 × 107 to 1.9 × 108 ions s−1. This paper presents the measured β+-activity profiles as well as d...

  14. Expression and characterization of recombinant single-chain salmon class I MHC fused with beta2-microglobulin with biological activity

    DEFF Research Database (Denmark)

    Zhao, Heng; Stet, René J M; Skjødt, Karsten;


    Heterodimeric class I major histocompatibility complex (MHC) molecules consist of a putative 45-kDa heavy chain and a 12-kDa beta2-microglobulin (beta2m) light chain. The knowledge about MHC genes in Atlantic salmon accumulated during the last decade has allowed us to generate soluble and stable...... antibodies were successfully produced against both the MHC class I heavy chain and beta(2)m, and showed binding to the recombinant molecule. The recombinant complex Sasabeta2mUBA*0301 was expressed and isolated; the production was scaled up by adjusting to its optimal conditions. Subsequently...

  15. Activation of PPARd and RXRa stimulates fatty acid oxidatin and insulin secretion inpancreatic beta-cells

    DEFF Research Database (Denmark)

    Børgesen, Michael; Ravnskjær, Kim; Frigerio, Francesca;

    ACTIVATION OF PPARd AND RXRa STIMULATES FATTY ACID OXIDATION AND INSULIN SECRETION IN PANCREATIC b-CELLS Michael Boergesen1, Kim Ravnskjaer2, Francesca Frigerio3, Allan E. Karlsen4, Pierre Maechler3 and Susanne Mandrup1 1 Department of Biochemistry and Molecular Biology, University of Southern...... oxidation and dissipation of lipids particularly in skeletal muscle. Here we show that PPARd at the RNA as well as protein level is the most abundant PPAR subtype in the rat pancreatic ß-cell line INS-1E and in isolated rat pancreatic islets. In keeping with that, a large number of PPAR target genes...... involved in fatty acid uptake and oxidation. This correlates with a 5-fold induction of 14C-Oleate ß-oxidation when INS-1E cells are exposed to PPARd and RXR agonists. Notably, culture of INS-1E cells with oleate and other unsaturated fatty acids in the presence of an RXR agonist induces the same subset...

  16. The crystal structure of tryptophan hydroxylase with bound amino acid substrate

    DEFF Research Database (Denmark)

    Windahl, Michael Skovbo; Petersen, Charlotte Rode; Christensen, Hans Erik Mølager;


    acid hydroxylase with bound natural amino acid substrate. The iron coordination can be described as distorted trigonal bipyramidal coordination with His273, His278, and Glu318 (partially bidentate) and one imidazole as ligands. The tryptophan stacks against Pro269 with a distance of 3.9 Å between...... of the neurotransmitter and hormone serotonin (5-hydroxytryptamine). We have determined the 1.9 Å resolution crystal structure of the catalytic domain (Δ1−100/Δ415−445) of chicken TPH isoform 1 (TPH1) in complex with the tryptophan substrate and an iron-bound imidazole. This is the first structure of any aromatic amino......124−Asp139 and Ile367−Thr369 close around the active site. Similar structural changes are seen in the catalytic domain of phenylalanine hydroxylase (PAH) upon binding of substrate analogues norleucine and thienylalanine to the PAH·BH4 complex. In fact, the chicken TPH1·Trp·imidazole structure...

  17. A rapid assay for 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D 24-hydroxylase

    International Nuclear Information System (INIS)

    A rapid method for the measurement of the 24-hydroxylated metabolites of 25-hydroxy[26,27-3H]vitamin D3 and 1,25-dihydroxy[26,27-3H]vitamin D3 has been developed. This measurement has, in turn, made possible a rapid assay for the 24-hydroxylases of the vitamin D system. The assay involves the use of 26,27-3H-labeled 1,25-dihydroxyvitamin D3 or 25-hydroxyvitamin D3 as the substrate and treatment of the enzyme reaction mixture with sodium periodate, which specifically cleaves the 24-hydroxylated products between carbons 24 and 25, releasing tritiated acetone. The acetone is measured after its separation from the labeled substrate by using a reversed-phase cartridge. The results obtained with this assay were validated by comparison with the results obtained with a well-established high-performance liquid chromatography assay. The activity of the enzyme determined by both methods was equal. This assay has been successfully used for the rapid screening of column fractions during purification of the enzyme and in the screening for monoclonal antibodies to the 24-hydroxylase

  18. RNAi down-regulation of cinnamate-4-hydroxylase increases artemisinin biosynthesis in Artemisia annua. (United States)

    Kumar, Ritesh; Vashisth, Divya; Misra, Amita; Akhtar, Md Qussen; Jalil, Syed Uzma; Shanker, Karuna; Gupta, Madan Mohan; Rout, Prashant Kumar; Gupta, Anil Kumar; Shasany, Ajit Kumar


    Cinnamate-4-hydroxylase (C4H) converts trans-cinnamic acid (CA) to p-coumaric acid (COA) in the phenylpropanoid/lignin biosynthesis pathway. Earlier we reported increased expression of AaCYP71AV1 (an important gene of artemisinin biosynthesis pathway) caused by CA treatment in Artemisia annua. Hence, AaC4H gene was identified, cloned, characterized and silenced in A. annua with the assumption that the elevated internal CA due to knock down may increase the artemisinin yield. Accumulation of trans-cinnamic acid in the plant due to AaC4H knockdown was accompanied with the reduction of p-coumaric acid, total phenolics, anthocyanin, cinnamate-4-hydroxylase (C4H) and phenylalanine ammonia lyase (PAL) activities but increase in salicylic acid (SA) and artemisinin. Interestingly, feeding trans-cinnamic acid to the RNAi line increased the level of artemisinin along with benzoic (BA) and SA with no effect on the downstream metabolites p-coumaric acid, coniferylaldehyde and sinapaldehyde, whereas p-coumaric acid feeding increased the content of downstream coniferylaldehyde and sinapaldehyde with no effect on BA, SA, trans-cinnamic acid or artemisinin. SA is reported earlier to be inducing the artemisinin yield. This report demonstrates the link between the phenylpropanoid/lignin pathway with artemisinin pathway through SA, triggered by accumulation of trans-cinnamic acid because of the blockage at C4H. PMID:27220407

  19. The activity of serum beta-galactosidase in colon cancer patients with a history of alcohol and nicotine dependence: preliminary data

    Directory of Open Access Journals (Sweden)

    Napoleon Waszkiewicz


    Full Text Available Introduction: Beta-galactosidase (GAL is a lysosomal exoglycosidase involved in the catabolism of glycoconjugates through the sequential release of beta-linked terminal galactosyl residues. The stimulation of activity of exoglycosidases and other degradative enzymes has been noted in cancers as well as in alcohol and nicotine addiction separately. This is the first study to evaluate the activity of the serum senescence marker GAL in colon cancer patients with a history of alcohol and nicotine dependence, as a potential factor of worse cancer prognosis.Material and Methods: The material was serum of 18 colon cancer patients and 10 healthy volunteers. Ten colon cancer patients met alcohol and nicotine dependence criteria. The activity of beta-galactosidase (pkat/ml was determined by the colorimetric method. Comparisons between groups were made using the Kruskal-Wallis analysis and differences evaluated using the Mann-Whitney U test. Spearman’s rank correlation coefficient was used to measure the statistical dependence between two variables.Results: The activity of serum GAL was significantly higher in colon cancer patients with a history of alcohol and nicotine dependence, in comparison to colon cancer patients without a history of drinking/smoking (p=0.015; 46�0increase, and the controls (p=0.0002; 81�0increase. The activity of serum GAL in colon cancer patients without a history of alcohol/nicotine dependence was higher than the activity in the controls (p = 0.043; 24�0increase.Discussion/Conclusion: Higher activity of beta-galactosidase may potentially reflect the accelerated growth of the cancer, invasion, metastases, and maturation, when alcohol and nicotine dependence coincide with colon cancer. For a better prognosis of colon cancer, alcohol and nicotine withdrawal seems to be required.

  20. Isolation and structural elucidation of 4-(beta-D-glucopyranosyldisulfanyl)butyl glucosinolate from leaves of rocket salad (Eruca sativa L.) and its antioxidative activity. (United States)

    Kim, Sun-Ju; Jin, Shigeki; Ishii, Gensho


    A structurally unique glucosinolate (GSL) was identified to be 4-(beta-D-glucopyranosyldisulfanyl)butyl GSL in rocket leaves. The positive-ion electrospray ionization mass spectrometry (ESI-MS) data indicated that the new GSL had a molecular weight of 521 (m/z 522, [M+H](+), as desulfo-GSL). The molecular formula of the substance was determined to be C(17)H(32)O(11)NS(3) (m/z 522.1143, [M+H](+)) based on its positive-ion high-resolution fast atom bombardment mass spectrometry (HR-FAB-MS) data. For the further confirmation, desulfated GSL of 4-(beta-D-glucopyranosyldisulfanyl)butyl GSL was prepared by commercial 1-thio-beta-D-glucose and dimeric 4-mercaptobutyl desulfo-GSL, which was also isolated from rocket leaves, and its chemical structure was then confirmed by MS data and nuclear magnetic resonance (NMR) spectroscopy. In addition, the antioxidative activity of 4-(beta-D-glucopyranosyldisulfanyl)butyl desulfo-GSL was measured by means of chemiluminescence (CL) for evaluating the functional properties. The antioxidative activity (2.089 unit/g) was relatively higher than that of dimeric 4-mercaptobutyl desulfo-GSL (1.227).

  1. Activation of Cyclic AMP Synthesis by Full and Partial Beta-Adrenergic Receptor Agonists in Chicken Skeletal Muscle Cells (United States)

    Young, R. B.; Bridge, K. Y.


    Several beta-adrenergic receptor (bAR) agonists are known to cause hypertrophy of skeletal muscle tissue. Accordingly, five bAR agonists encompassing a range in activity from strong to weak were evaluated for their ability to stimulate CAMP accumulation in embryonic chicken skeletal muscle cells in culture. Two strong agonists (epinephrine and isoproterenol), one moderate agonist (albuterol), and two weak agonists known to cause hypertrophy in animals (clenbuterol and cimaterol) were studied. Dose response curves were determined over six orders of magnitude in concentration for each agonist, and values were determined for their maximum stimulation of CAMP synthesis rate (Bmax) and the agonist concentration at which 50% stimulation of CAMP synthesis (EC50) occurred. Bmax values decreased in the following order: isoproterenol, epinephrine, albuterol, cimaterol, clenbuterol. Cimaterol and clenbuterol at their Bmax concentrations were approximately 15-fold weaker than isoproterenol in stimulating the rate of CAMP synthesis. When cimaterol and clenbuterol were added to culture media at concentrations known to cause significant muscle hypertrophy in animals, there was no detectable effect on stimulation of CAMP synthesis. Finally, these same levels of cimaterol and clenbuterol did not antagonize the stimulation of CAMP by either epinephrine or isoproterenol.

  2. MMTV-Wnt1 and -DeltaN89beta-catenin induce canonical signaling in distinct progenitors and differentially activate Hedgehog signaling within mammary tumors.

    Directory of Open Access Journals (Sweden)

    Brigitte Teissedre

    Full Text Available Canonical Wnt/beta-catenin signaling regulates stem/progenitor cells and, when perturbed, induces many human cancers. A significant proportion of human breast cancer is associated with loss of secreted Wnt antagonists and mice expressing MMTV-Wnt1 and MMTV-DeltaN89beta-catenin develop mammary adenocarcinomas. Many studies have assumed these mouse models of breast cancer to be equivalent. Here we show that MMTV-Wnt1 and MMTV-DeltaN89beta-catenin transgenes induce tumors with different phenotypes. Using axin2/conductin reporter genes we show that MMTV-Wnt1 and MMTV-DeltaN89beta-catenin activate canonical Wnt signaling within distinct cell-types. DeltaN89beta-catenin activated signaling within a luminal subpopulation scattered along ducts that exhibited a K18(+ER(-PR(-CD24(highCD49f(low profile and progenitor properties. In contrast, MMTV-Wnt1 induced canonical signaling in K14(+ basal cells with CD24/CD49f profiles characteristic of two distinct stem/progenitor cell-types. MMTV-Wnt1 produced additional profound effects on multiple cell-types that correlated with focal activation of the Hedgehog pathway. We document that large melanocytic nevi are a hitherto unreported hallmark of early hyperplastic Wnt1 glands. These nevi formed along the primary mammary ducts and were associated with Hedgehog pathway activity within a subset of melanocytes and surrounding stroma. Hh pathway activity also occurred within tumor-associated stromal and K14(+/p63(+ subpopulations in a manner correlated with Wnt1 tumor onset. These data show MMTV-Wnt1 and MMTV-DeltaN89beta-catenin induce canonical signaling in distinct progenitors and that Hedgehog pathway activation is linked to melanocytic nevi and mammary tumor onset arising from excess Wnt1 ligand. They further suggest that Hedgehog pathway activation maybe a critical component and useful indicator of breast tumors arising from unopposed Wnt1 ligand.

