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Sample records for 12cgamma 3alpha ehnergeticheskoe

  1. Simultaneous quantification of GABAergic 3alpha,5alpha/3alpha,5beta neuroactive steroids in human and rat serum.

    PubMed

    Porcu, Patrizia; O'Buckley, Todd K; Alward, Sarah E; Marx, Christine E; Shampine, Lawrence J; Girdler, Susan S; Morrow, A Leslie

    2009-01-01

    The 3alpha,5alpha- and 3alpha,5beta-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone enhance GABAergic neurotransmission and produce inhibitory neurobehavioral and anti-inflammatory effects. Despite substantial information on the progesterone derivative (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha,5alpha-THP, allopregnanolone), the physiological significance of the other endogenous GABAergic neuroactive steroids has remained elusive. Here, we describe the validation of a method using gas chromatography-mass spectrometry to simultaneously identify serum levels of the eight 3alpha,5alpha- and 3alpha,5beta-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone. The method shows specificity, sensitivity and enhanced throughput compared to other methods already available for neuroactive steroid quantification. Administration of pregnenolone to rats and progesterone to women produced selective effects on the 3alpha,5alpha- and 3alpha,5beta-reduced neuroactive steroids, indicating differential regulation of their biosynthetic pathways. Pregnenolone administration increased serum levels of 3alpha,5alpha-THP (+1488%, p<0.001), (3alpha,5alpha)-3,21-dihydroxypregnan-20-one (3alpha,5alpha-THDOC, +205%, p<0.01), (3alpha,5alpha)-3-hydroxyandrostan-17-one (3alpha,5alpha-A, +216%, p<0.001), (3alpha,5alpha,17beta)-androstane-3,17-diol (3alpha,5alpha-A-diol, +190%, p<0.01). (3alpha,5beta)-3-hydroxypregnan-20-one (3alpha,5beta-THP) and (3alpha,5beta)-3-hydroxyandrostan-17-one (3alpha,5beta-A) were not altered, while (3alpha,5beta)-3,21-dihydroxypregnan-20-one (3alpha,5beta-THDOC) and (3alpha,5beta,17beta)-androstane-3,17-diol (3alpha,5beta-A-diol) were increased from undetectable levels to 271+/-100 and 2.4+/-0.9 pg+/-SEM, respectively (5/8 rats). Progesterone administration increased serum levels of 3alpha,5alpha-THP (+1806%, p<0.0001), 3alpha,5beta-THP (+575%, p<0.001), 3alpha,5alpha

  2. Activating STAT3 Alpha for Promoting Healing of Neurons

    NASA Technical Reports Server (NTRS)

    Conway, Greg

    2008-01-01

    A method of promoting healing of injured or diseased neurons involves pharmacological activation of the STAT3 alpha protein. Usually, injured or diseased neurons heal incompletely or not at all for two reasons: (1) they are susceptible to apoptosis (cell death); and (2) they fail to engage in axogenesis that is, they fail to re-extend their axons to their original targets (e.g., muscles or other neurons) because of insufficiency of compounds, denoted neurotrophic factors, needed to stimulate such extension. The present method (see figure) of treatment takes advantage of prior research findings to the effect that the STAT3 alpha protein has anti-apoptotic and pro-axogenic properties.

  3. Correlation of serum 3 alpha-androstanediol glucuronide with acne and chest hair density in men.

    PubMed

    Lookingbill, D P; Egan, N; Santen, R J; Demers, L M

    1988-11-01

    Serum 3 alpha-androstanediol glucuronide (3 alpha-Adiol-G) is considered to be an indicator of peripheral tissue androgen metabolism. Precursor circulating androgens are converted in peripheral tissue to dihydrotestosterone (DHT), which is ultimately metabolized to 3 alpha-Adiol-G and secreted from the cell. Elevated serum 3 alpha-Adiol-G concentrations have been reported in women in hyperandrogenic states. We studied 44 consecutive male medical students for chest hair density, acne, and serum dehydroepiandrosterone sulfate (DHEA-S), total testosterone (total T), free and albumin-bound (bioavailable) T (bio T), and 3 alpha-Adiol-G concentrations. Although there was considerable overlap of serum 3 alpha-Adiol-G values among the groups defined by hair density or acne scores, we found statistically significant correlations between serum 3 alpha-Adiol-G and chest hairiness (P = 0.0034), acne (P = 0.0005), and a combined chest hairiness and acne score (P = 0.0018). There was no significant correlation between these clinical parameters and the levels of precursor androgens. There was, however, a strong correlation between serum 3 alpha-Adiol-G and bio T (P = 0.0005), suggesting that in men serum 3 alpha-Adiol-G levels may be dependent upon available free and albumin-bound T. The correlations in men of serum 3 alpha-Adiol-G with chest hair density, acne, and the hairiness and acne index supports the hypothesis that the serum levels of 3 alpha-Adiol-G reflect the extent of androgen action in peripheral tissues. PMID:2972739

  4. Determination of proteinase 3-alpha 1-antitrypsin complexes in inflammatory fluids.

    PubMed

    Dolman, K M; van de Wiel, B A; Kam, C M; Abbink, J J; Hack, C E; Sonnenberg, A; Powers, J C; von dem Borne, A E; Goldschmeding, R

    1992-12-14

    Physiological inhibitors were tested for their in vitro interaction with neutrophil proteinase 3 (PR3). The major plasma proteinase inhibitor of PR3 is alpha 1AT. We have developed a radioimmunoassay (RIA) for quantitative detection of PR3-alpha 1AT complexes formed in vivo in inflammatory exudates such as synovial fluid and plasma from patients with sepsis. Levels of PR3-alpha 1AT complexes correlated significantly with levels of human neutrophil elastase (HNE)-alpha 1AT complexes. Thus, in vivo alpha 1AT not only protects against excessive HNE activity, but also against excessive PR3 activity.

  5. Effects of a new 3-alpha reaction on the s-process in massive stars

    SciTech Connect

    Kikuch, Yukihiro; Ono, Masaomi; Matsuo, Yasuhide; Hashimoto, Masa-aki; Fujimoto, Shin-ichiro

    2012-11-12

    Effect of a new 3-alpha reaction rate on the s-process during the evolution of a massive star of 25 solar mass is investigated for the first time, because the s-process in massive stars have been believed to be established with only minor change. We find that the s-process with use of the new rate during the core helium burning is very inefficient compared to the case with the previous 3-alpha rate. However, the difference of the overproduction is found to be largely compensated by the subsequent carbon burning. Since the s-process in massive stars has been attributed so far to the neutron irradiation during core helium burning, our finding reveals for the first time the importance of the carbon burning for the s-process during the evolution of massive stars.

  6. Detection and localization of Mip-3alpha/LARC/Exodus, a macrophage proinflammatory chemokine, and its CCR6 receptor in human pancreatic cancer.

    PubMed

    Kleeff, J; Kusama, T; Rossi, D L; Ishiwata, T; Maruyama, H; Friess, H; Büchler, M W; Zlotnik, A; Korc, M

    1999-05-17

    Macrophage Proinflammatory Human Chemokine-3alpha (Mip-3alpha/LARC/Exodus) belongs to a large family of chemotactic cytokines, which participate in directing inflammatory cell migration and in modulating angiogenesis. Mip-3alpha signals through a recently identified G-protein linked 7-transmembrane receptor, CCR6. In this study, we have characterized the expression of Mip-3alpha and CCR6 in 12 normal and 16 cancerous human pancreatic tissues and in 4 cultured pancreatic cancer cell lines, and assessed the effects of Mip-3alpha on growth and invasion of these cell lines. Pancreatic cancer tissues markedly overexpressed Mip-3alpha in comparison with normal pancreatic samples. By in situ hybridization Mip-3alpha and CCR6 mRNA moieties were present in cancer cells within the tumors. In addition, Mip-3alpha was abundant in the macrophages infiltrating the tumor mass. Mip-3alpha and its receptor CCR6 were expressed in all 4 tested pancreatic cancer cell lines. Mip-3alpha stimulated the growth of one cell line, enhanced the migration of another cell line, and was without effect in the other 2 cell lines. Together, our findings suggest that Mip-3alpha has the potential to act via autocrine and paracrine mechanisms to contribute to the pathobiology of human pancreatic cancer.

  7. Aqueous chemical growth of alpha-Fe2O3-alpha-Cr203 nanocompositethin films

    SciTech Connect

    Vayssieres, Lionel; Guo, Jinghua; Nordgren, Joseph

    2001-06-30

    We are reporting here on the inexpensive fabrication and optical properties of an iron(III) oxide chromium(III) oxide nanocomposite thin film of corundum crystal structure. Its novel and unique-designed architecture consists of uniformed, well-defined and oriented nanorods of Hematite (alpha-Fe2O3) of 50 nm in diameter and 500nm in length and homogeneously distributed nonaggregated monodisperse spherical nanoparticles of Eskolaite (alpha-Cr2O3) of 250 nm in diameter. This alpha-Fe2O3 alpha-Cr2O3 nanocomposite thin film is obtained by growing, directly onto transparent polycrystalline conducting substrate, an oriented layer of hematite nanorods and growing subsequently, the eskolaite layer. The synthesis is carried out by a template-free, low-temperature, multilayer thin film coating process using aqueous solution of metal salts as precursors. Almost 100 percent of the light is absorbed by the composite film between 300 and 525 nm and 40 percent at 800 nm which yields great expectations as photoanode materials for photovoltaic cells and photocatalytic devices.

  8. Mechanisms regulating male sexual behavior in the rat: role of 3 alpha- and 3 beta-androstanediols.

    PubMed

    Morali, G; Oropeza, M V; Lemus, A E; Perez-Palacios, G

    1994-09-01

    To assess whether naturally occurring 5 alpha-androstanediols (5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-androstane-3 beta, 17 beta-diol) play a role in the regulation of male sexual behavior in the rat, their capability to restore copulatory behavior in castrated animals was evaluated. Androstanediols were chronically administered either alone or in combination with 5 alpha-dihydrotestosterone (DHT) or with estradiol-17 beta (E2). Animals treated with testosterone (T), DHT, E2, and vehicle, either alone or in different combinations, served as controls. The occurrence of mounting, intromission, and ejaculation as well as detailed parameters of copulatory behavior were recorded twice per week for 3 weeks. At the end of treatments, the weights of sex accessory organs were also recorded. When 3 beta, 5 alpha-androstanediol (3 beta-diol; 500 micrograms/day) was administered in combination with DHT (300 micrograms/day), full copulatory behavior was restored in all subjects in a manner similar to that obtained with E2 plus DHT or T plus DHT combinations, thus indicating an estrogen-like behavioral effect of 3 beta-diol. Administration of 3 alpha, 5 alpha-androstanediol (3 alpha-diol; 500 micrograms/day) combined with DHT also restored sexual behavior, though to a lesser extent. When 3 alpha-diol (500 micrograms/day) was simultaneously administered with E2 (5 micrograms/day), the copulatory behavior of castrated animals was fully restored in a fashion similar to that observed after administration of DHT plus E2 and T plus E2 combinations, indicating a potent androgen-like effect of 3 alpha-diol.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7803627

  9. Rat NAD+-dependent 3alpha-hydroxysteroid dehydrogenase (AKR1C17): a member of the aldo-keto reductase family highly expressed in kidney cytosol.

    PubMed

    Sanai, Masaharu; Endo, Satoshi; Matsunaga, Toshiyuki; Ishikura, Shuhei; Tajima, Kazuo; El-Kabbani, Ossama; Hara, Akira

    2007-08-01

    Mammalian 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs) have been divided into two types: Cytosolic NADP(H)-dependent 3alpha-HSDs belonging to the aldo-keto reductase family, and mitochondrial and microsomal NAD(+)-dependent 3alpha-HSDs belonging to the short-chain dehydrogenase/reductase family. In this study, we characterized a rat aldo-keto reductase (AKR1C17), whose functions are unknown. The recombinant AKR1C17 efficiently oxidized 3alpha-hydroxysteroids and bile acids using NAD(+) as the preferred coenzyme at an optimal pH of 7.4-9.5, and was inhibited by ketamine and organic anions. The mRNA for AKR1C17 was detected specifically in rat kidney, where the enzyme was more highly expressed as a cytosolic protein than NADP(H)-dependent 3alpha-HSD (AKR1C9). Thus, AKR1C17 represents a novel NAD(+)-dependent type of cytosolic 3alpha-HSD with unique inhibitor sensitivity and tissue distribution. In addition, the replacement of Gln270 and Glu276 of AKR1C17 with the corresponding residues of NADP(H)-dependent 3alpha-HSD resulted in a switch in favor of NADP(+) specificity, suggesting their key roles in coenzyme specificity.

  10. 24xi-Methyl 5 alpha-cholestane-3 alpha,6 beta,9 alpha,25-tetrol 24-monoacetate, a novel polyhydroxylated steroid from the soft coral Sarcophyton tortuosum.

    PubMed

    Su, J Y; Peng, T S; Long, K H; Zeng, L M

    1986-01-01

    A novel polyhydroxylated steroid, named sartortuosterol A, with rare 3 alpha- and 6-hydroxyl groups, was isolated from the South China Sea soft coral Sarcophyton tortuosum Tixier-Durivault, and its structure was established as 24xi-methyl 5 alpha-cholestane-3 alpha, 6 beta, 9 alpha,25-tetrol 25-monoacetate from spectroscopic data and chemical conversions.

  11. Comparison of 3 alpha-hydroxysteroid dehydrogenase activities in the microsomal fractions of hyperplastic, malignant and normal human prostatic tissues.

    PubMed

    Hudson, R W

    1984-04-01

    The conversion of dihydrotestosterone (DHT) to 3 alpha-androstanediol (3 alpha-adiol) was studied using the microsomal fractions of 15 hyperplastic, 5 malignant and 6 normal human prostatis tissues. Standard assay conditions were: 0.2 microM DHT, 1.0 mM NADPH, 1.0 mM NADH, 2.0 mM EDTA and the microsomal fractions equivalent to 200 mg of prostatic tissue, in 0.1 M MES buffer, pH 6.5. Under the conditions of this assay, the back-conversion of 3 alpha-adiol to DHT or the conversion of DHT to androstanediol were negligible. Optimum enzyme activity was achieved under standard assay conditions. In the absence of EDTA: enzyme activity was 65% of the standard assay; activity was diminished further by 2 mM Ca2+ and virtually eliminated by 2 mM Mg2+ or 2 microM Zn2+. Activity in the absence of either NADPH or NADH was only 50% of the activities seen in the presence of both cofactors. The pH optimum of the enzyme was between 6.0 and 6.5. The apparent Km values of the enzymes in hyperplastic, malignant and normal tissues were 0.03, 0.02 and 0.03 microM, respectively. The Vmax values for these tissues were 6.0 +/- 2.1, 1.6 +/- 0.5 and 14.0 +/- 3.0 pmol/mg protein/20 min incubation, respectively. The results of these experiments offer further explanation for the differences in DHT and 3 alpha-adiol levels seen in the 3 prostatic tissues.

  12. Simultaneous inhibition of GSK3alpha and GSK3beta using hairpin siRNA expression vectors.

    PubMed

    Yu, Jenn-Yah; Taylor, Jennifer; DeRuiter, Stacy L; Vojtek, Anne B; Turner, David L

    2003-02-01

    Short interfering RNAs (siRNAs) can mediate sequence-specific inhibition of gene expression in mammalian cells. We and others have recently developed expression vector-based systems for synthesizing siRNAs or hairpin siRNAs in mammalian cells. Expression vector-based RNA interference (RNAi) effectively suppresses expression of target genes and is likely to be a powerful tool for analysis of gene function. Here we compare inhibition by vectors expressing hairpin siRNA designs either with different loop sequences connecting the two siRNA strands, or with duplex regions of different lengths. Our results suggest that lengthening the 19-nucleotide duplex region of a relatively ineffective hairpin siRNA can increase inhibition, but increasing the length of an effective 19-nt hairpin siRNA does not increase inhibition. We also demonstrate that hairpin siRNA vectors can be used to inhibit two target genes simultaneously. We have targeted glycogen synthase kinase-3alpha (GSK-3alpha) and GSK-3beta, two related kinases involved in the regulation of a variety of cellular processes and also implicated in the pathogenesis of several human diseases. Inhibition of either GSK-3alpha or GSK-3beta by transfection of hairpin siRNA vectors leads to elevated expression of the GSK-3 target beta-catenin, whereas inhibition of both kinases further increases beta-catenin expression. Our results suggest that vector-based siRNA inhibition may be useful for dissecting the functional roles of GSK-3alpha and GSK-3beta in somatic cells. The ability to inhibit two or more genes simultaneously with hairpin siRNA expression vectors should facilitate studies of gene function in mammalian cells.

  13. Cloning, sequencing, and functional analysis of the 5'-flanking region of the rat 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase gene.

    PubMed

    Lin, H K; Penning, T M

    1995-09-15

    Rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase (3 alpha-HSD/DD) is a member of the aldo-keto reductase gene superfamily. It displays high constitutive expression and inactivates circulating steroid hormones and suppresses the formation of polycyclic aromatic hydrocarbon anti- and syn-diol-epoxides (ultimate carcinogens). To elucidate mechanisms responsible for constitutive expression of the 3 alpha-HSD/DD gene a rat genomic library obtained from adult Sprague-Dawley female liver (HaeIII partial digest) was screened, using a probe corresponding to the 5'-end of the cDNA (-15 to +250), and a 15.8-kb genomic clone was isolated. Sequencing revealed that 6.3 kb contained exon 1 (+16 to +138 bp) plus additional introns and exons. The transcription start site (+1) was located by primer extension analysis, and the initiation codon, ATG, was located at +55 bp. The remaining 9.5 kb represented the 5'-flanking region of the rat 3 alpha-HSD/DD gene. A 1.6-kb fragment of this region was sequenced. A TATTTAA sequence (TATA box) was found at 33 bp upstream from the major transcription start site. cis-acting elements responsible for the constitutive expression of the rat 3 alpha-HSD/DD gene were located on the 5'-flanking region by transient transfection of reporter-gene (chloramphenicol acetyl transferase, CAT) constructs into human hepatoma cells (HepG2). CAT assays identified the basal promoter between (-199 and +55 bp), the presence of a proximal enhancer (-498 to -199 bp) which stimulated CAT activity 6-fold, the existence of a powerful silencer (-755 to -498 bp), and a strong distal enhancer (-4.0 to -2.0 kb) which increased CAT activity by 20-40-fold. A computer search of available consensus sequences for trans-acting factors revealed that a cluster of Oct-sites were uniquely located in the silencer region. Using the negative response element (-797 to -498 bp) as a probe and nuclear extracts from HepG2 cells, three bands were identified by gel mobility shift

  14. Overexpression of Reg3alpha increases cell growth and the levels of cyclin D1 and CDK4 in insulinoma cells.

    PubMed

    Cui, Wei; De Jesus, Kristine; Zhao, Hong; Takasawa, Shin; Shi, Bingyin; Srikant, Coimbatore B; Liu, Jun-Li

    2009-06-01

    Regenerating gene (Reg) family protein Reg3alpha is normally expressed in pancreatic acinar and endocrine cells. In order to explore its effect on islet beta-cell replication, insulinoma MIN6 cells were stably transfected with murine Reg3alpha cDNA. Determined using real-time PCR and Western blots, the levels of Reg3alpha mRNA and protein in Reg3alpha-transfected clones were increased 10- and 6-fold, respectively. Western blots also revealed that the protein was released into the culture medium, consistent with an endocrine effect. In MTT cell proliferation assay, Reg3alpha-overexpressing cells exhibited a 2-fold increase in the rate of cell growth. In order to investigate the intracellular mechanism, we studied cell cycle regulatory proteins. In Reg3alpha-expressing cells, we detected 2.2- and 2.5-fold increased levels of cyclin D1 and CDK4, respectively, which paralleled a 1.8-fold increase in the rate of Akt phosphorylation. It is established that beta-cell replication is associated with increased cyclin D1 and CDK4 levels; deficiency in CDK4 or cyclin D2 results in reduced beta-cell mass and diabetes. Our results suggest that Reg3alpha stimulates beta-cell replication, by activating Akt kinase and increasing the levels of cyclin D1/CDK4.

  15. Decreased expression of hepatocyte nuclear factor 3 alpha during the acute-phase response influences transthyretin gene transcription.

    PubMed Central

    Qian, X; Samadani, U; Porcella, A; Costa, R H

    1995-01-01

    Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins (alpha, beta, and gamma) are known to regulate the transcription of numerous liver-specific genes. The HNF-3 proteins bind to DNA as monomers through a winged-helix motif, which is also utilized by a number of developmental regulators, including the Drosophila homeotic fork head (fkh) protein. We have previously characterized a strong-affinity HNF-3S site in the transthyretin (TTR) promoter region which is essential for expression in human hepatoma (HepG2) cells. In the current study, we identify an activating protein 1 (AP-1) site which partially overlaps the HNF-3S sequence in the TTR promoter. We show that in HepG2 cells the AP-1 sequence confers 12-O-tetradecanoylphorbol-13-acetate inducibility to the TTR promoter and contributes to normal TTR transcriptional activity. We also demonstrate that the HNF-3 proteins and AP-1 bind independently to the TTR AP-1-HNF-3 site, and cotransfection experiments suggest that they do not cooperate to activate an AP-1-HNF-3 reporter construct. In addition, 12-O-tetradecanoylphorbol-13-acetate exposure of HepG2 cells results in a reciprocal decrease in HNF-3 alpha and -3 gamma expression which may facilitate interaction of AP-1 with the TTR AP-1-HNF-3 site. In order to explore the role of HNF-3 in the liver, we have examined expression patterns of TTR and HNF-3 during the acute-phase response and liver regeneration. Partial hepatectomy produced minimal fluctuation in HNF-3 and TTR expression, suggesting that HNF-3 expression is not influenced by proliferative signals induced during liver regeneration. In acute-phase livers, we observed a dramatic reduction in HNF-3 alpha expression which correlates with a decrease in the expression of its target gene, the TTR gene. Furthermore, consistent with previous studies, the acute-phase livers are induced for c-jun but not c-fos expression. We propose that the reduction in TTR gene expression during the acute phase is likely due

  16. Interactions across the interface contribute the stability of homodimeric 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase.

    PubMed

    Hwang, Chi-Ching; Hsu, Chao-Nan; Huang, Tzu-Jung; Chiou, Shean-Jaw; Hong, Yi-Ren

    2009-10-01

    The dimerization of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase was studied by interrupting the salt bridge interactions between D249 and R167 in the dimeric interface. Substitution of alanine, lysine and serine for D249 decreased catalytic efficiency 30, 1400 and 1.4-fold, and lowered the melting temperature 6.9, 5.4 and 7.6 degrees C, respectively. The mutated enzymes have the dimeric species but the equilibrium between monomer and dimer for these mutants varies from each other, implying that these residues might contribute differently to the dimer stability. Thermal and urea-induced unfolding profiles for wild-type and mutant enzymes appeared as a two-state transition and three-state transition, respectively. In addition, mutation on D249 breaks the salt bridges and causes different effects on the loss of enzymatic activity for D249A, D249K and D249S mutants in the urea-induced unfolding profiles. Hence, D249 at the dimeric interface in 3alpha-HSD/CR is essential for conformational stability, oligomeric integrity and enzymatic activity. PMID:19683506

  17. Relevance of classic anti-neutrophil cytoplasmic autoantibody (C-ANCA)-mediated inhibition of proteinase 3-alpha 1-antitrypsin complexation to disease activity in Wegener's granulomatosis.

    PubMed

    Dolman, K M; Stegeman, C A; van de Wiel, B A; Hack, C E; von dem Borne, A E; Kallenberg, C G; Goldschmeding, R

    1993-09-01

    In the sera of patients with Wegener's granulomatosis (WG), C-ANCA can be detected that are directed against proteinase 3 (PR3). We have previously observed that C-ANCA interfere with PR3 proteolytic activity and with complexation of PR3 with its major physiologic inhibitor, alpha 1-antitrypsin (alpha 1AT). In the present study we investigated whether this inhibitory effect of C-ANCA on PR3-alpha 1AT complexation correlates with clinical activity of WG. Serial serum samples of eight consecutive patients with histologically proven relapses of WG were tested. At the moment of relapse all sera revealed inhibitory activity towards PR3-alpha 1AT complexation (median 22%, range 10-59%). Disease activity score (r = 0.87, P < 0.02) and C-reactive protein (CRP) levels (r = 0.66, P < 0.1) correlated with C-ANCA inhibition of PR3-alpha 1AT complexation, while they did not correlate with the C-ANCA titre detected by indirect immunofluorescence (IIF) nor with IgG anti-PR3 antibody level measured by ELISA. The inhibitory effect of C-ANCA on PR3-alpha 1AT complexation had risen significantly at the moment of relapse compared with values 3 months (P < 0.05) and 6 months (P < 0.01) before relapse. Eight patients with established WG and positive for C-ANCA but without clinical evidence of relapse served as controls. In this group no inhibitory effect of C-ANCA on PR3-alpha 1AT complexation was observed in 7/8 patients sera. Sera of one control patient contained moderate C-ANCA inhibitory activity towards PR3-alpha 1AT complexation, which remained at a constant level during the 6 months period of observation. Thus, disease activity in WG appears to be more closely related to C-ANCA inhibitory activity towards PR3-alpha 1AT complexation.

  18. Behavioral action of ethanol in Porsolt's forced swim test: modulation by 3 alpha-hydroxy-5 alpha-pregnan-20-one.

    PubMed

    Hirani, K; Khisti, R T; Chopde, C T

    2002-12-01

    Ethanol is known to increase cortical and plasma content of GABAergic neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP) which is responsible for some of its behavioral and electrophysiological effects. We have previously demonstrated the antidepressant like effect of 3alpha,5alpha-THP in mice. This study investigated the role of 3alpha,5alpha-THP in acute, chronic and withdrawal effects of ethanol using mouse forced swim test (FST) paradigm. While acute systemic ethanol (2 or 2.5 g/kg) administration exhibited an antidepressant like effect, its prolonged consumption produced tolerance to this effect and its withdrawal, on the other hand, elicited enhanced behavioral despair (depression). The antidepressant like effect of ethanol was potentiated by GABA(A) receptor agonist, muscimol (0.5 mg/kg, i.p.), 3alpha,5alpha-THP (0.5, 1 or 2 microg/mouse, i.c.v.) and by neurosteroidogenic drugs viz. selective serotonin reuptake inhibitor (SSRI), fluoxetine (5 or 20 mg/kg, i.p.), agonist at mitochondrial diazepam binding inhibitor receptor, FGIN 1-27 (0.5 or 1 microg/mouse, i.c.v.), or 11beta-hydroxylase inhibitor, metyrapone (0.5 or 1 microg/mouse, i.c.v.) which are known to increase endogenous 3alpha,5alpha-THP content. Furthermore, inhibition of the endogenous neurosteroid biosynthesis by drugs like 5alpha-reductase inhibitor, finasteride (50 mg/kg, s.c.), 3beta-hydroxysteroid dehydrogenase inhibitor, trilostane (30 mg/kg i.p.) or 3alpha-hydroxysteroid dehydrogenase inhibitor, indomethacin (5 mg/kg, i.p.) and GABA(A) receptor antagonist, bicuculline (1 mg/kg, i.p.) blocked the antidepressant like effect of ethanol. Withdrawal of ethanol from mice consuming it chronically displayed enhanced behavioral despair and elicited tolerance to antidepressant like action of acute ethanol (2.5, 3 or 3.5 g/kg). Moreover, sub-antidepressant doses (0.25 or 0.5 microg/mouse, i.c.v.) of 3alpha,5alpha-THP and fluoxetine (5 mg/kg, i.p.) but not imipramine (1 mg/kg, i

  19. Association of protein kinase FA/GSK-3alpha (a proline-directed kinase and a regulator of protooncogenes) with human cervical carcinoma dedifferentiation/progression.

    PubMed

    Yang, S D; Yu, J S; Lee, T T; Ni, M H; Yang, C C; Ho, Y S; Tsen, T Z

    1995-10-01

    Computer analysis of protein phosphorylation-sites sequence revealed that most transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3alpha (kinase FA/GSK-3alpha) (a particular member of PDPK family) has been optimized for human cervical tissue and used to demonstrate for the first time significantly increased (P < 0.001) activity in poorly differentiated cervical carcinoma (82.8 +/- 6.6 U/mg of protein), moderately differentiated carcinoma (36.2 +/- 3.4 U/mg of protein), and well-differentiated carcinoma (18.3 +/- 2.4 U/mg of protein) from 36 human cervical carcinoma samples when compared to 12 normal controls (4.9 +/- 0.6 U/mg of protein). Immunoblotting analysis further revealed that increased activity of kinase FA/GSK-3alpha in cervical carcinoma is due to overexpression of protein synthesis of the kinase. Taken together, the results provide initial evidence that overexpression of protein synthesis and cellular activity of kinase FA/GSK-3alpha may be involved in human cervical carcinoma dedifferentiation/progression, supporting an association of proline-directed protein kinase with neoplastic transformation and tumorigenesis. Since protein kinase FA/GSK-3alpha may function as a possible regulator of transcription factors/proto-oncogenes, the results further suggest that kinase FA/GSK-3alpha may play a potential role in human cervical carcinogenesis, especially in its dedifferentiation and progression.

  20. Aqueous chemical growth of alpha-Fe2O3-alpha-Cr2O3 nanocomposite thin films.

    PubMed

    Vayssieres, L; Guo, J; Nordgren, J

    2001-12-01

    We are reporting here on the inexpensive fabrication and optical properties of an iron(III) oxide-chromium(III) oxide nanocomposite thin film of corundum crystal structure. Its novel and unique-designed architecture consists of uniformed, well-defined and oriented nanorods of Hematite (alpha-Fe2O3) of 50 nm in diameter and 500 nm in length and homogeneously distributed nonaggregated monodisperse spherical nanoparticles of Eskolaite (alpha-Cr2O3) of 250 nm in diameter. This alpha-Fe2O3-alpha-Cr2O3 nanocomposite thin film is obtained by growing, directly onto transparent polycrystalline conducting substrate, an oriented layer of hematite nanorods and growing subsequently, the eskolaite layer. The synthesis is carried out by a template-free, low-temperature, multilayer thin film coating process using aqueous solution of metal salts as precursors. Almost 100% of the light is absorbed by the composite film between 300 and 525 nm and 40% at 800 nm which yields great expectations as photoanode materials for photovoltaic cells and photocatalytic devices. PMID:12914078

  1. Identification and characterization of a novel translational repressor of the steroid-inducible 3 alpha-hydroxysteroid dehydrogenase/carbonyl reductase gene in Comamonas testosteroni.

    PubMed

    Xiong, Guangming; Martin, Hans-Jörg; Maser, Edmund

    2003-11-28

    Comamonas testosteroni 3 alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3 alpha-HSD/CR) is a key enzyme in the degradation of steroid compounds in soil and may therefore play a significant role in the bioremediation of hormonally active compounds in the environment. The enzyme is also involved in the degradation of the steroid antibiotic fusidic acid. In addition, 3 alpha-HSD/CR mediates the carbonyl reduction of non-steroidal aldehydes and ketones. Because the gene of 3 alpha-HSD/CR (hsdA) is inducible by steroids, we were interested in the mode of its molecular regulation. Recently, we could identify the first molecular determinant in procaryotic steroid signaling, i.e. a repressor protein (RepA), which acts as a negative regulator by binding to upstream operator sequences of hsdA, thereby blocking hsdA transcription. In this work, we identified and cloned a second novel regulator gene that we named repB. The gene locates 932 bp downstream from hsdA on the C. testosteroni chromosome with an orientation opposite to that of hsdA. The open reading frame of repB consists of 237 bp and translates into a protein of 78 amino acids that was found to act as a repressor that regulates hsdA expression on the translational level. Northern blot analysis, UV-cross linking, gel-shift assays, and competition experiments proved that RepB binds to a 16-nucleotide sequence downstream of AUG at the 5' end of the 3 alpha-HSD/CR mRNA, thereby blocking hsdA translation. Testosterone, on the other hand, was shown to specifically bind to RepB, thereby yielding the release of RepB from the 3 alpha-HSD/CR mRNA such that hsdA translation could proceed. Data bank searches with the RepB primary structure yielded a 46.2% identity to the regulator of nucleoside diphosphate kinase, a formerly unknown protein from Escherichia coli that can restore a growth defect in alginate production in Pseudomonas aeruginosa. In conclusion, the induction of hsdA by steroids in fact is a derepression

  2. Synthesis, aggregation behavior and cholesterol solubilization studies of 16-epi-pythocholic acid (3 alpha,12 alpha,16 beta-trihydroxy-5 beta-cholan-24-oic acid).

    PubMed

    Nonappa; Maitra, Uday

    2010-07-01

    Synthesis, aggregation behavior and in vitro cholesterol solubilization studies of 16-epi-pythocholic acid (3 alpha,12 alpha,16 beta-trihydroxy-5 beta-cholan-24-oic acid, EPCA) are reported. The synthesis of this unnatural epimer of pythocholic acid (3 alpha,12 alpha,16 alpha-trihydroxy-5 beta-cholan-24-oic acid, PCA) involves a series of simple and selective chemical transformations with an overall yield of 21% starting from readily available cholic acid (CA). The critical micellar concentration (CMC) of 16-epi-pythocholate in aqueous media was determined using pyrene as a fluorescent probe. In vitro cholesterol solubilization ability was evaluated using anhydrous cholesterol and results were compared with those of other natural di- and trihydroxy bile acids. These studies showed that 16-epi-pythocholic acid (16 beta-hydroxy-deoxycholic acid) behaves similar to cholic acid (CA) and avicholic acid (3 alpha,7 alpha,16 alpha-trihydroxy-5 beta-cholan-24-oic acid, ACA) in its aggregation behavior and cholesterol dissolution properties.

  3. Estren (4-estren-3alpha,17beta-diol) is a prohormone that regulates both androgenic and estrogenic transcriptional effects through the androgen receptor.