  3. Cytocompatibility, antibacterial activity and biodegradability of self-assembling beta-hairpin peptide-based hydrogels for tissue regenerative applications (United States)

    Salick, Daphne Ann

    Every year, millions of people suffer from tissue loss or failure. One approach to repair damaged or diseased tissue is through tissue/organ transplantation. However, one of the major problems which exist with this approach is that there are more people in need of a transplant than there are donors. Over the past several decades, scientists and doctors have come together to find a way to overcome this challenge. This collaboration has led to the development of biomimetic scaffolds, which closely mimic the desired tissue of interest to act as a substitute for the unfunctional tissue, with hopes to improve the quality of life. The Schneider and Pochan labs have developed a biomimetic scaffold using self-assembling beta-hairpin peptides. The self-assembly event can be triggered in response to physiological conditions, which is dictated by the monomer, to form non covalently crosslinked mechanically rigid hydrogels. In vitro studies showed that hydrogels were cytocompatible and may not elicit a pro-inflammatory response from murine macrophages. These material properties show promise for the use of these hydrogels in tissue engineering. When implanting a material into a host, a major concern is the introduction of infection. Infection, if not prevented or halted, results in poor tissue integration and function, ultimately leading to implant removal from the host. Interestingly, the beta-hairpin hydrogels were shown to exhibit antibacterial properties against pathogens commonly found in hospital environments. This inherently antibacterial hydrogel is advantageous because it may help decrease or diminish bacterial contamination when implanted in vivo, which may help to increase the success of implants. Also, a unique and exciting feature of these peptide-based hydrogels is their ability to shear-thin and self-heal. Hydrogels can be directly formed in a syringe and be subsequently delivered to a tissue defect in a minimally invasive manner where they will recover to their

  4. Antibiogram Studies and Extended Spectrum Beta-lactamase Activity Profile of Salmonella-like Species Isolated from Poultry Soil of the University of Uyo, Nigeria

    Directory of Open Access Journals (Sweden)

    Ikpeme, E. M.


    Full Text Available Aims: The contribution of beta-lactamase activity of various bacterial species to the increased antimicrobial resistance being experienced worldwide is very scanty in the literature. This study was undertaken to investigate the antibiotic resistance pattern (antibiogram of Salmonella-like bacterial species against some antibiotics, and the role beta-lactamase assumably produced by the Salmonella-like species, played in producing resistance.Methodology and Results: The antimicrobial sensitivity test and the beta-lactamase test of the Salmonella-like species were carried out using the methods of Kirby Bauer sensitivity test and the Double Disk Synergy test respectively, following isolation and identification of the organisms from poultry soil. Results revealed that Salmonella-like species were most highly resistant to Nalidixic acid (20, 66.66%, followed by Tetracycline (19, 63.33%, Cotrimoxazole, Amoxicillin and Augmentin (18, 60%, while the least was Ofloxacin (8, 26.66%. Multiple resistance of 4 or more antibiotics among the isolates from the soil outside the broilers enclosure was observed, while there was a significant difference (P <0.05 between poultry soil and control soil. This implied that the antibiotics with the highest resistance were most often applied to the birds, the droppings of which contaminated the soil. The resistant pattern of the isolates from the control soil is lower than that from the poultry soil. Extended Spectrum Beta-lactamase activity was expressed by all the isolates against Cefotazime, while the least resistance was against mostly Cefotazime.Conclusion, significance and impact of study: It is concluded that there is a widespread Beta-lactamase activity causing antibiotic resistance by many species of bacteria as well as poultry Salmonella, thus exacerbating the global problem of antibiotic resistance and a serious health related implication for antibiotic use in poultry.

  5. beta-Naphthoflavone protects from peritonitis by reducing TNF-alpha-induced endothelial cell activation. (United States)

    Hsu, Sheng-Yao; Liou, Je-Wen; Cheng, Tsung-Lin; Peng, Shih-Yi; Lin, Chi-Chen; Chu, Yuan-Yuan; Luo, Wei-Cheng; Huang, Zheng-Kai; Jiang, Shinn-Jong


    β-Naphthoflavone (β-NF), a ligand of the aryl hydrocarbon receptor, has been shown to possess anti-oxidative properties. We investigated the anti-oxidative and anti-inflammatory potential of β-NF in human microvascular endothelial cells treated with tumor necrosis factor-alpha (TNF-α). Pretreatment with β-NF significantly inhibited TNF-α-induced intracellular reactive oxygen species, translocation of p67(phox), and TNF-α-induced monocyte binding and transmigration. In addition, β-NF significantly inhibited TNF-α-induced ICAM-1 and VCAM-1 expression. The mRNA expression levels of the inflammatory cytokines TNF-α and IL-6 were reduced by β-NF, as was the infiltration of white blood cells, in a peritonitis model. The inhibition of adhesion molecules was associated with suppressed nuclear translocation of NF-κB p65 and Akt, and suppressed phosphorylation of ERK1/2 and p38. The translocation of Egr-1, a downstream transcription factor involved in the MEK-ERK signaling pathway, was suppressed by β-NF treatment. Our findings show that β-NF inhibits TNF-α-induced NF-kB and ERK1/2 activation and ROS generation, thereby suppressing the expression of adhesion molecules. This results in reduced adhesion and transmigration of leukocytes in vitro and prevents the infiltration of leukocytes in a peritonitis model. Our findings also suggest that β-NF might prevent TNF-α-induced inflammation.

  6. Beta1 integrins activate a MAPK signalling pathway in neural stem cells that contributes to their maintenance

    DEFF Research Database (Denmark)

    Campos, Lia S; Leone, Dino P; Relvas, Joao B;


    in the stem-cell containing regions of the embryonic CNS, with associated expression of the laminin alpha2 chain. Expression levels of laminin alpha2 are reduced in the postnatal CNS, but a population of cells expressing high levels of beta1 remains. Using neurospheres - aggregate cultures, derived from......The emerging evidence that stem cells develop in specialised niches highlights the potential role of environmental factors in their regulation. Here we examine the role of beta1 integrin/extracellular matrix interactions in neural stem cells. We find high levels of beta1 integrin expression...... single stem cells, that have a three-dimensional architecture that results in the localisation of the stem cell population around the edge of the sphere - we show directly that beta1 integrins are expressed at high levels on neural stem cells and can be used for their selection. MAPK, but not PI3K...

  7. Interaction of Protease-Activated Receptor 2 with G Proteins and Beta-Arrestin 1 Studied by Bioluminescence Resonance Energy Transfer

    Directory of Open Access Journals (Sweden)

    Mohammed Akli eAyoub


    Full Text Available G protein-coupled receptors (GPCRs are well recognized as being able to activate several signaling pathways through the activation of different G proteins as well as other signaling proteins such as beta-arrestins. Therefore, understanding how such multiple GPCR-mediated signaling can be integrated constitute an important aspect. Here, we applied bioluminescence resonance energy transfer (BRET to shed more light on the G protein coupling profile of trypsin receptor, or protease-activated receptor 2 (PAR2, and its interaction with beta-arrestin1. Using YFP and Rluc fusion constructs expressed in COS-7 cells, BRET data revealed a pre-assembly of PAR2 with both Galphai1 and Galphao and a rapid and transient activation of these G proteins upon receptor activation. In contrast, no preassembly of PAR2 with Galpha12 could be detected and their physical association can be measured with a very slow and sustained kinetics similar to that of beta-arrestin1 recruitment. These data demonstrate the coupling of PAR2 with Galphai1, Galphao and Galpha12 in COS-7 cells with differences in the kinetics of GPCR-G protein coupling, a parameter that very likely influences the cellular response. Moreover, this further illustrates that preassembly or agonist-induced G protein interaction depends on receptor-G protein pairs indicating another level of complexity and regulation of the signaling of GPCR-G protein complexes and its multiplicity.

  8. Glucose- and interleukin-1beta-induced beta-cell apoptosis requires Ca2+ influx and extracellular signal-regulated kinase (ERK) 1/2 activation and is prevented by a sulfonylurea receptor 1/inwardly rectifying K+ channel 6.2 (SUR/Kir6.2) selective potassium channel opener in human islets

    DEFF Research Database (Denmark)

    Maedler, Kathrin; Størling, Joachim; Sturis, Jeppe;


    -regulated kinase (ERK) 1/2, an effect that was abrogated by 3 micromol/l NN414. Similarly, 1 micromol/l of the mitogen-activated protein kinase/ERK kinase 1/2 inhibitor PD098059 or 1 micromol/l of the l-type Ca(2+) channel blocker nimodipine prevented glucose- and IL-1beta-induced ERK activation, beta......-cell apoptosis, and impaired function. Finally, islet release of IL-1beta in response to high glucose could be abrogated by nimodipine, NN414, or PD098059. Thus, in human islets, glucose- and IL-1beta-induced beta-cell secretory dysfunction and apoptosis are Ca(2+) influx and ERK dependent and can be prevented...

  9. Effect of clavulanic acid on the activities of ten beta-lactam agents against members of the Bacteroides fragilis group. (United States)

    Lamothe, F; Auger, F; Lacroix, J M


    Clavulanic acid reduced the MICs of amoxicillin, carbencillin , cefamandole, cefotaxime, ceftazidime, ceftizoxime, cephalothin, and penicillin G, but not of cefoxitin or moxalactam, against 77 isolates of the Bacteroides fragilis group, all rapidly beta-lactamase positive by the nitrocefin slide test. It had no effect on the susceptibilities of eight Bacteroides distasonis strains that were slowly beta-lactamase positive (18 h of incubation). PMID:6732233

  10. A macroporous bioreactor super activated by the recombinant human transforming growth factor-beta 3

    Directory of Open Access Journals (Sweden)

    Ugo eRipamonti


    Full Text Available Macroporous single-phase hydroxyapatite (HA and biphasic HA/β-tricalcium phosphate with 33% post-sinter hydroxyapatite (HA/β-TCP were combined with 25 or 125 µg recombinant human transforming growth factor-β3 (hTGF-β3 to engineer a super activated bioreactor implanted in orthotopic calvarial and heterotopic rectus abdominis muscle sites and harvested on day 30 and 90. Coral-derived calcium carbonate fully converted (100% and partially converted to 5% and 13% hydroxyapatite/calcium carbonate (HA/CC preloaded with 125 and 250 µg hTGF-β3, and 1:5 and 5:1 binary applications of hTGF-β3: hOP-1 by weight, were implanted in the rectus abdominis and harvested on day 20 and 30, respectively, to monitor spatial/temporal morphogenesis by high doses of hTGF-β3. Bone formation was assessed on decalcified paraffin-embedded sections by measuring the fractional volume of newly-formed bone. On day 30 and 90, single phase HA implants showed greater amounts of bone when compared to biphasic specimens; 5 % and 13 % HA/CC pre-loaded with 125 and 250 µg hTGF-β3 showed substantial induction of bone formation; 250 µg hTGF-β3 induced as yet unreported massive induction of bone formation as early as 20 days prominently outside the profile of the macroporous constructs. The induction of bone formation is controlled by the implanted ratio of the recombinant morphogens, i.e. the 1:5 hTGF-β3:hOP-1 ratio by weight was greater than the inverse ratio. The unprecedented tissue induction by single doses of 250 µg hTGF-β3 resulting in rapid bone morphogenesis of vast mineralized ossicles with multiple trabeculations surfaced by contiguous secreting osteoblasts is the novel molecular and morphological frontier for the induction of bone formation in clinical contexts.

  11. Increased cholesterol 7α-hydroxylase expression and size of the bile acid pool in the lactating rat (United States)

    Wooton-Kee, Clavia Ruth; Cohen, David E.; Vore, Mary


    Maximal bile acid secretory rates and expression of bile acid transporters in liver and ileum are increased in lactation, possibly to facilitate increased enterohepatic recirculation of bile acids. We determined changes in the size and composition of the bile acid pool and key enzymes of the bile acid synthetic pathway [cholesterol 7α-hydroxylase (Cyp7a1), sterol 27-hydroxylase (Cyp27a1), and sterol 12α-hydroxylase (Cyp8b1)] in lactating rats relative to female virgin controls. The bile acid pool increased 1.9 to 2.5-fold [postpartum (PP) days 10, 14, and 19–23], compared with controls. A 1.5-fold increase in cholic acids and a 14 to 20% decrease in muricholic acids in lactation significantly increased the hydrophobicity index. In contrast, the hepatic concentration of bile acids and small heterodimer partner mRNA were unchanged in lactation. A 2.8-fold increase in Cyp7a1 mRNA expression at 16 h (10 h of light) demonstrated a shift in the diurnal rhythm at day 10 PP; Cyp7a1 protein expression and cholesterol 7α-hydroxylase activity were significantly increased at this time and remained elevated at day 14 PP but decreased to control levels by day 21 PP. There was an overall decrease in Cyp27a1 mRNA expression and a 20% decrease in Cyp27a1 protein expression, but there was no change in Cyp8b1 mRNA or protein expression at day 10 PP. The increase in Cyp7a1 expression PP provides a mechanism for the increase in the bile acid pool. PMID:18292185

  12. Restricted expression of Neuroglobin in the mouse retina and co-localization with Melanopsin and Tyrosine Hydroxylase

    Energy Technology Data Exchange (ETDEWEB)

    Hundahl, C.A., E-mail: [Department of Clinical Biochemistry, Bispebjerg Hospital, University of Copenhagen, Copenhagen (Denmark); Centre of Excellence for Translational Medicine, University of Tartu, Tartu (Estonia); Department of Physiology, University of Tartu, Tartu (Estonia); Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen (Denmark); Fahrenkrug, J. [Department of Clinical Biochemistry, Bispebjerg Hospital, University of Copenhagen, Copenhagen (Denmark); Luuk, H. [Centre of Excellence for Translational Medicine, University of Tartu, Tartu (Estonia); Department of Physiology, University of Tartu, Tartu (Estonia); Hay-Schmidt, A. [Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen (Denmark); Hannibal, J. [Department of Clinical Biochemistry, Bispebjerg Hospital, University of Copenhagen, Copenhagen (Denmark)


    Highlights: Black-Right-Pointing-Pointer Restricted Neuroglobin expression in the mouse retina. Black-Right-Pointing-Pointer Antibody validation using Neuroglobin-null mice. Black-Right-Pointing-Pointer Co-expression of Neuroglobin with Melanopsin and tyrosine hydroxylase. Black-Right-Pointing-Pointer No effect of Neuroglobin deficiency on neuronal survival. -- Abstract: Neuroglobin (Ngb), a neuronal specific oxygen binding heme-globin, reported to be expressed at high levels in most layers of the murine retina. Ngb's function is presently unknown, but based on its high expression level and oxygen binding capabilities Ngb was proposed to function as an oxygen reservoir facilitating oxygen metabolism in highly active neurons or to function as a neuroprotectant. In the present study, we re-examined the expression pattern of Ngb in the retina using a highly validated antibody. Furthermore, intactness of retino-hypothalamic projections and the retinal expression level of Melanopsin and Tyrosine Hydroxylase were investigated in Ngb-null mice. Ngb-immunoreactivity was found in a few neurons of the ganglion cell and inner nuclear layers co-expressing Melanopsin and Tyrosine Hydroxylase, respectively. Ngb deficiency neither affected the level of Melanopsin and Tyrosine Hydroxylase proteins nor the intactness of PACAP-positive retinohypothalamic projections in the suprachiasmatic nucleus. Based on the present results, it seems unlikely that Ngb could have a major role in retinal oxygen homeostasis and neuronal survival under normal conditions. The present study suggests that a number of previously published reports have relied on antibodies with dubious specificity.

  13. Carbene vs olefin products of C-H activation on ruthenium via competing alpha- and beta-H elimination. (United States)

    Kuznetsov, Vladimir F; Abdur-Rashid, Kamaluddin; Lough, Alan J; Gusev, Dmitry G


    Bulky pincer complexes of ruthenium are capable of C-H activation and H-elimination from the pincer ligand backbone to produce mixtures of olefin and carbene products. To characterize the products and determine the mechanisms of the C-H cleavage, reactions of [RuCl(2)(p-cymene)](2) with N,N'-bis(di-tert-butylphosphino)-1,3-diaminopropane (L1) and 1,3-bis(di-tert-butylphosphinomethyl)cyclohexane (L2) were studied using a combination of X-ray crystallography, NMR spectroscopy, and DFT computational techniques. The reaction of L1 afforded a mixture of an alkylidene, a Fischer carbene, and two olefin isomers of the 16-e monohydride RuHCl[(t)Bu(2)PNHC(3)H(4)NHPBu(t)(2)] (2), whereas the reaction of L2 gave two olefin and two alkylidene isomers of 16-e RuHCl[2,6-(CH(2)PBu(t)(2))(2)C(6)H(8)] (3), all resulting from dehydrogenations of the ligand backbone of L1 and L2. The key intermediates implicated in the C-H activation reactions were identified as 14-electron paramagnetic species RuCl(PCP), where PCP = cyclometalated L1 or L2. Thus the alpha- and beta-H elimination reactions of RuCl(PCP) involved spin change and were formally spin-forbidden. Hydrogenation of 2 and 3 afforded 16-electron dihydrides RuH(2)Cl(PCP) distinguished by a large quantum exchange coupling between the hydrides. PMID:17076513


    Directory of Open Access Journals (Sweden)

    Seham Ragab


    Full Text Available Background  :Serum haptoglobin (Hp is a reliable marker for hemolysis regardless the inflammatory state.  Objective: We investigated the possible relation between Hp depletion and hemolysis severity, hepatitis C virus (HCV infection and iron load in β-thalassemia children. Methods: Twenty  two β-thalassemia major (TM ,20 β-thalassemia  intermedia (TI children with 20 age and sex matched healthy controls were involved. Pre-transfusion hemoglobin level was considered . Serum ferritin , Hp  and transferrin receptor  levels (sTfR  (by ELISA , alanine aminotransferase (ALT and  aspartate aminotransferase (AST  (by colorimetric method were assayed. Markers of hepatitis C virus  (HCV  were done by PCR. Results:  The mean Hp levels among the studied groups were as follows; 8.02 ± 0.93 (mg/dl , 8.6 ±0.72 (mg/dl  and 122  ± 18.5(mg/dl   for TM ,TI and the controls respectively . Both patient groups had significantly lower Hp level compared to the controls (P<0.0001  with significant lower level in TM compared to TI  children ( P= 0.034  .Significant inverse correlations were  found between serum Hp and sTfR levels in thalassemia children combined and in each group (TM and TI as well as among HCV infected children. STfR   was the only significant independent predictor for  serum Hp level (t= -5.585 , P<0.0001 . Among  HCV infected patients , no significant correlation was found between serum Hp and serum transaminases  .Conclusion:  Serum Hp depletion in thalassemia had significant relation to disease severity and correlated   well with their erythropoietic activity, as assessed by the measurement of  sTfR without significant relation  HCV infection . Large sample  multicenter studies are  recommended.

  15. [Various aspects of IL-1 biological activity. II. IL-1 beta in diseases and the Central Nervous System]. (United States)

    Wieczorek, Marek


    Precise understanding of the mechanisms of reciprocal relations between the nervous and the immune systems, has been the subject of numerous studies for the recent two decades. These mechanisms are significant, particularly at the stage of early response to bacterial, parasite, or viral infections. They are also essential from the medical point of view, as they may help in the development of the new methods of treatment of infectious diseases, and also may provide better methods to neutralize possible side effects of the therapy. As it is commonly understood, both forms of IL-1 (alpha and beta), play an important role as a signaling molecules in these mechanisms. Regardless of the route of administration, they cause to the activation of the brain neurotransmitters, and the hypothalamo-pituitary-adrenal-axis (HPA). The HPA response induced by activity of the immune system is a normal, physiological phenomenon with essential meaning. It gives the negative feedback where glucocorticoids, released from the adrenal cortex, inhibit activity of the immune system, and by this reduce the probability of the over-stimulation of this system and its self-aggression. Therefore, precise recognition of the mechanism which is the indicator of influence of cytokines on the brain and also leads to initiate that response has a significant scientific and practical meaning. Also, the two mechanisms are probably the most important, and under appropriate conditions could complement each other. These are enzymatic and neural ways by which immune system influences the brain. The former predicts, that Il-1 influences the tissue, stimulating them to the synthesis, via the cyclooxygenases (COX) activation, and release molecules such as prostaglandines (especially PGE2), which have the ability to penetrate the brain barrier. The latter assumes that IL-1, directly or indirectly, can influence the peripheral nerves (the most important is probably the vagus nerve), which afferent sensory endings

  16. Differential effects of bone morphogenetic protein-2 and transforming growth factor-beta1 on gene expression of collagen-modifying enzymes in human adipose tissue-derived mesenchymal stem cells. (United States)

    Knippenberg, Marlene; Helder, Marco N; Doulabi, Behrouz Zandieh; Bank, Ruud A; Wuisman, Paul I J M; Klein-Nulend, Jenneke


    Adipose tissue-derived mesenchymal stem cells (AT-MSCs) in combination with bone morphogenetic protein-2 (BMP-2) or transforming growth factor-beta1 (TGF-beta1) are under evaluation for bone tissue engineering. Posttranslational modification of type I collagen is essential for functional bone tissue with adequate physical and mechanical properties. We investigated whether BMP-2 (10-100 ng/mL) and/or TGF-beta1 (1-10 ng/mL) affect gene expression of alpha2(I) procollagen and collagen-modifying enzymes, that is, lysyl oxidase and lysyl hydroxylases 1, 2, and 3 (encoded by PLOD1, 2, and 3), by human AT-MSCs. BMP-2, but not TGF-beta1, increased alkaline phosphatase activity after 28 days, indicating osteogenic differentiation of AT-MSCs. At day 4, both BMP-2 and TGF-beta1 upregulated alpha2(I) procollagen and PLOD1, which was downregulated at day 28. TGF-beta1, but not BMP-2, downregulated PLOD3 at day 28. Lysyl oxidase was upregulated by TGF-beta1 at day 4 and by BMP-2 at day 7. Neither BMP-2 nor TGF-beta1 affected PLOD2. In conclusion, these results suggest that AT-MSCs differentially respond to BMP-2 and TGF-beta1 with changes in gene expression of collagen-modifying enzymes. AT-MSCs may thus be able to appropriately modify type I collagen to form a functional bone extracellular matrix for tissue engineering, dependent on the growth factor added. PMID:19231972

  17. Curcumin Attenuates Beta-Amyloid-Induced Neuroinflammation via Activation of Peroxisome Proliferator-Activated Receptor-Gamma Function in a Rat Model of Alzheimer's Disease. (United States)

    Liu, Zun-Jing; Li, Zhong-Hao; Liu, Lei; Tang, Wen-Xiong; Wang, Yu; Dong, Ming-Rui; Xiao, Cheng


    Neuroinflammation is known to have a pivotal role in the pathogenesis of Alzheimer's disease (AD), and curcumin has been reported to have therapeutical effects on AD because of its anti-inflammatory effects. Curcumin is not only a potent PPARγ agonist, but also has neuroprotective effects on cerebral ischemic injury. However, whether PPARγ activated by curcumin is responsible for the anti-neuroinflammation and neuroprotection on AD remains unclear, and needs to be further investigated. Here, using both APP/PS1 transgenic mice and beta-amyloid-induced neuroinflammation in mixed neuronal/glial cultures, we showed that curcumin significantly alleviated spatial memory deficits in APP/PS1 mice and promoted cholinergic neuronal function in vivo and in vitro. Curcumin also reduced the activation of microglia and astrocytes, as well as cytokine production and inhibited nuclear factor kappa B (NF-κB) signaling pathway, suggesting the beneficial effects of curcumin on AD are attributable to the suppression of neuroinflammation. Attenuation of these beneficial effects occurred when co-administrated with PPARγ antagonist GW9662 or silence of PPARγ gene expression, indicating that PPARγ might be involved in anti-inflammatory effects. Circular dichroism and co-immunoprecipitation analysis showed that curcumin directly bound to PPARγ and increased the transcriptional activity and protein levels of PPARγ. Taking together, these data suggested that PPARγ might be a potential target of curcumin, acting to alleviate neuroinflammation and improve neuronal function in AD. PMID:27594837

  18. Requirement of catalytically active Tyk2 and accessory signals for the induction of TRAIL mRNA by IFN-beta. (United States)

    Rani, M R Sandhya; Pandalai, Sudha; Shrock, Jennifer; Almasan, Alex; Ransohoff, Richard M


    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) mRNA was induced preferentially by interferon (IFN)-beta but not IFN-alpha in human fibrosarcoma and primary fibroblast cells. To characterize the signaling components mediating the IFN subtype-specific induction of this gene, we used mutant cell lines lacking individual components involved in signaling by type I IFNs. TRAIL was not induced by IFN-beta in mutant cell lines U2A, U3A, U4A, U5A, and U6A, which lack, respectively, IFN regulatory factor-9 (IRF-9), Stat1, Jak1, IFNAR-2.2, and Stat2, indicating transcription factor IFN-stimulated gene factor 3 (ISGF3) was essential for the induction of this gene. TRAIL was not induced by IFN-beta in U1A (Tyk2 null) or U1A.R930 cells (that express a kinase-deficient point mutant of Tyk2) but was induced in U1A.wt-5 cells (U1A cells expressing wild-type Tyk2), indicating that Tyk2 protein and kinase activity were both required for induction of the gene. Biochemical and genetic analyses revealed the requirement of transcription factor NF-kappa B and phosphoinositide 3-kinase (PI3K) but not extracellular signal-regulated kinase (ERK) for the induction of TRAIL by IFN-beta. Furthermore, the antiproliferative but not antiviral effects of IFN-beta required catalytically active Tyk2, suggesting that expression of genes, such as TRAIL, may play an important role in mediating the biologic effects of IFNs.

  19. Transforming growth factor-beta activities in 'in vivo' lines of hormone-dependent and independent mammary adenocarcinomas induced by medroxyprogesterone acetate in BALB/c mice. (United States)

    Elizalde, P V; Lanari, C; Kordon, E; Tezón, J; Charreau, E H


    We have determined the presence of transforming growth factor-beta (TGF-beta)-like polypeptides in mammary adenocarcinomas induced by medroxyprogesterone acetate (MPA) in BALB/c mice. In hormone-dependent tumors (HD) from nontreated and MPA-treated mice a high molecular weight (43 kDa) transforming activity was purified by Bio-Gel P-60 chromatography. This TGF was able to confer the neoplastic phenotype on NRK-49F cells without the addition of epidermal growth factor (EGF), though its activity was potentiated by EGF. It did not compete for binding to the EGF receptor, had no mitogenic activity on monolayer cultures of NRK fibroblasts, and was a potent inhibitor of DNA synthesis induced in these cells by EGF and insulin. In HD and hormone-independent tumors (HI) another TGF with a Mr of 13 kDa was isolated. This transforming activity showed the same biological properties as 43 kDa TGF, with the exception that in the absence of EGF it did not stimulate soft agar growth of NRK-49F cells. The synthesis of both factors in 'in vivo' HD tumors seems to be under MPA control, since it is much lower in HD tumors from MPA-treated mice. Further purification of the 13 and 43 kDa TGFs by hydrophobic interaction HPLC demonstrated that each one eluted in a different position, and that their elution profile differed from the TGF-beta from human platelets. The biological activity of the 13 and 43 kDa TGFs was not neutralized by a specific anti-TGF-beta antibody. PMID:2145045

  20. GDP beta S enhances the activation of phospholipase C caused by thrombin in human platelets: evidence for involvement of an inhibitory GTP-binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Oberdisse, E.; Lapetina, E.G.