    PubMed

    Centrella, Michael; McCarthy, Thomas L; Chang, Wei-Zhong; Labaree, David C; Hochberg, Richard B

    2004-05-01

    Alternative mechanisms of steroid action, through both traditional nuclear receptors and indirect pathways of gene activation, are emerging. Recent studies suggest that the synthetic steroid, 4-estrene-3alpha,17beta-diol (estren), has nongenotropic as well as sex-nonspecific osteogenic effects in ovariectomized and orchidectomized mice. We found limited estrogen receptor-dependent effects by estren on gene expression in primary osteoblast cultures and showed that it binds poorly to estrogen and androgen receptors in vitro. However, estren potently regulated direct and indirect androgen receptor-dependent effects on gene expression by osteoblasts. Consistent with this, osteoblasts produced the potent androgen 19-nortestosterone from estren by way of a 3alpha-hydroxysteroid dehydrogenase-like activity. Moreover, recombinant 3alpha-hydroxysteroid dehydrogenase (AKR1C9) and osteoblast-derived cell lysate each effectively converted estren to 19-nortestosterone in vitro, and mRNA encoding this enzyme occurs in osteoblasts. In addition to its androgenic activity, estren potently stimulated androgen receptor-dependent effects on gene expression through conventional estrogen-sensitive transcriptional elements in osteoblasts. Therefore, through local metabolism, estren indirectly activates the androgen receptor to regulate both androgen- and estrogen-like transcriptional responses by bone-forming cells.

  4. 17alpha-ethyl-5beta-estrane-3alpha, 17beta-diol, a biological marker for the abuse of norethandrolone and ethylestrenol in slaughter cattle.

    PubMed

    Van Puymbroeck, M; Kuilman, M E; Maas, R F; Witkamp, R F; Leyssens, L; Van Miert, A S; Hendriks, L; Vanderzande, D; Adriaensens, P; Jacobs, M P; Raus, J

    1999-05-28

    The metabolism of the illegal growth promoter ethylestrenol (EES) was evaluated in bovine liver cells and subcellular fractions of bovine liver preparations. Incubations with bovine microsomal preparations revealed that EES is extensively biotransformed into norethandrolone (NE), another illegal growth promoter. Furthermore, incubations of monolayer cultures of hepatocytes with NE indicated that NE itself is rapidly reduced to 17alpha-ethyl-5beta-estrane-3alpha, 17beta-diol (EED). In vivo tests confirmed that, after administration of either EES or NE, EED is excreted as a major metabolite. Therefore, it was concluded that, both in urine and faeces samples, EED can be used as a biological marker for the illegal use of EES and/or NE. Moreover, by monitoring EED in urine or faeces samples, the detection period after NE administration is significantly prolonged. These findings were further confirmed by three cases of norethandrolone abuse in a routine screening program for forbidden growth promoters.

  5. 17alpha-ethyl-5beta-estrane-3alpha, 17beta-diol, a biological marker for the abuse of norethandrolone and ethylestrenol in slaughter cattle.

    PubMed

    Van Puymbroeck, M; Kuilman, M E; Maas, R F; Witkamp, R F; Leyssens, L; Van Miert, A S; Hendriks, L; Vanderzande, D; Adriaensens, P; Jacobs, M P; Raus, J

    1999-05-28

    The metabolism of the illegal growth promoter ethylestrenol (EES) was evaluated in bovine liver cells and subcellular fractions of bovine liver preparations. Incubations with bovine microsomal preparations revealed that EES is extensively biotransformed into norethandrolone (NE), another illegal growth promoter. Furthermore, incubations of monolayer cultures of hepatocytes with NE indicated that NE itself is rapidly reduced to 17alpha-ethyl-5beta-estrane-3alpha, 17beta-diol (EED). In vivo tests confirmed that, after administration of either EES or NE, EED is excreted as a major metabolite. Therefore, it was concluded that, both in urine and faeces samples, EED can be used as a biological marker for the illegal use of EES and/or NE. Moreover, by monitoring EED in urine or faeces samples, the detection period after NE administration is significantly prolonged. These findings were further confirmed by three cases of norethandrolone abuse in a routine screening program for forbidden growth promoters. PMID:10406207

  6. The selenium-75-homocholic acid taurine test reevaluated: combined measurement of fecal selenium-75 activity and 3 alpha-hydroxy bile acids in 211 patients

    SciTech Connect

    van Tilburg, A.J.; de Rooij, F.W.; van den Berg, J.W.; Kooij, P.P.; van Blankenstein, M. )

    1991-06-01

    The recommended reference values for the selenium-75-homocholic acid taurine (75SeHCAT) test, used in the analysis of chronic diarrhea, were evaluated in 211 patients by comparing simultaneous measurements of 3 alpha-hydroxy bile acids and 75Se activity in daily collected stools. An initial evaluation in 11 patients showed that the fecal collection method, which allows inspection and additional analysis of stools, was equivalent to the abdominal retention method. Selenium-75-HCAT whole-body retention half-life (WBR50) was greater than 2.8 days in less than 10% of the patients with bile acid malabsorption and less than 1.7 days in less than 10% of the normals. We recommend that a 75SeHCAT WBR50 less than 1.7 days is abnormal, a WBR50 greater than 2.8 days is normal, and a WBR50 in the range 1.7-2.8 days is equivocal, which was the case in 48% (94/195) of the patients in this study.

  7. Identification and characterization of cis elements in the STAT3 gene regulating STAT3 alpha and STAT3 beta messenger RNA splicing.

    PubMed

    Shao, H; Quintero, A J; Tweardy, D J

    2001-12-15

    Signal transducer and activator of transcription 3 (STAT3) is an oncogene and a critical regulator of multiple cell-fate decisions, including myeloid cell differentiation. Two isoforms of STAT3 have been identified: alpha (p92) and beta (p83). These differ structurally in their C-terminal transactivation domains, resulting in distinct functional activities. The cis genetic elements that regulate the ratio of alpha to beta messenger RNA (mRNA) are unknown. In this study, cloning, sequencing, and splicing analysis of the human and murine STAT3 genes revealed a highly conserved 5' donor site for generation of both alpha and beta mRNA and distinct branch-point sequences, polypyrimidine tracts, and 3' acceptor sites (ASs) for each. The beta 3' AS was found to be located 50 nucleotides downstream of the alpha 3' AS in exon 23. Two additional cryptic 3' ASs (delta and epsilon) were also identified. Thus, we identified for the first time the cis regulatory sequences responsible for generation of STAT3 alpha and STAT3 beta mRNA.

  8. Effects of 4'-chloro-3 alpha-(diphenylmethoxy)-tropane on mesostriatal, mesocortical, and mesolimbic dopamine transmission: comparison with effects of cocaine.

    PubMed

    Tanda, Gianluigi; Ebbs, Aaron; Newman, Amy H; Katz, Jonathan L

    2005-05-01

    Increase in dopamine (DA) neurotransmission resulting from blockade of the DA transporter (DAT) after administration of cocaine is believed to play a major role in mediating its behavioral and reinforcing effects. Since it was hypothesized that drugs that block the DAT have cocaine-like behavioral effects, it was of interest to study in the present article the stimulant effects of cocaine on locomotor activity and on pattern of activation of DA neurotransmission in different DAergic terminal areas in rats and compare these effects with those of 4'-chloro-3alpha-(diphenylmethoxy)-tropane (4-Cl-BZT), a benztropine analog showing higher affinity for the DAT, but reduced behavioral effects compared with cocaine. Administration of cocaine resulted in a dose-dependent stimulation of locomotor activity and DA neurotransmission in the nucleus accumbens shell and core, dorsal caudate, and in the medial prefrontal cortex (PFCX) measured by microdialysis. At comparable doses, the effects of 4-Cl-BZT on DA levels in all brain areas except the PFCX were generally reduced compared with those of cocaine, as were the effects on locomotor activity. The differences in behavioral effects corresponded generally to differences between the drugs with regard to their stimulation of extracellular DA levels, although the mechanism(s) for the differences in extracellular DA may involve effects mediated by sites other than the DAT or differences in the efficiency of the two drugs in blocking DA uptake. Nonetheless, the present results suggest that the differences in behavioral effects between cocaine and 4-Cl-BZT are related to differences in their patterns of activation of DA transmission.

  9. The dopamine uptake inhibitor 3 alpha-[bis(4'-fluorophenyl)metoxy]-tropane reduces cocaine-induced early-gene expression, locomotor activity, and conditioned reward.

    PubMed

    Velázquez-Sánchez, Clara; Ferragud, Antonio; Hernández-Rabaza, Vicente; Nácher, Amparo; Merino, Virginia; Cardá, Miguel; Murga, Juan; Canales, Juan J

    2009-11-01

    Benztropine (BZT) analogs, a family of high-affinity dopamine transporter ligands, are molecules that exhibit pharmacological and behavioral characteristics predictive of significant therapeutic potential in cocaine addiction. Here, we examined in mice the effects of 3 alpha-[bis(4'-fluorophenyl)metoxy]-tropane (AHN-1055) on motor activity, conditioned place preference (CPP) and c-Fos expression in the striatum. AHN-1055 produced mild attenuation of spontaneous locomotor activity at a low dose (1 mg/kg) and weak stimulation at a higher dose (10 mg/kg). In parallel, the BZT analog significantly increased c-Fos expression in the dorsolateral caudoputamen at the high dose, whereas producing marginal decreases at low and moderate doses (1, 3 mg/kg) in both dorsal and ventral striatum. Interaction assays showed that cocaine's ability to stimulate locomotor activity was decreased by AHN-1055 treatment, but not by treatment with D-amphetamine. Such reduced ability did not result from an increase in stereotyped behavior. Another dopamine uptake inhibitor, nomifensine, decreased cocaine-induced locomotor activity but evoked by itself intense motor stereotypies. Remarkably, the BZT analog dose-dependently blocked cocaine-induced CPP without producing CPP when given alone, and blocked in conditioned mice cocaine-stimulated early-gene activation in the nucleus accumbens and dorsomedial striatum. These observations provide evidence that AHN-1055 does not behave as a classical psychomotor stimulant and that some of its properties, including attenuation of cocaine-induced striatal c-Fos expression, locomotor stimulation, and CPP, support its candidacy, and that of structurally related molecules, as possible pharmacotherapies in cocaine addiction.

  10. Postnatal changes of nicotinic acetylcholine receptor alpha 2, alpha 3, alpha 4, alpha 7 and beta 2 subunits genes expression in rat brain.

    PubMed

    Zhang, X; Liu, C; Miao, H; Gong, Z H; Nordberg, A

    1998-10-01

    Postnatal changes of nicotinic acetylcholine receptor (nAChR) alpha 2, alpha 3, alpha 4, alpha 7 and beta 2 subunits mRNAs were investigated in rat brain using ribonuclease protection assay. Multiple developmental patterns were observed: (1) transient expression during the first few postnatal weeks; alpha 2 in the hippocampus and brain stem, alpha 3 in the striatum, cerebellum and cortex, alpha 4 in the hippocampus, striatum and cerebellum, alpha 7 in the cerebellum and beta 2 in the striatum. (2) Constant expression across development; alpha 2 and alpha 3 in the thalamus, alpha 4 in the cortex, thalamus and brain stem, alpha 7 in the thalamus and brain stem and beta 2 in all brain regions except striatum. (3) Non-detection across development; alpha 2 in the cortex, striatum and cerebellum. (4) Increase with age; alpha 7 in the cortex and hippocampus. (5) Bell-shaped development; alpha 7 in the striatum. Postnatal changes of nAChR isoforms in different brain regions of rat were investigated by receptor binding assays. The developmental patterns of [3H]epibatidine and (-)-[3H]nicotine binding sites were similar to each other in each brain region, but different from that of [3H] alpha-bungarotoxin binding sites. No obvious correlation was observed between the developmental patterns of [3H] alpha-bungarotoxin, [3H]epibatidine and (-)-[3H]nicotine binding sites and corresponding subunits mRNAs. These results indicate that multiple mechanisms are involved in changes of gene expression of nAChRs subunits in the brain of developing rats.

  11. Chemical modification of 3 alpha,20 beta-hydroxysteroid dehydrogenase with diethyl pyrocarbonate. Evidence for an essential, highly reactive, lysyl residue.

    PubMed

    Pasta, P; Mazzola, G; Carrea, G

    1987-03-10

    Diethyl pyrocarbonate inactivated the tetrameric 3 alpha,20 beta-hydroxysteroid dehydrogenase with second-order rate constants of 1.63 M-1 s-1 at pH 6 and 25 degrees C or 190 M-1 s-1 at pH 9.4 and 25 degrees C. The activity was slowly and partially restored by incubation with hydroxylamine (81% reactivation after 28 h with 0.1 M hydroxylamine, pH 9, 25 degrees C). NADH protected the enzyme against inactivation with a Kd (10 microM) very close to the Km (7 microM) for the coenzyme. The ultraviolet difference spectrum of inactivated vs. native enzyme indicated that a single histidyl residue per enzyme subunit was modified by diethyl pyrocarbonate, with a second-order rate constant of 1.8 M-1 s-1 at pH 6 and 25 degrees C. The histidyl residue, however, was not essential for activity because in the presence of NADH it was modified without enzyme inactivation and modification of inactivated enzyme was rapidly reversed by hydroxylamine without concomitant reactivation. Progesterone, in the presence of NAD+, protected the histidyl residue against modification, and this suggests that the residue is located in or near the steroid binding site of the enzyme. Diethyl pyrocarbonate also modified, with unusually high reaction rate, one lysyl residue per enzyme subunit, as demonstrated by dinitrophenylation experiments carried out on the treated enzyme. The correlation between inactivation and modification of lysyl residues at different pHs and the protection by NADH against both inactivation and modification of lysyl residues indicate that this residue is essential for activity and is located in or near the NADH binding site of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. The dopamine uptake inhibitor 3 alpha-[bis(4'-fluorophenyl)metoxy]-tropane reduces cocaine-induced early-gene expression, locomotor activity, and conditioned reward.

    PubMed

    Velázquez-Sánchez, Clara; Ferragud, Antonio; Hernández-Rabaza, Vicente; Nácher, Amparo; Merino, Virginia; Cardá, Miguel; Murga, Juan; Canales, Juan J

    2009-11-01

    Benztropine (BZT) analogs, a family of high-affinity dopamine transporter ligands, are molecules that exhibit pharmacological and behavioral characteristics predictive of significant therapeutic potential in cocaine addiction. Here, we examined in mice the effects of 3 alpha-[bis(4'-fluorophenyl)metoxy]-tropane (AHN-1055) on motor activity, conditioned place preference (CPP) and c-Fos expression in the striatum. AHN-1055 produced mild attenuation of spontaneous locomotor activity at a low dose (1 mg/kg) and weak stimulation at a higher dose (10 mg/kg). In parallel, the BZT analog significantly increased c-Fos expression in the dorsolateral caudoputamen at the high dose, whereas producing marginal decreases at low and moderate doses (1, 3 mg/kg) in both dorsal and ventral striatum. Interaction assays showed that cocaine's ability to stimulate locomotor activity was decreased by AHN-1055 treatment, but not by treatment with D-amphetamine. Such reduced ability did not result from an increase in stereotyped behavior. Another dopamine uptake inhibitor, nomifensine, decreased cocaine-induced locomotor activity but evoked by itself intense motor stereotypies. Remarkably, the BZT analog dose-dependently blocked cocaine-induced CPP without producing CPP when given alone, and blocked in conditioned mice cocaine-stimulated early-gene activation in the nucleus accumbens and dorsomedial striatum. These observations provide evidence that AHN-1055 does not behave as a classical psychomotor stimulant and that some of its properties, including attenuation of cocaine-induced striatal c-Fos expression, locomotor stimulation, and CPP, support its candidacy, and that of structurally related molecules, as possible pharmacotherapies in cocaine addiction. PMID:19606084

  13. Nicotine enhances expression of the alpha 3, alpha 4, alpha 5, and alpha 7 nicotinic receptors modulating calcium metabolism and regulating adhesion and motility of respiratory epithelial cells.

    PubMed

    Zia, S; Ndoye, A; Nguyen, V T; Grando, S A

    1997-09-01

    The purpose of this study was to investigate the possibility of direct toxic effects of nicotine (Nic) on human bronchial epithelial cells (BEC) suggested by our previous findings of functional nicotinic acetylcholine receptors (nAChRs) in the epithelial cells lining mucocutaneous membranes. We now demonstrate for the first time that human and murine BEC both in vivo and in vitro express functional nAChRs, and that classic alpha 3, alpha 4, alpha 5 and alpha 7 subunits can contribute to formation of these acetylcholine-gated ion channels. In human bronchial and mouse lung tissues, and in cultures of human BEC, the nAChRs were visualized by subunit-specific antibodies on the cell membranes, particularly at the sites of cell-to-cell contacts. The epithelial cells of submucosal glands abundantly expressed alpha 7 nAChRs. Smoking significantly (p < 0.05) increased the relative numbers of nAChRs, and this effects could be reproduced in cultures of BEC exposed to 10 microM Nic. At a higher dose, Nic decreased the relative numbers of alpha 5-containing nAChRs, suggesting a role for receptor desensitization. The function of the nAChR channels expressed by BEC was demonstrated by biphasic increase in the concentrations of intracellular calcium ([Ca++]i) in response to activation of the channel by Nic and fluctuations of [Ca++]i due to channel blockade by mecamylamine (Mec). Long-term exposure to milimolar concentrations of Nic resulted in a steady increase of [Ca++]i, which may lead to cell damage. The biological roles of epithelial nAChRs apparently involve regulation of cell-to-cell communications, adhesion and motility, because Mec caused rapid and profound changes in these cell functions which were reversible by Nic. An over exposure of BEC to Nic, however, produced an antagonist-like effect, suggesting that the pathobiological effects of Nic toxicity might result from both activation of nAChR channels and nAChR desensitization. We conclude that medical consequences of

  14. pha-4 is Ce-fkh-1, a fork head/HNF-3alpha,beta,gamma homolog that functions in organogenesis of the C. elegans pharynx.

    PubMed

    Kalb, J M; Lau, K K; Goszczynski, B; Fukushige, T; Moons, D; Okkema, P G; McGhee, J D

    1998-06-01

    The C. elegans Ce-fkh-1 gene has been cloned on the basis of its sequence similarity to the winged-helix DNA binding domain of the Drosophila fork head and mammalian HNF-3alpha,beta,gamma genes, and mutations in the zygotically active pha-4 gene have been shown to block formation of the pharynx (and rectum) at an early stage in embryogenesis. In the present paper, we show that Ce-fkh-1 and pha-4 are the same gene. We show that PHA-4 protein is present in nuclei of essentially all pharyngeal cells, of all five cell types. PHA-4 protein first appears close to the point at which a cell lineage will produce only pharyngeal cells, independently of cell type. We show that PHA-4 binds directly to a 'pan-pharyngeal enhancer element' previously identified in the promoter of the pharyngeal myosin myo-2 gene; in transgenic embryos, ectopic PHA-4 activates ectopic myo-2 expression. We also show that ectopic PHA-4 can activate ectopic expression of the ceh-22 gene, a pharyngeal-specific NK-2-type homeodomain protein previously shown to bind a muscle-specific enhancer near the PHA-4 binding site in the myo-2 promoter. We propose that it is the combination of pha-4 and regulatory molecules such as ceh-22 that produces the specific gene expression patterns during pharynx development. Overall, pha-4 can be described as an 'organ identity factor', completely necessary for organ formation, present in all cells of the organ from the earliest stages, capable of integrating upstream developmental pathways (in this case, the two distinct pathways that produce the anterior and posterior pharynx) and participating directly in the transcriptional regulation of organ specific genes. Finally, we note that the distribution of PHA-4 protein in C. elegans embryos is remarkably similar to the distribution of the fork head protein in Drosophila embryos: high levels in the foregut/pharynx and hindgut/rectum; low levels in the gut proper. Moreover, we show that pha-4 expression in the C. elegans gut

  15. Novel 3alpha-methoxyserrat-14-en-21beta-ol (PJ-1) and 3beta-methoxyserrat-14-en-21beta-ol (PJ-2)-curcumin, kojic acid, quercetin, and baicalein conjugates as HIV agents.

    PubMed

    Tanaka, Reiko; Tsujii, Hiroko; Yamada, Takeshi; Kajimoto, Tetsuya; Amano, Fumio; Hasegawa, Junya; Hamashima, Yoshio; Node, Manabu; Katoh, Kayoko; Takebe, Yutaka

    2009-07-15

    Sixteen novel compounds; 3alpha-methoxyserrat-14-en-21beta-ol (1) and 3beta-methoxyserrat-14-en-21beta-ol (2) and their curcumin, kojic acid, quercetin, and baicalein conjugates (3)-(18) were designed, synthesized, and evaluated for in vitro anti-HIV-1 reverse transcriptase (RT) activity in infected C8166-CCR5 cells, a human CD4(+) T-lymphocyte cell line. Among them, kojic acid derivatives, 9-12 showed significant biological activity. In particular, the compound 13, the conjugate of two molecules of 3alpha-methoxyserrat-14-en-21beta-ol (1) and one molecule of kojic acid, exerted significant anti-HIV activity with an EC50 value of 0.12microg/mL.

  16. 3-(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N-alkyl-N-arylbenzamides: potent, non-peptidic agonists of both the micro and delta opioid receptors.

    PubMed

    Bishop, Michael J; Garrido, Dulce M; Boswell, G Evan; Collins, Mark A; Harris, Philip A; McNutt, Robert W; O'Neill, Scott J; Wei, Ke; Chang, Kwen-Jen

    2003-02-13

    Opioid analgesics with both micro and delta opioid receptor activation represent a new approach to the treatment of severe pain with an improved safety profile. Compounds with this profile may exhibit strong analgesic properties due to micro agonism, with a reduced side effect profile resulting from delta agonism. Replacing the p-diethylamide of the known potent delta opioid receptor selective agonist BW373U86 with a m-diethylamide resulted in a compound with agonist activity at both the micro and delta opioid receptors. Modifying the amide to an N-methyl-N-phenylamide increased agonist potency at both receptors. A series of 3-(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N-alkyl-N-arylbenzamides have been made to explore the structure-activity relationship (SAR) around the N-methyl-N-phenylamide. Several potent agonists of both the micro and delta opioid receptors have been identified, including (+)-3-((alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N-(4-fluorophenyl)-N-methylbenzamide (23), which has EC50 values of 0.67 and 1.1 nM at the micro (guinea pig ileum assay) and delta (mouse vas deferens assay) opioid receptors, respectively.

  17. Chemical synthesis and hepatic biotransformation of 3 alpha,7 alpha-dihydroxy-7 beta-methyl-24-nor-5 beta-cholan-23-oic acid, a 7-methyl derivative of norchenodeoxycholic acid: studies in the hamster.

    PubMed

    Yoshii, M; Mosbach, E H; Schteingart, C D; Hagey, L R; Hofmann, A F; Cohen, B I; McSherry, C K

    1991-11-01

    A new bile acid analogue, 3 alpha,7 alpha-dihydroxy-7 beta-methyl-24-nor-5 beta-cholan-23-oic acid (7-Me-norCDCA) was synthesized from the methyl ester of norursodeoxycholic acid, and its hepatic biotransformation was defined in the hamster. To synthesize 7-Me-norCDCA, the 3 alpha-hydroxyl group of methyl norursodeoxycholate was protected as the hemisuccinate, and the 7 beta-hydroxyl group was oxidized with CrO3 to form the 7-ketone. A Grigard reaction with methyl magnesium iodide followed by alkaline hydrolysis gave 7-Me-norCDCA (greater than 70% yield). The structure of the new compound was confirmed by proton magnetic resonance and mass spectrometry. After intraduodenal administration of the 14C-labeled compound into the anesthetized biliary fistula hamster, it was rapidly and efficiently secreted into the bile; 80% of radioactivity was recovered in 2 h. After intravenous infusion, the compound was efficiently extracted by the liver and secreted into the bile (greater than 75% in 3 h). Most (93%) of the biliary radioactivity was present in biotransformation products. The major biotransformation product (48.7 +/- 6.0%) was a new compound, assigned the structure of 3 alpha,5 beta,7 alpha- trihydroxy-7 beta-methyl-24-nor-5 beta-cholan-23-oic acid (5 beta-hydroxy-7- Me-norCDCA). In addition, conjugates of 7-Me-norCDCA with taurine (13.7 +/- 5.0%), sulfate (10.3 +/- 3.0%), or glucuronide (5.1 +/- 1.7%) were formed. 7-Me-norCDCA was strongly choleretic in the hamster; during its intravenous infusion, bile flow increased 2 to 3 times above the basal level, and the calculated choleretic activity of the compound (and its metabolic products) was much greater than that of many natural bile acids, indicating that the compound induced hypercholeresis. It is concluded that the biotransformation and physiological properties of 7-Me-norCDCA closely resemble those of norCDCA. Based on previous studies, the major biological effect of the 7-methyl group in 7-Me-norCDCA is to

  18. Phenotypic consequences of deletion of the {gamma}{sub 3}, {alpha}{sub 5}, or {beta}{sub 3} subunit of the type A {gamma}-aminobutyric acid receptor in mice

    SciTech Connect

    Culia, C.T.; Stubbs, L.J.; Montgomery, C.S.; Russell, L.B.; Rinchik, E.M.

    1994-03-29

    Three genes (Gabrg3, Gabra5, and Gabrb3) encoding the {gamma}{sub 3}, {alpha}{sub 5}, and {beta}{sub 3} subunits of the type A {gamma}-aminobutyric acid receptor, respectively, are known to map near the pink-eyed dilution (p) locus in mouse chromosome 7. This region shares homology with a segment of human chromosome 15 that is implicated in Angelman syndrome, an inherited neurobehavioral disorder. By mapping Gabrg3-Gabra5-Gabrb3-telomere. Like Gabrb3, neither the Gabra5 nor Gabrg3 gene is functionally imprinted in adult mouse brain. Mice deleted for all three subunits die at birth with a cleft palate, although there are rare survivors ({approximately} 5%) that do not have a cleft palate but do exhibit a neurological abnormality characterized by tremor, jerky gait, and runtiness. The authors have previously suggested that deficiency of the {beta}{sub 3} subunit may be responsible for the clefting defect. Most notably, however, in this report they describe mice carrying two overlapping, complementing p deletions that fail to express the {gamma}{sub 3} transcript, as well as mice from another line that express neither the {gamma}{sub 3} nor {alpha}{sub 5} transcripts. Surprisingly, mice from both of these lines are phenotypically normal and do not exhibit any of the neurological symptoms characteristic of the rare survivors that are deleted for all three ({gamma}{sub 3}, {alpha}{sub 5}, and {beta}{sub 3}) subunits. These mice therefore provide a whole-organism type A {gamma}-aminobutyric-acid receptor background that is devoid of any receptor subtypes that normally contain the {gamma}{sub 3} and/or {alpha}{sub 5} subunits. The absence of an overt neurological phenotype in mice lacking the {gamma}{sub 3} and/or {alpha}{sub 5} subunits also suggests that mutations in these genes are unlikely to provide useful animal models for Angelman syndrome in humans.

  19. 3{alpha}-6{alpha}-Dihydroxy-7{alpha}-fluoro-5{beta}-cholanoate (UPF-680), physicochemical and physiological properties of a new fluorinated bile acid that prevents 17{alpha}-ethynyl-estradiol-induced cholestasis in rats

    SciTech Connect

    Clerici, Carlo . E-mail: clerici@unipg.it; Castellani, Danilo; Asciutti, Stefania; Pellicciari, Roberto; Setchell, Kenneth D.R. |; O'Connell, Nancy C. |; Sadeghpour, Bahman; Camaioni, Emidio; Fiorucci, Stefano; Renga, Barbara; Nardi, Elisabetta; Sabatino, Giuseppe; Clementi, Mattia; Giuliano, Vittorio; Baldoni, Monia; Orlandi, Stefano; Mazzocchi, Alessandro; Morelli, Antonio; Morelli, Olivia

    2006-07-15

    3{alpha}-6{alpha}-Dihydroxy-7{alpha}-fluoro-5{beta}-cholanoate (UPF-680), the 7{alpha}-fluorine analog of hyodeoxycholic acid (HDCA), was synthesized to improve bioavailability and stability of ursodeoxycholic acid (UDCA). Acute rat biliary fistula and chronic cholestasis induced by 17{alpha}-ethynyl-estradiol (17EE) models were used to study and compare the effects of UPF-680 (dose range 0.6-6.0 {mu}mol/kg min) with UDCA on bile flow, biliary bicarbonate (HCO{sub 3} {sup -}), lipid output, biliary bile acid composition, hepatic enzymes and organic anion pumps. In acute infusion, UPF-680 increased bile flow in a dose-related manner, by up to 40.9%. Biliary HCO{sub 3} {sup -} output was similarly increased. Changes were observed in phospholipid secretion only at the highest doses. Treatment with UDCA and UPF-680 reversed chronic cholestasis induced by 17EE; in this model, UDCA had no effect on bile flow in contrast to UPF-680, which significantly increased bile flow. With acute administration of UPF-680, the biliary bile acid pool became enriched with unconjugated and conjugated UPF-680 (71.7%) at the expense of endogenous cholic acid and muricholic isomers. With chronic administration of UPF-680 or UDCA, the main biliary bile acids were tauro conjugates, but modification of biliary bile acid pool was greater with UPF-680. UPF-680 increased the mRNA for cytochrome P450 7A1 (CYP7A1) and cytochrome P450 8B (CYP8B). Both UDCA and UPF-680 increased the mRNA for Na{sup +} taurocholate co-transporting polypeptide (NCTP). In conclusion, UPF-680 prevented 17EE-induced cholestasis and enriched the biliary bile acid pool with less detergent and cytotoxic bile acids. This novel fluorinated bile acid may have potential in the treatment of cholestatic liver disease.

  20. Mechanism of potentiation of a dysfunctional epilepsy-linked mutated GABA(A) receptor by a neurosteroid (3alpha, 21-dihydroxy-5alpha-pregnan-20-one): transient kinetic investigations.

    PubMed

    Ramakrishnan, Latha; Hess, George P

    2010-09-14

    The malfunction of a mutated GABA(A) receptor (alpha1beta2gamma2L(K289M)) in an inheritable form of epilepsy (GEFS+, generalized epilepsy with febrile seizures plus) in humans [Baulac, S., Huberfeld, G., Gourfinkel-An, I., Mitropoulou, G., Beranger, A., Prud'homme, J. F., Baulac, M., Brice, A., Bruzzone, R., and LeGuern, E. (2001) Nat. Genet. 28, 46-48] has been accounted for by a 5-fold decrease in the channel-opening equilibrium of the mutated receptor compared to the wild type [Ramakrishnan, L., and Hess, G. P. (2004) Biochemistry 43, 7534-7540]. Here we describe the mechanism by which the neurosteroid 3alpha, 21-dihydroxy-5alpha-pregnan-20-one (5alpha-THDOC) alleviates this malfunction of the mutated receptor transiently expressed in HEK293 cells. Two rapid reaction techniques, the cell-flow and the laser-pulse photolysis methods, were used in combination with whole-cell current recordings. 150-muM 5alpha-THDOC does not affect the rate constant for channel opening (k(op)) of approximately 250 s(-1) but does decrease the rate constant for channel closing (k(cl)) from 121 +/- 11 s(-1) to 56 +/- 21 s(-1). This results in an increase in the channel-opening equilibrium constant ((Phi(-1) = k(op)/k(cl)) by a factor of about 2, leading to about 50% alleviation of the malfunction of the inheritable mutated (alpha1beta2gamma2L(K289M)) GABA(A) receptor linked to GEFS+.

  1. Structural identification of a major cytokinin in coconut milk as 14-O-(3-O-[beta-D-galactopyranosyl-(1-->2)-alpha-D-galactopyranosyl- (1-->3)-alpha-L-arabinofuranosyl]-4-O-(alpha-L-arabinofuranosyl)- beta-d-galactopyranosyl)-trans-zeatin riboside.

    PubMed

    Kobayashi, H; Morisaki, N; Tago, Y; Hashimoto, Y; Iwasaki, S; Kawachi, E; Nagata, R; Shudo, K

    1997-02-01

    A cytokinin isolated from the fluid endosperm of Cocos mucifera L. (coconut milk), accounting for more than 20% of the total cytokinin activity, was structurally analyzed by NMR techniques, mass spectrometry, and sugar analysis by high performance liquid chromatography (HPLC). The planar structure of the cytokinin was deduced from its NMR and mass spectrometric data. The structure of the sugar moiety, including its absolute structure, was determined by HPLC analysis of alditol acetates and aldononitrile acetates derived from the cytokinin. The configuration of the sugar-sugar bonds was determined by NMR, and the structure was finally identified as 14-O-(3-O-[-beta-D-galactopyranosyl-(1-->2)-alpha-galactopyranosyl-(1--> 3)- alpha-L-arabinofuranosyl]-4-O-(alpha-L-arabino-furanosyl)-beta-D- galactopyranosyl)-trans-zeatin riboside. PMID:9118441

  2. Structural identification of a major cytokinin in coconut milk as 14-O-(3-O-[beta-D-galactopyranosyl-(1-->2)-alpha-D-galactopyranosyl- (1-->3)-alpha-L-arabinofuranosyl]-4-O-(alpha-L-arabinofuranosyl)- beta-d-galactopyranosyl)-trans-zeatin riboside.

    PubMed

    Kobayashi, H; Morisaki, N; Tago, Y; Hashimoto, Y; Iwasaki, S; Kawachi, E; Nagata, R; Shudo, K

    1997-02-01

    A cytokinin isolated from the fluid endosperm of Cocos mucifera L. (coconut milk), accounting for more than 20% of the total cytokinin activity, was structurally analyzed by NMR techniques, mass spectrometry, and sugar analysis by high performance liquid chromatography (HPLC). The planar structure of the cytokinin was deduced from its NMR and mass spectrometric data. The structure of the sugar moiety, including its absolute structure, was determined by HPLC analysis of alditol acetates and aldononitrile acetates derived from the cytokinin. The configuration of the sugar-sugar bonds was determined by NMR, and the structure was finally identified as 14-O-(3-O-[-beta-D-galactopyranosyl-(1-->2)-alpha-galactopyranosyl-(1--> 3)- alpha-L-arabinofuranosyl]-4-O-(alpha-L-arabino-furanosyl)-beta-D- galactopyranosyl)-trans-zeatin riboside.