    Guanosine 5'-O-thiotriphosphate (GTP gamma S) and thrombin stimulate the activity of phospholipase C in platelets that have been permeabilized with saponin and whose inositol phospholipids have been prelabeled with (/sup 3/H)inositol. Ca/sup 2 +/ has opposite effects on the formation of (/sup 3/H)inositol phosphates induced by thrombin or GTP gamma S. While the action of GTP gamma S on the formation of (/sup 3/H)inositol phosphates is inhibited by Ca/sup 2 +/, action of thrombin is stimulated by Ca/sup 2 +/. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits the function of GTP-binding proteins, also inhibits the effect of GTP gamma S on phospholipase C stimulation but, surprisingly, increases the effect of thrombin. Ca/sup 2 +/ increases the inhibitory effect of GDP beta S on GTP gamma S activation of phospholipase C, but Ca/sup 2 +/ further enhances the stimulatory effect of GDP beta S on the thrombin activation of phospholipase C. This indicates that two mechanisms are responsible for the activation of phospholipase C in platelets. A GTP-binding protein is responsible for regulation of phospholipase C induced by GTP gamma S, while the effect of thrombin on the stimulation of phospholipase C is independent of GTP-binding proteins. However, the effect of thrombin may be modulated by the action of an inhibitory GTP-binding protein.

  1. Activation of PPAR{delta} up-regulates fatty acid oxidation and energy uncoupling genes of mitochondria and reduces palmitate-induced apoptosis in pancreatic {beta}-cells

    Energy Technology Data Exchange (ETDEWEB)

    Wan, Jun; Jiang, Li; Lue, Qingguo; Ke, Linqiu [Department of Endocrinology, West China Hospital of Sichuan University, 37 Guoxue Lane, Chengdu, Sichuan 610041 (China); Li, Xiaoyu [State Key Laboratory of Oral Diseases, Sichuan University, No. 14, 3rd Section, Renmin South Road, Chengdu, Sichuan 610041 (China); Tong, Nanwei, E-mail: [Department of Endocrinology, West China Hospital of Sichuan University, 37 Guoxue Lane, Chengdu, Sichuan 610041 (China)


    Recent evidence indicates that decreased oxidative capacity, lipotoxicity, and mitochondrial aberrations contribute to the development of insulin resistance and type 2 diabetes. The goal of this study was to investigate the effects of peroxisome proliferator-activated receptor {delta} (PPAR{delta}) activation on lipid oxidation, mitochondrial function, and insulin secretion in pancreatic {beta}-cells. After HIT-T15 cells (a {beta}-cell line) were exposed to high concentrations of palmitate and GW501516 (GW; a selective agonist of PPAR{delta}), we found that administration of GW increased the expression of PPAR{delta} mRNA. GW-induced activation of PPAR{delta} up-regulated carnitine palmitoyltransferase 1 (CPT1), long-chain acyl-CoA dehydrogenase (LCAD), pyruvate dehydrogenase kinase 4 (PDK4), and uncoupling protein 2 (UCP2); alleviated mitochondrial swelling; attenuated apoptosis; and reduced basal insulin secretion induced by increased palmitate in HIT cells. These results suggest that activation of PPAR{delta} plays an important role in protecting pancreatic {beta}-cells against aberrations caused by lipotoxicity in metabolic syndrome and diabetes.

  2. Aging and a long-term diabetes mellitus increase expression of 1 α-hydroxylase and vitamin D receptors in the rat liver. (United States)

    Vuica, Ana; Ferhatović Hamzić, Lejla; Vukojević, Katarina; Jerić, Milka; Puljak, Livia; Grković, Ivica; Filipović, Natalija


    Diabetes mellitus (DM) is a metabolic disorder associated with serious liver complications. As a metabolic chronic disease, DM is very common in the elderly. Recent studies suggest ameliorating effects of vitamin D on metabolic and oxidative stress in the liver tissue in an experimental model of DM. The aim of this study was to investigate the expression of vitamin D receptors (VDRs) and 1α-hydroxylase, the key enzyme for the production of active vitamin D form (calcitriol) in the liver during long-term diabetes mellitus type 1 (DM1) in aging rats. We performed immunohistochemical analysis of liver expression of 1α-hydroxylase and VDRs during aging in long-term streptozotocin-induced DM1. 1α-Hydroxylase was identified in the monocyte/macrophage system of the liver. In addition to the nuclear expression, we also observed the expression of VDR in membranes of lipid droplets within hepatocytes. Aging and long-term DM1 resulted in significant increases in the number of 1α-hydroxylase immunoreactive cells, as well as the percentage of strongly positive VDR hepatocytes. In conclusion, the liver has the capacity for active vitamin D synthesis in its monocyte/macrophage system that is substantially increased in aging and long-term diabetes mellitus. These conditions are also characterized by significant increases in vitamin D receptor expression in hepatocytes. The present study suggests that VDR signaling system could be a potential target in prevention of liver complications caused by diabetes and aging.

  3. Supermassive Black Holes with High Accretion Rates in Active Galactic Nuclei. IV. H$\\beta$ Time Lags and Implications for Super-Eddington Accretion

    CERN Document Server

    Du, Pu; Lu, Kai-Xing; Huang, Ying-Ke; Cheng, Cheng; Qiu, Jie; Li, Yan-Rong; Zhang, Yang-Wei; Fan, Xu-Liang; Bai, Jin-Ming; Bian, Wei-Hao; Yuan, Ye-Fei; Kaspi, Shai; Ho, Luis C; Netzer, Hagai; Wang, Jian-Min


    We have completed two years of photometric and spectroscopic monitoring of a large number of active galactic nuclei (AGNs) with very high accretion rates. In this paper, we report on the result of the second phase of the campaign, during 2013--2014, and the measurements of five new H$\\beta$ time lags out of eight monitored AGNs. All five objects were identified as super-Eddington accreting massive black holes (SEAMBHs). The highest measured accretion rates for the objects in this campaign are $\\dot{\\mathscr{M}}\\gtrsim 200$, where $\\dot{\\mathscr{M}}= \\dot{M}_{\\bullet}/L_{\\rm Edd}c^{-2}$, $\\dot{M}_{\\bullet}$ is the mass accretion rates, $L_{\\rm Edd}$ is the Eddington luminosity and $c$ is the speed of light. We find that the H$\\beta$ time lags in SEAMBHs are significantly shorter than those measured in sub-Eddington AGNs, and the deviations increase with increasing accretion rates. Thus, the relationship between broad-line region size ($R_{_{\\rm H\\beta}}$) and optical luminosity at 5100\\AA, $R_{_{\\rm H\\beta}}-L...

  4. Modulation of tyrosine hydroxylase gene expression in the central nervous system visualized by in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Berod, A.; Biguet, N.F.; Dumas, S.; Bloch, B.; Mallet, J.


    cDNA probe was used for in situ hybridization studies on histological sections through the locus coeruleus, substantia nigra, and the ventral tegmental area of the rat brain. Experimental conditions were established that yielded no background and no signal when pBR322 was used as control probe. Using the tyrosine hydroxylase probe, the authors ascertained the specificity of the labeling over catecholaminergic cells by denervation experiments and comparison of the hybridization pattern with that of immunoreactivity. The use of /sup 35/S-labeled probe enabled the hybridization signal to be resolved at the cellular level. A single injection of reserpine into the rat led to an increase of the intensity of the autoradiographic signal over the locus coeruleus area, confirming an RNA gel blot analysis. The potential of in situ hybridization to analyze patterns of modulation of gene activity as a result of nervous activity is discussed.

  5. Elevated blood plasma levels of epinephrine, norepinephrine, tyrosine hydroxylase, TGFβ1, and TNFα associated with high-altitude pulmonary edema in an Indian population (United States)

    Pandey, Priyanka; Ali, Zahara; Mohammad, Ghulam; Pasha, M A Qadar


    Biomarkers are essential to unravel the locked pathophysiology of any disease. This study investigated the role of biomarkers and their interactions with each other and with the clinical parameters to study the physiology of high-altitude pulmonary edema (HAPE) in HAPE-patients (HAPE-p) against adapted highlanders (HLs) and healthy sojourners, HAPE-controls (HAPE-c). For this, seven circulatory biomarkers, namely, epinephrine, norepinephrine, tyrosine hydroxylase, transforming growth factor beta 1, tumor necrosis factor alpha (TNFα), platelet-derived growth factor beta beta, and C-reactive protein (CRP), were measured in blood plasma of the three study groups. All the subjects were recruited at ~3,500 m, and clinical features such as arterial oxygen saturation (SaO2), body mass index, and mean arterial pressure were measured. Increased levels of epinephrine, norepinephrine, tyrosine hydroxylase, transforming growth factor-beta 1, and TNFα were observed in HAPE-p against the healthy groups, HAPE-c, and HLs (P0.01). Correlation analysis revealed a negative correlation between epinephrine and norepinephrine (P=4.6E−06) in HAPE-p and positive correlation in HAPE-c (P=0.004) and HLs (P=9.78E−07). A positive correlation was observed between TNFα and CRP (P=0.004) in HAPE-p and a negative correlation in HAPE-c (P=4.6E−06). SaO2 correlated negatively with platelet-derived growth factor beta beta (HAPE-p; P=0.05), norepinephrine (P=0.01), and TNFα (P=0.005) and positively with CRP (HAPE-c; P=0.02) and norepinephrine (HLs; P=0.04). Body mass index correlated negatively with epinephrine (HAPE-p; P=0.001) and positively with norepinephrine and tyrosine hydroxylase in HAPE-c (P0.70, P<0.05). The results clearly suggest that increased plasma levels of these circulatory biomarkers associated with HAPE. PMID:27540296

  6. Antitumor effect of beta2-microglobulin in leukemic cell-bearing mice via apoptosis-inducing activity: activation of caspase-3 and nuclear factor-kappaB. (United States)

    Mori, M; Terui, Y; Tanaka, M; Tomizuka, H; Mishima, Y; Ikeda, M; Kasahara, T; Uwai, M; Ueda, M; Inoue, R; Itoh, T; Yamada, M; Hayasawa, H; Furukawa, Y; Ishizaka, Y; Ozawa, K; Hatake, K


    We have reported previously that beta2-microglobulin (beta2m) induces apoptosis in leukemic cells in vitro, and that an interaction between beta2m and HLA class I antigen induces apoptosis. Here we examined whether beta2m can induce apoptosis in leukemic cells in vivo and whether it has an antitumor effect in tumor-bearing mice. Daily administration of 50 or 250 microg of beta2m induced apoptosis and an antitumor effect on K562 leukemia cell-bearing mice in the same manner as tumor necrosis factor-alpha. In tumor tissues in beta2m-treated mice, both caspase-3 and nuclear factor-kappaB (NF-kappaB) were stained more strongly than in control mice by anti-caspase-3 and anti-NF-kappaB p65/Rel A polyclonal antibodies. We also observed the in vivo immunological effects of beta2m on lymphoid and hematopoietic organs, such as thymus, bone marrow, Peyer's patches, liver, and spleen in normal mice. Using antibodies against caspase-3 and NF-kappaB, immunohistochemical staining showed that no specific tissues were damaged or stained in normal mice. We conclude that beta2m stimulates caspase-3 and NF-kappaB pathways to induce apoptosis, making it a useful approach to a new therapy for leukemia.

  7. Ultrasound-assisted synthesis of 1-N-{beta}-D-glucopyranosyl-1H-1,2,3-triazole benzoheterocycles and their anti-inflammatory activities

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Gilson B. da; Guimaraes, Bruna M.; Oliveira, Ronaldo N. de, E-mail: [Universidade Federal Rural de Pernambuco (UFRPE), Recife, PE (Brazil). Departamento de Ciencias Moleculares; Assis, Shalom P.O.; Lima, Vera L.M. [Universidade Federal Rural de Pernambuco (UFRPE), Recife, PE (Brazil). Departamento de Bioquimica. Laboratorio de Quimica e Metabolismo de Lipideos e Lipoproteinas


    In this work, the preparation of various glucosyl triazoles from a reaction between 2,3,4,6-tetra-O-acetyl-{beta}-D-glucopyranosyl azide and terminal alkynes was developed in moderate to excellent yields (63-99%). Ultrasound energy was applied at each step of the reaction to increase chemical reactivity. In addition, the compounds conjugated with benzoheterocycles moieties revealed potent anti-inflammatory activity. (author)

  8. Cefotetan, a new cephamycin: comparison of in vitro antimicrobial activity with other cephems, beta-lactamase stability, and preliminary recommendations for disk diffusion testing.


    Ayers, L W; Jones, R N; Barry, A. L.; Thornsberry, C; Fuchs, P C; Gavan, T L; Gerlach, E H; Sommers, H M


    Cefotetan is a new, potent, 7 alpha-methoxy cephalosporin (cephamycin). The in vitro activity of cefotetan tested in a multiphasic, collaborative study against 12,260 consecutive clinical isolates and 448 selected isolates showed 93% of Enterobacteriaceae, 90% of methicillin-susceptible Staphylococcus aureus (broth dilution), 83% of Bacteroides fragilis, and 72% of non-enterococcal streptococci to be inhibited by less than or equal to 8 micrograms/ml. Beta-Lactamase-producing and -nonproducin...