  3. The efficacy of serum carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen 15-3 (CA15-3), alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG) levels in determining the malignancy of solitary pulmonary nodules.

    PubMed

    Bekci, T T; Senol, T; Maden, E

    2009-01-01

    We investigated the utility of the tumour markers carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen 15-3 (CA15-3), alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG) for the differential diagnosis of benign and malignant solitary pulmonary nodules in 42 hospitalized patients. Routine medical history and physical examination of each patient was performed and each patient also had a chest X-ray and a thoracic computed tomography scan. The following diagnostic procedures were also undertaken: bronchoscopy, transthoracic needle aspiration biopsy, sputum cytology and culture, analysis of sputum acid-fast bacilli and thoracotomy. Measurement of serum levels of tumour antigens by Immulite 2000 radioimmunoassay found that three tumour markers, CEA, CA125 and CA15-3, could be used in the diagnosis of malignant solitary pulmonary nodules. More research is now required involving a larger group of patients.

  4. FT-EPR study of the photochemical homolysis of Re- and Ru-alkyl bonds in Re(R)(CO){sub 3}({alpha}-diimine) and Ru(I)(R)(CO){sub 2}({alpha}-diimine) complexes. Evidence for the reactive {sigma}(M-R){pi}{sup *} state

    SciTech Connect

    Kleverlaan, C.J.; Stufkens, D.J.; Oskam, A.; Martino, D.M.; Willigen, H. van

    1996-11-28

    A Fourier transform EPR (FT-EPR) study was made of the photochemistry of Re(R)(CO){sub 3}({alpha}-diimine) and Ru(I)(R)(CO){sub 2}({alpha}-diimine) complexes, where R = benzyl, 2-propyl, or ethyl and {alpha}-diimine = 4,4`-dimethyl-2, 2`-bipyridine or N,N`-diisopropyl-1,4-diazabutadiene. The FT-EPR technique was used to monitor the formation and decay of alkyl radicals generated by metal-R bound homolysis. The spectra showed pronounced chemically induced dynamic electron polarization (CIDEP) effects with polarization patterns that depend strongly on metal ion, alkyl substituent, and solvent. To varying degrees, all spectra shows a contribution form ST{sub 0} radical pair mechanism (RPM) CIDEP. From the low-field-emission/high-field-adsorption polarization pattern given by this mechanism, it can be deduced that the free radicals are formed via a triplet-state precursor, confirming conclusions reached in earlier spectroscopic studies. In addition to the RPM contribution, some spectra also show a net-absorptive CIDEP contribution while others show a net-emissive contribution. These net spin polarization contributions are attributed tentatively to triplet mechanism and spin-orbit coupling CIDEP. From the fact that the alkyl radical spectra exhibit strong spin polarization, it is concluded that metal ion atomic orbital contributions to the electronic states of precursor triplet and radical pair must be small. 17 refs., 4 figs.

  5. Abnormalities in brain structure and behavior in GSK-3alpha mutant mice

    PubMed Central

    2009-01-01

    Background Glycogen synthase kinase-3 (GSK-3) is a widely expressed and highly conserved serine/threonine protein kinase encoded by two genes that generate two related proteins: GSK-3α and GSK-3β. Mice lacking a functional GSK-3α gene were engineered in our laboratory; they are viable and display insulin sensitivity. In this study, we have characterized brain functions of GSK-3α KO mice by using a well-established battery of behavioral tests together with neurochemical and neuroanatomical analysis. Results Similar to the previously described behaviours of GSK-3β+/-mice, GSK-3α mutants display decreased exploratory activity, decreased immobility time and reduced aggressive behavior. However, genetic inactivation of the GSK-3α gene was associated with: decreased locomotion and impaired motor coordination, increased grooming activity, loss of social motivation and novelty; enhanced sensorimotor gating and impaired associated memory and coordination. GSK-3α KO mice exhibited a deficit in fear conditioning, however memory formation as assessed by a passive avoidance test was normal, suggesting that the animals are sensitized for active avoidance of a highly aversive stimulus in the fear-conditioning paradigm. Changes in cerebellar structure and function were observed in mutant mice along with a significant decrease of the number and size of Purkinje cells. Conclusion Taken together, these data support a role for the GSK-3α gene in CNS functioning and possible involvement in the development of psychiatric disorders. PMID:19925672

  6. ENEA extremity dosemeter based on LiF(Mg,Cu,P) to evaluate Hp(3,alpha).

    PubMed

    Mariotti, F; Fantuzzi, E; Morelli, B; Gualdrini, G; Botta, M C; Uleri, G; Bordy, J M; Denoziere, M

    2011-03-01

    Recent epidemiological studies suggest a rather low-dose threshold (<0.5 Gy) for the induction of a cataract of the eye lens. Some other studies even assume that there is no threshold at all. Therefore, protection measures have to be optimised and current dose limits for the eye lens may be reduced in the future. ICRP Publication 103 on H(p)(d), in §(136), reads that '… a depth d = 3 mm has been proposed for the rare case of monitoring the dose to the lens of the eye. In practice, however, H(p)(3) has rarely been monitored and H(p)(0.07) can be used for the same monitoring purpose… '. As recommended on the EU 'Technical recommendations for monitoring individuals occupationally exposed to external radiation', a test on the ENEA TL extremity dosemeter is herein reported. The results within the actual EU founded Optimization of RAdiation protection for MEDical staff (ORAMED) Project, whose WP2 is aimed at the quantity H(p)(3) and eye lens dosimetry in practice, are taken into account. The paper summarises the main aspects of the study carried out at ENEA-Radiation Protection Institute (Bologna, Italy) to provide practical solutions (in the use and the design) to evaluate the response of the ENEA TL extremity dosemeter in terms of H(p)(3).

  7. [Temperature effect correction for Chang'E-3 alpha particle X-ray spectrometer].

    PubMed

    Wu, Ming-Ye; Wang, Huan-Yu; Peng, Wen-Xi; Zhang, Cheng-Mo; Zhang, Jia-Yu; Cui, Xing-Zhu; Liang, Xiao-Hua; Wang, Jin-Zhou; Yang, Jia-Wei; Fan, Rui-Rui; Liu, Ya-Qing; Dong, Yi-Fan; Wu, Feng; Zhao, Xiao-Yun

    2012-07-01

    Alpha particle X-ray spectrometer (APXS) is one of the payloads of Chang'E-3 lunar rover of China's Lunar Exploration Project. The present paper introduces briefly the components of APXS, how it works and its working environment on the lunar surface. The environmental temperature effect has been studied with simulations and experiments, and the results show that the temperature of the APXS sensor will be varying during the measuring on the lunar surface. And another experiment reveals that the energy resolution becomes worse if the sensor's temperature is varying. In this paper, a correction method based on Pearson's chi-squared test is presented. The method can improve the energy resolution when the sensor's temperature is varying. We have tested the method with the spectra acquired by APXS in the temperature varying period of Temperature Cycling Test, and the results show that the method is efficient and reliable.

  8. Macrophage Inflammatory Protein-3 Alpha (MIP-3α)/CCL20 in HIV-1-Infected Individuals

    PubMed Central

    Aziz, Najib; Detels, Roger; Chang, L Cindy; Butch, Anthony W

    2016-01-01

    Objective Uncontrolled HIV infection progresses to the depletion of systemic and mucosal CD4 and AIDS. Early HIV infection may be associated with increases in the concentration of MIP-3α in the blood and gut fluids. MIP-3α/CCL20 is the only chemokine known to interact with CCR6 receptors which are expressed on immature dendritic cells and both effector and memory CD8+ and CD4+ T cells. The role and prognostic value of blood levels of MIP-3α in HIV-infected individuals has yet to be described. Methods We determined the serum levels of MIP-3α, and IFN-γ, in 167 HIV-1-infected and 27 HIV-1-uninfected men participating in the Multicenter AIDS Cohort Study (MACS). The blood biomarkers were measured using enzyme-linked immunosorbent assays (ELISA) and the cell phenotypes using flow cytometry. Results Median serum levels of MIP-3α in HIV-1-infected and uninfected men was significantly different (p<0.0001) and were 21.3 pg/mL and 6.4 pg/mL respectively. The HIV-1-infected men with CD4+ T cell count <200 cells/μL showed the highest median serum MIP-3α (23.1 pg/mL). Serum levels of MIP-3α in HIV-1 infected (n=167) were negatively correlated with absolute number of CD4+ T cell (p=0.01) and were positively correlated with CD38 molecules on CD8+ T cells (p=0.0002) and with serum levels of IFN-γ (0.006). Conclusion Serum levels of MIP-3α concomitantly increase with plasma levels of IFN-γ, CD38 expression on CD8+ T cells, and decreased of absolute CD4+ T cells in HIV-1-infected men. A higher blood level of MIP-3α may be representation of locally high level of MIP-3α and more recruitment of immature dendritic cell at site of infection. Involvement of CCR6/CCL20 axis and epithelial cells at the recto-colonel level may enhance sexual transmission of HIV-1 in MSM and may be useful as a prognostic marker in HIV-1-infection and AIDS. PMID:27617163

  9. Functional bread with n-3 alpha linolenic acid from whole chia (Salvia hispanica L.) flour.

    PubMed

    Luna Pizarro, Patricia; Almeida, Eveline Lopes; Coelho, Alessandra Silva; Sammán, Norma Cristina; Hubinger, Miriam Dupas; Chang, Yoon Kil

    2015-07-01

    This work proposed to study the effects of the addition of whole chia flour (WCF) on the technological, nutritional and sensory qualities of bread. Different WCF contents (0 and 20 %) and vital gluten (VG) (0 and 4 %) were added to bread according to a 2(2) central composite rotational design. WCF decreased the specific volume, lightness and hue angle of the bread loaves, but did not affect the chroma values. WCF and VG contributed to maintenance of the moisture content of the loaves during the storage period. The increased firmness found with the addition of high levels of WCF (more than 10 %) was countered by larger amounts of VG (more than 2 %). The optimum loaf (10 % WCF and 2 % VG) showed 26 % more lipids, 19 % more protein and 11 % more ash than the standard loaf (0 % WCF and 0 % VG). A better lipid profile was also found (higher omega-3 fatty acid content and a better omega-6/omega-3 ratio). Both breads were positively rated in the sensory profile analysis.

  10. Macrophage Inflammatory Protein-3 Alpha (MIP-3α)/CCL20 in HIV-1-Infected Individuals

    PubMed Central

    Aziz, Najib; Detels, Roger; Chang, L Cindy; Butch, Anthony W

    2016-01-01

    Objective Uncontrolled HIV infection progresses to the depletion of systemic and mucosal CD4 and AIDS. Early HIV infection may be associated with increases in the concentration of MIP-3α in the blood and gut fluids. MIP-3α/CCL20 is the only chemokine known to interact with CCR6 receptors which are expressed on immature dendritic cells and both effector and memory CD8+ and CD4+ T cells. The role and prognostic value of blood levels of MIP-3α in HIV-infected individuals has yet to be described. Methods We determined the serum levels of MIP-3α, and IFN-γ, in 167 HIV-1-infected and 27 HIV-1-uninfected men participating in the Multicenter AIDS Cohort Study (MACS). The blood biomarkers were measured using enzyme-linked immunosorbent assays (ELISA) and the cell phenotypes using flow cytometry. Results Median serum levels of MIP-3α in HIV-1-infected and uninfected men was significantly different (p<0.0001) and were 21.3 pg/mL and 6.4 pg/mL respectively. The HIV-1-infected men with CD4+ T cell count <200 cells/μL showed the highest median serum MIP-3α (23.1 pg/mL). Serum levels of MIP-3α in HIV-1 infected (n=167) were negatively correlated with absolute number of CD4+ T cell (p=0.01) and were positively correlated with CD38 molecules on CD8+ T cells (p=0.0002) and with serum levels of IFN-γ (0.006). Conclusion Serum levels of MIP-3α concomitantly increase with plasma levels of IFN-γ, CD38 expression on CD8+ T cells, and decreased of absolute CD4+ T cells in HIV-1-infected men. A higher blood level of MIP-3α may be representation of locally high level of MIP-3α and more recruitment of immature dendritic cell at site of infection. Involvement of CCR6/CCL20 axis and epithelial cells at the recto-colonel level may enhance sexual transmission of HIV-1 in MSM and may be useful as a prognostic marker in HIV-1-infection and AIDS.

  11. AIRWAY EPITHELIAL CELLS RELEASE MIP-3 ALPHA TO CYTOKINES AND AMBIENT PARTICULATE MATTER. (R827351C003)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  12. Assessment of the influence of histaminergic actions on cocaine-like effects of 3alpha-diphenylmethoxytropane analogs.

    PubMed

    Campbell, Vera C; Kopajtic, Theresa A; Newman, Amy Hauck; Katz, Jonathan L

    2005-11-01

    Previous studies demonstrated that analogs of benztropine (BZT) possess high affinity for the dopamine (DA) transporter (DAT) but generally have behavioral effects different from those of cocaine, suggesting either unique actions at the DA transporter or that another action of these drugs interferes with cocaine-like effects. Because the parent compound has histamine-antagonistic effects, the affinity of its analogs for histamine H(1), H(2), and H(3) receptors were compared with DA transporter affinity to assess whether those differences predicted the amount of cocaine-like activity. All of the compounds displaced [(3)H]mepyramine from H(1), [(125)I]iodoaminopotentidine from H(2), and [(3)H]N-alpha-methylhistamine from H(3) histamine receptors with affinities ranging from 15.7 to 37,600, 218 to >4430, and 4040 to >150,000 nM, respectively. Affinities at histamine H(1) receptors were, respectively, approximately 25- or 300-fold greater than those at H(2) or H(3) histamine receptors. Relative affinities for H(1) and DAT binding did not reliably predict the degree of cocaine-like stimulation of locomotor activity. In addition, interactions of various histaminic agents with cocaine assessed whether an action at any of the histamine sites could interfere with cocaine-like effects. None of the histaminic agents fully substituted for cocaine in rats trained to discriminate 10 mg/kg cocaine from saline nor did any of the compounds antagonize or otherwise diminish the discriminative stimulus effects of cocaine. The results suggest that affinity for histamine receptors cannot account for the diminished cocaine-like effects of the BZT analogs and suggest alternatively that these compounds have actions different from those of cocaine but likely mediated by their interaction with the DAT.

  13. Reduction of 3 alpha-hydroxy-5 beta-chol-6-en-24-oic acid to lithocholic acid in rats

    SciTech Connect

    Kimura, K.; Ogura, M.

    1988-03-01

    After (24-/sup 14/C)delta 6-lithocholic acid was injected into the cecum of rats, (/sup 14/C)lithocholic acid was identified as a metabolite in feces. When the labeled delta 6-bile acid was injected intraperitoneally into bile-fistula rats, radioactivity excreted in bile was contained most abundantly in the taurine-conjugated fraction of bile acids. In the fraction, taurine conjugate of (/sup 14/C)delta 6-lithocholic acid but of neither (/sup 14/C)lithocholic acid nor other bile acids was found. The results showed that (24-/sup 14/C)delta 6-lithocholic acid was reduced to (/sup 14/C)lithocholic acid by the intestinal flora but not by the liver, which, however, was capable of conjugating delta 6-lithocholic acid with taurine.

  14. Crystallographic Analysis of Murine Constitutive Androstane Receptor Ligand-Binding Domain Complexed with 5[alpha]-androst-16-en-3[alpha]-ol

    SciTech Connect

    Vincent, J.; Shan, L.; Fan, M.; Brunzelle, J.S.; Forman, B.M.; Fernandez, E.J.

    2010-03-08

    The constitutive androstane receptor (CAR) is a member of the nuclear receptor superfamily. In contrast to classical nuclear receptors, which possess small-molecule ligand-inducible activity, CAR exhibits constitutive transcriptional activity in the apparent absence of ligand. CAR is among the most important transcription factors; it coordinately regulates the expression of microsomal cytochrome P450 genes and other drug-metabolizing enzymes. The murine CAR ligand-binding domain (LBD) was coexpressed with the steroid receptor coactivator protein (SRC-1) receptor-interacting domain (RID) in Escherichia coli. The mCAR LBD subunit was purified away from SRC-1 by affinity, anion-exchange and size-exclusion chromatography, crystallized with androstenol and the structure of the complex determined by molecular replacement.

  15. Hepatobiliary disposition of 3alpha,6alpha,7alpha,12alpha-tetrahydroxy-cholanoyl taurine: a substrate for multiple canalicular transporters.

    PubMed

    Megaraj, Vandana; Iida, Takashi; Jungsuwadee, Paiboon; Hofmann, Alan F; Vore, Mary

    2010-10-01

    Tetrahydroxy bile acids become major biliary bile acids in Bsep(-/-) mice and Fxr(-/-) mice fed cholic acid; we characterized disposition of these novel bile acids that also occur in patients with cholestasis. We investigated mouse Mrp2 (mMrp2) and P-glycoprotein [(P-gp) mMdr1a]-mediated transport of a tetrahydroxy bile acid, 6α-OH-taurocholic acid (6α-OH-TC), and its biliary excretion in wild-type and Mrp2(-/-) mice in the presence or absence of N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918), a P-gp and breast cancer resistance protein inhibitor. 6α-OH-TC was rapidly excreted into bile of wild-type mice (78% recovery); coinfusion of GF120918 had no significant effect. In Mrp2(-/-) mice, biliary excretion was decreased (52% recovery) and coinfusion of GF120918 further decreased these values (34% recovery). In wild-type, but not Mrp2(-/-), mice, 6α-OH-TC increased bile flow 2.5-fold. Membrane vesicle transport studies of 6α-OH-TC (0.05-0.75 mM) yielded saturation kinetics with a higher apparent affinity for mMrp2 (K(m) = 0.13 mM) than for mMdr1a (K(m) = 0.33 mM); mBsep transported 6α-OH-TC with positive cooperativity (Hill slope = 2.1). Human multidrug resistance-associated protein (MRP) 2 and P-gp also transported 6α-OH-TC but with positive cooperativity (Hill slope = 3.6 and 1.6, respectively). After intraileal administration, the time course of 6α-OH-TC biliary recovery was similar to that of coinfused taurocholate, implying that 6α-OH-TC can undergo enterohepatic cycling. Thus, Mrp2 plays a key role in 6α-OH-TC biliary excretion, whereas P-glycoprotein plays a secondary role; Bsep likely mediates excretion of 6α-OH-TC in the absence of Mrp2 and P-gp. In Bsep(-/-) mice, efficient synthesis of tetrahydroxy bile acids that are Mrp2 and P-gp substrates can explain the noncholestatic phenotype.

  16. Platelet activating factor antagonist design. 2. X-ray structure of dimethyl 2,3,4,5-tetrahydro-5 beta-(3,4-methylenedioxyphenyl)-2-oxo-3 beta-(3,4,5-trimethoxybenzoyl)-3 alpha,4 alpha-furandicarboxylate.

    PubMed

    Peterson, J R; Do, H D; Rogers, R D

    1989-07-15

    C25H24O12, Mr = 516.46, triclinic, P-1, a = 8.780 (3), b = 11.298 (4), c = 13.271 (6) A, alpha = 71.77 (4), beta = 70.31 (3), gamma = 72.66 (3) degrees, V = 1189 A3, Z = 2, Dx = 1.44 g cm-3, lambda (Mo K alpha) = 0.71073 A, mu = 0.74 cm-1, F(000) = 540, T = 293 K, final R = 0.046 for 2495 observed [Fo greater than or equal to 5 sigma (Fo)] reflections. The observed structure reveals a trans disposition for the methoxycarbonyl and aryl substituents at positions 4 and 5 of the heterocycle and a cis-3,4-bis(methoxycarbonyl) relationship. There is no crystallographically imposed symmetry. Several intermolecular van der Waals interactions occur in the cell lattice of this compound. PMID:2610989

  17. Platelet activating factor antagonist design. 2. X-ray structure of dimethyl 2,3,4,5-tetrahydro-5 beta-(3,4-methylenedioxyphenyl)-2-oxo-3 beta-(3,4,5-trimethoxybenzoyl)-3 alpha,4 alpha-furandicarboxylate.

    PubMed

    Peterson, J R; Do, H D; Rogers, R D

    1989-07-15

    C25H24O12, Mr = 516.46, triclinic, P-1, a = 8.780 (3), b = 11.298 (4), c = 13.271 (6) A, alpha = 71.77 (4), beta = 70.31 (3), gamma = 72.66 (3) degrees, V = 1189 A3, Z = 2, Dx = 1.44 g cm-3, lambda (Mo K alpha) = 0.71073 A, mu = 0.74 cm-1, F(000) = 540, T = 293 K, final R = 0.046 for 2495 observed [Fo greater than or equal to 5 sigma (Fo)] reflections. The observed structure reveals a trans disposition for the methoxycarbonyl and aryl substituents at positions 4 and 5 of the heterocycle and a cis-3,4-bis(methoxycarbonyl) relationship. There is no crystallographically imposed symmetry. Several intermolecular van der Waals interactions occur in the cell lattice of this compound.

  18. Multivalent II [beta-D-Galp-(1-->4)-beta-D-GlcpNAc] and Talpha [beta-D-Galp-(1-->3)-alpha-D-GalpNAc] specific Moraceae family plant lectin from the seeds of Ficus bengalensis fruits.

    PubMed

    Singha, Biswajit; Adhya, Mausumi; Chatterjee, Bishnu P

    2007-06-11

    A galactose specific lectin was isolated from the seeds of Ficus bengalensis (Moraceae) fruits and designated as F. bengalensis agglutinin (FBA). The lectin was purified by affinity repulsion chromatography on fetuin-agarose and was a monomer of molecular mass 33kDa. Like other Moraceae family lectins, carbohydrate-binding activity of FBA was independent of any divalent cation. FBA did not bind with simple saccharides, however sugar ligands with aromatic aglycons showed pronounced binding. The combining site of FBA recognized preferably Galbeta1,4GlcNAcbeta1-(II) followed by Galbeta1,3GalNAcalpha1-(Talpha) containing glycotopes. Interaction with saccharides revealed that the combining site of FBA could well accommodate a tetrasaccharide, asialo GM1 glycan (Galbeta1,3GalNAcbeta1,4Galbeta1,4Glcbeta1-), whereas polyvalent Tn (GalNAcalpha1-Ser/Thr), one of the well-recognized ligands of Moraceae family lectin, did not interact well with FBA.

  19. Structural characterization (1->2)-beta-xylose-(1->3)-alpha-arabinose-containing oligosaccharide products of extracted switchgrass (Panicum virgatum, L.) xylan treatment with alpha-arabinofuranosidase and beta-endo-xylanase.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Switchgrass (Panicum virgatum, L.) is a potential dedicated biomass crop for use in biocatalytic conversion systems to biofuels. Nearly 30% of switchgrass cell wall material is xylan. The complete depolymerization of xylan is desirable both as an additional carbon source for microbial fermentation a...

  20. Tirucallane triterpenes from the roots of Ozoroa insignis.

    PubMed

    Liu, Yonghong; Abreu, Pedro

    2006-07-01

    Eight tirucallane triterpenes, methyl 3alpha,24S-dihydroxytirucalla-8,25-dien-21-oate (2), methyl 3alpha-hydroxy-24-oxotirucalla-8,25-dien-21-oate (3), methyl 3alpha-hydroxy-25,26,27-trinor-24-oxotirucall-8-en-21-oate (4), 3alpha,25-dihydroxy-24-(2-hydroxyethyl)-tirucall-8-en-21-oic acid (5), 3alpha,24S,25-trihydroxytirucall-8-en-21-oic acid (6), 3alpha,24R,25-trihydroxytirucall-8-en-21-oic acid (7), 3alpha,25-dihydroxytirucall-8-en-21-oic acid (8), and methyl 3alpha,25-dihydroxytirucall-8-en-21-oate (9), together with alpha-elemolic acid methyl ester (1), were isolated from the roots of Ozoroa insignis. Their structures were elucidated on the basis of spectroscopic evidence.

  1. Bis-spirolabdane-type diterpenoids from Leonurus sibiricus.

    PubMed

    Moon, Hyun Teak; Jin, Qinglong; Shin, Ji Eun; Choi, Eun Jin; Han, Hyo-Kyung; Kim, Yeong Shik; Woo, Eun-Rhan

    2010-02-26

    Six new bis-spirolabdane-type diterpenoids, leosibirinone A (1), 3alpha-acetoxyleoheteronone C (2), leosibirinone B (3), 3alpha-hydroxyleoheteronone A (4), 3alpha-acetoxyleoheteronone E (5), and 3alpha-acetoxy-15-epileoheteronone E (6), were isolated from the aerial parts of Leonurus sibiricus. Their structures were identified on the basis of 1D and 2D NMR, including (1)H-(1)H COSY, HSQC, HMBC, and NOESY spectroscopic analyses.

  2. New triterpenoids of mallotus repandus

    PubMed

    Huang; Wang; Lin

    1999-06-01

    Three new triterpenoids-3alpha-hydroxy-13alpha-ursan-28,12beta-olide 3-benzoate (1), 3alpha-hydroxy-28beta-methoxy-13alpha-ursan-28, 12beta-epoxide 3-benzoate (2), and 3alpha-hydroxy-13alpha-ursan-28-oic acid (3)-and four known compounds were isolated from the stem and root bark of Mallotus repandus.

  3. A new acylated flavonol glycoside from Derris triofoliata.

    PubMed

    Xu, Lu-Rong; Wu, Jun; Zhang, Si

    2006-01-01

    A new acylated flavonol glycoside, kaempferol 3-O-[(6''''-feruloyl)-beta-D-glucopyranosyl-(1 --> 3)]-[alpha-L-rhamnopyranosyl-(1 --> 6)]-beta-D-glucopyranoside and two known cyclolignan glycosides, (+)-lyoniresinol-3alpha-O-beta-D-glucopyranoside and ( - )-lyoniresinol-3alpha-O-beta-D-glucopyranoside were isolated from n-BuOH extracts of the aerial parts of Derris triofoliata, their structures were determined from spectroscopic and chemical evidences.

  4. Saponins from Fagonia arabica.

    PubMed

    Miyase, T; Melek, F R; El-Gindi, O D; Abdel-Khalik, S M; El-Gindi, M R; Haggag, M Y; Hilal, S H

    1996-03-01

    Seven new triterpenoid saponins were isolated and identified from the aerial parts of Fagonia arabica. They were characterized as 3-O-beta-D-xylopyranosyl(1-->2)-[beta-D-glucopyranosyl(1-->3)]-alpha-L- arabinopyranosyl oleanolic acid 28-O-beta-D-glucopyranoside, 3-O-beta-D-glucopyranosyl(1-->2)-[beta-D-glucopyranosyl(1-->3)]-alpha-L- arabino pyranosyl oleanolic acid 28-O-beta-D-glucopyranoside, 3-O-beta-D-xylopyranosyl(1-->2)-[beta-D-glucopyranosyl (1-->3)]-alpha- L-arabinopyranosyl oleanolic acid, 3-O-beta-D-glucopyranosyl(1-->2)-[beta-D-glucopyranosyl(1-->3)]-alpha-L- arabino pyranosyl oleanolic acid, 3-O-beta-D-xylopyranosyl(1-->2)-[beta-D-glucopyranosyl (1-->3)]-alpha-L-arabinopyranosyl 27-hydroxyoleanolic acid, 28-O-beta-D-glucopyranoside, 3-O-beta-D-xylopyranosyl(1-->2)-[beta-D-glucopyranosyl(1-->3)]-alpha-L- arabinopyranosyl ursolic acid 28-O-beta-D-glucopyranoside and 3-O-beta-D-xylopyranosyl (1-->2)-[beta-D-glucopyranosyl (1-->3)-alpha-L-arabinopyranosyl 27-hydroxyursolic acid 28-O-beta-D-glucopyranoside. The structures of the saponins were established by analyses of their 1H and 13C NMR spectra with the aid of 2D experiments. The two genins, 27-hydroxyoleanolic acid and 27-ursolic acid, are new. PMID:8728716

  5. Structural and functional characterization of polyphenols isolated from acerola (Malpighia emarginata DC.) fruit.

    PubMed

    Hanamura, Takayuki; Hagiwara, Toshihiko; Kawagishi, Hirokazu

    2005-02-01

    Two anthocyanins, cyanidin-3-alpha-O-rhamnoside (C3R) and pelargonidin-3-alpha-O-rhamnoside (P3R), and quercitrin (quercetin-3-alpha-O-rhamnoside), were isolated from acerola (Malpighia emarginata DC.) fruit. These polyphenols were evaluated based on the functional properties associated with diabetes mellitus or its complications, that is, on the radical scavenging activity and the inhibitory effect on both alpha-glucosidase and advanced glycation end product (AGE) formation. C3R and quercitrin revealed strong radical scavenging activity. While the inhibitory profiles of isolated polyphenols except quercitrin towards alpha-glucosidase activity were low, all polyphenols strongly inhibited AGE formation.

  6. Microbial transformation of the diterpene 7-epi-foliol by Fusarium fujikuroi.

    PubMed

    Fraga, Braulio M; Bressa, Carlo; González, Pedro; Guillermo, Ricardo

    2014-08-01

    The incubation of 3alpha,7alpha,18-trihydroxy-ent-kaur-16-ene (7-epi-foliol) with the fungus Fusarium fujikuroi gave 3alpha,7alpha,18-trihydroxy-ent-kaur-16-en-18-al as the sole product. The biotransformation of other 7alpha- or 7beta-hydroxy derivatives had led to the oxidation of C-19, which is a main step in the biosynthesis of gibberellins and kaurenolides. Now, the presence of the 3alpha-hydroxyl impedes that oxidation, which is directed to the adjacent C-18 hydroxymethyl forming the corresponding aldehyde.

  7. Triterpenoid saponins from Fagonia indica.

    PubMed

    Shaker, K H; Bernhardt, M; Elgamal, M H; Seifert, K

    1999-08-01

    Two new triterpenoid saponins, 3-O-{[beta-D-glucopyranosyl-(1-->2)]-[alpha-L-arabinopyranosyl-(1- ->3)]- alpha-L-arabinopyranosyl}-ursolic acid-28-O-[beta-D-glucopyranosyl] ester (indicasaponin A), 3-O-{[beta-D-glucopyranosyl-(1-->2)]-[alpha-L-arabinopyranosyl-(1- ->3)]- alpha-L-arabinopyranosyl}-oleanolic acid-28-O-[beta-D-glucopyranosyl] ester (indicasaponin B) and two known triterpenoid saponins, 3-O-[beta-D-glucopyranosyl-(1-->3)-alpha-L-arabinopyranosyl]-ur solic acid-28-O-[beta-D-glucopyranosyl] ester, 3-O-[beta-D-glucopyranosyl-(1-->3)-alpha-L-arabinopyranosyl]-olean olic acid-28-O-[beta-D-glucopyranosyl] ester have been isolated from Fagonia indica. The structures were determined primarily by NMR spectroscopy. The assignment of NMR signals was performed by means of 1H-1H COSY, NOESY, ROESY, TOCSY, HMQC and HMBC experiments. PMID:10444859

  8. Involvement of N-linked carbohydrate chains of pig zona pellucida in sperm-egg binding.

    PubMed

    Yonezawa, N; Aoki, H; Hatanaka, Y; Nakano, M

    1995-10-01

    The sperm receptor activity of pig zona pellucida has been previously shown to exist in one of the components, pig zona protein 3 alpha (PZP3 alpha), that can be purified after the removal of sialylated and/or sulfated N-acetylpoly(lactosamine) by digestion with endo-beta-galactosidase. In this study, we examined whether N-linked or O-linked carbohydrate chains are involved in the sperm receptor activity of pig zona pellucida. The elimination of N-linked carbohydrate chains from endo-beta-galactosidase-digested PZP3 alpha by digestion with N-glycanase markedly reduced its inhibitory effect on sperm-egg binding in an in vitro competition assay, whereas the elimination of O-linked carbohydrate chains by alkali treatment hardly reduced the inhibitory effect. These results indicate that N-linked carbohydrate chains of PZP3 alpha play a major role in mediating the sperm binding of zona pellucida in pig.

  9. Stat3 and G-CSF-induced myeloid differentiation.

    PubMed

    Chakraborty, A; Tweardy, D J

    1998-08-01

    Granulocyte colony-stimulating factor (G-CSF) is the cytokine critical for directing neutrophilic granulocyte differentiation. Early G-CSF signaling events in myeloid cells involves activation of STATs, proteins that serve the dual function of signal transduction and activation of transcription, especially the activation of Stat3. A dominant-negative mutant construct of Stat3 inhibited G-CSF-mediated neutrophilic differentiation indicating that Stat3 is a essential component for driving the G-CSF-mediated differentiation program in myeloid cells. Three isoforms of Stat3 have been identified, alpha(p92), beta(p83) and gamma(p72) each derived from a single gene. Stat3alpha is the predominant isoform expressed in most cells. Stat3beta is derived from Stat3alpha by alternative RNA splicing. Stat3gamma is derived from Stat3alpha by limited proteolysis. Mapping of Stat3alpha and Stat3beta activation in M1 murine myeloid leukemia cells revealed that their optimal activation required G-CSFR constructs containing both Y704 and Y744. These amino acid residues has previously been demonstrated to be essential for G-CSF-induced differentiation in this cells. Phosphopeptide affinity and phosphopeptide inhibition studies indicate that Stat3alpha and Stat3beta are recruited to the G-CSF receptor complex through their interaction with the receptor at phosphotyrosines Y704 and Y744. Y744 is followed at the +3 position by Cys (C). This sequence YXXC, represents a novel motif implicated in the recruitment and activation of Stat3alpha, Stat3beta and Stat3gamma by the hG-CSFR. Structurally, Stat3alpha, Stat3beta and Stat3gamma differ from each other in their C-terminal transactivation domain. In the beta isoform, the Stat3alpha transactivation domain is replaced by 7 amino acid residues which enable Stat3beta to interact with c-Jun. In the gamma isoform, the Stat3alpha transactivation domain is removed by limited proteolysis creating a dominant negative isoform. In immature human

  10. Stat3 isoforms, alpha and beta, demonstrate distinct intracellular dynamics with prolonged nuclear retention of Stat3beta mapping to its unique C-terminal end.