  9. Anti-fibrotic role of Ac SDKP through inhibition of P38MAPK pathway activity mediated transforming growth beta recepters in rat with silicosis

    Institute of Scientific and Technical Information of China (English)



    Objective To investigate the distribution and expression of transforming growth factor beta(TGF-β)receptorsⅠandⅡ,p38 mitogen-activated protein kinase(p38 MAPK),and typeⅠand typeⅢcollagen in the lungs of rats with silicosis and cultured pulmonary fibroblasts,and to investigate the relationship of the anti-fibrosis effect of N-acetyl-sery-aspartyl-lysy-proline(Ac SDKP)with its inhibition of TGF-βreceptor-mediated p38

  10. HIF prolyl hydroxylase inhibition augments dopamine release in the rat brain in vivo. (United States)

    Witten, Louise; Sager, Thomas; Thirstrup, Kenneth; Johansen, Jens Leander; Larsen, Dorrit Bjerg; Montezinho, Liliana P; Mørk, Arne


    The transcription factor hypoxia-inducible factor (HIF) is essential for the activation of several genes that promote the survival of cells exposed to oxidative stress. Expression of tyrosine hydroxylase (TH), which is the rate-limiting enzyme in the dopamine (DA) synthesis, is one of the genes that are positively regulated by HIF. Accordingly, HIF induction results in elevated DA release in various cell lines in vitro. HIF prolyl hydroxylase (HPH) is critically involved in the negative regulation of HIF levels. We investigated the in vivo effects of the HPH inhibitor FG0041 on brain DA function in rats by microdialysis in freely moving rats, locomotor activity, and Western blot analysis. Administration of FG0041 (10 mg/kg i.p.), as an acute (single injection), or as subchronic (once daily for 6 days) treatment and cobalt chloride (CoCl2) (60 mg/kg s.c.) potentiated potassium (K+) induced increases in extracellular levels of DA levels in the rat striatum. The increase in extracellular DA of freely moving rats was sought in relationship to locomotor activity in rats. A significant increase in locomotor activity was observed in FG0041-treated rats compared with vehicle on a cocaine challenge. In support of these findings, protein levels of TH in the rat brain stem were increased after treatment with FG0041. These data indicate that FG0041 augments DA function in the rat brain. Inhibition of HPH enhances DA function by increasing DA release, which has implications for the use of HIF induction in the treatment of neurodegenerative diseases. PMID:19156859

  11. Dihydromyricetin protects neurons in an MPTP-induced model of Parkinson's disease by suppressing glycogen synthase kinase-3 beta activity (United States)

    Ren, Zhao-xiang; Zhao, Ya-fei; Cao, Ting; Zhen, Xue-chu


    Aim: It is general believed that mitochondrial dysfunction and oxidative stress play critical roles in the pathology of Parkinson's disease (PD). Dihydromyricetin (DHM), a natural flavonoid extracted from Ampelopsis grossedentata, has recently been found to elicit potent anti-oxidative effects. In the present study, we explored the role of DHM in protecting dopaminergic neurons. Methods: Male C57BL/6 mice were intraperitoneally injected with 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 7 d to induce PD. Additionally, mice were treated with either 5 or 10 mg/kg DHM for a total of 13 d (3 d before the start of MPTP, during MPTP administration (7 d) and 3 d after the end of MPTP). For the saline or DHM alone treatment groups, mice were injected with saline or DHM for 13 d. On d 14, behavioral tests (locomotor activity, the rotarod test and the pole test) were administered. After the behavioral tests, the mice were sacrificed, and brain tissue was collected for immunofluorescence staining and Western blotting. In addition, MES23.5 cells were treated with MPP+ and DHM, and evaluated using cell viability assays, reactive oxygen species (ROS) measurements, apoptosis analysis and Western blotting. Results: DHM significantly attenuated MPTP-induced mouse behavioral impairments and dopaminergic neuron loss. In the MES23.5 cells, DHM attenuated MPP+-induced cell injury and ROS production in a dose-dependent manner. In addition, DHM increased glycogen synthase kinase-3 beta phosphorylation in a dose- and time-dependent manner, which may be associated with DHM-induced dopaminergic neuronal protection. Conclusion: The present study demonstrated that DHM is a potent neuroprotective agent for DA neurons by modulating the Akt/GSK-3β pathway, which suggests that DHM may be a promising therapeutic candidate for PD. PMID:27374489

  12. Evidence for a Dual Role of an Active Site Histidine in [alpha]-Amino-[beta]-carboxymuconate-[epsilon]-semialdehyde Decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Huo, Lu; Fielding, Andrew J.; Chen, Yan; Li, Tingfeng; Iwaki, Hiroaki; Hosler, Jonathan P.; Chen, Lirong; Hasegawa, Yoshie; Que, Jr., Lawrence; Liu, Aimin (GSU); (Kansai); (UMMC); (UMM)


    The previously reported crystal structures of {alpha}-amino-{beta}-carboxymuconate-{epsilon}-semialdehyde decarboxylase (ACMSD) show a five-coordinate Zn(II)(His){sub 3}(Asp)(OH{sub 2}) active site. The water ligand is H-bonded to a conserved His228 residue adjacent to the metal center in ACMSD from Pseudomonas fluorescens (PfACMSD). Site-directed mutagenesis of His228 to tyrosine and glycine in this study results in a complete or significant loss of activity. Metal analysis shows that H228Y and H228G contain iron rather than zinc, indicating that this residue plays a role in the metal selectivity of the protein. As-isolated H228Y displays a blue color, which is not seen in wild-type ACMSD. Quinone staining and resonance Raman analyses indicate that the blue color originates from Fe(III)-tyrosinate ligand-to-metal charge transfer. Co(II)-substituted H228Y ACMSD is brown in color and exhibits an electron paramagnetic resonance spectrum showing a high-spin Co(II) center with a well-resolved {sup 59}Co (I = 7/2) eight-line hyperfine splitting pattern. The X-ray crystal structures of as-isolated Fe-H228Y (2.8 {angstrom}) and Co-substituted (2.4 {angstrom}) and Zn-substituted H228Y (2.0 {angstrom} resolution) support the spectroscopic assignment of metal ligation of the Tyr228 residue. The crystal structure of Zn-H228G (2.6 {angstrom}) was also determined. These four structures show that the water ligand present in WT Zn-ACMSD is either missing (Fe-H228Y, Co-H228Y, and Zn-H228G) or disrupted (Zn-H228Y) in response to the His228 mutation. Together, these results highlight the importance of His228 for PfACMSD's metal specificity as well as maintaining a water molecule as a ligand of the metal center. His228 is thus proposed to play a role in activating the metal-bound water ligand for subsequent nucleophilic attack on the substrate.

  13. Structure-activity relationships in cinnamamides. 1. Synthesis and pharmacological evaluation of some (E)- and (Z)-N-alkyl-alpha,beta-dimethylcinnamamides. (United States)

    Balsamo, A; Barili, P L; Crotti, P; Macchia, D; Macchia, F; Pecchia, A; Cuttica, A; Passerini, N


    Two series of (E)- and (Z)-N-alkyl-alpha,beta-dimethylcinnamamide derivatives were prepared and the biological activity of these compounds was investigated in a series of pharmacological tests. All compounds tested had clear activity on the CNS; generally, this was depressant with E isomers, while Z isomers always caused marked stimulation (tremors and convulsions). Some of the E isomers also had a clear-cut anticonvulsant activity as shown by the antagonistic effect on pentylenetetrazole-induced seizures in the mouse. The NMR spectra of these compounds, which confirm their configurations, are discussed. PMID:1159703

  14. A large duplication in the gene for lysyl hydroxylase accounts for the type VI variant of Ehlers-Danlos syndrome in two siblings

    Energy Technology Data Exchange (ETDEWEB)

    Hautala, T.; Heikkinen, J.; Kivirikko, K.I.; Myllylae, R. (Univ. of Oulu (Finland))


    Ehlers-Danlos syndrome is a deterogeneous disorder characterized by joint hypermobility, skin hyperextensibility, fragility, and other sign of connective tissue involvement. In addition to these, the type VI variant of the disease has some special characteristics such as kyphoscoliosis and ocular abnormalities. The biochemical abnormality in most patients with this autosomal recessively inherited type IV variant is a deficiency in the activity of lysyl hydroxylase (EC 1.14,11.4), the enzyme catalyzing the formation of hydroxylysine in collagens and other proteins with collagen-like amino acid sequences. The type VI variant of Ehlers-Danlos syndrome was first identified in two sisters with a reduced amount of lysyl hydroxylase activity in their skin fibroblasts (S.R. Pinnell, S.M. Krane, J.E. Kenzora, and M.J. Glimcher (1972) N. Engl. J. Med. 286; 1013-1020). Our recent molecular cloning of lysyl hydroxylase has now made it possible to study the mutations leading to the deficiency in lysyl dydroxylase activity in these cells. Our data indicate that the mRNA for lysyl hydroxylase produced in the affected cells is about 4 kb in size, whereas it is 3.2 kb in the control cells. The sequencing of the cDNA for lysyl hydroxylase from the affected cells revealed an apparently homozygous duplication rearrangement of nucleotides 1176 to 1955, corresponding to amino acids 326 to 585 in the normal sequence. From Southern blotting data, the duplicated area in the gene equals about 6-9 kb and corresponds to seven exons. 35 refs., 4 figs.

  15. TGF-ß induces Lysyl hydroxylase 2b in human synovial osteoarthritic fibroblasts through ALK5 signaling. (United States)

    Remst, Dennis F G; Blaney Davidson, Esmeralda N; Vitters, Elly L; Bank, Ruud A; van den Berg, Wim B; van der Kraan, Peter M


    Lysyl hydroxylase 2b (LH2b) is known to increase pyridinoline cross-links, making collagen less susceptible to enzymatic degradation. Previously, we observed a relationship between LH2b and osteoarthritis-related fibrosis in murine knee joint. For this study, we investigate if transforming growth factor-beta (TGF-ß) and connective tissue growth factor (CTGF) regulate procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (gene encoding LH2b) and LH2b expression differently in osteoarthritic human synovial fibroblasts (hSF). Furthermore, we investigate via which TGF-ß route (Smad2/3P or Smad1/5/8P) LH2b is regulated, to explore options to inhibit LH2b during fibrosis. To answer these questions, fibroblasts were isolated from knee joints of osteoarthritis patients. The hSF were stimulated with TGF-ß with or without a kinase inhibitor of ALK4/5/7 (SB-505124) or ALK1/2/3/6 (dorsomorphin). TGF-ß, CTGF, constitutively active (ca)ALK1 and caALK5 were adenovirally overexpressed in hSF. The gene expression levels of PLOD1/2/3, CTGF and COL1A1 were analyzed with Q-PCR. LH2 protein levels were determined with western blot. As expected, TGF-ß induced PLOD2/LH2 expression in hSF, whereas CTGF did not. PLOD1 and PLOD3 were not affected by either TGF-ß or CTGF. SB-505124 prevented the induction of TGF-ß-induced PLOD2, CTGF and COL1A1. Surprisingly, dorsomorphin completely blocked the induction of CTGF and COL1A1, whereas TGF-ß-induced PLOD2 was only slightly reduced. Overexpression of caALK5 in osteoarthritic hSF significantly induced PLOD2/LH2 expression, whereas caALK1 had no effect. We showed, in osteoarthritic hSF, that TGF-ß induced PLOD2/LH2 via ALK5 Smad2/3P. This elevation of LH2b in osteoarthritic hSF makes LH2b an interesting target to interfere with osteoarthritis-related persistent fibrosis. PMID:24192939

  16. Study of a 4{pi}{beta}-{gamma} coincidence system for absolute radionuclide activity measurement using plastic scintillators; Estudo de um sistema de coincidencias 4{pi}{beta}-{gamma} para a medida absoluta de atividade de radionuclideos empregando cintiladores plasticos

    Energy Technology Data Exchange (ETDEWEB)

    Piuvezam Filho, Helio


    The present work was intended to study a coincidence system 4{pi}(PS){beta}-{gamma} for absolute activity measurement using plastic scintillators in 4{pi} geometry. Along with experiments on the coincidence system, simulations were also performed applying the Monte Carlo Method, by means of codes PENELOPE and ESQUEMA. These simulations were performed in order to calculate the extrapolation curve of the coincidence system 4{pi}(PS){beta}-{gamma} and compare it to experimental data. A new geometry was proposed to the coincidence system adding up a second photomultiplier tube to the previous system for improving light collection from the plastic scintillator, as this system presented limitations in the minimum detected energy due to the presence of electronic noise and low gain. The results show that an improvement in the signal-to-noise ratio was obtained, as well as in the minimum detected energy. Moreover, there was an increase in the detection efficiency. With these modifications, it is now possible to calibrate radionuclides which emit low energy electrons or X-rays, increasing the number of radionuclides that can be standardized with this type of system.(author)

  17. Adeno-Associated Virus Vectors (AAV Expressing Phenylalanine Hydroxylase (PAH

    Directory of Open Access Journals (Sweden)

    Ayşegül Akbay Yarpuzlu


    Full Text Available Recent articles have appeared in the literature reporting use of adeno-associated virus vectors (AAV expressing phenylalanine hydroxylase in animal trials and suggesting its use in treatment of phenylketonuria (PKU as a form of gene therapy However, agents used in gene therapy to deliver genes are not site-specific and DNA is may be put in the wrong place, causing damage to the organism. The adverse immunogenicity of AAVs also needs to be reconsidered. This letter is written to discuss present unreadiness for Phase 1 clinical trials of gene therapy of PKU. Turk Jem 2009; 13: 18-9

  18. Cardiac metaiodobenzylguanidine activity can predict the long-term efficacy of angiotensin-converting enzyme inhibitors and/or beta-adrenoceptor blockers in patients with heart failure

    Energy Technology Data Exchange (ETDEWEB)

    Nakata, Tomoaki; Wakabayashi, Takeru; Kyuma, Michifumi; Takahashi, Toru; Tsuchihashi, Kazufumi; Shimamoto, Kazuaki [Sapporo Medical University School of Medicine, Second Department of Internal Medicine (Cardiology), Sapporo (Japan)


    Although the benefits of treatment with angiotensin-converting enzyme (ACE) inhibitors and beta-blockers are well known, no method has as yet been established to predict the efficacy of drug therapy. This study tested whether cardiac{sup 123}I-metaiodobenzylguanidine (MIBG) activity is of prognostic value and can predict the improvement in heart failure patients resulting from treatment with ACE inhibitors and/or beta-blockers. Following quantification of the heart-to-mediastinum ratio (HMR) of MIBG activity, 88 patients with heart failure who were treated with ACE inhibitors and/or beta-blockers (treated group) and 79 patients with heart failure who were treated conventionally without the aforementioned agents, and who served as controls, were followed up for 43 months with a primary endpoint of cardiac death. The treated group had a significantly lower prevalence of cardiac death and a significantly lower mortality at 5 years compared with the control group (15% vs 37% and 21% vs 42%, p<0.05, respectively). Multivariate analysis revealed that significant predictors were HMR, age, nitrate use and ventricular tachycardia for the treated group, and HMR, nitrate use and NYHA class for the control group. The drug treatment significantly reduced mortality from 36% to 12% when HMR was 1.53 or more and from 53% to 37% when HMR was less than 1.53. The reduction in risk of mortality within 5 years in patients without a severe MIBG defect (67%) was twice that in patients with such a defect (32%) (p<0.05). The reduction in mortality risk achieved by using ACE inhibitors and/or beta-blockers is associated with the severity of impairment of cardiac MIBG uptake. Cardiac MIBG activity can consequently be of long-term prognostic value in predicting the effectiveness of such treatment in patients with heart failure. (orig.)