    PubMed

    Huang, Ying; Qiu, Jihui; Dong, Shuo; Redell, Michele S; Poli, Valeria; Mancini, Michael A; Tweardy, David J

    2007-11-30

    Two isoforms of Stat3 (signal transducer and activator of transcription 3) are expressed in cells, alpha (p92) and beta (p83), both derived from a single gene by alternative mRNA splicing. The 55-residue C-terminal transactivation domain of Stat3alpha is deleted in Stat3beta and replaced by seven unique C-terminal residues (CT7) whose function remains uncertain. We subcloned the open reading frames of Stat3alpha and Stat3beta into the C terminus of green fluorescent protein (GFP). Fluorescent microscopic analysis of HEK293T cells transiently transfected with GFP-Stat3alpha or GFP-Stat3beta revealed similar kinetics and cytokine concentration dependence of nuclear accumulation; these findings were confirmed by high throughput microscope analysis of murine embryonic fibroblasts that lacked endogenous Stat3 but stably expressed either GFP-Stat3alpha or GFP-Stat3beta. However, although time to half-maximal cytoplasmic reaccumulation after cytokine withdrawal was 15 min for GFP-Stat3alpha, it was >180 min for GFP-Stat3beta. Furthermore, although the intranuclear mobility of GFP-Stat3alpha was rapid and increased with cytokine stimulation, the intranuclear mobility of GFP-Stat3beta in unstimulated cells was slower than that of GFP-Stat3alpha in unstimulated cells and was slowed further following cytokine stimulation. Deletion of the unique CT7 domain from Stat3beta eliminated prolonged nuclear retention but did not alter its intranuclear mobility. Thus, Stat3alpha and Stat3beta have distinct intracellular dynamics, with Stat3beta exhibiting prolonged nuclear retention and reduced intranuclear mobility especially following ligand stimulation. Prolonged nuclear retention, but not reduced intranuclear mobility, mapped to the CT7 domain of Stat3beta.

  11. Synthesis of 24-nor-5 beta-cholan-23-oic acid derivatives: a convenient and efficient one-carbon degradation of the side chain of natural bile acids.

    PubMed

    Schteingart, C D; Hofmann, A F

    1988-10-01

    An efficient procedure for obtaining nor-bile acids from natural (C24) bile acids is described. Treatment of formylated bile acids with sodium nitrite in a mixture of trifluoroacetic anhydride with trifluoroacetic acid gives, through a "second order" Beckmann rearrangement, 24-nor-23-nitriles. These compounds, on alkaline hydrolysis, afford the corresponding nor-bile acids in high yields. The sequence was successfully applied to the synthesis of 3 alpha-hydroxy-24-nor-5 beta-cholan-23-oic (norlithocholic) acid, 3 alpha,6 alpha- (norhyodeoxycholic), 3 alpha,7 alpha- (norchenodeoxycholic), 3 alpha,7 beta- (norursodeoxycholic), and 3 alpha,12 alpha-dihydroxy-24-nor-5 beta-cholan-23-oic (nordeoxycholic) acids, as well as 3 alpha,7 alpha,12 alpha-trihydroxy-24-nor-5 beta-cholan-23-oic (norcholic) acid. 13C-NMR spectra of their methyl esters are reported. The procedure provides a more rapid alternative to the Barbier-Wieland degradation for shortening by one methylene group the side chain of natural (C24) bile acids.

  12. Dihydrotestosterone is a peripheral paracrine hormone.

    PubMed

    Horton, R

    1992-01-01

    Androgen action in sexual tissues, especially skin and the prostate, is expressed by dihydrotestosterone (DHT) acting at the nuclear level. Dihydrotestosterone in the circulation and target tissues is almost solely derived from the peripheral conversion of secreted testosterone (T) in men and androstenedione in women. The general pathway is testosterone----DHT in equilibrium with androstanediol (3 alpha diol). However, a number of studies suggest that blood DHT or 3 alpha diol are not reliable indicators of peripheral DHT formation. This is particularly suggested by discrepancies in the specific activity of DHT in blood and urine following infusion of labeled DHT, suggesting that total body DHT formation is not reflected by blood levels. Thus, DHT should be thought of as a paracrine hormone formed and acting primarily in target tissues. 3 alpha androstanediol glucuronide (3 alpha diol G) is a major metabolite of DHT. An important site of its formation is the skin. Levels in blood and urine are increased in hirsutism and acne, and blood levels closely parallel pubertal development. 3 alpha diol G levels are especially increased in adrenal disorders of androgenicity such as andrenogenital syndrome; it is also a good marker of response to therapy. Levels are reduced in various forms of male pseudohermaphroditism. 3 alpha androstanediol glucuronide appears to be the best marker available of DHT formation in target tissues such as skin. PMID:1551803

  13. Solubility of calcium salts of unconjugated and conjugated natural bile acids.

    PubMed

    Gu, J J; Hofmann, A F; Ton-Nu, H T; Schteingart, C D; Mysels, K J

    1992-05-01

    The approximate solubility products of the calcium salts of ten unconjugated bile acids and several taurine conjugated bile acids were determined. The formation of micelles, gels, and/or precipitates in relation to Ca2+,Na+, and bile salt concentration was summarized by "phase maps." Because the ratio of Ca2+ to bile salt in the precipitates was ca. 1:2, and the activity of Ca2+ but not that of bile salt (BA-) could be measured, the ion product of aCa2+ [BA-]2 was calculated. The ion product (= Ksp) ranged over nine orders of magnitude and the solubility thus ranged over three orders of magnitude; its value depended on the number and orientation of the hydroxyl groups in the bile acid. Ion products (in units of 10(-9) mol/l)3 were as follows: cholic (3 alpha OH,7 alpha OH,12 alpha OH) 640; ursocholic (3 alpha OH,7 beta OH,12 alpha OH) 2300; hyocholic (3 alpha OH,6 alpha OH,7 alpha OH) 11; ursodeoxycholic (3 alpha OH,7 beta OH) 91; chenodeoxycholic (3 alpha OH,7 alpha OH) 10; deoxycholic (3 alpha OH,12 alpha OH) 1.5; 12-epideoxycholic (lagodeoxycholic, 3 alpha OH,12 beta OH) 2.2; hyodeoxycholic (3 alpha OH,6 alpha OH) 0.7; and lithocholic (3 alpha OH) 0.00005. The critical micellization temperature of the sodium salt of murideoxycholic acid (3 alpha OH,6 beta OH) was greater than 100 degrees C, and its Ca2+ salt was likely to be very insoluble. Taurine conjugates were much more soluble than their corresponding unconjugated derivatives: chenodeoxycholyltaurine, 384; deoxycholyltaurine, 117; and cholyltaurine, greater than 10,000. Calcium salts of unconjugated bile acids precipitated rapidly in contrast to those of glycine conjugates which were metastable for months. Thus, hepatic conjugation of bile acids with taurine or glycine not only enhances solubility at acidic pH, but also at Ca2+ ion concentrations present in bile and intestinal content.

  14. Structure of alpha6 beta3 delta GABA(A) receptors and their lack of ethanol sensitivity.

    PubMed

    Baur, Roland; Kaur, Kuldeep H; Sigel, Erwin

    2009-12-01

    Delta (delta) subunit containing GABA(A) receptors are expressed extra-synaptically and mediate tonic inhibition. In cerebellar granule cells, they often form a receptor together with alpha(6) subunits. We were interested to determine the architecture of these receptors. We predefined the subunit arrangement of 24 different GABA(A) receptor pentamers by subunit concatenation. These receptors (composed of alpha(6), beta(3) and delta subunits) were expressed in Xenopus oocytes and their electrophysiological properties analyzed. Currents elicited in response to GABA were determined in presence and absence of 3alpha, 21-dihydroxy-5alpha-pregnan-20-one and to 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol. alpha(6)-beta(3)-alpha(6)/delta receptors showed a substantial response to GABA alone. Three receptors, beta(3)-alpha(6)-delta/alpha(6)-beta(3), alpha(6)-beta(3)-alpha(6)/beta(3)-delta and beta(3)-delta-beta(3)/alpha(6)-beta(3), were only uncovered in the combined presence of the neurosteroid 3alpha, 21-dihydroxy-5alpha-pregnan-20-one with GABA. All four receptors were activated by 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol. None of the functional receptors was modulated by physiological concentrations (up to 30 mM) of ethanol. GABA concentration response curves indicated that the delta subunit can contribute to the formation of an agonist site. We conclude from the investigated receptors that the delta subunit can assume multiple positions in a receptor pentamer composed of alpha(6), beta(3) and delta subunits.

  15. The dark and the bright side of Stat3: proto-oncogene and tumor-suppressor.

    PubMed

    Ecker, Andrea; Simma, Olivia; Hoelbl, Andrea; Kenner, Lukas; Beug, Hartmut; Moriggl, Richard; Sexl, Veronika

    2009-01-01

    Stat transcription factors have been implicated in tumorigenesis in mice and men. Stat3 and Stat5 are considered powerful proto-oncogenes, whereas Stat1 has been demonstrated to suppress tumor formation. We demonstrate here for the first time that a constitutive active version of Stat3alpha (Stat3alphaC) may also suppress transformation. Mouse embryonic fibroblasts (MEFs) deficient for p53 can be transformed with either c-myc or with rasV12 alone. Interestingly, transformation by c-myc is efficiently suppressed by co-expression of Stat3alphaC, but Stat3alphaC does not interfere with transformation by the rasV12-oncogene. In contrast, transplantation of bone marrow cells expressing Stat3alphaC induces the formation of a highly aggressive T cell leukemia in mice. The leukemic cells invaded multiple organs including lung, heart, salivary glands, liver and kidney. Interestingly, transplanted mice developed a similar leukemia when the bone marrow cells were transduced with Stat3beta, which is also constitutively active when expressed at significant levels. Our experiments demonstrate that Stat3 has both - tumor suppressing and tumor promoting properties.

  16. Bile acids of snakes of the subfamily Viperinae and the biosynthesis of C-23-hydroxylated bile acids in liver homogenate fractions from the adder, Vipera berus (Linn.).

    PubMed Central

    Ikawa, S; Tammar, A R

    1976-01-01

    1. Analysis of bile salts of four snakes of the subfamily Viperinae showed that their bile acids consisted mainly of C-23-hydroxylated bile acids. 2. Incubations of 14C-labelled sodium cholate (3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholan-24-oate) and deoxycholate (3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oate) with whole and fractionated adder liver homogenates were carried out in the presence of molecular oxygen and NADPH or an NADPH-generating system. The formation of C-23-hydroxylated bile acids, namely bitocholic acid (3 alpha, 12 alpha, 23xi-trihydroxy-5 beta-cholan-24-oic acid) and 3 alpha, 7 alpha, 12 alpha, 23 xi-tetrahydroxy-cholanic acid (3 alpha, 7 alpha, 12 alpha, 23 xi-tetrahydroxy-5 beta-cholan-24-oic acid), was observed mainly in the microsomal fraction and partly in the mitochondrial fraction. 3. Biosynthetic pathways of C-23-hydroxylated bile acids are discussed. PMID:6007

  17. Saponins from Fagonia glutinosa.

    PubMed

    Melek, F R; Miyase, T; el-Gindy, M R; Abdel-Khalik, S M; Ghaly, N S; el-Kady, M

    2000-10-01

    Twelve triterpenoid saponins, including six new, were isolated and identified from the aerial parts of Fagonia glutinosa. The new saponins were characterised as 3-O-[beta-D-glucopyranosyl(1-->2)][beta-D-glucopyranosyl(1-->3)]-alpha-L - arabinopyranosyl-27-hydroxy oleanolic acid 28-O-beta-D-glucopyranosyl ester, 3-O-[beta-D-glucopyranosyl(1-->3)]-alpha-L-arabinopyranosyl ursolic acid, 3-O-alpha-L-arabinopyranosyl ursolic acid 28-O-beta-D-glucopyranosyl ester, 3-O-[beta-D-xylopyranosyl(1-->2)][beta- D-glucopyranosyl(1-->3)]-alpha-L-arabinopyranosyl ursolic acid, 3-O-[beta-D-glucopyranosyl(1-->2)][beta-D- glucopyranosyl(1-->3)]-alpha-L-arabinopyranosyl ursolic acid 28-O-beta-D-glucopyranosyl ester and 3-O-[beta-D-glucopyranosyl(1-->2)][beta-D-glucopyranosyl(1-->3)]-alpha-L - arabinopyranosyl-27-hydroxy ursolic acid 28-O-beta-D-glucopyranosyl ester. The structures of the saponins were established by spectral and chemical evidences. The assignments of the NMR signals were performed by means of HOHAHA, 1H-1H COSY, ROE, HMQC and HMBC experiments. PMID:11082842

  18. [Triterpenes from aerial parts of Clematoclethra scandens subsp. actinidioides].

    PubMed

    Xiao, Shi-Ji; He, Da-Hai; Ding, Li-Sheng; Zhou, Yan; Chen, Fang

    2013-02-01

    To study the chemical constituents of Clematoclethra scandens subsp. actinidioides, chromatographic methods such as silica gel and MCI column chromatographic technology, and preparative HPLC were used and sixteen compounds were isolated from the aerial parts of this plant. By using spectroscopic techniques including 1H, 13C-NMR, HMBC and ESI-MS, these compounds were identified as betulinic acid (1), ursolic acid (2), oleanic acid (3), corosolic acid (4), 3beta-(trans-p-coumaroyloxy)-2alpha, 23-dihydroxyurs-12-en-28-oic acid (5), 3beta-(trans-p-coumaroyloxy)-2alpha, 23-dihydroxyurs-12, 20 (30)-dien-28-oic acid (6), 2alpha, 3alpha, 23-trihydroxyurs-12, 20 (30)-dien-28-oic acid (7), 2alpha, 3alpha, 23-trihydroxyurs-12-en-28-oic acid (8), asiatic acid (9), 2alpha, 3alpha, 24-tri-hydroxyurs-12-en-28-oic acid (10), 2alpha, 3beta, 23-trihydroxyurs-12, 20 (30)-dien-28-oic acid (11), 2alpha, 3beta, 19alpha, 24-tetrahydroxyurs-12-en-28-oic acid (12), 2alpha, 3alpha, 19alpha, 24-tetrahydroxyurs-12-en-28-oic acid (13), 2alpha, 3beta, 23, 24-tetrahydroxyurs-12-en-28-oic acid (14), 2alpha, 3alpha, 19alpha, 23, 24-pentahydroxyurs-12-en-28-oic acid (15) and daucosterol (16). Among them, compounds 3-6, 11-12, 14 and 15 were isolated from this endemic plant for the first time. PMID:23668009

  19. Acylated triterpene saponins from the roots of Securidaca longepedunculata.

    PubMed

    Mitaine-Offer, Anne-Claire; Pénez, Nicolas; Miyamoto, Tomofumi; Delaude, Clément; Mirjolet, Jean-François; Duchamp, Olivier; Lacaille-Dubois, Marie-Aleth

    2010-01-01

    Four triterpene saponins, 3-O-beta-D-glucopyranosylpresenegenin 28-O-beta-D-apiofuranosyl-(1-->3)-beta-d-xylopyranosyl-(1-->4)-[beta-D-apiofuranosyl-(1-->3)]-alpha-L-rhamnopyranosyl-(1-->2)-{4-O-[(E)-3,4,5-trimethoxycinnamoyl]}-beta-D-fucopyranosyl ester, 3-O-beta-D-glucopyranosylpresenegenin 28-O-beta-D-apiofuranosyl-(1-->3)-beta-D-xylopyranosyl-(1-->4)-[beta-D-apiofuranosyl-(1-->3)]-alpha-L-rhamnopyranosyl-(1-->2)-[(6-O-acetyl)-beta-D-glucopyranosyl-(1-->3)]-{4-O-[(E)-3,4,5-trimethoxycinnamoyl]}-beta-D-fucopyranosyl ester, 3-O-beta-D-glucopyranosylpresenegenin 28-O-beta-D-apiofuranosyl-(1-->3)-beta-D-xylopyranosyl-(1-->4)-[beta-D-apiofuranosyl-(1-->3)]-alpha-L-rhamnopyranosyl-(1-->2)-[beta-D-galactopyranosyl-(1-->3)]-{4-O-[(E)-3,4,5-trimethoxycinnamoyl]}-beta-D-fucopyranosyl ester, and 3-O-beta-D-glucopyranosylpresenegenin 28-O-beta-D-apiofuranosyl-(1-->3)-[alpha-L-arabinopyranosyl-(1-->4)]-beta-D-xylopyranosyl-(1-->4)-[beta-D-apiofuranosyl-(1-->3)]-alpha-L-rhamnopyranosyl-(1-->2)-{4-O-[(E)-3,4,5-trimethoxycinnamoyl]}-beta-D-fucopyranosyl ester, were isolated from the roots of Securidaca longepedunculata, together with three known compounds. Their structures were established mainly by 2D NMR techniques and mass spectrometry. PMID:19863977

  20. Triple-alpha reaction rate studied with the Faddeev three-body formalism

    SciTech Connect

    Ishikawa, Souichi

    2012-11-12

    The triple-alpha (3{alpha}) reaction, {sup 4}He+{sup 4}He+{sup 4}He{yields}{sup 12}C+{gamma}, which plays a significant role in the stellar evolution scenarios, is studied in terms of a three-alpha (3-{alpha}) model. The reaction rate of the process is calculated via an inverse process, 3-{alpha} photodisintegration of a {sup 12}C nucleus. Both of 3-{alpha} bound and-continuum states are calculated by a Faddeev method with accommodating the long range Coulomb interaction. With being adjusted to the empirical E2-strength for {sup 12}C(0{sub 2}{sup +}){yields}{sup 12}C(2{sub 1}{sup +}) transition, results of the 3{alpha} reaction rate <{alpha}{alpha}{alpha}> at higher temperature (T > 10{sup 8} K), where the reaction proceeds mainly through the {sup 8}Be and {sup 12}C(0{sub 2}{sup +}) resonant states, almost agree with those of the Nuclear Astrophysics Compilation of Reaction Rates (NACRE). On the other hand, calculated values of <{alpha}{alpha}{alpha}> are about 10{sup 3} times larger than the NACRE rate at a low temperature (T= 10{sup 7} K), which means our results are remarkably smaller than recent CDCC results.

  1. Kaempferol acetylrhamnosides from the rhizome of Dryopteris crassirhizoma and their inhibitory effects on three different activities of human immunodeficiency virus-1 reverse transcriptase.

    PubMed

    Min, B S; Tomiyama, M; Ma, C M; Nakamura, N; Hattori, M

    2001-05-01

    Three new kaempferol glycosides, called crassirhizomosides A (1), B (2) and C (3), were isolated from the rhizome of Dryopteris crassirhizoma (Aspidiaceae), together with the known kaempferol glycoside, sutchuenoside A (4). The structures of 1-3 were determined as kaempferol 3-alpha-L-(2,4-di-O-acetyl)rhamnopyranoside-7-alpha-L-rhamnopyranoside, kaempferol 3-alpha-L-(3,4-di-O-acetyl)rhamnopyranoside, and kaempferol 3-alpha-L-(2,3-di-O-acetyl)rhamnopyranosside-7-alpha-L-rhamnopyranoside, respectively, by chemical and spectroscopic means. Inhibitory effects of 1-4 and kaempferol on human immunodeficiency virus reverse transcriptase-associated DNA polymerase (RNA-dependent DNA polymerase and DNA-dependent DNA polymerase) and RNase H activities were investigated.

  2. Triterpenoids from the leaves of Diospyros kaki (persimmon) and their inhibitory effects on protein tyrosine phosphatase 1B.

    PubMed

    Thuong, Phuong Thien; Lee, Chul Ho; Dao, Trong Tuan; Nguyen, Phi Hung; Kim, Wan Gi; Lee, Sang Jun; Oh, Won Keun

    2008-10-01

    Phytochemical study on a methanol-soluble extract of the leaves of persimmon (Diospyros kaki) resulted in the isolation of two new ursane-type triterpenoids, 3alpha,19alpha-dihydroxyurs-12,20(30)-dien-24,28-dioic acid (1) and 3alpha,19alpha-dihydroxyurs-12-en-24,28-dioic acid (2), together with 12 known ursane- and oleanane-type triterpenoids (3-14). Triterpenoids with a 3beta-hydroxy group were found to inhibit protein tyrosine phosphatase 1B (PTP1B) activity, with IC50 values ranging from 3.1+/-0.2 to 18.8+/-1.3 microM, whereas those with a 3alpha-hydroxy moiety were not active.

  3. Novel O-antigen of Hafnia alvei PCM 1195 lipopolysaccharide with a teichoic acid-like structure.

    PubMed

    Niedziela, Tomasz; Kenne, Lennart; Lugowski, Czeslaw

    2010-01-26

    The lipopolysaccharide (LPS) of Hafnia alvei strain PCM 1195 was obtained by the hot phenol/water method. The O-specific polysaccharide was released by mild acidic hydrolysis and isolated by gel filtration. The structure of the O-specific polysaccharide was investigated by (1)H, (13)C, and (31)P NMR spectroscopy, MALDI-TOF MS, and GC-MS, accompanied by monosaccharide and methylation analysis. It was concluded that the O-specific polysaccharide is composed of a hexasaccharide repeating units interlinked with a phosphate group: {-->4-alpha-D-Glcp-(1-->3)-alpha-L-FucpNAc-(1-->3)-[alpha-D-Glcp-(1-->4)]-alpha-D-GlcpNAc-(1-->3)-alpha-L-FucpNAc-(1-->4)-alpha-D-Glcp-(1-->P}(n). PMID:20003963

  4. Revised structure of a homonojirimycin isomer from Aglaonema treubii: first example of a naturally occurring alpha-homoallonojirimycin.

    PubMed

    Martin, O R; Compain, P; Kizu, H; Asano, N

    1999-11-01

    The structure of a homonojirimycin isomer isolated from Aglaonema treublii and originally proposed as alpha-3,4-di-epi-homonojirimycin was revised to alpha-4-epi-homonojirimycin 3 ("alpha-homoallonojirimycin") on the basis of NMR analysis and synthetic studies. Its activity as a glycosidase inhibitor is compared to that of other homonojirimycin isomers.

  5. Microbial and chemical transformations of some 12,13-epoxytrichothec-9,10-enes.

    PubMed

    Claridge, C A; Schmitz, H

    1978-07-01

    Resting cells of Streptomyces griseus, Mucor mucedo, and a growing culture of Acinetobacter calcoaceticus when mixed with compounds related to 12,13-epoxytrichothec-9-ene-4beta,15-diacetoxy-3alpha-ol(anguidine) produced a series of derivatives that were either partially hydrolyzed or selectively acylated. These derivatives showed marked differences in activities as assayed by antifungal and tissue culture cytotoxicity tests.

  6. Vasodilating effect of norethisterone and its 5 alpha metabolites: a novel nongenomic action.

    PubMed

    Perusquía, Mercedes; Villalón, Carlos M; Navarrete, Erika; García, Gustavo A; Pérez-Palacios, Gregorio; Lemus, Ana E

    2003-08-15

    Estrogens are generally administered in hormone replacement therapy in combination with synthetic progestins. Studies of cardiovascular risk factors in postmenopausal women have shown a variety of responses according to the molecular structure of the progestin used in hormone replacement therapy schemes. The present study sets out to determine the vasoactive effects of norethisterone and its 5alpha-dihydro (5alpha-norethisterone) and -tetrahydro (3alpha,5alpha-norethisterone and 3beta,5alpha-norethisterone) metabolites in isolated precontracted rat thoracic aorta. The addition of norethisterone and 3alpha,5alpha-norethisterone in rat aorta exhibited a potent, concentration-response inhibition of noradrenaline-induced contraction, while 5alpha- and 3beta,5alpha-norethisterone had very little, if any, vasorelaxing effect. Relaxation to norethisterone and 3alpha,5alpha-norethisterone had very rapid time-courses and it was neither affected by the absence of endothelium nor by the inhibitor of nitric oxide synthase, Nomega-nitro-L-arginine methyl ester (L-NAME). The addition of specific anti-androgen, anti-progestin and anti-estrogen compounds and protein synthesis inhibitors did not preclude the vasorelaxing effect of norethisterone and its 3alpha,5alpha-reduced metabolite. The results strongly suggest that these effects are not mediated by nuclear sex steroid hormone receptors. The overall data document a novel nongenomic endothelium-independent vasorelaxing action of a 19-nor synthetic progestin and one of its A-ring-reduced derivatives.

  7. A new multiflorane triterpenoid ester from Momordica cochinchinensis Spreng.

    PubMed

    De Shan, M; Hu, L H; Chen, Z L

    2001-01-01

    A new triterpenoid ester 3,29-di-O-(p-methoxy)benzoylmultiflora-8-ene-3 alpha,29-diol-7-one from the seeds of Momordica cochinchinensis Spreng has been isolated. Its structure was elucidated by spectroscopic evidence and confirmed by X-ray crystallography. PMID:11561447

  8. On the Extraction of Components and the Applicability of the Factor Model.

    ERIC Educational Resources Information Center

    Dziuban, Charles D.; Harris, Chester W.

    A reanalysis of Shaycroft's matrix of intercorrelations of 10 test variables plus 4 random variables is discussed. Three different procedures were used in the reanalysis: (1) Image Component Analysis, (2) Uniqueness Rescaling Factor Analysis, and (3) Alpha Factor Analysis. The results of these analyses are presented in tables. It is concluded from…

  9. Effect of isotretinoin on serum levels of precursor and peripherally derived androgens in patients with acne.

    PubMed

    Lookingbill, D P; Demers, L M; Tigelaar, R E; Shalita, A R

    1988-04-01

    Sebaceous glands are stimulated by androgens and can convert them to more active forms. Isotretinoin, however, has a profound inhibitory effect on sebaceous gland size and function. This study evaluated the effect of isotretinoin on serum levels of precursor and tissue-derived androgens. Twenty-four subjects (15 men and nine women) were treated for 20 weeks with 1 mg/kg/d of isotretinoin. Serum samples were obtained at baseline, 8, 16, and 24 weeks, and assayed for precursor androgens--total testosterone (TT), free testosterone (free T), dehydroepiandrosterone (DHEA), dehydroepiandrosterone-sulfate (DHEA-S); and tissue androgens--dihydrotestosterone (DHT), and its metabolite, 3 alpha-androstanediol glucuronide (3 alpha-diol G). Isotretinoin had no meaningful effects on precursor androgens, except for producing an elevation of free T in women. In contrast, isotretinoin produced depressions in the serum levels of DHT and 3 alpha-diol G in women and in 3 alpha-diol G in men. These decreases are believed to be the result, rather than the cause, of a reduction in the size of the sebaceous glands: The magnitude of the observed decreases may represent the amount of tissue-derived androgens that sebaceous glands normally contribute to the circulating pool. PMID:2965551

  10. Partial purification and characterization of 7 alpha-hydroxysteroid dehydrogenase from rat liver microsomes.

    PubMed

    Amuro, Y; Yamade, W; Yamamoto, T; Maebo, A; Hada, T; Higashino, K

    1987-01-13

    An NADPH-dependent 7 alpha-hydroxysteroid dehydrogenase acting on 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid was partially purified 160-fold with a yield of 13% from rat liver microsomes using DEAE-cellulose, hydroxyapatite and Affi-Gel Blue column chromatography. The specific activity of the purified enzyme was 91.3 nmol chenodeoxycholic acid formed/min per mg of protein. The reaction was reversible, and the optimum pH of the enzyme for the oxidation was about 8.5, whereas that for the reduction was about 5.0 A molecular weight of the enzyme was estimated to be about 130,000 by Superose 6TM gel filtration chromatography. The apparent Km value for 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid was 35.7 microM and that for NADPH was 90.9 microM. The preferred substrate for the enzyme was 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid rather than 3 alpha,12 alpha-dihydroxy-7-keto-5 beta-cholanoic acid, a 7-keto-bile acid analogue. The enzyme also preferred the unconjugated form to the conjugated forms. The enzyme activity was inhibited by p-chloromercuribenzoate; however, the inhibition was prevented by addition of reduced form of glutathione to the reaction mixture, indicating that the enzyme requires a sulfhydryl group for activity. PMID:3466650

  11. A new multiflorane triterpenoid ester from Momordica cochinchinensis Spreng.

    PubMed

    De Shan, M; Hu, L H; Chen, Z L

    2001-01-01

    A new triterpenoid ester 3,29-di-O-(p-methoxy)benzoylmultiflora-8-ene-3 alpha,29-diol-7-one from the seeds of Momordica cochinchinensis Spreng has been isolated. Its structure was elucidated by spectroscopic evidence and confirmed by X-ray crystallography.

  12. A new 24-nor-oleanane triterpenoid from Salvia carduacea.

    PubMed

    Ballesta-Acosta, María C; Pascual-Villalobos, María J; Rodríguez, Benjamín

    2002-10-01

    A new triterpenoid (1) has been isolated from the acetone extract of the leaves of Salvia carduacea, together with other compounds described previously. The structure of 1 [2alpha,3alpha-dihydroxy-24-nor-4(23),12-oleanadien-28-oic acid] was elucidated by spectroscopic methods, particularly by extensive 1D and 2D NMR studies.

  13. Structure and heterogeneity of the O-antigen chain of Salmonella Agona lipopolysaccharide.

    PubMed

    Szafranek, Janusz; Kumirska, Jolanta; Czerwicka, Małgorzata; Kunikowska, Danuta; Dziadziuszko, Halina; Głośnicka, Renata

    2006-11-01

    Lipopolysaccharide of Salmonella Agona smooth-type cells was obtained from bacteria by a hot phenol-water extraction procedure. Mild acid hydrolysis of lipopolysaccharide, followed by gel filtration, yielded the pure O-polysaccharide. Abequose, rhamnose, mannose, galactose and glucose in the molar ratio 0.8 : 1.0 : 1.0 : 1.1 : 0.5 were detected, and their linkages were established. Sugar configurations were determined by gas chromatography. Two repeating units, namely -->2)-[alpha-Abep-(1-->3)-]-alpha-d-Manp-(1-->4)-alpha-l-Rhap-(1-->3)-alpha-d-Galp-(1-->and -->2)-[alpha-Abep-(1-->3)-]-alpha-d-Manp-(1-->4)-alpha-l-Rhap-(1-->3)-[alpha-d-Glcp-(1-->4)-]-alpha-d-Galp-(1-->, were deduced from nuclear magnetic resonance studies. The effort to separate them was unsuccessful. An immunochemical test performed by means of Western blotting with anti O12 serum demonstrated that glucose was present in the longer lipopolysaccharide chains, at some distance from the core region.

  14. Ubiquitin conjugation by the N-end rule pathway and mRNAs for its components increase in muscles of diabetic rats

    NASA Technical Reports Server (NTRS)

    Lecker, S. H.; Solomon, V.; Price, S. R.; Kwon, Y. T.; Mitch, W. E.; Goldberg, A. L.

    1999-01-01

    Insulin deficiency (e.g., in acute diabetes or fasting) is associated with enhanced protein breakdown in skeletal muscle leading to muscle wasting. Because recent studies have suggested that this increased proteolysis is due to activation of the ubiquitin-proteasome (Ub-proteasome) pathway, we investigated whether diabetes is associated with an increased rate of Ub conjugation to muscle protein. Muscle extracts from streptozotocin-induced insulin-deficient rats contained greater amounts of Ub-conjugated proteins than extracts from control animals and also 40-50% greater rates of conjugation of (125)I-Ub to endogenous muscle proteins. This enhanced Ub-conjugation occurred mainly through the N-end rule pathway that involves E2(14k) and E3alpha. A specific substrate of this pathway, alpha-lactalbumin, was ubiquitinated faster in the diabetic extracts, and a dominant negative form of E2(14k) inhibited this increase in ubiquitination rates. Both E2(14k) and E3alpha were shown to be rate-limiting for Ub conjugation because adding small amounts of either to extracts stimulated Ub conjugation. Furthermore, mRNA for E2(14k) and E3alpha (but not E1) were elevated 2-fold in muscles from diabetic rats, although no significant increase in E2(14k) and E3alpha content could be detected by immunoblot or activity assays. The simplest interpretation of these results is that small increases in both E2(14k) and E3alpha in muscles of insulin-deficient animals together accelerate Ub conjugation and protein degradation by the N-end rule pathway, the same pathway activated in cancer cachexia, sepsis, and hyperthyroidism.

  15. Effect of UVB on hydrolysis of alpha-tocopherol acetate to alpha-tocopherol in mouse skin.

    PubMed

    Kramer-Stickland, K; Liebler, D C

    1998-08-01

    We have assessed the hydrolysis of alpha-tocopherol acetate (alpha-TAc) to the active antioxidant alpha-tocopherol (alpha-TH) in mouse epidermis and in supernatant from epidermal homogenates. Topically administered alpha-TH prevents UVB photocarcinogenesis in C3H mice, whereas alpha-TAc does not. Hydrolysis in skin was monitored in mice treated topically with deuterium labeled alpha-TAc (d3-alpha-TAc). Epidermal samples were isolated from mice and analyzed for endogenous (d0-alpha-TAc) and d3-alpha-TH by gas chromatography-mass spectrometry. Within 24 h, the levels of d3-alpha-TH increased up to 10-fold over endogenous d0-alpha-TH levels; however, in mice irradiated with UVB prior to the application of d3-alpha-TAc, levels of d3-alpha-TH increased up to 30-40-fold over endogenous d0-alpha-TH. This enhancement of alpha-TAc hydrolysis increased with increasing UVB dose. Prior UVB exposure may increase hydrolysis of alpha-TAc by increasing epidermal esterase activity. Nonspecific esterase activity was measured in the 2000 x g supernatant from epidermis of unirradiated and irradiated mice. Alpha-napthyl acetate, a nonspecific esterase substrate, was converted to alpha-napthol in supernatants from unirradiated mice. Hydrolysis to alpha-napthol increased approximately 3-fold in supernatants from irradiated mice. Hydrolysis of alpha-TAc to alpha-TH also occurred in supernatant from unirradiated mice, and this hydrolysis increased approximately 3-fold in supernatant from irradiated animals. These data indicate that nonspecific esterase activity was increased by UVB in the skin, that alpha-TAc is converted to alpha-TH in the homogenate fraction containing nonspecific esterase, and that UVB exposure modulates the metabolism of alpha-TAc to alpha-TH in vivo.

  16. Synthesis of testosterone and 5alpha-androstanediols during nutritionally stimulated gonadal growth in Lytechinus variegatus lamarck (Echinodermata:Echinoidea).

    PubMed

    Wasson, K M; Hines, G A; Watts, S A

    1998-08-01

    Although sex steroids and steroid converting enzymes have been found in echinoids, the relationship between steroids and reproduction has not been demonstrated. On days 0, 4, 8, 16, 32, and 48 of feeding, the gonads of previously starved Lytechinus variegatus were excised and incubated with [3H]androstenedione for 0.5 h to determine if changes in steroidogenic capacity are correlated with gonadal growth. Total rates of androstenedione conversion in the testes and ovaries increased significantly during feeding. In addition, the types and relative quantities of metabolites synthesized varied, suggesting that androstenedione metabolism is influenced by nutritional status. Both testes and ovaries synthesized testosterone, 5alpha-androstane-3alpha,17beta-diol, and 5alpha-androstane-3beta, 17beta-diol (5alpha-adiols), 5alpha-androstanedione, epiandrosterone, and androsterone on all days of feeding. In the testes, the relative quantities of testosterone and 5alpha-adiols increased greatly on day 4 of feeding. In contrast, in the ovaries testosterone synthesis was not detectable on day 4, although the relative quantities of 5alpha-adiols increased threefold. The sex-specific changes in the synthesis of these metabolites reflect a shift in the metabolic pathway indicated by changes in the relative enzyme activity indices for 5alpha-reductase (5alpha-R) (necessary for the synthesis of 5alpha-reduced androgens) and 3alpha/beta-hydroxysteroid dehydrogenase (3alpha/beta-HSDs, necessary for the synthesis of 3alpha- or 3beta-hydroxylated androgens). In both testes and ovaries the relative activities of 5alpha-R and 3alpha/beta-HSD increased on day 4 of feeding. The physiological significance of changes in androstenedione metabolism may be associated with the initiation of biosynthetic processes associated with gametogenesis. PMID:9679091

  17. THE IMPACT OF HELIUM-BURNING REACTION RATES ON MASSIVE STAR EVOLUTION AND NUCLEOSYNTHESIS

    SciTech Connect

    West, Christopher; Heger, Alexander; Austin, Sam M. E-mail: alexander.heger@monash.edu

    2013-05-20

    We study the sensitivity of presupernova evolution and supernova nucleosynthesis yields of massive stars to variations of the helium-burning reaction rates within the range of their uncertainties. The current solar abundances from Lodders are used for the initial stellar composition. We compute a grid of 12 initial stellar masses and 176 models per stellar mass to explore the effects of independently varying the {sup 12}C({alpha}, {gamma}){sup 16}O and 3{alpha} reaction rates, denoted R{sub {alpha},12} and R{sub 3{alpha}}, respectively. The production factors of both the intermediate-mass elements (A = 16-40) and the s-only isotopes along the weak s-process path ({sup 70}Ge, {sup 76}Se, {sup 80}Kr, {sup 82}Kr, {sup 86}Sr, and {sup 87}Sr) were found to be in reasonable agreement with predictions for variations of R{sub 3{alpha}} and R{sub {alpha},12} of {+-}25%; the s-only isotopes, however, tend to favor higher values of R{sub 3{alpha}} than the intermediate-mass isotopes. The experimental uncertainty (one standard deviation) in R{sub 3{alpha}}(R{sub {alpha},12}) is approximately {+-}10%({+-}25%). The results show that a more accurate measurement of one of these rates would decrease the uncertainty in the other as inferred from the present calculations. We also observe sharp changes in production factors and standard deviations for small changes in the reaction rates, due to differences in the convection structure of the star. The compactness parameter was used to assess which models would likely explode as successful supernovae, and hence contribute explosive nucleosynthesis yields. We also provide the approximate remnant masses for each model and the carbon mass fractions at the end of core-helium burning as a key parameter for later evolution stages.

  18. The N-end rule pathway catalyzes a major fraction of the protein degradation in skeletal muscle

    NASA Technical Reports Server (NTRS)

    Solomon, V.; Lecker, S. H.; Goldberg, A. L.

    1998-01-01

    In skeletal muscle, overall protein degradation involves the ubiquitin-proteasome system. One property of a protein that leads to rapid ubiquitin-dependent degradation is the presence of a basic, acidic, or bulky hydrophobic residue at its N terminus. However, in normal cells, substrates for this N-end rule pathway, which involves ubiquitin carrier protein (E2) E214k and ubiquitin-protein ligase (E3) E3alpha, have remained unclear. Surprisingly, in soluble extracts of rabbit muscle, we found that competitive inhibitors of E3alpha markedly inhibited the 125I-ubiquitin conjugation and ATP-dependent degradation of endogenous proteins. These inhibitors appear to selectively inhibit E3alpha, since they blocked degradation of 125I-lysozyme, a model N-end rule substrate, but did not affect the degradation of proteins whose ubiquitination involved other E3s. The addition of several E2s or E3alpha to the muscle extracts stimulated overall proteolysis and ubiquitination, but only the stimulation by E3alpha or E214k was sensitive to these inhibitors. A similar general inhibition of ubiquitin conjugation to endogenous proteins was observed with a dominant negative inhibitor of E214k. Certain substrates of the N-end rule pathway are degraded after their tRNA-dependent arginylation. We found that adding RNase A to muscle extracts reduced the ATP-dependent proteolysis of endogenous proteins, and supplying tRNA partially restored this process. Finally, although in muscle extracts the N-end rule pathway catalyzes most ubiquitin conjugation, it makes only a minor contribution to overall protein ubiquitination in HeLa cell extracts.

  19. 5alpha-Reduced androgens block estradiol-BSA-stimulated release of oxytocin.

    PubMed

    Caldwell, Jack D; Song, Yan; Englöf, Ila; Höfle, Simone; Key, Mary; Morris, Mariana

    2003-06-27

    In this study we test the postulate that estradiol conjugated to bovine serum albumin (E-BSA) acts via receptors for the steroid-binding protein sex hormone binding globulin (SHBG) by attempting to block E-BSA-stimulated release of oxytocin with two antagonists of SHBG receptor actions: the 5alpha-reduced androgens dihydrotestosterone (DHT) and 3alpha-diol. Simultaneous superfusion with either DHT or 3alpha-diol significantly blocked E-BSA-stimulated release of oxytocin. We also found that a wide range of free 17beta-estradiol was unable to stimulate oxytocin release, suggesting that E-BSA stimulates receptors other than those for free estradiol to release oxytocin, perhaps SHBG receptors.

  20. Primordial lithium - New reaction rates, new abundances, new constraints

    NASA Technical Reports Server (NTRS)

    Kawano, Lawrence; Schramm, David; Steigman, Gary

    1988-01-01

    Newly measured nuclear reaction rates for H-3(alpha, gamma)Li-7 (higher than previous values) and Li-7(p, alpha)He-4 (lower than previous values) are shown to increase the Li-7 yield from big band nucleosynthesis for lower baryon-to-photon ratio (less than about 4 x 10 to the 10th). Recent revisions in the He-3(alpha, gamma)Be-7 and the D(p, gamma)He-3 rates enhance the high (greater than 4 x 10 to the 10th) Li-7(Be) production. New, independent determinations of Li abundances in extreme population II stars are in excellent agreement with the work of Spites and give continued confidence in the use of Li-7 in big bang baryon density determinations.

  1. Logarithms of alpha in QED bound states from the renormalization group

    PubMed

    Manohar; Stewart

    2000-09-11

    The velocity renormalization group is used to determine lnalpha contributions to QED bound state energies. The leading-order anomalous dimension for the potential gives the alpha(5)lnalpha Lamb shift. The next-to-leading-order anomalous dimension determines the alpha(6)lnalpha, alpha(7)ln (2)alpha, and alpha(8)ln (3)alpha corrections to the energy. These are used to obtain the alpha(8)ln (3)alpha Lamb shift and alpha(7)ln (2)alpha hyperfine splitting for hydrogen, muonium, and positronium, as well as the alpha(2)lnalpha and alpha(3)ln (2)alpha corrections to the ortho- and parapositronium lifetimes. This shows for the first time that these logarithms can be computed from the renormalization group.

  2. In search of the active PZP epitope in white-tailed deer immunocontraception.

    PubMed

    Miller, Lowell A; Killian, Gary J

    2002-06-21

    Native porcine zona pellucida (PZP) has been shown to be highly effective as an immunocontraceptive in white-tailed deer. However, the immunogenicity of PZP extracted from pig ovaries may vary from lot to lot and the extract has the potential of containing either viral or pathogenic material. Determination of the immunocontraceptive epitopes of PZP would allow portions of the molecule to be synthesized or inserted into a recombinant system for production of a consistent and safe vaccine. In this study, epitopes of PZP were selected and tested by in vitro binding, immunogenicity in rabbits, immunogenicity and immunocontraception in deer. Sera from PZP immunocontracepted deer were tested on ELISA plates containing immobilized peptides from ZP1 and ZP3alpha. Peptides with which sera from infertile deer reacted (six peptides from ZP1 and six peptides from ZP3alpha) were selected, synthesized and tested for immunogenicity in rabbits. Deer were then immunized with combinations of peptides from either the ZP1 or ZP3alpha groups. ZP3alpha peptides induced high immune titers against native PZP, but did not induce infertility in the deer. Although ZP1 peptides induced lower titers, deer immunized with two ZP1 peptides exhibited multiple estrus events and infertility, typical of that for deer immunized with native PZP vaccine. Competitive inhibition assays using the ZP1 peptides demonstrated that the peptide comprising pins 10-16 was most effective in blocking binding by the serum antibody of native PZP immunized deer. This peptide was used to immunocontracept deer, resulting in a significant reduction in fawning for 1 year.

  3. Conformation of heparin pentasaccharide bound to antithrombin III.

    PubMed Central

    Hricovíni, M; Guerrini, M; Bisio, A; Torri, G; Petitou, M; Casu, B

    2001-01-01

    The interaction, in aqueous solution, of the synthetic pentasaccharide AGA*IA(M) (GlcN,6-SO(3)alpha 1-4GlcA beta 1-4GlcN,3,6-SO(3)alpha 1-4IdoA,2-SO(3)alpha 1-4GlcN,6-SO(3)alpha OMe; where GlcN,6-SO(3) is 2-deoxy-2-sulphamino-alpha-D-glucopyranosyl 6-sulphate, IdoA is l-iduronic acid and IdoA2-SO(3) is L-iduronic acid 2-sulphate), which exactly reproduces the structure of the specific binding sequence of heparin and heparan sulphate for antithrombin III, has been studied by NMR. In the presence of antithrombin there were marked changes in the chemical shifts and nuclear Overhauser effects (NOEs), compared with the free state. On the basis of the optimized geometry of the pentasaccharide the transferred NOEs were interpreted with full relaxation and conformational exchange matrix analysis. An analysis of the three-dimensional structures of the pentasaccharide in the free state, and in the complex, revealed the binding to be accompanied by dihedral angle variation at the A-G and I-A(M) (where G, I, A and A(M) are beta-d-glucuronic acid, 2-O-sulphated alpha-L-iduronic acid, N,6-O-sulphated alpha-D-glucosamine and the alpha-methyl-glycoside of A respectively) glycosidic linkages. Evidence is also provided that the protein drives the conformation of the 2-O-sulphated iduronic acid residue towards the skewed (2)S(0) form. PMID:11583572

  4. Place conditioning and locomotor effects of N-substituted, 4',4''-difluorobenztropine analogs in rats.

    PubMed

    Li, Su-Min; Newman, Amy Hauck; Katz, Jonathan L

    2005-06-01

    Previous studies demonstrated that analogs of benztropine [3alpha-(diphenyl-methoxy)tropane (BZT)] bind to the dopamine (DA) transporter with high affinity, inhibit DA uptake, but do not maintain rates of responding in self-administration procedures comparable with those maintained by cocaine. Some BZT analogs have an onset of action that is slower than that for cocaine that may contribute to this decreased effectiveness. In addition, some BZT analogs have affinity for muscarinic-M1 receptors that may interfere with reinforcing effects. The present study assessed effects of BZT analogs in place-conditioning procedures designed to accommodate variations in onset of effect. BZT analogs with variations in relative affinities for the DA transporter over M1 receptors from equal [AHN 1-055 (3alpha-[bis(4'-fluorophenyl)methoxy]-tropane)] to 16-fold [JHW 007 (N-(n-butyl)-3alpha-[bis(4'-fluorophenyl)methoxy]-tropane)] were compared with cocaine and the muscarinic antagonist, atropine. Cocaine (10-20 mg/kg) but not atropine (1.0-5.6 mg/kg) produced dose-related place conditioning. The N-methyl-substituted BZT analog, AHN 1-055, was without significant effects at doses that ranged from 0.3 to 3.0 mg/kg and when administered up to 90 min before conditioning trials. In contrast, effects of AHN 2-005 (N-allyl-3alpha-[bis(4'-fluorophenyl)methoxy]-tropane; 0.1-10.0 mg/kg) were significant, and those of JHW 007 approached significance when administered 45 min but not immediately or 90 min before trials. Atropine blocked the effect of AHN 2-005 and approached significant antagonism of cocaine. The present study further supports and extends previous results showing minimal preclinical indications of abuse liability of BZT analogs and suggests that these differences from cocaine are not entirely accounted for by a slower onset of action or muscarinic M1 receptor affinity.

  5. Microbial and chemical transformations of some 12,13-epoxytrichothec-9,10-enes.

    PubMed Central

    Claridge, C A; Schmitz, H

    1978-01-01

    Resting cells of Streptomyces griseus, Mucor mucedo, and a growing culture of Acinetobacter calcoaceticus when mixed with compounds related to 12,13-epoxytrichothec-9-ene-4beta,15-diacetoxy-3alpha-ol(anguidine) produced a series of derivatives that were either partially hydrolyzed or selectively acylated. These derivatives showed marked differences in activities as assayed by antifungal and tissue culture cytotoxicity tests. PMID:100053

  6. In vivo and vitro studies on formation of bile acids in patients with Zellweger syndrome. Evidence that peroxisomes are of importance in the normal biosynthesis of both cholic and chenodeoxycholic acid.

    PubMed Central

    Kase, B F; Pedersen, J I; Strandvik, B; Björkhem, I

    1985-01-01

    The last step in bile acid formation involves conversion of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) into cholic acid and 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA) into chenodeoxycholic acid. The peroxisomal fraction of rat and human liver has the highest capacity to catalyze these reactions. Infants with Zellweger syndrome lack liver peroxisomes, and accumulate 5 beta-cholestanoic acids in bile and serum. We recently showed that such an infant had reduced capacity to convert a cholic acid precursor, 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol into cholic acid. 7 alpha-Hydroxy-4-cholesten-3-one is a common precursor for both cholic acid and chenodeoxycholic acid. Intravenous administration of [3H]7 alpha-hydroxy-4-cholesten-3-one to an infant with Zellweger syndrome led to a rapid incorporation of 3H into biliary THCA but only 10% of 3H was incorporated into cholic acid after 48 h. The incorporation of 3H into DHCA was only 25% of that into THCA and the incorporation into chenodeoxycholic acid approximately 50% of that in cholic acid. The conversion of intravenously administered [3H]THCA into cholic acid in another infant with Zellweger syndrome was only 7%. There was a slow conversion of THCA into 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-C29-dicarboxylic acid. The pool size of both cholic- and chenodeoxycholic acid was markedly reduced. Preparations of liver from two patients with Zellweger syndrome had no capacity to catalyze conversion of THCA into cholic acid. There was, however, a small conversion of DHCA into chenodeoxycholic acid and into THCA. It is concluded that liver peroxisomes are important both for the conversion of THCA into cholic acid and DHCA into chenodeoxycholic acid. PMID:4077985

  7. [Chemical constituents of Myristica fragrans Houttuyn seed and their physiological activities].

    PubMed

    Maeda, Aki; Tanimoto, Shinichi; Abe, Tomo; Kazama, Shunsuke; Tanizawa, Hisayuki; Nomura, Masato

    2008-01-01

    The antioxidative activity of phenylpropanoid compound extracts from nutmeg (Myristica fragragrans Houttuyn) seed was determined. The antioxidant activity was evaluated using the 1,1-diphenyl-2-picrylhydrazyl radical-scavenging method, superoxide disumutase assay, ferric thiocyanate assay, and radical-scavenging effect assay with electron-spin resonance. High antioxidant activity was found in monoterpenoid extracts including terpinene-4-ol (3), alpha-terpineol (4), and 4-allyl-2,6-dimethoxyphenol (12). Compound, 12 expressed particularly high antioxidant activity.

  8. Glucuronidation of anabolic androgenic steroids by recombinant human UDP-glucuronosyltransferases.

    PubMed

    Kuuranne, Tiia; Kurkela, Mika; Thevis, Mario; Schänzer, Wilhelm; Finel, Moshe; Kostiainen, Risto

    2003-09-01

    A multidimensional study on the glucuronidation of anabolic androgenic steroids and their phase I metabolites by 11 recombinant human UDP-glucuronosyltransferases (UGTs) was carried out using liquid chromatographic-tandem mass spectrometric analyses. Large differences between the enzymes with respect to the conjugation profiles of the 11 tested aglycones were detected. Two UGTs, 1A6 and 1A7, did not exhibit measurable activity toward any of the aglycones that were examined in this study. Regioselectivity was demonstrated by UGTs 1A8, 1A9, and 2B15 that preferentially catalyzed hydroxyl glucuronidation at the 17beta-position. Most of the other enzymes glucuronidated hydroxyl groups at both the 3alpha- and the 17beta-positions. Clear stereoselectivity was observed in glucuronidation of diastereomeric nandrolone metabolites (5alpha-estran-3alpha-ol-17-one and 5beta-estran-3alpha-ol-17-one), whereas such specificity was not seen when analogous methyltestosterone metabolites were assayed. UGTs 1A1, 1A3, 1A4, 1A8, 1A9, 1A10, 2B4, 2B7, and 2B15 readily glucuronidated 5alpha-androstane-3alpha,17beta-diol, but none of them exhibited methyltestosterone glucuronidation activity. In agreement with the latter observations, we found that the methyltestosterone glucuronidation activity of human liver microsomes is extremely low, whereas in induced rat liver microsomes it was significantly higher. The homology among UGTs 1A7 to 1A10 at the level of amino acid sequence is very high, and it was thus surprising to find large differences in their activity toward this set of aglycones. Furthermore, the high activity of UGT1A8 and 1A10 toward some of the substrates indicates that extrahepatic enzymes might play a role in the metabolism of anabolic androgenic steroids. PMID:12920167

  9. Synthesis of di- to penta-saccharides related to the O-specific polysaccharide of Shigella dysenteriae type 1, and their nuclear magnetic resonance study.

    PubMed

    Pozsgay, V; Coxon, B; Yeh, H

    1993-10-01

    The syntheses of oligosaccharide fragments of the O-specific polysaccharide of the lipopolysaccharide of Shigella dysenteriae type 1 are described, including disaccharides methyl O-alpha-D-mannopyranosyl-(1-->2)-alpha-D-galactopyranoside (1), and methyl O-(2-deoxy-2-propionamido-alpha-D-glucopyranosyl)-(1-->3)-alpha-L- rhamnopyranoside (2), trisaccharide methyl O-alpha-D-galactopyranosyl-(1-->3)-O-(2-acetamido-2-deoxy-alpha-D- glucopyranosyl)-(1-->3)-alpha-L-rhamnopyranoside (3), tetrasaccharide methyl O-alpha-L-rhamnopyranosyl-(1-->2)-O-alpha-D-galactopyranosyl-(1-->3)- O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-(1-->3)-alpha-L-rhamno -pyranoside (4), and pentasaccharide methyl O-alpha-L-rhamnopyranosyl-(1-->3)-O-alpha-L- rhamnopyranosyl-(1-->2)-O-alpha-D-galactopyranosyl- (1-->3)-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-(1-->3)-alpha-L- rhamnopyranoside (5). The following monosaccharide building blocks were used as starting compounds: methyl 6-O-tert-butyldiphenylsilyl-3,4-O-isopropylidene-alpha-D-galact opy ranoside (8), methyl 3,4,6-tri-O-benzyl-2-O-(4-methoxybenzyl)-1-thio-beta-D- galactopyranoside (11), methyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-1-thio-alpha- D-glucopyranoside (16), methyl 2-azido-4,6-O-benzylidene-2-deoxy-1-thio-alpha-D- glucopyranoside (18), methyl 2,4-di-O-benzyl-alpha-L-rhamnopyranoside (21), methyl 2,3,4-tri-O-benzoyl-1-thio-alpha-L-rhamnopyranoside (22), 2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl bromide (23), and methyl 4-O-benzyl-alpha-L-rhamnopyranoside (24). Nuclear magnetic resonance data indicate that oligosaccharides 4 and 5 partially mimic the conformation of the O-specific polysaccharide of S. dys. type 1. PMID:7521746

  10. On the equation of an improper convex affine sphere: a generalization of a theorem of Joergens

    SciTech Connect

    Kokarev, V N

    2003-12-31

    It is proved that if a function {phi}(t) of variable t>0 belongs to the class C{sup 3,{alpha}} and satisfies for sufficiently small positive {epsilon} ({epsilon}<10{sup -4}) certain conditions, then every complete solution z(x,y) of the equation z{sub xx}z{sub yy}-z{sub xy}{sup 2}={phi}(z{sub xx}+z{sub yy}) is a quadratic polynomial.

  11. Sulfated fucans from the egg jellies of the closely related sea urchins Strongylocentrotus droebachiensis and Strongylocentrotus pallidus ensure species-specific fertilization.

    PubMed

    Vilela-Silva, Ana-Cristina E S; Castro, Michelle O; Valente, Ana-Paula; Biermann, Christiane H; Mourao, Paulo A S

    2002-01-01

    Sulfated polysaccharides from egg jelly are the molecules responsible for inducing the sperm acrosome reaction in sea urchins. This is an obligatory event for sperm binding to, and fusion with, the egg. The sulfated polysaccharides from sea urchins have simple, well defined repeating structures, and each species represents a particular pattern of sulfate substitution. Here, we examined the egg jellies of the sea urchin sibling species Strongylocentrotus droebachiensis and Strongylocentrotus pallidus. Surprisingly, females of S. droebachiensis possess eggs containing one of two possible sulfated fucans, which differ in the extent of their 2-O-sulfation. Sulfated fucan I is mostly composed of a regular sequence of four residues ([4-alpha-l-Fucp-2(OSO3)-1-->4-alpha-l-Fucp-2(OSO3)-1-->4-alpha-l-Fucp-1-->4-alpha-l-Fucp-1]n), whereas sulfated fucan II is a homopolymer of 4-alpha-l-Fucp-2(OSO3)-1 units. Females of S. pallidus contain a single sulfated fucan with the following repeating structure: [3-alpha-l-Fucp-2(OSO3)-1-->3-alpha-l-Fucp-2(OSO3)-1-->3-alpha-l-Fucp-4(OSO3)-1-->3-alpha-l-Fucp-4(OSO3)-1]n. The egg jellies of these two species of sea urchins induce the acrosome reaction in homologous (but not heterologous) sperm. Therefore, the fine structure of the sulfated alpha-fucans from the egg jellies of S. pallidus and S. droebachiensis, which differ in their sulfation patterns and in the position of their glycosidic linkages, ensures species specificity of the sperm acrosome reaction and prevents interspecies crosses. In addition, our observations allow a clear appreciation of the common structural features among the sulfated polysaccharides from sea urchin egg jelly and help to identify structures that confer finer species specificity of recognition in the acrosome reaction.

  12. Expression of two different sulfated fucans by females of Lytechinus variegatus may regulate the seasonal variation in the fertilization of the sea urchin.

    PubMed

    Cinelli, Leonardo P; Castro, Michelle O; Santos, Livia L; Garcia, Clarice R; Vilela-Silva, Ana-Cristina E S; Mourão, Paulo A S

    2007-08-01

    The egg jellies of sea urchins contain sulfated polysaccharides with unusual structures, composed of linear chains of l-fucose or l-galactose with well-defined repetitive units. The specific pattern of sulfation and the position of the glycosidic bond vary among sulfated polysaccharides from different species. These polysaccharides show species specificity in inducing the acrosome reaction, which is a critical event for fertilization. Females of the sea urchin Lytechinus variegatus spawn eggs containing a sulfated fucan with the repetitive sequence [3-alpha-L-Fucp-2(OSO(3))-1 --> 3-alpha-L-Fucp-4(OSO(3))-1 --> 3-alpha-L-Fucp-2,4(OSO(3))-1 --> 3-alpha-L-Fucp-2(OSO(3))-1](n). We now observe that, close to winter, a period of decreased fertility for the sea urchin, the females synthesize a distinct sulfated fucan with a simple structure, composed of 4-sulfated, 3-linked alpha-fucose residues. This sulfated fucan is inactive when tested in vitro for the acrosome reaction using homologous sperm. The amount of egg jellies spawned by females (and their constituent sulfated polysaccharides) varied greatly throughout the year. Apparently, there is a correlation between the temperature of the sea water and the expression of the 4-sulfated, 3-linked sulfated fucan. Overall, we described the occurrence of two isotypes of sulfated fucan in the egg jelly of the sea urchin L. variegatus, which differ in their biological activity and may be involved in the periodicity of the reproductive cycle of the invertebrate.

  13. Modulation of neuropeptide Y and Y1 receptor expression in the amygdala by fluctuations in the brain content of neuroactive steroids during ethanol drinking discontinuation in Y1R/LacZ transgenic mice.

    PubMed

    Eva, Carola; Mele, Paolo; Collura, Devis; Nai, Antonella; Pisu, Maria Giuseppina; Serra, Mariangela; Biggio, Giovanni

    2008-02-01

    Previous studies have shown that GABAergic neuroactive steroids increase Y1 receptor (Y1R) gene expression in the amygdala of Y1R/LacZ transgenic mice, harbouring the murine Y1R gene promoter linked to a LacZ reporter gene. As ethanol is known to increase GABAergic neuroactive steroids, we investigated the relationship between fluctuations in the brain content of neuroactive steroids induced by chronic voluntary ethanol consumption or ethanol discontinuation and both the level of neuropeptide Y (NPY) immunoreactivity and Y1R gene expression in the amygdala of Y1R/LacZ transgenic mice. Ethanol discontinuation (48 h) after voluntary consumption of consecutive solutions of 3%, 6%, 10% and 20% (v/v) ethanol over 4 weeks produced an anxiety-like behaviour as measured by elevated plus maze. Voluntary ethanol intake increased the cerebrocortical concentration of the progesterone metabolite 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG) that returned to control level 48 h after discontinuation of ethanol intake. Ethanol discontinuation significantly decreased NPY immunoreactivity and concomitantly increased Y1R/LacZ transgene expression in the amygdala, whereas chronic ethanol intake failed to affect these parameters. The 5alpha-reductase inhibitor finasteride prevented both the increase in the cerebrocortical concentration of 3alpha,5alpha-TH PROG apparent after 4 weeks of ethanol intake and the changes in NPY immunoreactivity and transgene expression induced by ethanol discontinuation. Data suggest that 3alpha,5alpha-TH PROG plays an important role in the changes in NPY-Y1R signalling in the amygdala during ethanol discontinuation.

  14. Quality experimental and calculated powder x-ray diffraction

    SciTech Connect

    Sullenger, D.B.; Cantrell, J.S.; Beiter, T.A.; Tomlin, D.W.

    1996-08-01

    For several years, we have submitted quality powder XRD patterns to the International Centre for Diffraction Data for inclusion as reference standards in their Powder Diffraction File. The procedure followed is described; examples used are {beta}-UH{sub 3}, {alpha}- BaT{sub 2}, alpha-lithium disilicate ({alpha}-Li{sub 2}Si{sub 2}O{sub 5}), and 2,2`,4,4`,6,6`hexanitroazobenzene-III (HNAB-III).

  15. Demonstration of 26-hydroxylation of C27-steroids in human skin fibroblasts, and a deficiency of this activity in cerebrotendinous xanthomatosis.

    PubMed Central

    Skrede, S; Björkhem, I; Kvittingen, E A; Buchmann, M S; Lie, S O; East, C; Grundy, S

    1986-01-01

    26-Hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and other C27-steroids was demonstrated in cultured skin fibroblasts from healthy individuals. Activities in skin fibroblasts were approximately 5-10% of those previously found in human liver homogenates, and were inhibited by CO. The apparent Km was lowest for 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol (1.3 mumol/liter) and highest for 5-cholestene-3 beta, 7 alpha-diol (12 mumol/liter). The rate of 26-hydroxylation was highest with 7 alpha-hydroxy-4-cholesten-3-one. These characteristics are similar to those of hepatic mitochondrial C27-steroid 26-hydroxylase. In skin fibroblasts from three patients with cerebrotendinous xanthomatosis (CTX), 26-hydroxylation of C27-steroids proceeded at a rate of only 0.2-2.5% of healthy controls. No accumulation of endogenous 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol could be demonstrated in these cells, and the lowered formation of radioactive, 26-hydroxylated products could not be explained by dilution of the labeled exogenous substrate. The present results add strong evidence to the concept that the primary metabolic defect in CTX is a deficiency of C27-steroid 26-hydroxylase. PMID:3745434

  16. Metabolism of methyltestosterone in the greyhound.

    PubMed

    Biddle, S T B; O'Donnell, A; Houghton, E; Creaser, C

    2009-03-01

    Gas chromatography/mass spectrometry and selective derivatisation techniques have been used to identify urinary metabolites of methyltestosterone following oral administration to the greyhound. Several metabolites were identified including reduced, mono-, di- and trihydroxylated steroids. The major metabolites observed were 17alpha-methyl-5beta-androstane-3alpha-17beta-diol, 17alpha-methyl-5beta-androstane-3alpha,16alpha,17beta-triol, and a further compound tentatively identified as 17alpha-methyl-5z-androstane-6z,17beta-triol. The most abundant of these was the 17alpha-methyl-5beta-androstane-3alpha,16alpha,17beta-triol. This metabolite was identified by comparison with a reference standard synthesised using a Grignard procedure and characterised using trimethylsilyl (TMS) and acetonide-TMS derivatisation techniques. There did not appear to be any evidence for 16beta-hydroxylation as a phase I metabolic transformation in the greyhound. However, significant quantities of 16alpha-hydroxy metabolites were detected. Selective enzymatic hydrolysis procedures indicated that the major metabolites identified were excreted as glucuronic acid conjugates. Metabolic transformations observed in the greyhound have been compared with those of other mammalian species and are discussed here.

  17. Similarity of "core" structures in two different glycans of tyrosine-linked eubacterial S-layer glycoproteins.

    PubMed Central

    Messner, P; Christian, R; Neuninger, C; Schulz, G

    1995-01-01

    Previously, the repeating-unit structure of the S-layer glycoprotein from the eubacterium Bacillus alvei CCM 2051 has been determined to be [-->3)-beta-D-Galp-(1-->4)-[alpha-D-Glcp-(1-->6)-]-beta-D-ManpNAc- (1-->]n (E. Altman, J.-R. Brisson, P. Messner, and U. B. Sleytr, Biochem. Cell Biol. 69:72-78, 1991). Nuclear magnetic resonance spectroscopic reexamination of this glycan reveals that the O-antigen-like domain of the polysaccharide is [see text] connected with the S-layer polypeptide through the "core" structure -->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-alpha-L-R hap-(1-->3)-beta-D-Galp-(1-->O)-Tyr. Except for the substitution in position 4 of the nonreducing rhamnose with the modified glyceric acid phosphate residue GroA-2-->OPO2-->4-beta-D-ManpNAc-(1-->, this core is identical to the core of the tyrosine-linked glycan from the S-layer glycoprotein of Thermoanaerobacter thermohydrosulfuricus L111-69 (K. Bock, J. Schuster-Kolbe, E. Altman, G. Allmaier, B. Stahl, R. Christian, U. B. Sleytr, and P. Messner, J. Biol. Chem. 269:7137-7144, 1994). PMID:7721708

  18. Complete structure of the tyrosine-linked saccharide moiety from the surface layer glycoprotein of Clostridium thermohydrosulfuricum S102-70.