  19. Rotavirus NSP1 inhibits NFkappaB activation by inducing proteasome-dependent degradation of beta-TrCP: a novel mechanism of IFN antagonism.

    Directory of Open Access Journals (Sweden)

    Joel W Graff


    Full Text Available Mechanisms by which viruses counter innate host defense responses generally involve inhibition of one or more components of the interferon (IFN system. Multiple steps in the induction and amplification of IFN signaling are targeted for inhibition by viral proteins, and many of the IFN antagonists have direct or indirect effects on activation of latent cytoplasmic transcription factors. Rotavirus nonstructural protein NSP1 blocks transcription of type I IFNalpha/beta by inducing proteasome-dependent degradation of IFN-regulatory factors 3 (IRF3, IRF5, and IRF7. In this study, we show that rotavirus NSP1 also inhibits activation of NFkappaB and does so by a novel mechanism. Proteasome-mediated degradation of inhibitor of kappaB (IkappaBalpha is required for NFkappaB activation. Phosphorylated IkappaBalpha is a substrate for polyubiquitination by a multisubunit E3 ubiquitin ligase complex, Skp1/Cul1/F-box, in which the F-box substrate recognition protein is beta-transducin repeat containing protein (beta-TrCP. The data presented show that phosphorylated IkappaBalpha is stable in rotavirus-infected cells because infection induces proteasome-dependent degradation of beta-TrCP. NSP1 expressed in isolation in transiently transfected cells is sufficient to induce this effect. Targeted degradation of an F-box protein of an E3 ligase complex with a prominent role in modulation of innate immune signaling and cell proliferation pathways is a unique mechanism of IFN antagonism and defines a second strategy of immune evasion used by rotaviruses.

  20. Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex

    Directory of Open Access Journals (Sweden)

    Lin Yuheng


    Full Text Available Abstract Background Caffeic acid (3,4-dihydroxycinnamic acid is a natural phenolic compound derived from the plant phenylpropanoid pathway. Caffeic acid and its phenethyl ester (CAPE have attracted increasing attention for their various pharmaceutical properties and health-promoting effects. Nowadays, large-scale production of drugs or drug precursors via microbial approaches provides a promising alternative to chemical synthesis and extraction from plant sources. Results We first identified that an Escherichia coli native hydroxylase complex previously characterized as the 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H was able to convert p-coumaric acid to caffeic acid efficiently. This critical enzymatic step catalyzed in plants by a membrane-associated cytochrome P450 enzyme, p-coumarate 3-hydroxylase (C3H, is difficult to be functionally expressed in prokaryotic systems. Moreover, the performances of two tyrosine ammonia lyases (TALs from Rhodobacter species were compared after overexpression in E. coli. The results indicated that the TAL from R. capsulatus (Rc possesses higher activity towards both tyrosine and L-dopa. Based on these findings, we further designed a dual pathway leading from tyrosine to caffeic acid consisting of the enzymes 4HPA3H and RcTAL. This heterologous pathway extended E. coli native tyrosine biosynthesis machinery and was able to produce caffeic acid (12.1 mg/L in minimal salt medium. Further improvement in production was accomplished by boosting tyrosine biosynthesis in E. coli, which involved the alleviation of tyrosine-induced feedback inhibition and carbon flux redirection. Finally, the titer of caffeic acid reached 50.2 mg/L in shake flasks after 48-hour cultivation. Conclusion We have successfully established a novel pathway and constructed an E. coli strain for the production of caffeic acid. This work forms a basis for further improvement in production, as well as opens the possibility of microbial synthesis

  1. Recent Advances in Developing Inhibitors for Hypoxia-Inducible Factor Prolyl Hydroxylases and Their Therapeutic Implications

    Directory of Open Access Journals (Sweden)

    So Yeon Kim


    Full Text Available Hypoxia-inducible factor (HIF prolyl hydroxylases (PHDs are members of the 2-oxoglutarate dependent non-heme iron dioxygenases. Due to their physiological roles in regulation of HIF-1α stability, many efforts have been focused on searching for selective PHD inhibitors to control HIF-1α levels for therapeutic applications. In this review, we first describe the structure of PHD2 as a molecular basis for structure-based drug design (SBDD and various experimental methods developed for measuring PHD activity. We further discuss the current status of the development of PHD inhibitors enabled by combining SBDD approaches with high-throughput screening. Finally, we highlight the clinical implications of small molecule PHD inhibitors.

  2. Expression, purification and enzymatic characterization of the catalytic domains of human tryptophan hydroxylase isoforms

    DEFF Research Database (Denmark)

    Windahl, Michael Skovbo; Boesen, Jane; Karlsen, Pernille Efferbach;


    Tryptophan hydroxylase exists in two isoforms: Isoform 1 catalyses the first and rate-limiting step in the synthesis of serotonin in the peripheral parts of the body while isoform 2 catalyses this step in the brain. The catalytic domains of human tryptophan hydroxylase 1 and 2 have been expressed...

  3. Biochemical and catalytic properties of two intracellular beta-glucosidases from the fungus Penicillium decumbens active on flavonoid glucosides

    DEFF Research Database (Denmark)

    Mamma, D.; Hatzinikolaou, D.G.; Christakopoulos, Paul


    In the presence of rutin as sole carbon source, Penicillium decumbens produces two intracellular beta-glucosidases named G(I) and G(II), with molecular masses of 56,000 and 460,000 Da, respectively. The two proteins have been purified to homogeneity. G(I) and G(II) composed of two and four equal...

  4. Synthesis and biological activity of beta-glucuronyl carbamate-based prodrugs of paclitaxel as potential candidates for ADEPT

    NARCIS (Netherlands)

    deBont, DBA; Leenders, RGG; Haisma, HJ; vanderMeulenMuileman, [No Value; Scheeren, HW


    The syntheses of prodrugs of paclitaxel, which can be used in ADEPT in order to target paclitaxel towards tumor cells, are described. The prodrugs 1 and 2a,b consist of a spacer molecule connected via a carbamate linkage to a beta-glucuronic acid. The spacer molecule is also connected via an ester l

  5. In-vitro activity and beta-lactamase stability of methicillin, isoxazolyl penicillins and cephalothin against coagulase-negative staphylococci

    DEFF Research Database (Denmark)

    Jarløv, J O; Rosdahl, V T; Mortensen, I;


    the isoxazolyl penicillins showed a high degree of uniformity. However more strains were resistant to cloxacillin and oxacillin than to dicloxacillin and flucloxacillin. Only a weak correlation was found between beta-lactamase production, and resistance to the six antibiotics. Methicillin was the most...

  6. Enzymatic Hydrolysis of Wheat Arabinoxylan by a Recombinant "Minimal" Enzyme Cocktail Containing beta-Xylosidase and Novel endo-1,4-beta-Xylanase and alpha-L-Arabinofuranosidase Activities

    DEFF Research Database (Denmark)

    Sørensen, Hanne R.; Pedersen, Sven; Jørgensen, Christel T.;


    24 h at pH 5, 50 degrees C. A 10%:40%:50% mixture of Abf II, Abf III, and beta-xyl released 56 mg of arabinose and 91 mg of xylose per gram of vinasse dry matter after 24 h at pH 5, 50 degrees C. The optimal dosages of the "minimal" enzyme cocktails were determined to be 0.4, 0.3, and 0.2 g enzyme......This study describes the identification of the key enzyme activities required in a "minimal" enzyme cocktail able to catalyze hydrolysis of water-soluble and water-insoluble wheat arabinoxylan and whole vinasse, a fermentation effluent resulting from industrial ethanol manufacture from wheat...

  7. Activation of human monocytes by live Borrelia burgdorferi generates TLR2-dependent and -independent responses which include induction of IFN-beta.

    Directory of Open Access Journals (Sweden)

    Juan C Salazar


    Full Text Available It is widely believed that innate immune responses to Borrelia burgdorferi (Bb are primarily triggered by the spirochete's outer membrane lipoproteins signaling through cell surface TLR1/2. We recently challenged this notion by demonstrating that phagocytosis of live Bb by peripheral blood mononuclear cells (PBMCs elicited greater production of proinflammatory cytokines than did equivalent bacterial lysates. Using whole genome microarrays, we show herein that, compared to lysates, live spirochetes elicited a more intense and much broader transcriptional response involving genes associated with diverse cellular processes; among these were IFN-beta and a number of interferon-stimulated genes (ISGs, which are not known to result from TLR2 signaling. Using isolated monocytes, we demonstrated that cell activation signals elicited by live Bb result from cell surface interactions and uptake and degradation of organisms within phagosomes. As with PBCMs, live Bb induced markedly greater transcription and secretion of TNF-alpha, IL-6, IL-10 and IL-1beta in monocytes than did lysates. Secreted IL-18, which, like IL-1beta, also requires cleavage by activated caspase-1, was generated only in response to live Bb. Pro-inflammatory cytokine production by TLR2-deficient murine macrophages was only moderately diminished in response to live Bb but was drastically impaired against lysates; TLR2 deficiency had no significant effect on uptake and degradation of spirochetes. As with PBMCs, live Bb was a much more potent inducer of IFN-beta and ISGs in isolated monocytes than were lysates or a synthetic TLR2 agonist. Collectively, our results indicate that the enhanced innate immune responses of monocytes following phagocytosis of live Bb have both TLR2-dependent and -independent components and that the latter induce transcription of type I IFNs and ISGs.

  8. Bioactivity-guided isolation of beta-sitosterol and some fatty acids as active compounds in the anxiolytic and sedative effects of Tilia americana var. mexicana. (United States)

    Aguirre-Hernández, Eva; Rosas-Acevedo, Hortensia; Soto-Hernández, Marcos; Martínez, Ana Laura; Moreno, Julia; González-Trujano, Ma Eva


    Tilia species have been used as anxiolytics for many years. In a previous study anxiolytic-like effects of a hexane extract of Tilia americana var. mexicana inflorescences were observed in experimental models in mice. To get additional insights into the neuroactive actions of this particular Tilia species, in this study we report a bioactivity guided-fractionation of the extract and separation by column chromatographic methods to isolate three fatty acids and a triterpene identified as beta-sitosterol as major constituents. Our results revealed that the crude extract at 10 and 30 mg/kg I. P. and some pooled fractions at the same dosages potentiated sodium pentobarbital-induced sleeping time and caused a significant increase in the time spent at the open-arm sides in the plus-maze test. A reduction in the exploratory behavioral pattern manifested as ambulatory activity, as well as head dipping and rearing tests was also observed. Further fractionation and purification yielded four major fractions containing fatty acids and beta-sitosterol as the active compounds. A dose-response curve of beta-sitosterol in the range 1 to 30 mg/kg doses indicated that this compound produced an anxiolytic-like action from 1 to 10 mg/kg and a sedative response when the dose was increased to 30 mg/kg, these effects resemble those produced by diazepam (0.1 mg/kg). Our results suggest that hexane extract of Tilia americana var. mexicana produces depressant actions on the central nervous system, at least in part, because of the presence of beta-sitosterol and some fatty acids that remain to be identified.

  9. Diphenyleneiodonium inhibits NF-kappaB activation and iNOS expression induced by IL-1beta: involvement of reactive oxygen species


    Mendes, A. Ferreira; Carvalho, A. Pato; Caramona, M. Margarida; Lopes, M. Celeste


    AIMS: In this work, we studied the mechanisms by which diphenyleneiodonium chloride (DPI) inhibits nitric oxide (NO) synthesis induced by the proinflammatory cytokine interleukin-1beta (IL-1) in bovine articular chondrocytes. To achieve this, we evaluated the ability of DPI to inhibit the expression and activity of the inducible isoform of the NO synthase (iNOS) induced by IL-1. We also studied the ability of DPI to prevent IL-1-induced NF-kappaB activation and reactive oxygen species (ROS) p...