    PubMed Central

    Christian, R; Schulz, G; Schuster-Kolbe, J; Allmaier, G; Schmid, E R; Sleytr, U B; Messner, P

    1993-01-01

    In this study, we have extended and completed a previous investigation (P. Messner, R. Christian, J. Kolbe, G. Schulz, and U. B. Sleytr, J. Bacteriol. 174:2236-2240, 1992) in which we demonstrated for the first time in prokaryotic organisms the presence of a novel O-glycosidic linkage via tyrosine. The surface layer glycoprotein of the eubacterium Clostridium thermohydrosulfuricum S102-70 is arranged in a hexagonal lattice, with center-to-center spacings of approximately 16.3 nm. Molecular weight determination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both glycosylated and chemically deglycosylated surface layer glycoprotein showed values for the monomeric subunits of 94,000 and 87,500, respectively. Glycopeptide fractions obtained after exhaustive pronase digestion of purified, intact glycoprotein were isolated by reversed-phase liquid chromatography. One- and two-dimensional nuclear magnetic resonance studies, together with chemical analyses and plasma desorption time-of-flight mass spectrometry, were used to elucidate the structure of the hexasaccharide moiety linked by the novel O-glycosidic linkage to tyrosine. The combined evidence suggests the following structure: beta-D-Galf-(1-->3)-alpha-D-Galp- (1-->2)-alpha-L-Rhap-(1-->3)-alpha-D-Manp-(1--3)-alpha-L- Rhap-(1-->3)-beta- D-Glcp-(1-->4)-L-Tyr. Images PMID:8444787

  19. Absorption and transport of deuterium-substituted 2R,4'R,8'R-alpha-tocopherol in human lipoproteins

    SciTech Connect

    Traber, M.G.; Ingold, K.U.; Burton, G.W.; Kayden, H.J.

    1988-08-01

    Oral administration of a single dose of tri- or hexadeuterium substituted 2R,4'R,8'R-alpha-tocopheryl acetate (d3- or d6-alpha-T-Ac) to humans was used to follow the absorption and transport of vitamin E in plasma lipoproteins. Three hr after oral administration of d3-alpha-T-Ac (15 mg) to 2 subjects, plasma levels of d3-alpha-T were detectable; these increased up to 10 hr, reached a plateau at 24 hr, then decreased. Following administration of d6-alpha-T-Ac (15-16 mg) to 2 subjects, the percentage of deuterated tocopherol relative to the total tocopherol in chylomicrons increased more rapidly than the corresponding percentage in whole plasma. Chylomicrons and plasma lipoproteins were isolated from 2 additional subjects following administration of d3-alpha-T-Ac (140 or 60 mg). The percentage of deuterated tocopherol relative to the total tocopherol increased most rapidly in chylomicrons, then in very low density lipoproteins (VLDL), followed by essentially identical increases in low and high density lipoproteins (LDL and HDL, respectively) and lastly, in the red blood cells. This pattern of appearance of deuterated tocopherol is consistent with the concept that newly absorbed vitamin E is secreted by the intestine into chylomicrons; subsequently, chylomicron remnants are taken up by the liver from which the vitamin E is secreted in VLDL. The metabolism of VLDL in the circulation results in the simultaneous delivery of vitamin E into LDL and HDL.

  20. Phytochemical and molluscicidal investigations of Fagonia arabica.

    PubMed

    El-Wakil, Eman A

    2007-01-01

    The aqueous methanolic extract of the aerial parts of Fagonia arabica L. (family Zygophyllaceae) was successively fractionated using certain organic solvents. From the ethyl acetate fraction, two flavonoid glycosides were isolated and identified as kaempferol-7-O-rhamnoside and acacetin-7-O-rhamnoside. Four triterpenoidal glycosides were isolated from the butanolic layer. Their structures were elucidated on the basis of the spectral and chemical data as 3-O-beta-D-glucopyranosyl-(1-->3)-alpha-L-arabinopyranoside oleanolic acid (1), 3-O-alpha-L-arabinopyranosyl quinovic acid 28-O-beta-D-glucopyranoside (2), 3-O-[beta-D-glucopyranosyl-(1-->2)]-beta-D-glucopyranosyl-(1-->3)-alpha-L-arabinosyl oleanolic acid (3) and 3-O-beta-D-glucopyranosyl-(1-->3)-alpha-L-arabino-pyranosyl quinovic acid 28-O-beta-D-glucopyranoside (4). The two monodesmosidic saponins 1 and 3 were found to possess strong molluscicidal activity against Biomphalaria alexandrina snails, the intermediate host of Schistosoma mansoni in Egypt (LC90 = 13.33 and 16.44 microM), whereas the other two bidesmosidic saponins 2 and 4 as well as the two flavonoid glycosides were inactive up to 50 microM. PMID:18069237

  1. Be-{alpha} correlations in the linear-chain structure of C isotopes

    SciTech Connect

    Suhara, Tadahiro; Kanada-En'yo, Yoshiko

    2011-08-15

    We investigate the linear-chain structures in highly excited states of {sup 14}C using a generalized molecular-orbital model, by which we incorporate an asymmetric configuration of three {alpha} clusters in the linear-chain states. By applying this model to the {sup 14}C system, we study the {sup 10}Be+{alpha} correlation in the linear-chain state of {sup 14}C. To clarify the origin of the {sup 10}Be+{alpha} correlation in the {sup 14}C linear-chain state, we analyze linear 3 {alpha} and 3{alpha} + n systems in a similar way. We find that a linear 3{alpha} system prefers the asymmetric 2{alpha} + {alpha} configuration, whose origin is the many-body correlation incorporated by the parity projection. This configuration causes an asymmetric mean field for two valence neutrons, which induces the concentration of valence neutron wave functions around the correlating 2{alpha}. A linear-chain structure of {sup 16}C is also discussed.

  2. {sup 12}C formation: A classical quest in new light

    SciTech Connect

    Tengblad, O.; Alcorta, M.; Borge, M. J. G.; Madurga, M.; Perea, A.; Cubero, M.; Fynbo, H. O. U.; Riisager, K.; Kirsebom, O.; Hyldegaard, S.; Jonson, B.; Nyman, G.; Nilsson, T.; Diget, D. G.; Fulton, B.

    2011-10-28

    In this work we have studied the break-up of {sup 12}C following the reactions {sup 10}B({sup 3}He,p{alpha}{alpha}{alpha}) and {sup 11}B({sup 3}He,d{alpha}{alpha}{alpha}). The study was performed at the 5 MV tandem in Madrid. The break-up gives us information on excited states in {sup 12}C from the famous Hoyle state up to an energy of almost 18 MeV. Using a highly segmented experimental set-up the simultaneous detection of the three alpha particles in coincidence with a proton or deuteron respectively made possible a full kinematic reconstruction of the break-up. On the basis of the energies of the 3 {alpha} particles and their angular correlations it has been possible to determine the spin and parity of states for cases in which the assignment has been doubtful. Some of these levels will also de-excite via electromagnetic emission. The comparison between the energy of proton that populate a state of {sup 12}C and the sum of the energies of the 3{alpha} emitted from the same state makes possible to determine the presence of electromagnetic disintegration ({gamma}) to lower states within {sup 12}C followed by the 3{alpha} break-up.

  3. Functional Expression of Two Neuronal Nicotinic Acetylcholine Receptors from cDNA Clones Identifies a Gene Family

    NASA Astrophysics Data System (ADS)

    Boulter, Jim; Connolly, John; Deneris, Evan; Goldman, Dan; Heinemann, Steven; Patrick, Jim

    1987-11-01

    A family of genes coding for proteins homologous to the α subunit of the muscle nicotinic acetylcholine receptor has been identified in the rat genome. These genes are transcribed in the central and peripheral nervous systems in areas known to contain functional nicotinic receptors. In this paper, we demonstrate that three of these genes, which we call alpha3, alpha4, and beta2, encode proteins that form functional nicotinic acetylcholine receptors when expressed in Xenopus oocytes. Oocytes expressing either alpha3 or alpha4 protein in combination with the beta2 protein produced a strong response to acetylcholine. Oocytes expressing only the alpha4 protein gave a weak response to acetylcholine. These receptors are activated by acetylcholine and nicotine and are blocked by Bungarus toxin 3.1. They are not blocked by α -bungarotoxin, which blocks the muscle nicotinic acetylcholine receptor. Thus, the receptors formed by the alpha3, alpha4, and beta2 subunits are pharmacologically similar to the ganglionic-type neuronal nicotinic acetylcholine receptor. These results indicate that the alpha3, alpha4, and beta2 genes encode functional nicotinic acetylcholine receptor subunits that are expressed in the brain and peripheral nervous system.

  4. Lanostanes from Phellinus igniarius and their iNOS inhibitory activities.

    PubMed

    Wang, Guei-Jane; Tsai, Tung-Hu; Chang, Tun-Tschu; Chou, Cheng-Jen; Lin, Lie-Chwen

    2009-12-01

    Four new lanostanol-type triterpenoids, igniarens A - D ( 1- 4), were isolated from the fruit body of Phellinus igniarius together with two known triterpenoids, and two known ergostanes. These four new compounds were identified by spectroscopic analysis as 22 R-hydroxy-24-methylene-29-norlanost-7, 9(11)-dien-3-one (1), 3alpha,22 R-dihydroxy-24-methylene-29-norlanost-7, 9(11)-diene (2), 3alpha,22 R-dihydroxy-24-methylene-29-norlanost-8-ene ( 3), and 3alpha,22 R-dihydroxy-24-methylenelanost-8-ene ( 4). Their effects on NO production in lipopolysaccharide (LPS)-activated macrophages were assessed. Compounds 1- 8 inhibited NO production in activated RAW 264.7 cells to various degrees. The most potent compound 5alpha,8alpha-epidioxy-22 E-ergosta-6,22-dien-3beta-ol ( 7) significantly inhibited LPS-induced NO production in a concentration-dependent manner without affecting the cellular viability, with an IC (50) of 37.57 +/- 1.38 microM. PMID:19557671

  5. Antioxidant activities and antitumor screening of extracts from cranberry fruit (Vaccinium macrocarpon).

    PubMed

    Yan, Xiaojun; Murphy, Brian T; Hammond, Gerald B; Vinson, Joe A; Neto, Catherine C

    2002-10-01

    Polyphenolic compounds in cranberries have been investigated to determine their role in protection against cardiovascular disease and some cancers. Extracts of whole fruit were assayed for radical-scavenging activity and tumor growth inhibition using seven tumor cell lines. Selective inhibition of K562 and HT-29 cells was observed from a methanolic extract in the range of 16-125 microg/mL. Radical-scavenging activity was greatest in an extract composed primarily of flavonol glycosides. Seven flavonol glycosides were isolated and purified from whole fruit for further evaluation; the anthocyanin cyanidin 3-galactoside was also purified for comparison with the flavonoids. Three flavonol monoglycosides were newly identified by (13)C NMR as myricetin 3-alpha-arabinofuranoside, quercetin 3-xyloside, and 3-methoxyquercetin 3-beta-galactoside (isorhamnetin); the other four isolated were the previously identified myricetin 3-beta-galactoside, quercetin 3-beta-galactoside, quercetin 3-alpha-arabinofuranoside, and quercetin 3-alpha-rhamnopyranoside. These compounds were evaluated for 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity and ability to inhibit low-density lipoprotein oxidation in vitro. Most of the flavonol glycosides showed antioxidant activity comparable or superior to that of vitamin E; cyanidin 3-galactoside showed activity superior to that of the flavonoids as well as vitamin E or Trolox in both antioxidant assays.

  6. Increased 21-hydroxylase and shutdown of C(17,20) lyase activities in testicular tissues of the grouper (Epinephelus coioides) during 17alpha-methyltestosterone-induced sex inversion.

    PubMed

    Lee, S T; Lam, T J; Tan, C H

    2002-05-01

    The metabolism in vitro of [(3)H]17-hydroxyprogesterone by gonadal tissues of the grouper (Epinephelus coioides) during 17alpha-methyltestosterone (MT)-induced female-to-male sex inversion was examined. In the female phase, C(17,20) lyase, 5beta-reductase, 3alpha/beta-HSD, 20beta-HSD, and 17beta-HSD activities resulted in the biosynthesis of 5beta-pregnans and 5beta-androstanes (including 5beta-androstane-3alpha/beta, 17beta-diol, 3alpha/beta, 17alpha-dihydroxy-5beta-pregnen-20-one, and 5beta-androstane-3,17-dione). In the MT-induced male phase, however, the abrogation of C(17,20) lyase activity and the concomitant activation of 21alpha-hydroxylase/11beta-hydroxylase resulted in the preferential synthesis of polar 21alpha-hydroxlyated 5beta-pregnans (5beta-pregnan-3beta,17alpha,20beta,21alpha-tetrol and 3beta,20beta,21alpha-trihydroxy-5beta-pregnan-3-one) and corticosteroids (11-deoxycortisol and cortisol). Interestingly, synthesis of these 21alpha-hydroxylated 5beta-pregnans and corticosteroids was uniquely compartmentalized in only testicular tissues of the MT-induced males. This study shows that there is selective activation of specific steroidogenic enzymes in the different sexual phases leading to the synthesis of metabolites that may be involved in regulating sex inversion of the grouper.

  7. Isotretinoin, tetracycline and circulating hormones in acne.

    PubMed

    Palatsi, R; Ruokonen, A; Oikarinen, A

    1997-09-01

    Isotretinoin, used to treat severe acne, has been shown to induce hormonal changes, especially to reduce 5 alpha-reductase in the production of the tissue-derived dihydrotestosterone (DHT) metabolite 3 alpha-Adiol G. However, the effects of isotretinoin on other pituitary, adrenal or gonadal hormones have not been thoroughly elucidated. In the present study, isotretinoin administered at a dose of 0.5 mg/kg/day for 4 weeks caused no marked changes in the serum levels of pituitary, adrenal or gonadal hormones or 3 alpha-Adiol G in patients with severe papulopustulotic acne (n = 19). After 12 weeks of therapy, there was a decrease in the levels of the precursor androgens androstenedione, testosterone and 3 alpha-Adiol G in 6/9 patients. Acne improved after 4.5 months in all but 2 male patients, who had very low serum hormone binding globulins (SHBG) and a high free androgen index (FAI). Isotretinoin did not affect the elevated LH/FSH ratio in a patient with the polycystic ovarian syndrome (PCOS); nor did it change the high FAI or low SHBG in the male patients. For comparison, tetracycline had no effects on the serum hormonal levels of patients with mild acne (n = 19) after 7 days of treatment. This study confirms that the effects of isotretinoin on the serum hormone levels are small and unlikely to be of relevance for the resolution of acne or the suppression of sebum excretion. PMID:9298137

  8. Implication of STAT3 signaling in human colonic cancer cells during intestinal trefoil factor 3 (TFF3) -- and vascular endothelial growth factor-mediated cellular invasion and tumor growth.

    PubMed

    Rivat, Christine; Christine, Rivat; Rodrigues, Sylvie; Sylvie, Rodrigues; Bruyneel, Erik; Erik, Bruyneel; Piétu, Geneviève; Geneviève, Piétu; Robert, Amélie; Amélie, Robert; Redeuilh, Gérard; Gérard, Redeuilh; Bracke, Marc; Marc, Bracke; Gespach, Christian; Christian, Gespach; Attoub, Samir; Samir, Attoub

    2005-01-01

    Signal transducer and activator of transcription (STAT) 3 is overexpressed or activated in most types of human tumors and has been classified as an oncogene. In the present study, we investigated the contribution of the STAT3s to the proinvasive activity of trefoil factors (TFF) and vascular endothelial growth factor (VEGF) in human colorectal cancer cells HCT8/S11 expressing VEGF receptors. Both intestinal trefoil peptide (TFF3) and VEGF, but not pS2 (TFF1), activate STAT3 signaling through Tyr(705) phosphorylation of both STAT3alpha and STAT3beta isoforms. Blockade of STAT3 signaling by STAT3beta, depletion of the STAT3alpha/beta isoforms by RNA interference, and pharmacologic inhibition of STAT3alpha/beta phosphorylation by cucurbitacin or STAT3 inhibitory peptide abrogates TFF- and VEGF-induced cellular invasion and reduces the growth of HCT8/S11 tumor xenografts in athymic mice. Differential gene expression analysis using DNA microarrays revealed that overexpression of STAT3beta down-regulates the VEGF receptors Flt-1, neuropilins 1 and 2, and the inhibitor of DNA binding/differentiation (Id-2) gene product involved in the neoplastic transformation. Taken together, our data suggest that TFF3 and the essential tumor angiogenesis regulator VEGF(165) exert potent proinvasive activity through STAT3 signaling in human colorectal cancer cells. We also validate new therapeutic strategies targeting STAT3 signaling by pharmacologic inhibitors and RNA interference for the treatment of colorectal cancer patients.

  9. Coincident expression of the chemokine receptors CCR6 and CCR7 by pathologic Langerhans cells in Langerhans cell histiocytosis.

    PubMed

    Fleming, Mark D; Pinkus, Jack L; Fournier, Marcia V; Alexander, Sarah W; Tam, Carmen; Loda, Massimo; Sallan, Stephen E; Nichols, Kim E; Carpentieri, David F; Pinkus, Geraldine S; Rollins, Barrett J

    2003-04-01

    It has been suggested that a switch in chemokine receptor expression underlies Langerhans cell migration from skin to lymphoid tissue. Activated cells are thought to down-regulate CCR6, whose ligand macrophage inflammatory protein-3 alpha (MIP-3 alpha)/CCL20 is expressed in skin, and up-regulate CCR7, whose ligands are in lymphoid tissues. In Langerhans cell histiocytosis (LCH), pathologic Langerhans cells (LCs) accumulate in several tissues, including skin, bone, and lymphoid organs. We have examined 24 LCH cases and find that pathologic LCs expressed CCR6 and CCR7 coincidentally in all cases. Furthermore, MIP-3 alpha/CCL20 is expressed by keratinocytes in involved skin and by macrophages and osteoblasts in involved bone. Expression of CCR6 by pathologic LCs may contribute to their accumulation in nonlymphoid organs such as skin and bone, whereas CCR7 expression may direct them to lymphoid tissue. Histiocytes in Rosai-Dorfman disease and hemophagocytic syndrome also coexpressed CCR6 and CCR7, suggesting that this may be a general attribute of abnormal histiocytes.

  10. A minimal sequence code for switching protein structure and function.

    PubMed

    Alexander, Patrick A; He, Yanan; Chen, Yihong; Orban, John; Bryan, Philip N

    2009-12-15

    We present here a structural and mechanistic description of how a protein changes its fold and function, mutation by mutation. Our approach was to create 2 proteins that (i) are stably folded into 2 different folds, (ii) have 2 different functions, and (iii) are very similar in sequence. In this simplified sequence space we explore the mutational path from one fold to another. We show that an IgG-binding, 4beta+alpha fold can be transformed into an albumin-binding, 3-alpha fold via a mutational pathway in which neither function nor native structure is completely lost. The stabilities of all mutants along the pathway are evaluated, key high-resolution structures are determined by NMR, and an explanation of the switching mechanism is provided. We show that the conformational switch from 4beta+alpha to 3-alpha structure can occur via a single amino acid substitution. On one side of the switch point, the 4beta+alpha fold is >90% populated (pH 7.2, 20 degrees C). A single mutation switches the conformation to the 3-alpha fold, which is >90% populated (pH 7.2, 20 degrees C). We further show that a bifunctional protein exists at the switch point with affinity for both IgG and albumin. PMID:19923431

  11. AKT/SGK-sensitive phosphorylation of GSK3 in the regulation of L-selectin and perforin expression as well as activation induced cell death of T-lymphocytes

    SciTech Connect

    Bhavsar, Shefalee K.; Merches, Katja; Bobbala, Diwakar; Lang, Florian

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer Akt/SGK dependent phosphorylation of GSK3{alpha},{beta} regulates T lymphocytes. Black-Right-Pointing-Pointer T cells from mice expressing Akt/SGK insensitive GSK3{alpha},{beta} (gsk3{sup KI}) release less IL-2. Black-Right-Pointing-Pointer CD4{sup +} cells from gsk3{sup KI} mice express less CD62L. Black-Right-Pointing-Pointer CD8{sup +} cells from gsk3{sup KI} mice are relatively resistant to activation induced cell death. Black-Right-Pointing-Pointer Perforin expression is enhanced in gsk3{sup KI} T cells. -- Abstract: Survival and function of T-lymphocytes critically depends on phosphoinositide (PI) 3 kinase. PI3 kinase signaling includes the PKB/Akt and SGK dependent phosphorylation and thus inhibition of glycogen synthase kinase GSK3{alpha},{beta}. Lithium, a known unspecific GSK3 inhibitor protects against experimental autoimmune encephalomyelitis. The present study explored, whether Akt/SGK-dependent regulation of GSK3 activity is a determinant of T cell survival and function. Experiments were performed in mutant mice in which Akt/SGK-dependent GSK3{alpha},{beta} inhibition was disrupted by replacement of the serine residue in the respective SGK/Akt-phosphorylation consensus sequence by alanine (gsk3{sup KI}). T cells from gsk3{sup KI} mice were compared to T cells from corresponding wild type mice (gsk3{sup WT}). As a result, in gsk3{sup KI} CD4{sup +} cells surface CD62L (L-selectin) was significantly less abundant than in gsk3{sup WT} CD4{sup +} cells. Upon activation in vitro T cells from gsk3{sup KI} mice reacted with enhanced perforin production and reduced activation induced cell death. Cytokine production was rather reduced in gsk3{sup KI} T cells, suggesting that GSK3 induces effector function in CD8{sup +} T cells. In conclusion, PKB/Akt and SGK sensitive phosphorylation of GSK3{alpha},{beta} is a potent regulator of perforin expression and activation induced cell death in T lymphocytes.

  12. The first biantennary bacterial secondary cell wall polymer and its influence on S-layer glycoprotein assembly.

    PubMed Central

    Steindl, Christian; Schäffer, Christina; Wugeditsch, Thomas; Graninger, Michael; Matecko, Irena; Müller, Norbert; Messner, Paul

    2002-01-01

    The cell surface of Aneurinibacillus thermoaerophilus DSM 10155 is covered with a square surface (S)-layer glycoprotein lattice. This S-layer glycoprotein, which was extracted with aqueous buffers after a freeze-thaw cycle of the bacterial cells, is the only completely water-soluble S-layer glycoprotein to be reported to date. The purified S-layer glycoprotein preparation had an overall carbohydrate content of 19%. Detailed chemical investigations indicated that the S-layer O-glycans of previously established structure accounted for 13% of total glycosylation. The remainder could be attributed to a peptidoglycan-associated secondary cell wall polymer. Structure analysis was performed using purified secondary cell wall polymer-peptidoglycan complexes. NMR spectroscopy revealed the first biantennary secondary cell wall polymer from the domain Bacteria, with the structure alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->4)-beta-L-Gal p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->4)-beta-L-Gal p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->4)-[alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->4)-beta-L-Gal p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->4)-beta-L-Gal p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)]-beta-L-Man p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->O)-PO(2)(-)-O-PO(2)(-)-(O-->6)-MurNAc- (where MurNAc is N -acetylmuramic acid). The neutral polysaccharide is linked via a pyrophosphate bond to the C-6 atom of every fourth N -acetylmuramic acid residue, in average, of the A1gamma-type peptidoglycan. In vivo, the biantennary polymer anchored the S-layer glycoprotein very effectively to the cell wall, probably due to the doubling of motifs for a proposed lectin-like binding between the polymer and the N-terminus of the S-layer protein. When the cellular support was removed during S-layer glycoprotein isolation, the co-purified polymer mediated the solubility of the S

  13. Prevention of DNA photodamage by vitamin E compounds and sunscreens: roles of ultraviolet absorbance and cellular uptake.

    PubMed

    McVean, M; Liebler, D C

    1999-03-01

    Topical application of alpha-tocopherol (alphaTH), the most prominent naturally occurring form of vitamin E, inhibits ultraviolet (UV) B-induced photocarcinogenesis and DNA photodamage in C3H mice in vivo. In this study, we compared alphaTH with other vitamin E compounds and with three commercial sunscreen compounds for their ability to inhibit DNA photodamage in C3H mouse skin in vivo. When applied in a 5% dispersion in a neutral cream vehicle, alpha-tocopherol (alphaTH), gamma-tocopherol (gammaTH), and delta-tocopherol (deltaTH) each produced a statistically significant inhibition of thymine dimer formation, whereas alpha-tocopherol acetate (alphaTAc) and alpha-tocopherol methyl ether (alphaTOMe) did not. Application of 5% dispersions of the commercial sunscreen agent octylmethoxycinnamate also inhibited dimer formation, whereas ethylhexyl salicylate and oxybenzone did not, despite their considerably greater UVB absorbances than alphaTH. To test the hypothesis that cellular uptake and distribution are necessary for optimal photoprotection by tocopherols, photoprotection was studied in mouse 308 keratinocyte cells in vitro. Preincubation of 308 cells with 1 microM alphaTH for at least 2 h before exposure to 2.5 J/m2/s UVB for 10 min significantly (P < 0.05) attenuated thymine dimer formation. Pre-incubation with 1 microM gammaTH, deltaTH, alphaTAc, or alphaTOMe for 2 h did not inhibit thymine dimer formation significantly. Uptake of alphaTH was measured after incubation with 1 microM [2H3]alphaTH (d3-alphaTH) and resulted in a time-dependent increase in alphaTH levels. Use of d3-alphaTH allowed separate, simultaneous measurement of added d3-alphaTH and unlabeled endogenous alphaTH by gas chromatography-mass spectrometry. Accumulation of 167 +/- 62 pmol d3-alphaTH/mg protein was measured within 1 h in whole-cell fractions. d3-AlphaTH in the nuclear fraction reached levels of 15 +/- 4 pmol d3-alphaTH/mg protein at 2 h. Accumulation of alphaTH in the whole cell and

  14. Defective peroxisomal cleavage of the C27-steroid side chain in the cerebro-hepato-renal syndrome of Zellweger.

    PubMed Central

    Kase, B F; Björkhem, I; Hågå, P; Pedersen, J I

    1985-01-01

    Based on in vitro work with rat liver, we recently suggested that the peroxisomal fraction is most important for the oxidation of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) into cholic acid. The cerebro-hepato-renal syndrome of Zellweger is a fatal recessive autosomal disorder, the most characteristic histological feature of which is a virtual absence of peroxisomes in liver and kidneys. This disease offers a unique opportunity to evaluate the relative importance of peroxisomes in bile acid biosynthesis. A child with Zellweger syndrome was studied in the present work. In accordance with previous work, there was a considerable accumulation of THCA, 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid (24-OH-THCA), 3 alpha, 7 alpha, 12 alpha-trihydroxy-27-carboxymethyl-5 beta-cholestan-26-oic acid (C29-dicarboxylic acid), and 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid in serum. In addition, a tetrahydroxylated 5 beta-cholestanoic acid with all the hydroxyl groups in the steroid nucleus was found. 3H-Labeled 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol was administered intravenously together with 14C-labeled cholic acid. There was a rapid incorporation of 3H in THCA and a slow incorporation into cholic acid. The specific radioactivity of 3H in THCA was about one magnitude higher than that in cholic acid. The conversion was evaluated by following the increasing ratio between 3H and 14C in biliary cholic acid. The rate of incorporation of 3H in cholic acid was considerably less than previously reported in experiments with healthy subjects, and the maximal conversion of the triol into cholic acid was only 15-20%. About the same rate of conversion was found after oral administration of 3H-THCA. Both in the experiment with 3H-5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and with 3H-THCA, there was an efficient incorporation of 3H in the above unidentified tetrahydroxylated 5 beta-cholestanoic acid. There was

  15. Prevention of DNA photodamage by vitamin E compounds and sunscreens: roles of ultraviolet absorbance and cellular uptake.

    PubMed

    McVean, M; Liebler, D C

    1999-03-01

    Topical application of alpha-tocopherol (alphaTH), the most prominent naturally occurring form of vitamin E, inhibits ultraviolet (UV) B-induced photocarcinogenesis and DNA photodamage in C3H mice in vivo. In this study, we compared alphaTH with other vitamin E compounds and with three commercial sunscreen compounds for their ability to inhibit DNA photodamage in C3H mouse skin in vivo. When applied in a 5% dispersion in a neutral cream vehicle, alpha-tocopherol (alphaTH), gamma-tocopherol (gammaTH), and delta-tocopherol (deltaTH) each produced a statistically significant inhibition of thymine dimer formation, whereas alpha-tocopherol acetate (alphaTAc) and alpha-tocopherol methyl ether (alphaTOMe) did not. Application of 5% dispersions of the commercial sunscreen agent octylmethoxycinnamate also inhibited dimer formation, whereas ethylhexyl salicylate and oxybenzone did not, despite their considerably greater UVB absorbances than alphaTH. To test the hypothesis that cellular uptake and distribution are necessary for optimal photoprotection by tocopherols, photoprotection was studied in mouse 308 keratinocyte cells in vitro. Preincubation of 308 cells with 1 microM alphaTH for at least 2 h before exposure to 2.5 J/m2/s UVB for 10 min significantly (P < 0.05) attenuated thymine dimer formation. Pre-incubation with 1 microM gammaTH, deltaTH, alphaTAc, or alphaTOMe for 2 h did not inhibit thymine dimer formation significantly. Uptake of alphaTH was measured after incubation with 1 microM [2H3]alphaTH (d3-alphaTH) and resulted in a time-dependent increase in alphaTH levels. Use of d3-alphaTH allowed separate, simultaneous measurement of added d3-alphaTH and unlabeled endogenous alphaTH by gas chromatography-mass spectrometry. Accumulation of 167 +/- 62 pmol d3-alphaTH/mg protein was measured within 1 h in whole-cell fractions. d3-AlphaTH in the nuclear fraction reached levels of 15 +/- 4 pmol d3-alphaTH/mg protein at 2 h. Accumulation of alphaTH in the whole cell and

  16. Riemannian geometry of thermodynamics and systems with repulsive power-law interactions.

    PubMed

    Ruppeiner, George

    2005-07-01

    A Riemannian geometric theory of thermodynamics based on the postulate that the curvature scalar R is proportional to the inverse free energy density is used to investigate three-dimensional fluid systems of identical classical point particles interacting with each other via a power-law potential energy gamma r(-alpha) . Such systems are useful in modeling melting transitions. The limit alpha-->infinity corresponds to the hard sphere gas. A thermodynamic limit exists only for short-range (alpha>3) and repulsive (gamma>0) interactions. The geometric theory solutions for given alpha>3 , gamma>0 , and any constant temperature T have the following properties: (1) the thermodynamics follows from a single function b (rho T(-3/alpha) ) , where rho is the density; (2) all solutions are equivalent up to a single scaling constant for rho T(-3/alpha) , related to gamma via the virial theorem; (3) at low density, solutions correspond to the ideal gas; (4) at high density there are solutions with pressure and energy depending on density as expected from solid state physics, though not with a Dulong-Petit heat capacity limit; (5) for 3<alpha<3.7913 , the solution goes from the low to the expected high density limit smoothly; (6) for alpha>3.7913 a phase transition is required to go between these regimes; (7) for any alpha>3 we may include a first-order phase transition, which is expected from computer simulations; and (8) if alpha-->infinity, the density approaches a finite value as the pressure increases to infinity, with the pressure diverging logarithmically in the density difference.

  17. Development of criteria for the detection of adrenosterone administration by gas chromatography-mass spectrometry and gas chromatography-combustion-isotope ratio mass spectrometry for doping control.

    PubMed

    Brooker, Lance; Parr, Maria Kristina; Cawley, Adam; Flenker, Ulrich; Howe, Christopher; Kazlauskas, Rymantas; Schänzer, Wilhelm; George, Adrian

    2009-11-01

    Adrenosterone (androst-4-ene-3,11,17-trione, 11-oxoandrostenedione) is an endogenous steroid hormone that has been promoted as a dietary supplement capable of reducing body fat and increasing muscle mass. It is proposed that adrenosterone may function as an inhibitor of the 11beta-hydroxysteroid dehydrogenase type 1 enzyme (11beta-HSD1), which is primarily responsible for reactivation of cortisol from cortisone. The urinary metabolism of adrenosterone was investigated, after a single oral administration in two male subjects, by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Substantially increased excretion of 11beta-hydroxyandrosterone, 11beta-hydroxyetiocholanolone, 11-oxoandrosterone and 11-oxoetiocholanolone was observed. Minor metabolites such as 3alpha,17beta-dihydroxy-5beta-androstan-11-one, 3alpha-hydroxyandrost-4-ene-11,17-dione and 3alpha,11beta-dihydroxyandrost-4-en-17-one were also identified. The exogenous origin of the most abundant adrenosterone metabolites was confirmed by GC-C-IRMS according to World Anti-Doping Agency criteria. Through analysis of a reference population data set obtained from urine samples provided by elite athlete volunteers (n = 85), GC-MS doping control screening criteria are proposed: 11beta-hydroxyandrosterone concentration greater than 10 000 ng/mL (specific gravity adjusted to 1.020) or 11beta-hydroxyandrosterone/11beta-hydroxyetiocholanolone ratio greater than 20.Urine samples fulfilling these screening criteria may be subjected to GC-C-IRMS analysis for confirmation of adrenosterone administration.

  18. Synthesis of deuterium-labeled 17-hydroxyprogesterone suitable as an internal standard for isotope dilution mass spectrometry

    SciTech Connect

    Shimizu, K.; Yamaga, N.; Kohara, H.

    1988-03-01

    A synthesis is reported of 17-hydroxyprogesterone, labeled with four atoms of deuterium at ring C and suitable for use as an internal standard for isotope dilution mass spectrometry. Base-catalyzed equilibration of methyl 3 alpha-acetoxy-12-oxo-cholanate (III) with /sup 2/H/sub 2/O, followed by reduction of the 12-oxo group by the modified Wolff-Kisher method using (/sup 2/H)diethylene glycol and (/sup 2/H)hydrazine hydrate afforded (11,11,12,12,23,23(-2)H)lithocholic acid (V). The Meystre-Miescher degradation of the side chain of V yielded 3 alpha-hydroxy-5 beta-(11,11,12,12(-2)H)pregnan-20-one (X). Oxidation of the 3,20-enol-diacetate of X with perbenzoic acid followed by saponification afforded 3 alpha,17-dihydroxy-5 beta-(11,11,12,12(-2)H)pregnan-20-one (XI). Oxidation of XI with N-bromoacetamide yielded 17-hydroxy-5 beta-(11,11,12,12(-2)H)pregnane-3,20-dione (XII). Bromination of XII followed by dehydrobromination yielded 17-hydroxy-(11,11,12,12(-2)H) progesterone (XIV), consisting of 0.3% /sup 2/H0-, 1.1% /sup 2/H/sub 1/-, 8.6% /sup 2/H/sub 2/-, 37.1% /sup 2/H/sub 3/-, 52.1% /sup 2/H/sub 4/-, and 0.8% /sup 2/H/sub 5/-species.

  19. Characterization of homogeneous recombinant rat ovarian 20alpha-hydroxysteroid dehydrogenase: fluorescent properties and inhibition profile.

    PubMed Central

    Ma, H; Penning, T M

    1999-01-01

    In rat ovary, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), a member of the aldo-keto reductase (AKR) superfamily, converts progesterone into the inactive progestin 20alpha-hydroxyprogesterone and has been implicated in the termination of pregnancy. Here we report a convenient overexpression system that permits the purification of milligram quantities of homogeneous recombinant 20alpha-HSD with wild-type enzyme activity. The availability of this enzyme has permitted detailed kinetic, inhibition and fluorescence analyses. The enzyme exhibited narrow steroid specificity, catalysing reactions only at C-20; it reduced progesterone and 17alpha-hydroxyprogesterone and oxidized 20alpha-hydroxypregnanes. It also turned over common AKR substrates, such as 9, 10-phenanthrenequinone and 4-nitrobenzaldehyde. The intrinsic fluorescence spectrum of 20alpha-HSD was characterized and was quenched on the binding of NADP(H), yielding a KNADPd of 0.36 microM and a KNADPHd of 0.64 microM. NADP(H) binding generated an energy transfer band that could not be quenched by steroids. Inhibition studies conducted with non-steroidal and steroidal anti-inflammatory drugs and synthetic oestrogens indicated that even though rat ovarian 20alpha-HSD and rat liver 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) share more than 67% amino acid identity, their inhibition profiles are markedly different. Unlike 3alpha-HSD, most of these compounds did not inhibit 20alpha-HSD. Only meclofenamic acid and hexoestrol were potent competitive inhibitors for 20alpha-HSD, yielding K(i) values of 18.9 and 14.3 microM respectively. These studies suggest that selective non-steroidal AKR inhibitors could be developed for 20alpha-HSD that might be useful in maintaining pregnancy and that specific inhibitors might be developed from either N-phenylanthranilates or biphenols. PMID:10417353

  20. Characterization of homogeneous recombinant rat ovarian 20alpha-hydroxysteroid dehydrogenase: fluorescent properties and inhibition profile.

    PubMed

    Ma, H; Penning, T M

    1999-08-01

    In rat ovary, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), a member of the aldo-keto reductase (AKR) superfamily, converts progesterone into the inactive progestin 20alpha-hydroxyprogesterone and has been implicated in the termination of pregnancy. Here we report a convenient overexpression system that permits the purification of milligram quantities of homogeneous recombinant 20alpha-HSD with wild-type enzyme activity. The availability of this enzyme has permitted detailed kinetic, inhibition and fluorescence analyses. The enzyme exhibited narrow steroid specificity, catalysing reactions only at C-20; it reduced progesterone and 17alpha-hydroxyprogesterone and oxidized 20alpha-hydroxypregnanes. It also turned over common AKR substrates, such as 9, 10-phenanthrenequinone and 4-nitrobenzaldehyde. The intrinsic fluorescence spectrum of 20alpha-HSD was characterized and was quenched on the binding of NADP(H), yielding a KNADPd of 0.36 microM and a KNADPHd of 0.64 microM. NADP(H) binding generated an energy transfer band that could not be quenched by steroids. Inhibition studies conducted with non-steroidal and steroidal anti-inflammatory drugs and synthetic oestrogens indicated that even though rat ovarian 20alpha-HSD and rat liver 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) share more than 67% amino acid identity, their inhibition profiles are markedly different. Unlike 3alpha-HSD, most of these compounds did not inhibit 20alpha-HSD. Only meclofenamic acid and hexoestrol were potent competitive inhibitors for 20alpha-HSD, yielding K(i) values of 18.9 and 14.3 microM respectively. These studies suggest that selective non-steroidal AKR inhibitors could be developed for 20alpha-HSD that might be useful in maintaining pregnancy and that specific inhibitors might be developed from either N-phenylanthranilates or biphenols. PMID:10417353

  1. The role of alpha-methylacyl-CoA racemase in bile acid synthesis.

    PubMed

    Cuebas, Dean A; Phillips, Christopher; Schmitz, Werner; Conzelmann, Ernst; Novikov, Dmitry K

    2002-05-01

    According to current views, the second peroxisomal beta-oxidation pathway is responsible for the degradation of the side chain of bile acid intermediates. Peroxisomal multifunctional enzyme type 2 [peroxisomal multifunctional 2-enoyl-CoA hydratase/(R)-3-hydroxyacyl-CoA dehydrogenase; MFE-2] catalyses the second (hydration) and third (dehydrogenation) reactions of the pathway. Deficiency of MFE-2 leads to accumulation of very-long-chain fatty acids, 2-methyl-branched fatty acids and C(27) bile acid intermediates in plasma, but bile acid synthesis is not blocked completely. In this study we describe an alternative pathway, which allows MFE-2 deficiency to be overcome. The alternative pathway consists of alpha-methylacyl-CoA racemase and peroxisomal multifunctional enzyme type 1 [peroxisomal multifunctional 2-enoyl-CoA hydratase/(S)-3-hydroxyacyl-CoA dehydrogenase; MFE-1]. (24E)-3alpha,7alpha,12alpha-Trihydroxy-5beta-cholest-24-enoyl-CoA, the presumed physiological isomer, is hydrated by MFE-1 with the formation of (24S,25S)-3alpha,7alpha,12alpha,24-tetrahydroxy-5beta-cholestanoyl-CoA [(24S,25S)-24-OH-THCA-CoA], which after conversion by a alpha-methylacyl-CoA racemase into the (24S,25R) isomer can again be dehydrogenated by MFE-1 to 24-keto-3alpha,7alpha,12alpha-trihydroxycholestanoyl-CoA, a physiological intermediate in cholic acid synthesis. The discovery of the alternative pathway of cholesterol side-chain oxidation will improve diagnosis of peroxisomal deficiencies by identification of serum 24-OH-THCA-CoA diastereomer profiles.

  2. Progesterone metabolism by the hypothalamus, pituitary, and uterus of the rat during pregnancy

    SciTech Connect

    Marrone, B.L.; Karavolas, H.J.

    1981-07-01

    Metabolites of (/sup 3/H)progesterone were quantitated from incubations of hypothalamus, pituitary, and uterus of rats during different stages of pregnancy. The hypothalamus, anterior pituitary, and a section of uterus from five rats on Days 1, 8, 15, and 21 of pregnancy were incubated individually with (3H)progesterone and analyzed for metabolite formation by reverse isotopic dilution analysis. The radioactive metabolites present were 5 alpha-pregnane-3,20-dione (5 alpha-DHP), 3 alpha-hydroxy-5 alpha-pregnan-20-one, 20 alpha-hydroxy-4-pregnen-3-one, 20 alpha-hydroxy-5 alpha-pregnan-3-one, and 5 alpha-pregnane-3 alpha, 20 alpha-diol. The major metabolite formed by the hypothalamus and pituitary was 5 alpha-DHP. In the pituitary samples, formation of 5 alpha-DHP was decreased on Days 15 and 21 of pregnancy compared to Day 1, and formation of 20 alpha-hydroxy-5 alpha-pregnan-3-one was decreased on Day 21 compared to Day 1. In the uterine samples, 3 alpha-hydroxy-5 alpha-pregnan-20-one was the major metabolite formed at all stages of pregnancy. The formation of all metabolic products of progesterone by the uterus was increased on Day 21 compared to Days 1, 8, and 15 of pregnancy. No changes in the formation of progesterone metabolites were observed in the hypothalamic samples during pregnancy. It is concluded that there are different profiles in the in vitro metabolism of (3H)progesterone by the hypothalamus, pituitary, and uterus of the rat during the course of pregnancy.

  3. Riemannian geometry of thermodynamics and systems with repulsive power-law interactions.

    PubMed

    Ruppeiner, George

    2005-07-01

    A Riemannian geometric theory of thermodynamics based on the postulate that the curvature scalar R is proportional to the inverse free energy density is used to investigate three-dimensional fluid systems of identical classical point particles interacting with each other via a power-law potential energy gamma r(-alpha) . Such systems are useful in modeling melting transitions. The limit alpha-->infinity corresponds to the hard sphere gas. A thermodynamic limit exists only for short-range (alpha>3) and repulsive (gamma>0) interactions. The geometric theory solutions for given alpha>3 , gamma>0 , and any constant temperature T have the following properties: (1) the thermodynamics follows from a single function b (rho T(-3/alpha) ) , where rho is the density; (2) all solutions are equivalent up to a single scaling constant for rho T(-3/alpha) , related to gamma via the virial theorem; (3) at low density, solutions correspond to the ideal gas; (4) at high density there are solutions with pressure and energy depending on density as expected from solid state physics, though not with a Dulong-Petit heat capacity limit; (5) for 3<alpha<3.7913 , the solution goes from the low to the expected high density limit smoothly; (6) for alpha>3.7913 a phase transition is required to go between these regimes; (7) for any alpha>3 we may include a first-order phase transition, which is expected from computer simulations; and (8) if alpha-->infinity, the density approaches a finite value as the pressure increases to infinity, with the pressure diverging logarithmically in the density difference. PMID:16090049

  4. Progesterone reduces depression-like behavior in a murine model of Alzheimer's Disease.

    PubMed

    Frye, Cheryl A; Walf, Alicia A

    2009-06-01

    Although anxiety and depression are not the core symptoms of Alzheimer's Disease (AD), there are changes observed in mood in those with AD, as well as in the aging population. Anxiety and depression may be influenced by progesterone P(4) and/or its neuroactive metabolites, dihydroprogesterone (DHP) and 5 alpha-pregnan-3 alpha-ol-20-one (3 alpha,5 alpha-THP). To begin to investigate progestogens' role in AD, a double transgenic mouse model of early-onset familial AD that co-overexpresses mutant forms of amyloid precursor protein (APPswe) and presenilin 1 Delta exon 9 mutation was utilized. As such, the effects of long-term (from 6 to 12 months of age) administration of P(4) to ovariectomized (ovx) wildtype and APPswe+PSEN1 Delta e9 mice for changes in affective behavior was investigated. APPswe+PSEN1 Delta 9 mutant mice had increased anxiety-like (i.e., increased emergence latencies, decreased time spent on the open quadrants of the elevated zero maze) and increased depressive-like behavior (i.e., increased time spent immobile) than did wildtype mice. Compared to vehicle-administration, P(4) administration (which produced physiological circulating P(4), DHP, and 3 alpha,5 alpha-THP levels, particularly in the wildtype mice) decreased depressant-like behavior in the forced swim test. These effects occurred independent of changes in general motor behavior/coordination, pain threshold, and plasma corticosterone levels. Thus, the APPswe+PSEN1 Delta 9 mutation alters affective behavior, and P(4) treatment reversed depressive-like behavior.

  5. Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans.

    PubMed

    Alves, A P; Mulloy, B; Moy, G W; Vacquier, V D; Mourão, P A

    1998-09-01

    The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p -2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.

  6. Neuroactive steroids and peripheral myelin proteins.

    PubMed

    Magnaghi, V; Cavarretta, I; Galbiati, M; Martini, L; Melcangi, R C

    2001-11-01

    The present review summarizes observations obtained in our laboratories which underline the importance of neuroactive steroids (i.e., progesterone (PROG), dihydroprogesterone (5alpha-DH PROG), tetrahydroprogesterone (3alpha, 5alpha-TH PROG), testosterone (T), dihydrotestosterone (DHT) and 5alpha-androstan-3alpha,17beta-diol (3alpha-diol)) in the control of the gene expression of myelin proteins (i.e. glycoprotein Po (Po) and the peripheral myelin protein 22 (PMP22)) in the peripheral nervous system. Utilizing different in vivo (aged and adult male rats) and in vitro (Schwann cell cultures) experimental models, we have observed that neuroactive steroids are able to stimulate the mRNA levels of Po and PMP22. The effects of these neuroactive steroids, which are able to interact with classical (progesterone receptor, PR, and androgen receptor, AR) and non-classical (GABA(A) receptor) steroid receptors is further supported by our demonstration in sciatic nerve and/or Schwann cells of the presence of these receptors. On the basis of the observations obtained in the Schwann cells cultures, we suggest that the stimulatory effect of neuroactive steroids on Po is acting through PR, while that on PMP22 needs the GABA(A) receptor. The present findings might be of importance for the utilization of specific receptor ligands as new therapeutical approaches for the rebuilding of the peripheral myelin, particularly in those situations in which the synthesis of Po and PMP22 is altered (i.e. demyelinating diseases like Charcot-Marie-Tooth type 1A and type 1B, hereditary neuropathy with liability to pressure palsies and the Déjérine-Sottas syndrome, aging, and after peripheral injury). PMID:11744100

  7. Further characterization of the saccharide specificity of peanut (Arachis hypogaea) agglutinin.

    PubMed

    Swamy, M J; Gupta, D; Mahanta, S K; Surolia, A

    1991-06-25

    2-Dansylamino-2-deoxy-D-galactose (GalNDns) has been shown to bind to peanut (Arachis hypogaea) agglutinin (PNA) in a saccharide-specific manner. This binding was accompanied by a five-fold increase in the fluorescence of GalNDns. The interaction was characterized by an association constant of 0.15 mM at 15 degrees and delta H and delta S values of -57.04 kJ.mol-1 and -118.1J.mol-1.K-1, respectively. Binding of a variety of other mono-, di- and oligo-saccharides to PNA, studied by monitoring their ability to dissociate the PNA GalNDns complex, revealed that PNA interacts with several T-antigen-related structures, such as beta-D-Galp-(1----3)-D-GalNAc, beta-D-Galp-(1----3)-alpha-D-GalpNAcOMe, and beta-D-Galp-(1----3)-alpha-D-GalpNAc-(1----3)-Ser, as well as the asialo-GM1 tetrasaccharide, with comparable affinity, thus showing that this lectin does not discriminate between saccharides in which the penultimate sugar of the beta-D-Galp-(1----3)-D-GalNAc unit is the alpha or beta anomer, in contrast to jacalin (Artocarpus integrifolia agglutinin), another anti T-lectin which preferentially binds to beta-D-Galp-(1----3)-alpha-D-GalNAc and does not recognize beta-D-Galp-(1----3)-beta-D-GalNAc or the related asialo-GM1 oligosaccharide. These studies also indicated that, in the extended combining region of PNA which accommodates a disaccharide, the primary subsite (subsite A) is highly specific for D-galactose, whereas the secondary subsite (subsite B) is less specific and can accommodate various structures, such as D-galactose, 2-acetamido-2-deoxy-D-galactose, D-glucose, and 2-acetamido-2-deoxy-D-glucose.

  8. Metabolism of C26 bile alcohols in the bullfrog, Rana catesbeiana

    SciTech Connect

    Noma, Y.; Kihira, K.; Kuramoto, T.; Hoshita, T.

    1988-03-01

    Metabolism of C26 bile alcohols in the bullfrog, Rana catesbeiana, was studied. (24-14C)-24-Dehydro-26-deoxy-5 beta-ranol (3 alpha,7 alpha,12 alpha-trihydroxy-27-nor-5 beta-cholestan-24-one) was chemically synthesized from (24-14C)cholic acid and incubated with bullfrog liver homogenate fortified with NADPH. 24-Dehydro-26-deoxy-5 beta-ranol was shown to be converted into both 26-deoxy-5 beta-ranol and 24-epi-26-deoxy-5 beta-ranol ((24S)- and (24R)-27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24-tetrols) in addition to 5 beta-ranol ((24R)-27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,26-pentol), which is the major bile alcohol of the bullfrog. (24-3H)-26-Deoxy-5 beta-ranol and (24-3H)-24-epi-26-deoxy-5 beta-ranol were prepared from 24-dehydro-26-deoxy-5 beta-ranol by reduction with sodium (3H) borohydride and administered respectively to two each of four bullfrogs by intraperitoneal injection. After 24 h, labeled 5 beta-ranol was isolated from the bile of the bullfrogs that received (24-3H)-26-deoxy-5 beta-ranol. In contrast little if any radioactivity could be detected in 5 beta-ranol or its 24-epimer after administration of (24-3H)-24-epi-26-deoxy-5 beta-ranol.

  9. Definition of the surface antigens of Mycobacterium malmoense and use in studying the etiology of a form of mycobacteriosis.

    PubMed Central

    McNeil, M; Tsang, A Y; McClatchy, J K; Stewart, C; Jardine, I; Brennan, P J

    1987-01-01

    Mycobacterium malmoense is the latest of a roster of atypical mycobacteria implicated in pulmonary infections. Yet it lacks recognizable phenotypic features to allow its ready identification. Some 23 clinical isolates of M. malmoense were examined for homologous seroagglutination reactions and characteristic surface antigens. One group showed concordant agglutination interreactions and an identical spectrum of glycolipids and are regarded as M. malmoense sensu stricto. The glycolipids are of the newly found, trehalose-containing lipooligosaccharide class. De-O-acylation followed by high-pressure liquid chromatography revealed one major and several minor oligosaccharides. Partial acidic cleavage to release glycosidically linked trehalose, alpha-mannosidase digestion to demonstrate the presence of a non-reducing-end mannobiose, perdeuteriomethylation, partial acid hydrolysis, reduction, and O ethylation, combined with 1H nuclear magnetic resonance and electron impact and fast-atom bombardment mass spectrometry revealed the structure of the major oligosaccharide as alpha-D-Manp-(1----3) -alpha-D-Manp-(1----[2-alpha-L-Rhap-(1--]4--3)-alpha-L-Rh ap- (1----3)-alpha-D-Glcp-(1----1)-alpha-D-Glcp, in which two of the 2-alpha-L-Rhap residues are O methylated at C-3. (Man, mannose; Rha, rhamnose; Glc, glucose; p, pyranosyl). The structures of the minor oligosaccharides were also determined; they differ at the distal nonreducing end. The dominant oligosaccharide was acylated by octanoate, 2-methyleicosanoate, and 2,4-dimethylpentacosanoate to yield the major species-specific surface antigen of M. malmoense, which we regard as the most characteristic feature of the pathogen. Images PMID:3597323

  10. Bile acid N-acetylglucosaminidation. In vivo and in vitro evidence for a selective conjugation reaction of 7 beta-hydroxylated bile acids in humans.

    PubMed Central

    Marschall, H U; Matern, H; Wietholtz, H; Egestad, B; Matern, S; Sjövall, J

    1992-01-01

    The aim of this study was to define whether N-acetylglucosaminidation is a selective conjugation pathway of structurally related bile acids in humans. The following bile acids released enzymatically from N-acetylglucosaminides were identified: 3 alpha,7 beta-dihydroxy-5 beta-cholanoic (ursodeoxycholic), 3 beta, 7 beta-dihydroxy-5 beta-cholanoic (isoursodeoxycholic), 3 beta,7 beta-dihydroxy-5 alpha-cholanoic (alloisoursodeoxycholic), 3 beta,7 beta-dihydroxy-5-cholenoic, 3 alpha,7 beta,12 alpha-trihydroxy-5 beta-cholanoic, and 3 alpha,6 alpha,7 beta-trihydroxy-5 beta-cholanoic acids. The selectivity of conjugation was studied by administration of 0.5 g ursodeoxycholic (UDCA) or hyodeoxycholic (HDCA) acids, labeled with 13C, to patients with extrahepatic cholestasis, and of 0.5 g of 13C-labeled chenodeoxycholic acid (CDCA) to patients with extra- or intrahepatic cholestasis. After administration of [24-13C]-CDCA, labeled glucosides, and the glucuronide of CDCA were excreted in similar amounts. Labeled N-acetylglucosaminides of UDCA and isoUDCA were also formed. When [24-13C]-UDCA was given, 13C-label was detected in the N-acetylglucosaminide, the glucosides, and the glucuronide of UDCA, and in the N-acetylglucosaminide of isoUDCA. In the patient studied, 32% of the total UDCA excreted in urine was conjugated with N-acetylglucosamine. In contrast, 96% of the excreted amount of [24-13C]HDCA was glucuronidated, and 13C-labeled glucosides but no N-acetylglucosaminide were detected. The selectivity of N-acetylglucosaminidation towards bile acids containing a 7 beta-hydroxyl group was confirmed in vitro using human liver and kidney microsomes and uridine diphosphate glucose (UDP)-N-acetylglucosamine. These studies show that N-acetylglucosaminidation is a selective conjugation pathway for 7 beta-hydroxylated bile acids. PMID:1602004

  11. TorsinA binds the KASH domain of nesprins and participates in linkage between nuclear envelope and cytoskeleton.

    PubMed

    Nery, Flávia C; Zeng, Juan; Niland, Brian P; Hewett, Jeffrey; Farley, Jonathan; Irimia, Daniel; Li, Yuqing; Wiche, Gerhard; Sonnenberg, Arnoud; Breakefield, Xandra O

    2008-10-15

    A specific mutation (DeltaE) in torsinA underlies most cases of the dominantly inherited movement disorder, early-onset torsion dystonia (DYT1). TorsinA, a member of the AAA+ ATPase superfamily, is located within the lumen of the nuclear envelope (NE) and endoplasmic reticulum (ER). We investigated an association between torsinA and nesprin-3, which spans the outer nuclear membrane (ONM) of the NE and links it to vimentin via plectin in fibroblasts. Mouse nesprin-3alpha co-immunoprecipitated with torsinA and this involved the C-terminal region of torsinA and the KASH domain of nesprin-3alpha. This association with human nesprin-3 appeared to be stronger for torsinADeltaE than for torsinA. TorsinA also associated with the KASH domains of nesprin-1 and -2 (SYNE1 and 2), which link to actin. In the absence of torsinA, in knockout mouse embryonic fibroblasts (MEFs), nesprin-3alpha was localized predominantly in the ER. Enrichment of yellow fluorescent protein (YFP)-nesprin-3 in the ER was also seen in the fibroblasts of DYT1 patients, with formation of YFP-positive globular structures enriched in torsinA, vimentin and actin. TorsinA-null MEFs had normal NE structure, but nuclear polarization and cell migration were delayed in a wound-healing assay, as compared with wild-type MEFs. These studies support a role for torsinA in dynamic interactions between the KASH domains of nesprins and their protein partners in the lumen of the NE, with torsinA influencing the localization of nesprins and associated cytoskeletal elements and affecting their role in nuclear and cell movement.

  12. Spongian diterpenes from Australian Nudibranchs: an anatomically guided chemical study of Glossodoris atromarginata.

    PubMed

    Somerville, Michael J; Mollo, Ernesto; Cimino, Guido; Rungprom, Wimolpun; Garson, Mary J

    2006-07-01

    An Australian population of the nudibranch mollusk Glossodoris atromarginata has been found to contain furanoditerpenes of the spongian series. Spongia-13(16),14-dien-3-one (1) and 3alpha-acetoxy-19-hydroxyspongia-13(16),14-dien-2-one [corrected] (2) were isolated for the first time from a natural source, along with a series of known diterpenes (3-7). Anatomical dissection of the animals revealed the relative distribution and chemical variation of secondary metabolites. Structural studies have provided a basis for chemical comparisons between populations from different geographic locations. PMID:16872151

  13. Fused pyrimidines. Synthesis of new derivatives of potential diuretic activity.

    PubMed

    Eisa, H M; el-Ashmawy, M B; Tayel, M M; el-Magd, S A; el-Kashef, H A

    1996-11-01

    Certain derivatives of quinazoline and its bioisostere pyridopyrimidine carrying important structural features that contribute to diuretic activity, such as sulfonamido, morpholino and chlorophenyl, were prepared as potential diuretic agents. Likewise, some tricyclic 1,2,4-triazolo[3,4-b]quinazolines and pyrido[3,2-d][1,2,4]triazolo[4,3-alpha] pyrimidines with the same features were reported. Nine compounds were tested for the diuretic activity in rats and the results showed that the active compound is 7-chloro-2-methyl-3-phthalimido-4(3H)-quinazoline (4).

  14. Interactions of the alpha2A-adrenoceptor with multiple Gi-family G-proteins: studies with pertussis toxin-resistant G-protein mutants.

    PubMed Central

    Wise, A; Watson-Koken, M A; Rees, S; Lee, M; Milligan, G

    1997-01-01

    The alpha2A-adrenoceptor is the prototypic example of the family of G-protein-coupled receptors which function by activation of 'Gi-like' pertussis toxin-sensitive G-proteins. A number of members of this subfamily of G-proteins are often co-expressed in a single cell type. To examine the interaction of this receptor with individual Gi-family G-proteins the porcine alpha2A-adrenoceptor was transiently transfected into COS-7 cells either alone or with each of wild-type Gi1alpha, Gi2alpha and Gi3alpha or mutations of each of these G-proteins in which the cysteine residue which is the target for pertussis toxin-catalysed ADP-ribosylation was exchanged for a glycine residue. The alpha2-adrenoceptor agonist UK14304 stimulated both high-affinity GTPase activity and the binding of guanosine 5'-[gamma-35thio]-triphosphate (GTP[35S]), when expressed without any additional G-protein. These effects were greatly reduced by pretreatment of the cells with pertussis toxin. Co-expression of each of the wild-type Gi-like G-protein alpha-subunits resulted in enhanced agonist activation of the cellular G-protein population which was fully prevented by pretreatment with pertussis toxin. Co-expression of the receptor along with the cysteine-to-glycine mutations of Gi1alpha, Gi2alpha and Gi3alpha resulted in agonist stimulation of these G-proteins, which was as great as that of the wild type proteins, but now the agonist stimulation produced over that due to the activation of endogenously expressed Gi-like G-proteins was resistant to pertussis toxin treatment. The Cys --> Gly mutations of Gi1alpha, Gi2alpha and Gi3alpha were each also able to limit agonist-mediated stimulation of adenylate cyclase activity. The degree of agonist-mediated activation of the pertussis toxin-resistant mutant of Gi1alpha was correlated highly both with the level of expression of this G-protein and with the level of expression of the alpha2A-adrenoceptor. Half-maximal stimulation of high-affinity GTPase

  15. Differential expression of beta 1 integrins in nonneoplastic smooth and striated muscle cells and in tumors derived from these cells.

    PubMed Central

    Mechtersheimer, G.; Barth, T.; Quentmeier, A.; Möller, P.

    1994-01-01

    Integrins are a superfamily of transmembrane alpha beta heterodimers that play an important role in cell-matrix and cell-cell interactions by acting as receptors for extracellular matrix proteins and for cell adhesion molecules. Using monoclonal antibodies against beta 1, alpha 1 to alpha 6, and alpha v subunits, the in situ distribution pattern of beta 1 integrins was examined immunohistochemically in nonneoplastic smooth and striated muscle cells and in their tumors. Nonneoplastic smooth muscle cells were beta 1+, alpha 1+, alpha 3+, alpha v+ and, in diverse localizations, also alpha 5+ or even alpha 6+. The expression of the beta 1 chain was conserved in all leiomyomas and leiomyosarcomas. The distribution pattern of the alpha subunits by contrast underwent several changes during malignant transformation of smooth muscle cells. These alterations consisted in a neoexpression of alpha 2, alpha 4, and alpha 6 as well as in an abnormal abrogation of alpha 1 and alpha 3 in some leiomyosarcomas. Except for the absence of alpha 5 in the majority of epithelioid leiomyosarcomas, expression of the alpha 5 and alpha v subunits was mainly conserved. In addition, tumors with epithelioid differentiation differed from typical cases by the absence of alpha 1 and the simultaneous presence of alpha 4. Adult striated muscle cells were beta 1+ but alpha 1- to alpha 6- and alpha v-, whereas fetal striated muscle cells were not only beta 1+ but also alpha 3+/-, alpha 4+/-, alpha 5+ and alpha 6+. In all rhabdomyosarcomas the expression of beta 1 was retained. Furthermore, the majority of cases showed the expression of one or more alpha subunits most of which, ie, alpha 4, alpha 5, and alpha 6, were also found in fetal striated muscle cells. In conclusion, beta 1 integrins exhibited a differential expression pattern along the two lines of myogenic differentiation. This integrin profile underwent characteristic changes during malignant transformation. Nevertheless, the compiled

  16. Chemical composition of the essential oil of Croton gossypiifolius from Venezuela.

    PubMed

    Suárez, Alírica I; Oropeza, Marly; Vásquez, Luis; Tillett, Stephen; Compagnone, Reinaldo S

    2011-01-01

    The essential oil from leaves of Croton gossypiifolius Vahl. (Euphorbiaceae) was obtained by hydrodistillation, and analyzed by GC/FID and GC/MS. The constituents were identified by their mass spectra and Kovats' indices. Fifty-one compounds accounting for 92% of the oil were detected, and 47 of them were identified. The oil was dominated by oxygenated sesquiterpenes with the major presence of alpha-cedrene oxide (18.6%), spathulenol (16.3%), valencene (5.8%), geranyl-pentanoate (5.3%), alpha-cadinol (4.0%), germacrene D (3.5%) and longifolene (3.3%).

  17. New cytotoxic triterpene acids from aboveground parts of Manihot esculenta from the Suriname rainforest.

    PubMed

    Chaturvedula, V S; Schilling, Jennifer K; Malone, Stan; Wisse, Jan H; Werkhoven, Marga C; Kingston, David G

    2003-03-01

    Two novel triterpene acids, esculentoic acid A ( 1) and B ( 2) as well as the seven known compounds 3 - 9 were isolated from an EtOAc extract of leaves, stems, and twigs of Manihot esculenta by bioassay-guided fractionation for cytotoxic activity. The structures of the two new compounds were established as 3alpha-hydroxytaraxer-14-en-29-oic acid ( 1) and 3-oxotaraxer-14-en-29-oic acid ( 2) on the basis of 1D and 2D NMR spectroscopic data interpretation and chemical conversions. The two new compounds 1 and 2 showed moderate cytotoxicity against the A2780 human ovarian cancer cell line.

  18. Calystegins of Physalis alkekengi var. francheti (Solanaceae). Structure determination and their glycosidase inhibitory activities.

    PubMed

    Asano, N; Kato, A; Oseki, K; Kizu, H; Matsui, K

    1995-04-15

    Five calystegins were extracted from the roots of Physalis alkekengi var. francheti (Solanaceae) with hot water and purified to homogeneity by the combination of a variety of ion-exchange column chromatographies. Their structures have been determined from the 1H- and 13C-NMR spectral data, and two of the compounds were identified as calystegins A3 and B2, which have been isolated from the roots of Calystegia sepium (Convolvulaceae). Two of the remaining three were found to be 1 alpha, 3 alpha, 4 beta-trihydroxy-nor-tropane and 1 alpha, 2 alpha, 3 alpha, 4 beta-tetrahydroxy-nor-tropane and given the trivial name calystegins A5 and B3, respectively. The last calystegin was assigned as 1 alpha, 2 beta, 3 alpha, 6 alpha-tetrahydroxy-nor-tropane, which was the same as the relative configuration proposed in the literature for calystegin B1 isolated from C. sepium. However, the 13C-NMR spectral data for the compound from C. sepium differed substantially from our results. From a personal communication with the authors of the original paper on calystegins, it was clarified that the 13C-NMR chemical shifts of calystegin B1 in the original paper had been erroneous. Since their corrected 13C-NMR data of calystegin B1 and its 1H-NMR chemical shifts in the original paper are very close to our present data, we concluded that both compounds from C. sepium and P. alkekengi are identical. Calystegin B2 has been known to be a potent competitive inhibitor of almond beta-glucosidase (Ki = 1.2 microM) and coffee bean alpha-galactosidase (Ki = 0.86 microM). In this study calystegin B1 (1 alpha, 2 beta, 3 alpha, 6 alpha-tetrahydroxy-nor-tropane) proved to be a potent competitive inhibitor of almond beta-glucosidase (Ki = 1.9 microM) and bovine liver beta-galactosidase (Ki = 1.6 microM), but not an inhibitor of alpha-galactosidases. Calystegin A3 was found to be a weaker inhibitor compared to calystegin B2 but with the same inhibitory spectrum. Calystegin A5, a 2-deoxy derivative of

  19. Analysis of states in {sup 13}C populated in {sup 9}Be + {sup 4}He resonant scattering

    SciTech Connect

    Freer, M.; Ashwood, N. I.; Curtis, N.; Kokalova, Tz.; Wheldon, C.; Di Pietro, A.; Figuera, P.; Fisichella, M.; Scuderi, V.; Torresi, D.; Grassi, L.; Jelavic Malenica, D.; Koncul, M.; Mijatovic, T.; Prepolec, L.; Skukan, N.; Soic, N.; Szilner, S.; Tokic, V.; Milin, M.

    2011-09-15

    Measurements of {sup 9}Be + {alpha} resonant scattering have been performed using the thick-target approach with a {sup 4}He gas volume and a large-area silicon strip detector. {sup 9}Be beam energies in the range 12 to 21.4 MeV were used to measure the {sup 13}C excitation energy spectrum between 13.2 and 16.2 MeV. An R-matrix analysis has been performed to characterize the spins and widths of {sup 13}C resonances, some of which have been proposed to be associated with a 3{alpha}+n molecular band.

  20. A new lupane-triterpene glycoside from the leaves of Acanthopanax gracilistylus.

    PubMed

    Liu, Xiang-Qian; Chang, Seung-Yeup; Park, Sang-Yong; Nohara, Toshihiro; Yook, Chang-Soo

    2002-12-01

    A new and two known lupane-triterpene glycosides were isolated from the hot MeOH fraction of the leaves of Acanthopanax gracilistylus W. W. Smith. Based on the physical properties and spectroscopic data, their chemical structures were determined as acankoreoside A (1), acankoreoside D (2), and 3alpha-hydroxy-lup-23-al-20(29)-en-28-oic acid 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester (3), respectively. To our best knowledge, compound 3 appears to be novel, which was named as wujiapioside A. PMID:12510834

  1. Cytotoxic, antioxidant activities and structure activity relationship of some newly synthesized terpenoidal oxaliplatin analogs.