  10. Novel anthracycline-spacer-beta-glucuronide, -beta-glucoside, and -beta-galactoside prodrugs for application in selective chemotherapy

    NARCIS (Netherlands)

    Leenders, RGG; Damen, EWP; Bijsterveld, EJA; Scheeren, HW; Houba, PHJ; van der Meulen-Muileman, IH; Boven, E; Haisma, HJ


    A series of anthracycline prodrugs containing an immolative spacer was synthesized for application in selective chemotherapy. The prodrugs having the general structure anthracycline-spacer-beta-glycoside were designed to be activated by beta-glucuronidase or beta-galactosidase. Prodrugs with -chloro

  11. Regulation of gene expression by glucose in pancreatic beta -cells (MIN6) via insulin secretion and activation of phosphatidylinositol 3'-kinase. (United States)

    da Silva Xavier, G; Varadi, A; Ainscow, E K; Rutter, G A


    Increases in glucose concentration control the transcription of the preproinsulin (PPI) gene and several other genes in the pancreatic islet beta-cell. Although recent data have demonstrated that secreted insulin may regulate the PPI gene (Leibiger, I. B., Leibiger, B., Moede, T., and Berggren, P. O. (1998) Mol. Cell 1, 933-938), the role of insulin in the control of other beta-cell genes is unexplored. To study the importance of insulin secretion in the regulation of the PPI and liver-type pyruvate kinase (L-PK) genes by glucose, we have used intranuclear microinjection of promoter-luciferase constructs into MIN6 beta-cells and photon-counting imaging. The activity of each promoter was increased either by 30 (versus 3) mm glucose or by 1-20 nm insulin. These effects of insulin were not due to enhanced glucose metabolism since culture with the hormone had no impact on the stimulation of increases in intracellular ATP concentration caused by 30 mm glucose. Furthermore, the islet-specific glucokinase promoter and cellular glucokinase immunoreactivity were unaffected by 30 mm glucose or 20 nm insulin. Inhibition of insulin secretion with the Ca(2+) channel blocker verapamil, the ATP-sensitive K(+) channel opener diazoxide, or the alpha(2)-adrenergic agonist clonidine blocked the effects of glucose on L-PK gene transcription. Similarly, 30 mm glucose failed to induce the promoter after inhibition of phosphatidylinositol 3'-kinase activity with LY294002 and the expression of dominant negative-acting phosphatidylinositol 3'-kinase (Deltap85) or the phosphoinositide 3'-phosphatase PTEN (phosphatase and tensin homologue). LY294002 also diminished the activation of the L-PK gene caused by inhibition of 5'-AMP-activated protein kinase with anti-5'-AMP-activated protein kinase alpha2 antibodies. Conversely, stimulation of insulin secretion with 13 mm KCl or 10 microm tolbutamide strongly activated the PPI and L-PK promoters. These data indicate that, in MIN6 beta

  12. sup 86 Rb(K) influx and ( sup 3 H)ouabain binding by human platelets: Evidence for beta-adrenergic stimulation of Na-K ATPase activity

    Energy Technology Data Exchange (ETDEWEB)

    Turaihi, K.; Khokher, M.A.; Barradas, M.A.; Mikhailidis, D.P.; Dandona, P. (Royal Free Hospital and School of Medicine, London (England))


    Although active transport of potassium into human platelets has been demonstrated previously, there is hitherto no evidence that human platelets have an ouabain-inhibitable Na-K ATPase in their membrane. The present study demonstrates active rubidium (used as an index of potassium influx), {sup 86}Rb(K), influx into platelets, inhibitable by ouabain, and also demonstrates the presence of specific ({sup 3}H)ouabain binding by the human platelet. This {sup 86}Rb(K) influx was stimulated by adrenaline, isoprenaline, and salbutamol, but noradrenaline caused a mild inhibition. Active {sup 86}Rb(K) influx by platelets was inhibited markedly by timolol, mildly by atenolol, but not by phentolamine. Therefore, active {sup 86}Rb(K) influx in human platelets is enhanced by stimulation of beta adrenoceptors of the beta 2 subtype. The platelet may therefore replace the leukocyte in future studies of Na-K ATPase activity. This would be a considerable advantage in view of the ease and rapidity of preparation of platelets.

  13. N-acetyl cysteine, L-cysteine, and beta-mercaptoethanol augment selenium-glutathione peroxidase activity in glucose-6-phosphate dehydrogenase-deficient human erythrocytes. (United States)

    Alicigüzel, Y; Aslan, M


    In glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes, failure to maintain normal levels of reduced glutathione (GSH) due to decreased NADPH regeneration in the hexose monophosphate pathway results in acute hemolytic anemia following exposure to oxidative insults, such as ingestion of Vicia fava beans or use of certain drugs. GSH is a source of protection against oxidative attack, used by the selenium-dependent glutathione peroxidase (Se-GSH-Px)/reductase (GR) system to detoxify hydrogen peroxide and organic peroxides, provided that sufficient GSH is made available. In this study, Se-GSH-Px activity was analyzed in G6PD-deficient patients in the presence of reducing agents such as N-Acetyl cysteine, L-cysteine, and beta-mercaptoethanol. Se-GSH-Px activity was decreased in G6PD-deficient red blood cells (RBCs). N-Acetyl cysteine, L-cysteine, and beta-mercaptoethanol increased Se-GSH-Px activity in G6PD-deficient human erythrocytes, indicating that other reducing agents can be utilized to complement Se-GSH-Px activity in G6PD deficiency. Based on the increased susceptibility of G6PD-deficient patients to oxidative stress, the reported increase in Se-GSH-Px activity can facilitate the detoxification of reactive oxygen species. PMID:15598086

  14. Purification, characterization, and directed evolution study of a vitamin D{sub 3} hydroxylase from Pseudonocardia autotrophica

    Energy Technology Data Exchange (ETDEWEB)

    Fujii, Yoshikazu [Bioresource Laboratories, Mercian Corporation, 1808 Nakaizumi, Iwata, Shizuoka 438-0078 (Japan); Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589 (Japan); Kabumoto, Hiroki; Nishimura, Kenji; Fujii, Tadashi; Yanai, Satoshi [Bioresource Laboratories, Mercian Corporation, 1808 Nakaizumi, Iwata, Shizuoka 438-0078 (Japan); Takeda, Koji [BioTechnical Development Center, Mercian Corporation, 1808 Nakaizumi, Iwata, Shizuoka 438-0078 (Japan); Tamura, Noriko [Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517 (Japan); Arisawa, Akira, E-mail: [Bioresource Laboratories, Mercian Corporation, 1808 Nakaizumi, Iwata, Shizuoka 438-0078 (Japan); Tamura, Tomohiro, E-mail: [Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517 (Japan); Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589 (Japan)


    Vitamin D{sub 3} (VD{sub 3}) is a fat-soluble prohormone that plays a crucial role in bone metabolism, immunity, and control of cell proliferation and cell differentiation in mammals. The actinomycete Pseudonocardia autotrophica is capable of bioconversion of VD{sub 3} into its physiologically active forms, namely, 25(OH)VD{sub 3} or 1{alpha},25(OH){sub 2}VD{sub 3}. In this study, we isolated and characterized Vdh (vitamin D{sub 3} hydroxylase), which hydroxylates VD{sub 3} from P. autotrophica NBRC 12743. The vdh gene encodes a protein containing 403 amino acids with a molecular weight of 44,368 Da. This hydroxylase was found to be homologous with the P450 belonging to CYP107 family. Vdh had the same ratio of the V{sub max} values for VD{sub 3} 25-hydroxylation and 25(OH)VD{sub 3} 1{alpha}-hydroxylation, while other enzymes showed preferential regio-specific hydroxylation on VD{sub 3}. We characterized a collection of Vdh mutants obtained by random mutagenesis and obtained a Vdh-K1 mutant by the combination of four amino acid substitutions. Vdh-K1 showed one-order higher VD{sub 3} 25-hydroxylase activity than the wild-type enzyme. Biotransformation of VD{sub 3} into 25(OH)VD{sub 3} was successfully accomplished with a Vdh-expressed recombinant strain of actinobacterium Rhodococcus erythropolis. Vdh may be a useful enzyme for the production of physiologically active forms of VD{sub 3} by a single cytochrome P450.

  15. Deregulation of the lysyl hydroxylase matrix cross-linking system in experimental and clinical bronchopulmonary dysplasia. (United States)

    Witsch, Thilo J; Turowski, Pawel; Sakkas, Elpidoforos; Niess, Gero; Becker, Simone; Herold, Susanne; Mayer, Konstantin; Vadász, István; Roberts, Jesse D; Seeger, Werner; Morty, Rory E


    Bronchopulmonary dysplasia (BPD) is a common and serious complication of premature birth, characterized by a pronounced arrest of alveolar development. The underlying pathophysiological mechanisms are poorly understood although perturbations to the maturation and remodeling of the extracellular matrix (ECM) are emerging as candidate disease pathomechanisms. In this study, the expression and regulation of three members of the lysyl hydroxylase family of ECM remodeling enzymes (Plod1, Plod2, and Plod3) in clinical BPD, as well as in an experimental animal model of BPD, were addressed. All three enzymes were localized to the septal walls in developing mouse lungs, with Plod1 also expressed in the vessel walls of the developing lung and Plod3 expressed uniquely at the base of developing septa. The expression of plod1, plod2, and plod3 was upregulated in the lungs of mouse pups exposed to 85% O2, an experimental animal model of BPD. Transforming growth factor (TGF)-β increased plod2 mRNA levels and activated the plod2 promoter in vitro in lung epithelial cells and in lung fibroblasts. Using in vivo neutralization of TGF-β signaling in the experimental animal model of BPD, TGF-β was identified as the regulator of aberrant plod2 expression. PLOD2 mRNA expression was also elevated in human neonates who died with BPD or at risk for BPD, compared with neonates matched for gestational age at birth or chronological age at death. These data point to potential roles for lysyl hydroxylases in normal lung development, as well as in perturbed late lung development associated with BPD. PMID:24285264

  16. Alkane Hydroxylase Gene (alkB Phylotype Composition and Diversity in Northern Gulf of Mexico Bacterioplankton

    Directory of Open Access Journals (Sweden)

    Conor Blake Smith


    Full Text Available Natural and anthropogenic activities introduce alkanes into marine systems where they are degraded by alkane hydroxylases expressed by phylogenetically diverse bacteria. Partial sequences for alkB, one of the structural genes of alkane hydroxylase, have been used to assess the composition of alkane-degrading communities, and to determine their responses to hydrocarbon inputs. We present here the first spatially extensive analysis of alkB in bacterioplankton of the northern Gulf of Mexico (nGoM, a region that experiences numerous hydrocarbon inputs. We have analyzed 401 partial alkB gene sequences amplified from genomic extracts collected during March 2010 from 17 water column samples that included surface waters and bathypelagic depths. Previous analyses of 16S rRNA gene sequences for these and related samples have shown that nGoM bacterial community composition and structure stratify strongly with depth, with distinctly different communities above and below 100 m. Although we hypothesized that alkB gene sequences would exhibit a similar pattern, PCA analyses of operational protein units (OPU indicated that community composition did not vary consistently with depth or other major physical-chemical variables. We observed 22 distinct OPUs, one of which was ubiquitous and accounted for 57% of all sequences. This OPU clustered with alkB sequences from known hydrocarbon oxidizers (e.g., Alcanivorax and Marinobacter. Some OPUs could not be associated with known alkane degraders, however, and perhaps represent novel hydrocarbon-oxidizing populations or genes. These results indicate that the capacity for alkane hydrolysis occurs widely in the nGoM, but that alkane degrader diversity varies substantially among sites and responds differently than bulk communities to physical-chemical variables.

  17. CYP287A1 is a carotenoid 2-β-hydroxylase required for deinoxanthin biosynthesis in Deinococcus radiodurans R1. (United States)

    Zhou, Zhengfu; Zhang, Wei; Su, Shiyou; Chen, Ming; Lu, Wei; Lin, Min; Molnár, István; Xu, Yuquan


    The carotenoid deinoxanthin is a crucial resistance factor against various stresses in the radiation-resistant bacterium Deinococcus radiodurans. Disruption of the gene dr2473 encoding the cytochrome P450 CYP287A1 led to the accumulation of 2-deoxydeinoxanthin in D. radiodurans, demonstrating that CYP287A1 is a novel β-carotene 2-hydroxylase. The dr2473 knockout mutant was shown to be more sensitive to UV radiation and oxidative stress than the wild-type strain D. radiodurans R1, indicating that the C2 alcohol of deinoxanthin is important for antioxidant activity.

  18. Tumors initiated by constitutive Cdk2 activation exhibit transforming growth factor beta resistance and acquire paracrine mitogenic stimulation during progression

    DEFF Research Database (Denmark)

    Corsino, P.; Davis, B.; Law, M.;


    sites. Together, these results suggest that deregulation of the Cdk/Rb/E2F axis reprograms mammary epithelial cells to initiate a paracrine loop with tumor-associated fibroblasts involving TGF beta and HGF, resulting in desmoplasia. The MMTV-DIK2 mice should provide a useful model system...... for the development of therapeutic approaches to block the stromal desmoplastic reaction that likely plays an important role in the progression of multiple types of human tumors...

  19. Determination of total alpha and beta activity in water for human consumption by LSC(Liquid Scintillation Counter)

    International Nuclear Information System (INIS)

    The Ordinance Brazilian of Ministry of Health (MS 2914/2011) establishes the standards for quality of water intended for human consumption, being limits values of 5.0 Bq/L for gross alpha, and 1.0 Bq/L for gross beta radioactivity. The liquid scintillation spectrometry (LSC) technique has been presented as an alternative to conventional procedure using gas flow proportional counter. The present work shows a review of the methods for determination of gross alpha and gross beta in water by using LSC. Between the factors that influence the accuracy and repeatability of the analytical results we can highlight: thermal preconcentration, type of the acid and calibration standard. A procedure was established and carried out to samples of the National Program of Intercomparison of Radionuclides in Environmental Samples for evaluation of its performance. The gross alpha and gross beta analysis in samples of the public water supplies in the Metropolitan Region of Goiania, state of Goias was carried out. The results are consistent with the guideline values form the Ministry of Health concerning radioactivity. (author)

  20. Moderate Exercise Restores Pancreatic Beta-Cell Function and Autonomic Nervous System Activity in Obese Rats Induced by High-Fat Diet

    Directory of Open Access Journals (Sweden)

    Rodrigo Mello Gomes


    Full Text Available Background/Aims: Metabolic syndrome has been identified as one of the most significant threats to human health in the 21st century. Exercise training has been shown to counteract obesity and metabolic syndrome. The present study aimed to investigate the effects of moderate exercise training on pancreatic beta-cell function and autonomic nervous system (ANS activity in rats fed a high-fat diet (HFD. Methods: Weaning rats were divided into four groups: rats fed a standard chow or HFD (sedentary, Control-SED and HFD-SED; or exercised, Control-EXE and HFD-EXE, respectively. Exercised rats ran (from 21- to 91-days-old for 60 minutes (3 times/week over a 10-week period. Glucose and insulin tolerance tests were performed. Pancreatic islets were isolated to study glucose-induced insulin secretion (GIIS. Parasympathetic and sympathetic nerve electrical signals were measured, and liver samples were processed and histologically analyzed. Results: Exercise prevented obesity, insulin resistance, and liver steatosis as well as improved total cholesterol, ALT, and AST levels. Islets from HFD rats showed insulin hypersecretion which was ameliorated by exercise. Exercise decreased vagal nerve activity in the HFD-EXE group and increased the activity of the sympathetic nervous system in both exercised groups. Conclusion: Exercise prevents obesity and liver steatosis and restores pancreatic beta-cell function and ANS activity in HFD-obese rats.

  1. Impaired activation of adenylyl cyclase in lung of the Basenji-greyhound model of airway hyperresponsiveness: decreased numbers of high affinity beta-adrenoceptors.


    Emala, C. W.; Aryana, A.; Hirshman, C. A.


    1. To evaluate mechanisms involved in the impaired beta-adrenoceptor stimulation of adenylyl cyclase in tissues from the Basenji-greyhound (BG) dog model of airway hyperresponsiveness, we compared agonist and antagonist binding affinity of beta-adrenoceptors, beta-adrenoceptor subtypes, percentage of beta-adrenoceptors sequestered, and coupling of the beta-adrenoceptor to Gs alpha in lung membranes from BG and control mongrel dogs. We found that lung membranes from the BG dog had higher total...

  2. Structure and expression of the human Lysyl hydroxylase gene (PLOD): Introns 9 and 16 contain Alu sequences at the sites of recombination in Ehlers-Danlos syndrome type VI patients

    Energy Technology Data Exchange (ETDEWEB)

    Heikkinen, J.; Hautala, T.; Kivirikko, K.I. [Univ. of Oulu (Finland)] [and others


    Lysyl hydroxylase (EC catalyzes the formation of hydroxylysine in collagens by the hydroxylation of lysine residues in peptide linkages. This enzyme activity is known to be reduced in patients with the type VI variant of the Ehlers-Danlos syndrome, and the first mutations in the lysyl hydroxylase gene (PLOD) have recently been identified. We have now isolated genomic clones for human lysyl hydroxylase and determined the complete structure of the gene, which contains 19 exons and a 5{prime} flanking region with characteristics shared by housekeeping genes. The constitutive expression of the gene in different tissues further suggests that lysyl hydroxylase has an essential function. We have sequenced the introns of the gene in the region where many mutations and rearrangements analyzed to date are concentrated. Intron 9 and intron 16 show extensive homology resulting from the many Alu sequences found in these introns. Intron 9 contains five and intron 16 eight Alu sequences. The high homology and many short identical or complementary sequences in these introns generate many potential recombination sites with the gene. The delineation of the structure of the lysyl hydroxylase gene contributes significantly to our understanding of the rearrangements in the genome of Ehlers-Danlos type VI patients. 21 refs., 2 figs., 2 tabs.

  3. Structural and Kinetic Studies of Novel Cytochrome P450 Small-Alkane Hydroxylases

    Energy Technology Data Exchange (ETDEWEB)

    Arnold, Frances H.


    The goals of this project are to investigate (1) the kinetics and stabilities of engineered cytochrome P450 (P450) small alkane hydroxylases and their evolutionary intermediates, (2) the structural basis for catalytic proficiency on small alkanes of these engineered P450s, and (3) the changes in redox control resulting from protein engineering. To reach these goals, we have established new methods for determining the kinetics and stabilities of multicomponent P450s such as CYP153A6. Using these, we were able to determine that CYP153A6 is proficient for hydroxylation of alkanes as small as ethane, an activity that has never been observed previously in any natural P450. To elucidate the structures of the engineered P450s, we obtained x-ray diffraction data for two variants in the P450PMO (propane monooxygenase) lineage and a preliminary structure for the most evolved variant. This structure shows changes in the substrate binding regions of the enzyme and a reduction in active site volume that are consistent with the observed changes in substrate specificity from fatty acids in the native enzyme to small alkanes in P450PMO. We also constructed semi-rational designed libraries mutating only residues in the enzyme active site that in one round of mutagenesis and screening produced variants that achieved nearly half of the activity of the most evolved enzymes of the P450PMO lineage. Finally, we found that changes in redox properties of the laboratory-evolved P450 alkane hydroxylases did not reflect the improvement in their electron transfer efficiency. The heme redox potential remained constant throughout evolution, while activity increased and coupling efficiency improved from 10% to 90%. The lack of correlation between heme redox potential and enzyme activity and coupling efficiency led us to search for other enzyme properties that could be better predictors for activity towards small alkanes, specifically methane. We investigated the oxidation potential of the radical

  4. The Relationship Between Beta Endorphins and Emotional State in Physically Active Individuals Aged 45-55 (A Report on a Pilot Study

    Directory of Open Access Journals (Sweden)

    Kundziņa Ieva


    Full Text Available Introduction. This sports-science-related article heavily relies on studies that have reported an increase in beta-endorphin (â-EP concentration in plasma in response to physical activity. It examines the psychological and physiological effects of physical activity and exercise and reports on a research-experiment-based, endorphin-hypotheses-related pilot study aimed at exploring mood-related â-EP effects occurring in physically active male and female individuals aged 45-55 in response to physical load. Material and methods. Six 45 to 55-year-old individuals (3 males and 3 females rated as exhibiting moderate and high levels of physical activity in sport's laboratory. International Physical Activity Questionnaire (IPAQ was used to establish physical activity level. For facial expression analysis a short interview was applied, using software “FaceReader 3.0” (FR. As a load test a veloergometer exercise test was used, and Beta-endorphin (â-EP levels were measured from venous blood. Results. The findings demonstrated an increase in â-EP levels in 50% of the subjects. No positive relation between â-EP increase and happiness has been observed. In four subjects an increase in disgust was observed due to the laboratory conditions. Five minutes after the load test FR data recorded the reduction or disappearance of negative emotions for all research subjects. Conclusions. Further investigation into the relationship of plasma levels of â-EP and the emotional state of the individual involved in physical activities is needed. This necessitates a further insight into how exercise-elevated endorphins (â-EP affect mood state outside laboratory conditions. Therefore, a further investigation of people involved in physical recreation activities outdoors is envisaged.

  5. Evidence That the [beta] Subunit of Chlamydia trachomatis Ribonucleotide Reductase Is Active with the Manganese Ion of Its Manganese(IV)/Iron(III) Cofactor in Site 1

    Energy Technology Data Exchange (ETDEWEB)

    Dassama, Laura M.K.; Boal, Amie K.; Krebs, Carsten; Rosenzweig, Amy C.; Bollinger, Jr., J. Martin (NWU); (Penn)


    The reaction of a class I ribonucleotide reductase (RNR) begins when a cofactor in the {beta} subunit oxidizes a cysteine residue {approx}35 {angstrom} away in the {alpha} subunit, generating a thiyl radical. In the class Ic enzyme from Chlamydia trachomatis (Ct), the cysteine oxidant is the Mn{sup IV} ion of a Mn{sup IV}/Fe{sup III} cluster, which assembles in a reaction between O{sub 2} and the Mn{sup II}/Fe{sup II} complex of {beta}. The heterodinuclear nature of the cofactor raises the question of which site, 1 or 2, contains the Mn{sup IV} ion. Because site 1 is closer to the conserved location of the cysteine-oxidizing tyrosyl radical of class Ia and Ib RNRs, we suggested that the Mn{sup IV} ion most likely resides in this site (i.e., {sup 1}Mn{sup IV}/{sup 2}Fe{sup III}), but a subsequent computational study favored its occupation of site 2 ({sup 1}Fe{sup III}/{sup 2}Mn{sup IV}). In this work, we have sought to resolve the location of the Mn{sup IV} ion in Ct RNR-{beta} by correlating X-ray crystallographic anomalous scattering intensities with catalytic activity for samples of the protein reconstituted in vitro by two different procedures. In samples containing primarily Mn{sup IV}/Fe{sup III} clusters, Mn preferentially occupies site 1, but some anomalous scattering from site 2 is observed, implying that both {sup 1}Mn{sup II}/{sup 2}Fe{sup II} and {sup 1}Fe{sup II}/{sup 2}Mn{sup II} complexes are competent to react with O{sub 2} to produce the corresponding oxidized states. However, with diminished Mn{sup II} loading in the reconstitution, there is no evidence for Mn occupancy of site 2, and the greater activity of these 'low-Mn' samples on a per-Mn basis implies that the {sup 1}Mn{sup IV}/{sup 2}Fe{sup III}-{beta} is at least the more active of the two oxidized forms and may be the only active form.

  6. Characterization of paired mucoid/non-mucoid Pseudomonas aeruginosa isolates from Danish cystic fibrosis patients: antibiotic resistance, beta-lactamase activity and RiboPrinting

    DEFF Research Database (Denmark)

    Ciofu, O; Fussing, V; Bagge, N;


    The purpose of this study was to characterize 42 paired mucoid and non-mucoid Danish cystic fibrosis (CF) Pseudomonas aeruginosa isolates collected in 1997, by RiboPrinting, antibiotic susceptibility and beta-lactamase activity. Eight P. aeruginosa isolates collected before 1991 were included for...... before 1991 had an antibiotic susceptibility pattern similar to the 1997 isolates. Despite prolonged and intensive antibiotic treatment, susceptible mucoid isolates were isolated from the CF sputum, possibly because these bacteria are protected from the selective pressure of antibiotics by the resistant...

  7. A New Tyrosine Hydroxylase Genotype with Orofacial Dyskinaesia

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    Ahood M. Al-Muslamani


    Full Text Available Tyrosine hydroxylase (TH deficiency is a rare autosomal recessive and often treatable neurometabolic disorder with variable phenotypes. More than 20 pathological mutations have been identified in patients with TH deficiency. We report the case of a 10-month-old male patient who presented with developmental delay, hypotonia and oculogyric crises to the Salmaniya Medical Complex in Manama, Bahrain. At a later stage, he developed orofacial dyskinaesia and tremors with hyper-reflexia and clonus. A magnetic resonance imaging scan of the brain showed mild atrophy with widened ventricles and genetic testing revealed a novel homozygous mutation (c.938G>T; p.Arg313Leu in exon 9 of the TH gene. The patient showed a remarkable response to treatment using combined levodopa-carbidopa. In this case, the orofacial dyskinaesia may be a specific clinical association unique to this novel mutation, which is the first to be described in Bahrain and the Middle East.

  8. Casein kinase 2 down-regulation and activation by polybasic peptides are mediated by acidic residues in the 55-64 region of the beta-subunit. A study with calmodulin as phosphorylatable substrate

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Issinger, O G;


    The noncatalytic beta-subunit is responsible for the latency of casein kinase 2 (CK2) activity toward calmodulin. Twenty-one mutants of the beta-subunit bearing either deletions or Ala substitutions for charged residues in the 5-6, 55-70, and 171-178 sequences have been assayed for their ability...... insensitive to 42 nM polylysine, which conversely promotes a more than 10-fold increase of calmodulin phosphorylation with wild-type beta.(ABSTRACT TRUNCATED AT 250 WORDS)...

  9. Molecular cloning and characterization of a cytochrome P450 taxoid 9á-hydroxylase in Ginkgo biloba cells. (United States)

    Zhang, Nan; Han, Zhentai; Sun, Guiling; Hoffman, Angela; Wilson, Iain W; Yang, Yanfang; Gao, Qian; Wu, Jianqiang; Xie, Dan; Dai, Jungui; Qiu, Deyou


    Taxol is a well-known effective anticancer compound. Due to the inability to synthesize sufficient quantities of taxol to satisfy commercial demand, a biotechnological approach for a large-scale cell or cell-free system for its production is highly desirable. Several important genes in taxol biosynthesis are currently still unknown and have been shown to be difficult to isolate directly from Taxus, including the gene encoding taxoid 9α-hydroxylase. Ginkgo biloba suspension cells exhibit taxoid hydroxylation activity and provides an alternate means of identifying genes encoding enzymes with taxoid 9α-hydroxylation activity. Through analysis of high throughput RNA sequencing data from G. biloba, we identified two candidate genes with high similarity to Taxus CYP450s. Using in vitro cell-free protein synthesis assays and LC-MS analysis, we show that one candidate that belongs to the CYP716B, a subfamily whose biochemical functions have not been previously studied, possessed 9α-hydroxylation activity. This work will aid future identification of the taxoid 9α-hydroxylase gene from Taxus sp.

  10. A single gene directs synthesis of a precursor protein with beta- and alpha-amylase activities in Bacillus polymyxa.


    Uozumi, N; Sakurai, K.(Theoretical Particle Physics and Cosmology Group, Department of Physics, King’s College London, WC2R 2LS, London, UK); Sasaki, T.; Takekawa, S.; Yamagata, H; Tsukagoshi, N; Udaka, S


    The Bacillus polymyxa amylase gene comprises 3,588 nucleotides. The mature amylase comprises 1,161 amino acids with a molecular weight of 127,314. The gene appeared to be divided into two portions by the direct-repeat sequence located at almost the middle of the gene. The 5' region upstream of the direct-repeat sequence was shown to be responsible for the synthesis of beta-amylase. The 3' region downstream of the direct-repeat sequence contained four sequences homologous with those in other a...

  11. In cellulo examination of a beta-alpha hybrid construct of beta-hexosaminidase A subunits, reported to interact with the GM2 activator protein and hydrolyze GM2 ganglioside.

    Directory of Open Access Journals (Sweden)

    Incilay Sinici

    Full Text Available The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # (encoded by the HEXA and HEXB genes, respectively, and the GM2-activator protein (GM2AP, encoded by the GM2A gene. Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs diseas