    PubMed

    Amr, Abd El-Galil E; Ali, Korany A; Abdalla, Mohamed M

    2009-02-01

    The terpenoidal oxaliplatin derivatives (6) and (12) were newly synthesized using 2beta,3alpha-dihydroxy-11-oxo-18beta-olean-12-ene-30-oic acid (1) and 2alpha,2beta-dihydroxy-18beta-ursan-12-ene-28-oic acid (7) as starting materials. The synthesized compounds were evaluated for their cytotoxicity and antioxidant activities and were compared to Oxaliplatin and vitamin C as positive controls. Some of the compounds exhibited better cytotoxicity and antioxidant activities than the reference controls. The detailed synthesis, spectroscopic data, toxicity (LD(50)) and pharmacological screening for the synthesized compounds were reported.

  2. Molecular mechanism of antioxidant synergism of tocotrienols and carotenoids in palm oil.

    PubMed

    Schroeder, Maria T; Becker, Eleonora Miquel; Skibsted, Leif H

    2006-05-01

    During repeated deep-fat frying of potato slices at 163 degrees C in yellow or red palm olein of comparable fatty acid profiles, the oxidative stability (peroxide value and anisidine value) of the palm oleins was similar, and in yellow palm olein, the rate of antioxidant depletion decreased in the order gamma-T3 > alpha-T3 > delta-T3 (T3, tocotrienol). In red palm olein, which had a total tocopherol/tocotrienol content of 1260 vs 940 ppm in yellow palm olein and a corresponding longer induction period in the Rancimat stability test at 120 degrees C, only depletion of gamma-T3 was significant among the phenols during frying and slower as compared to that in yellow palm olein. The carotenes in the red palm olein were depleted linearly with the number of fryings, apparently yielding an overall protection of the phenols. In antioxidant-depleted palm olein and in phospholipid liposomes with added increasing concentrations of phenols, gamma-T3 was found to be a better antioxidant than alpha-T3. alpha-T3 and alpha-T (T, tocopherol) had a similar antioxidant effect in antioxidant-depleted palm olein in the Rancimat stability test, while in the liposomes the ordering as determined by induction period for the formation of conjugated dienes was gamma-T3 > alpha-T3 > alpha-T. The addition of 100-1000 ppm beta-carotene to antioxidant-depleted palm olein or liposomes (lycopene also tested) did not provide any protection against oxidation. In the liposomes, synergistic interactions were observed between beta-carotene or lycopene and alpha-T, alpha-T3, or gamma-T3 for carotene/phenol ratios of 1:10 and 1:2 but not for 1:1. In chloroform, carotenes were regenerated by tocopherols/tocotrienols from carotene radicals generated by laser flash photolysis as shown by transient absorption spectroscopy, suggesting that carotenes rather than phenols are the primary substrate for lipid-derived radicals in red palm olein, in effect depleting carotenes prior to phenols during frying

  3. Transcriptional up-regulation of inhibitory PAS domain protein gene expression by hypoxia-inducible factor 1 (HIF-1): a negative feedback regulatory circuit in HIF-1-mediated signaling in hypoxic cells.

    PubMed

    Makino, Yuichi; Uenishi, Rie; Okamoto, Kensaku; Isoe, Tsubasa; Hosono, Osamu; Tanaka, Hirotoshi; Kanopka, Arvydas; Poellinger, Lorenz; Haneda, Masakazu; Morimoto, Chikao

    2007-05-11

    The inhibitory PAS (Per/Arnt/Sim) domain protein (IPAS), a dominant negative regulator of hypoxia-inducible transcription factors (HIFs), is potentially implicated in negative regulation of angiogenesis in such tissues as the avascular cornea of the eye. We have previously shown IPAS mRNA expression is up-regulated in hypoxic tissues, which at least in part involves hypoxia-dependent alternative splicing of the transcripts from the IPAS/HIF-3alpha locus. In the present study, we demonstrate that a hypoxia-driven transcriptional mechanism also plays a role in augmentation of IPAS gene expression. Isolation and analyses of the promoter region flanking to the first exon of IPAS gene revealed a functional hypoxia response element at position -834 to -799, whereas the sequence upstream of the HIF-3alpha first exon scarcely responded to hypoxic stimuli. A transient transfection experiment demonstrated that HIF-1alpha mediates IPAS promoter activation via the functional hypoxia response element under hypoxic conditions and that a constitutively active form of HIF-1alpha is sufficient for induction of the promoter in normoxic cells. Moreover, chromatin immunoprecipitation and electrophoretic mobility shift assays showed binding of the HIF-1 complex to the element in a hypoxia-dependent manner. Taken together, HIF-1 directly up-regulates IPAS gene expression through a mechanism distinct from RNA splicing, providing a further level of negative feedback gene regulation in adaptive responses to hypoxic/ischemic conditions. PMID:17355974

  4. Purification and properties of bile acid sulfate sulfatase from Pseudomonas testosteroni.

    PubMed

    Tazuke, Y; Matsuda, K; Adachi, K; Tsukada, Y

    1994-05-01

    The bile acid sulfate sulfatase (BSS) produced by Pseudomonas testosteroni was purified and characterized. Chromatofocusing behavior and amino acid sequence over twelve amino acid residues from N-terminus of the enzyme indicated that BSS was composed of two isoforms of which molecular weights were 125,000 and 103,000. Each isoform was a homodimer of a subunit of which molecular weight was 53,000 or 51,000, respectively. The optimum pH was 8.5 and BSS was stable at pH 5.8-8.0. The thermostability above 32 degrees C was improved by the addition of polyols, such as sorbitol, sucrose, and glycerol. BSS was a Mn(2+)-dependent enzyme and contained 1-2 atoms of manganese in its own protein molecule. All 3 alpha-sulfate esters of the bile acids routinely appearing in human serum were hydrolyzed by BSS to 3 beta-hydroxyl iso-compounds corresponding to each bile acid and sulfuric acid. We tentatively named this novel enzyme BSS (bile acid 3 alpha-sulfate sulfohydrolase). PMID:7764976

  5. Studies on Hydrogen Extraction Characteristics of Proton-Conducting Ceramics and Their Applications to a Tritium Recovery System and a Tritium Monitor

    SciTech Connect

    Tanaka, M.; Asakura, Y.; Uda, T.; Katahira, K.; Iwahara, H.; Tsuji, N.; Yamamoto, I.

    2005-07-15

    For the purpose of the recovery of a hydrogen isotope exhausted from a fusion device and its application to a tritium monitor, hydrogen extraction properties using SrZr{sub 0.9}Yb{sub 0.1}O{sub 3-{alpha}} and CaZr{sub 0.9}In{sub 0.1}O{sub 3-{alpha}} and the effect of the electrode attachment method on the hydrogen extraction were evaluated under various atmospheres and temperatures. As a result, hydrogen could be extracted from mixed gases containing hydrogen, water vapor and methane. Furthermore, water vapor electrolysis for the tritium monitor was also evaluated under a wet atmosphere containing oxygen. From these results, it was revealed that a plated platinum electrode was suitable for mixed gases containing hydrogen, water vapor and methane, and that a porous pasted platinum electrode was suitable for water vapor electrolysis. From the findings obtained from the study of the hydrogen extraction properties, we described an optimum specification of the platinum electrode for a tritium recovery system and the number of proton-conducting ceramics for a tritium monitor.

  6. Determination of progesterone and some of its neuroactive ring A-reduced metabolites in human serum.

    PubMed

    Pearson Murphy, B E; Allison, C M

    2000-10-01

    A method for the separation and assay of some ring A-reduced metabolites of progesterone (pregnanediones and pregnanolones) is described. Serum was extracted with an organic solvent, and the extract chromatographed using high performance liquid chromatography (HPLC). A total of 50 fractions was collected for each sample and split using a stream splitter so that 30% was collected in counting vials for recovery while 70% was collected in test tubes which were assayed by radioimmunoassay. An antiserum raised in our laboratory to progesterone-3-CMO-BSA cross-reacted with five of these compounds (5alpha- and 5beta-dihydroprogesterone, 3alpha- and 3beta-5alpha-tetrahydroprogesterone, and 3beta, 5beta-tetrahydroprogesterone). Since pregnenolone eluted with 5alpha, 3beta-tetrahydroprogesterone, pregnenolone was assayed separately and its effect subtracted. Using this method it was shown that picogram to nanogram/ml amounts of these metabolites are present in all human sera. Levels in men were comparable to those of women in the follicular phase of the menstrual cycle. 5alpha-Dihydroprogesterone and 3alpha,5alpha-tetrahydroprogesterone rose substantially in the luteal phase of the menstrual cycle and all rose considerably during pregnancy.

  7. The organization, structure, and in vitro transcription of Alu family RNA polymerase III transcription units in the human alpha-like globin gene cluster: precipitation of in vitro transcripts by lupus anti-La antibodies.

    PubMed

    Shen, C K; Maniatis, T

    1982-01-01

    We have studied the location, structure, and in vitro transcription of repetitive DNA sequences within the human alpha-like globin gene cluster. At least eight different Alu family repeats were identified, each of which is transcribed in vitro to produce discrete RNA transcripts. The nucleotide sequence of one Alu repeat sequence, located on the 3' side of the alpha l globin gene (3'-alpha l), was determined and compared to published Alu repeat sequences. In vitro transcription of this repeat sequence generates RNA fragments of approximately 410, 260, 160, and 86 nucleotides. To determine whether these transcripts associate with specific proteins in vitro, we carried out immunoprecipitation experiments using an antiserum from systemic lupus erythematosus (SLE) patients. We find that the antiserum anti-La, which was shown to precipitate ribonucleoproteins (RNPs) containing the adenovirus VAI RNA from virus infected cells, preferentially precipitates the smallest two in vitro transcripts of the 3'-alpha l Alu repeat. These results suggest that the RNAs interact with specific factors in the in vitro transcription reaction mix to form RNP.

  8. Association of EEG, MRI, and regional blood flow biomarkers is predictive of prodromal Alzheimer’s disease

    PubMed Central

    Moretti, Davide Vito

    2015-01-01

    Background Thinning in the temporoparietal cortex, hippocampal atrophy, and a lower regional blood perfusion is connected with prodromal stage of Alzheimer’s disease (AD). Of note, an increase of electroencephalography (EEG) upper/low alpha frequency power ratio has also been associated with these major landmarks of prodromal AD. Methods Clinical and neuropsychological assessment, EEG recording, and high-resolution three-dimensional magnetic resonance imaging were done in 74 grown up subjects with mild cognitive impairment. This information was gathered and has been assessed 3 years postliminary. EEG recording and perfusion single-photon emission computed tomography assessment was done in 27 subjects. Alpha3/alpha2 frequency power ratio, including cortical thickness, was figured for every subject. Contrasts in cortical thickness among the groups were assessed. Pearson’s r relationship coefficient was utilized to evaluate the quality of the relationship between cortical thinning, brain perfusion, and EEG markers. Results The higher alpha3/alpha2 frequency power ratio group corresponded with more prominent cortical decay and a lower perfusional rate in the temporoparietal cortex. In a subsequent meetup after 3 years, these patients had AD. Conclusion High EEG upper/low alpha power ratio was connected with cortical diminishing and lower perfusion in the temporoparietal brain area. The increase in EEG upper/low alpha frequency power ratio could be helpful in recognizing people in danger of conversion to AD dementia and this may be quality information in connection with clinical assessment. PMID:26604762

  9. Long-term detection and identification of metandienone and stanozolol abuse in athletes by gas chromatography-high-resolution mass spectrometry.

    PubMed

    Schänzer, W; Delahaut, P; Geyer, H; Machnik, M; Horning, S

    1996-12-01

    The misuse of anabolic androgenic steroids (AAS) in human sports is controlled by gas chromatography-mass spectrometric analysis of urine specimens obtained from athletes. The analysis is improved with modern high-resolution mass spectrometry (HRMS). The detection and identification of metabolites of stanozolol (I) [3'-hydroxystanozolol (II) and 4 beta-hydroxystanozolol (III)] and metandienone (IV) I17 beta-methyl-5 beta-androst-1-ene-3 alpha,17 alpha-diol (V) and 18-nor-17,17-dimethyl-5 beta-androsta-1,13-dien-3 alpha-ol (VI)] with GC-HRMS at 3000 resolution yielded a large increase in the number of positive specimens. A total of 116 anabolic steroid positives were found in this laboratory in 1995 via GC-MS and GC-HRMS screening of 6700 human urine specimens collected at national and international sporting events and at out-of-competition testing. Of the 116 positive cases, 41 were detected using conventional (quadrupole) GC-MS screening. The other 75 positives were identified via GC-HRMS screening. To confirm the HRMS screening result, the urine sample was reanalyzed using a specific sample workup procedure to selectively isolate the metabolites of the identified substance. II and III were selectively isolated via immunoaffinity chromatography (IAC) using an antibody which was prepared for methyltestosterone and shows high cross reactivity to II and III. V and VI were isolated using high-performance liquid chromatography (HPLC) fractionation. PMID:9001957

  10. Skin-lightening effect of a polyphenol extract from Acerola (Malpighia emarginata DC.) fruit on UV-induced pigmentation.

    PubMed

    Hanamura, Takayuki; Uchida, Eriko; Aoki, Hitoshi

    2008-12-01

    To investigate the physiological functions of polyphenols from acerola (Malpighia emarginata DC.) fruit, the effects on melanogenesis were studied. The crude polyphenol concentrated extract from acerola (C-AP) was used to examine the skin-lightening effect on brownish guinea pigs which had been subjected to controlled UVB irradiation. The results show that C-AP significantly lightened the UVB-irradiated skin pigmentation. Furthermore, treatment with C-AP reduced the content of melanin in B16 melanoma cells, suggesting that the in vivo skin-lightening effect of C-AP was due to the suppression of melanin biosynthesis in melanocytes. In addition, we found that C-AP could effectively inhibit mushroom tyrosinase activity, the main constituents responsible for this effect being thought to be such anthocyanins as cyanidin-3-alpha-O-rhamnoside (C3R) and pelargonidin-3-alpha-O-rhamnoside (P3R). This result indicates that the skin-lightening effect of C-AP can be partly attributed to the suppression of melanogenesis through the inhibition of tyrosinase activity in melanocytes. An oral ingestion of C-AP may therefore be efficacious for reducing UVB-induced hyper-pigmentation by inhibiting the tyrosinase in melanocytes.

  11. Anticonvulsant and anesthetic effects of a fluorescent neurosteroid analog activated by visible light.

    PubMed

    Eisenman, Lawrence N; Shu, Hong-Jin; Akk, Gustav; Wang, Cunde; Manion, Brad D; Kress, Geraldine J; Evers, Alex S; Steinbach, Joe Henry; Covey, Douglas F; Zorumski, Charles F; Mennerick, Steven

    2007-04-01

    Most photoactivatable compounds suffer from the limitations of the ultraviolet wavelengths that are required for activation. We synthesized a neuroactive steroid analog with a fluorescent (7-nitro-2,1,3-benzoxadiazol-4-yl) amino (NBD) group in the beta configuration at the C2 position of (3alpha,5alpha)-3-hydroxypregnan-20-one (allopregnanolone, 3alpha5alphaP). Light wavelengths (480 nm) that excite compound fluorescence strongly potentiate GABAA receptor function. Potentiation is limited by photodepletion of the receptor-active species. Photopotentiation is long-lived and stereoselective and shows single-channel hallmarks similar to steroid potentiation. Other NBD-conjugated compounds also generate photopotentiation, albeit with lower potency. Thus, photopotentiation does not require a known ligand for neurosteroid potentiating sites on the GABAA receptor. Photoactivation of a membrane-impermeant, fluorescent steroid analog demonstrates that membrane localization is critical for activity. The photoactivatable steroid silences pathological spiking in cultured rat hippocampal neurons and anesthetizes tadpoles. Fluorescent steroids photoactivated by visible light may be useful for modulating GABAA receptor function in a spatiotemporally defined manner.

  12. Histo-blood group A/B versus H status of human carcinoma cells as correlated with haptotactic cell motility: approach with A and B gene transfection.

    PubMed

    Ichikawa, D; Handa, K; Withers, D A; Hakomori, S

    1997-08-01

    In a search for the molecular basis of ABH status of tumors as correlated with malignancy, we studied various malignancy-related phenotypes of high H/Le(y)-expressing tumor cell lines in comparison with phenotypes of the same lines transfected with histo-blood group A or B genes. A and B gene transfectants, prepared independently from different H-active parental cells, showed A or B activity and abolition of H activity. All A and B gene transfectants, regardless of source, were characterized by significantly reduced Matrigel-dependent haptotactic motility. The level of haptotaxis of all transfectants was similar to that of parental cells in the presence of antibodies against human integrin subunits alpha3, alpha6, or beta1. These subunits showed high expression of A or B epitope in the A and B gene transfectants. Enhancement versus reduction of malignancy, associated with deletion versus induction of A/B epitopes, may be due in part to enhanced haptotaxis sustained by alpha3, alpha6, and beta1 integrin receptors, the activities of which are regulated by H or A/B glycosylation. These phenotypic changes provide a rationale for the deletion of A and B epitopes as one criterion defining human tumor malignancy. PMID:9242430

  13. Characterization of flavonols in cranberry (Vaccinium macrocarpon) powder.

    PubMed

    Vvedenskaya, Irina O; Rosen, Robert T; Guido, Jane E; Russell, David J; Mills, Kent A; Vorsa, Nicholi

    2004-01-28

    Flavonoids were extracted from cranberry powder with acetone and ethyl acetate and subsequently fractionated with Sephadex LH-20 column chromatography. The fraction eluted with a 60% methanol solution was composed primarily of phenolic constituents with maximum absorbance at 340 nm. A high-performance liquid chromatography procedure was developed, which resolved 22 distinct peaks with UV/vis and mass spectra corresponding to flavonol glycoside conjugates. Six new constituents not previously reported in cranberry or in cranberry products were determined through NMR spectroscopy to be myricetin-3-beta-xylopyranoside, quercetin-3-beta-glucoside, quercetin-3-alpha-arabinopyranoside, 3'-methoxyquercetin-3-alpha-xylopyranoside, quercetin-3-O-(6' '-p-coumaroyl)-beta-galactoside, and quercetin-3-O-(6' '-benzoyl)-beta-galactoside. Quercetin-3-O-(6' '-p-coumaroyl)-beta-galactoside and quercetin-3-O-(6' '-benzoyl)-beta-galactoside represent a new class of cranberry flavonol compounds with three conjugated components consisting of a flavonol, sugar, and carboxylic acid (benzoic or hydroxycinnamic acids). This is also the first report identifying quercetin-3-arabinoside in both furanose and pyranose forms in cranberry. Elucidation of specific flavonol glycosides in cranberry is significant since the specificity of the sugar moiety may play a role in the bioavailability of the flavonol glycosides in vivo.

  14. 5 beta-hydroxylation by the liver. Identification of 3,5,7-trihydroxy nor-bile acids as new major biotransformation products of 3,7-dihydroxy nor-bile acids in rodents.

    PubMed

    Schteingart, C D; Hagey, L R; Setchell, K D; Hofmann, A F

    1993-05-25

    24-Norursodeoxycholic acid (nor-UDCA), when administered into the anesthetized biliary fistula hamster or injected into the perfusate of an isolated liver, was hydroxylated at C-5 to give 5 beta-hydroxynorursodeoxycholic acid 2 (3 alpha,5,7 beta-trihydroxy-24-nor-5 beta-cholan-23-oic acid), which was secreted into bile mainly as such. Similarly, 24-norchenodeoxycholic acid (nor-CDCA) was 5 beta-hydroxylated to give 5 beta-hydroxynor-chenodeoxycholic acid 4 (3 alpha,5,7 alpha-trihydroxy-24-nor-5 beta-cholan-23-oic acid), which was also secreted into bile without appreciable further biotransformation. The site of hydroxylation was assigned by 13C and 1H NMR and mass spectrometry. 5-Hydroxylation was a major biotransformation pathway at physiological bile acid loads. 5-Hydroxylation of UDCA also occurred in the perfused rat liver but to a lesser extent. 5-Hydroxylation of nor-UDCA was not observed in rabbit, dog, or man, indicating that its formation is species-specific. 5-Hydroxylation of nor-CDCA and nor-UDCA is the first reported example of hydroxylation of a tertiary carbon atom of bile acids. Nor-dihydroxy bile acids appear to be useful for the detection of minor hydroxylation pathways, because their prolonged hepatobiliary retention exposes them repeatedly to hydroxylases present in the hepatobiliary system.

  15. Development of an in vitro dual-chamber model of the female genital tract as a screening tool for epithelial toxicity.

    PubMed

    Gali, Youssef; Ariën, Kevin K; Praet, Marleen; Van den Bergh, Rafael; Temmerman, Marleen; Delezay, Olivier; Vanham, Guido

    2010-05-01

    Heterosexual transmission of human immunodeficiency virus (HIV-1) is the predominant mode of infection worldwide. However, the early steps of transepithelial infection still need to be clarified. Using epithelial cells, originating from the female genital tract, and peripheral blood mononuclear cells as subepithelial target cells, an in vitro dual-chamber model of the female genital tract was developed. Remarkably, an intact layer of some cell types (HEC-1A, CaSki and Ect1) served as a protective barrier against cell-free but not against cell-associated HIV-1 that crossed the epithelial barrier through transmigration. Furthermore, dysfunctions of the epithelial layers were assessed by monitoring transepithelial electric resistance and transepithelial passage of FluoSpheres and HIV-1 after treatment with nonoxynol-9 (N-9). Most of the functional assays showed dysfunction of the epithelial barrier at lower concentrations compared to a widely used colorimetric toxicity assay (WST-1). Finally, N-9 treatment caused a significant increase in the production of interleukin-8 (IL-8) and macrophage inflammatory protein-3alpha (MIP-3alpha) and a decrease of Secretory Leukocyte Protease Inhibitor (SLPI) and Monocyte Chemotactic Protein-1 (MCP-1) in this model. In conclusion, this model is a useful tool to (1) study HIV-1 transmission mechanisms and (2) evaluate epithelial toxicity of candidate microbicides. PMID:20138087

  16. The protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation and increases sarcoplasmic/endoplasmic reticulum calcium ATPase 2 levels

    SciTech Connect

    King, Taj D.; Gandy, Johanna C.; Bijur, Gautam N. . E-mail: gautam@uab.edu

    2006-11-01

    The ubiquitously expressed protein glycogen synthase kinase-3 (GSK3) is constitutively active, however its activity is markedly diminished following phosphorylation of Ser21 of GSK3{alpha} and Ser9 of GSK3{beta}. Although several kinases are known to phosphorylate Ser21/9 of GSK3, for example Akt, relatively much less is known about the mechanisms that cause the dephosphorylation of GSK3 at Ser21/9. In the present study KCl-induced plasma membrane depolarization of SH-SY5Y cells, which increases intracellular calcium concentrations caused a transient decrease in the phosphorylation of Akt at Thr308 and Ser473, and GSK3 at Ser21/9. Overexpression of the selective protein phosphatase-1 inhibitor protein, inhibitor-2, increased basal GSK3 phosphorylation at Ser21/9 and significantly blocked the KCl-induced dephosphorylation of GSK3{beta}, but not GSK3{alpha}. The phosphorylation of Akt was not affected by the overexpression of inhibitor-2. GSK3 activity is known to affect sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) levels. Overexpression of inhibitor-2 or treatment of cells with the GSK3 inhibitors lithium and SB216763 increased the levels of SERCA2. These results indicate that the protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation induced by KCl and that GSK3 activity regulates SERCA2 levels.

  17. Pressure-induced dissociation and denaturation of allophycocyanin at subzero temperatures.

    PubMed

    Foguel, D; Weber, G

    1995-12-01

    The thermodynamics of assembly of the allophycocyanin hexamer was examined employing hydrostatic pressures in the range of 1 bar to 2.4 kbar and temperatures of 20 to -12 degrees C, the latter made possible by the decrease of the freezing point of water under pressure. The existence of two processes, dissociation of the hexamer into dimers, (alpha beta)3-->3 (alpha beta), and dissociation of the alpha beta dimers into monomers, (alpha beta)-->alpha + beta have been recognized previously by changes in the absorbance and fluorescence of the tetrapyrrolic chromophores owing to added ligands. The same changes are observed in the absence of ligands at pressures of under 2.4 kbar and temperatures down to -12 degrees C. On decompression from 2.4 kbar at 0 degrees C, appreciable hysteresis and a persistent loss of 50% in the absorbance at 653 nm is observed. It results from the conformational drift of the isolated subunits and is reduced to 10% when the highest pressure is limited to 1.6 kbar. The thermodynamic parameters of the reaction alpha + beta-->alpha beta can be determined from pressure effects on perchlorate solutions of allophycocyanin, which consist of dimers alone. Their previous knowledge permits estimation, under suitable hypotheses, of the thermodynamic parameters of the reaction 3(alpha beta)-->(alpha beta)3 from the overall pressure effects on the hexamers. Both association reactions have positive enthalpy changes, and the whole hexamer assembly is made possible by the excess entropy.

  18. Disparate Degrees of Hypervariable Loop Flexibility Control T-Cell Receptor Cross-Reactivity, Specificity, and Binding Mechanism

    SciTech Connect

    Scott, Daniel R.; Borbulevych, Oleg Y.; Piepenbrink, Kurt H.; Corcelli, Steven A.; Baker, Brian M.

    2012-06-19

    {alpha}{beta} T-cell receptors (TCRs) recognize multiple antigenic peptides bound and presented by major histocompatibility complex molecules. TCR cross-reactivity has been attributed in part to the flexibility of TCR complementarity-determining region (CDR) loops, yet there have been limited direct studies of loop dynamics to determine the extent of its role. Here we studied the flexibility of the binding loops of the {alpha}{beta} TCR A6 using crystallographic, spectroscopic, and computational methods. A significant role for flexibility in binding and cross-reactivity was indicated only for the CDR3{alpha} and CDR3{beta} hypervariable loops. Examination of the energy landscapes of these two loops indicated that CDR3{beta} possesses a broad, smooth energy landscape, leading to rapid sampling in the free TCR of a range of conformations compatible with different ligands. The landscape for CDR3{alpha} is more rugged, resulting in more limited conformational sampling that leads to specificity for a reduced set of peptides as well as the major histocompatibility complex protein. In addition to informing on the mechanisms of cross-reactivity and specificity, the energy landscapes of the two loops indicate a complex mechanism for TCR binding, incorporating elements of both conformational selection and induced fit in a manner that blends features of popular models for TCR recognition.

  19. In situ deuteron NMR investigations of sheared liquid crystalline polymers.

    PubMed

    Siebert, Hartmut; Becker, Patrick; Quijada-Garrido, Isabel; Grabowski, David A; Schmidt, Claudia

    2002-01-01

    The flow behavior of nematic liquid crystalline polysiloxanes of the side-chain type is studied by in situ 2H NMR spectroscopy on samples under shear in a cone-and-plate cell. The director orientation as a function of applied shear rate is determined from the quadrupole splitting of the spectra. The data analysis yields the two Leslie viscosity coefficients alpha2 and alpha3 and the flow-alignment parameter lambda = -(alpha3 + alpha2)/(alpha3 - alpha2). The values of lambda were determined for several homopolymers with only one type of side chain and random copolymers containing two different side chains. The results show that the flow behavior is related to the phase structure of the polymers, which varies with their composition. Only polymers with large amounts of smectic clusters in the nematic state show the tumbling instability (absolute value(lambda) < 1); other polymers are flow aligning (absolute value(lambda) > or = 1). For some polymers, a transition from tumbling at low temperature to flow aligning at high temperatures was observed.

  20. Bisdesmosidic saponins from Securidaca longepedunculata roots: evaluation of deterrency and toxicity to Coleopteran storage pests.

    PubMed

    Stevenson, Philip C; Dayarathna, Thamara K; Belmain, Steven R; Veitch, Nigel C

    2009-10-14

    Powdered dry root bark of Securidaca longepedunculata was mixed with maize and cowpea and effectively reduced the numbers of Sitophilus zeamais and Callosobruchus maculatus emerging from these commodities, respectively, more than 9 months after treatment. This effect was reciprocated in grain treated with a methanol extract of the root bark, indicating that compounds were present that were oviposition deterrents or directly toxic to the adults or larvae. Two new bisdesmosidic saponins, 3-O-beta-D-glucopyranosyl-28-O-(alpha-L-arabinopyranosyl-(1 --> 3)-beta-D-xylopyranosyl-(1 --> 4)[beta-D-apiofuranosyl-(1 --> 3)]-alpha-L-rhamnopyranosyl-(1 --> 2)-[4-O-(4-methoxycinnamoyl-beta-D-fucopyranosyl)])-medicagenic acid (securidacaside A) and 3-O-beta-D-glucopyranosyl-28-O-(alpha-L-arabinopyranosyl-(1 --> 3)-beta-D-xylopyranosyl-(1 --> 4)[beta-D-apiofuranosyl-(1 --> 3)]-alpha-L-rhamnopyranosyl-(1 --> 2)-[4-O-(3,4,5-trimethoxy-(E)-cinnamoyl-beta-D-fucopyranosyl)])-medicagenic acid (securidacaside B), were isolated from the methanol extract of the roots of S. longepedunculata and characterized by spectroscopic methods. Securidacaside A, which occurred as (E)- and (Z)-regioisomers, showed deterrency and toxicity toward C. maculatus and S. zeamais and could contribute to the biological activity of the methanol extract. The potential to optimize the use of this plant for stored product protection using water extracts, which would be appropriate technology for target farmers, is discussed. PMID:19769365

  1. ICSBP is critically involved in the normal development and trafficking of Langerhans cells and dermal dendritic cells.

    PubMed

    Schiavoni, Giovanna; Mattei, Fabrizio; Borghi, Paola; Sestili, Paola; Venditti, Massimo; Morse, Herbert C; Belardelli, Filippo; Gabriele, Lucia

    2004-03-15

    Interferon consensus sequence-binding protein (ICSBP) is a transcription factor belonging to the interferon regulatory factor (IRF) family, recently shown to play a critical role in dendritic cell (DC) differentiation. Here, we analyzed the role of ICSBP in the development and trafficking of epidermal Langerhans cells (LCs) and dermal DCs and the implications for initiation of a competent immune response. ICSBP-/- mice exhibited a reduced frequency of LCs and a delayed mobility of DCs from skin that reflected a slower turnover rate in lymph nodes during steady-state conditions. Even under inflammatory changes, ICSBP-/- DCs displayed reduced mobility from skin to lymph nodes and, as a consequence, failed to induce a contact hypersensitivity (CHS) response, suggesting that these DCs were unable to initiate a competent antigen (Ag)-specific T-cell-mediated immunity. Moreover, bone marrow (BM)-derived DCs from ICSBP-/- mice exhibited an immature phenotype and a severe reduction of interleukin 12 (IL-12) expression. These BM DCs also showed a marked defect in their migratory response to macrophage inflammatory protein 3 alpha (MIP-3 alpha), MIP-3 beta, and the CC chemokine CCL21/6Ckine, which was paralleled by an impaired expression of the CC chemokine receptors, CCR6 and CCR7. Together, these results indicate that ICSBP is critically required for the development and trafficking of skin DCs, thus playing a critical role in the DC-mediated initiation of T-cell immunity. PMID:14615368

  2. Chemical constituents from the leaves of Boehmeria rugulosa with antidiabetic and antimicrobial activities.

    PubMed

    Semwal, Deepak Kumar; Rawat, Usha; Semwal, Ravindra; Singh, Randhir; Krishan, Pawan; Singh, Manjeet; Singh, Gur Jas Preet

    2009-12-01

    Three new flavonoid glycosides, named chalcone-6'-hydroxy-2',3,4-trimethoxy-4'-O-beta-D-glucopyranoside (1), isoflavone-3',4',5,6-tetrahydroxy-7-O-[beta-D-glucopyranosyl-(1-->3)-alpha-L-rhamnopyranoside] (2), and isoflavone-3',4',5,6-tetrahydroxy-7-O-[beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl-(1-->3)-alpha-L-rhamnopyranoside] (3), were isolated from the leaves of Boehmeria rugulosa, together with five known compounds, beta-sitosterol, quercetin, 3,4-dimethoxy-omega-(2'-piperidyl)-acetophenone (4), boehmeriasin A (5), and quercetin-7-O-beta-D-glucopyranoside. The structures of the isolated compounds were determined by means of chemical and spectral data including 2D NMR experiments. The ethanolic extract of leaves showed significant hypoglycemic activity on alloxan-induced diabetic mice. Glibenclamide, an oral hypoglycemic agent (5 mg/kg, p.o.), was used as a positive control. The ethanolic extract of the plant as well as the isolated compounds 1-3 (25 microg/ml) showed potent antimicrobial activity against two bacterial species (Staphylococcus aureus and Streptococcus mutans) and three fungus pathogens (Microsporum gypseum, Microsporum canis, and Trichophyton rubrum). The activities of the isolated compounds 1-3 have been compared with positive controls, novobiocin, and erythromycin (15 microg/ml). PMID:20183275

  3. Location of acyl groups of trehalose-containing lipooligosaccharides of mycobacteria.

    PubMed Central

    Camphausen, R T; McNeil, M; Jardine, I; Brennan, P J

    1987-01-01

    A variant of a Mycobacterium sp. originating in a patient with Crohn's disease, but not necessarily implicated in the disease, provided a simple version of a newer class of species-specific surface glycolipids, the trehalose-containing lipooligosaccharides. A combination of high-resolution 1H nuclear magnetic resonance, methylation, ethylation, and absolute configurational analysis established the structure of the oligosaccharide unit as beta-D-Glcp(1----3)-alpha-L-Rhap(1----3)-alpha-D-Glcp(1----1)-alph a-D-Glcp (where Glc is glucose, Rha is rhamnose, and p is pyranosyl), and gas chromatography-electron impact mass spectrometry allowed identification of the fatty acyl esters as primarily 2,4-dimethyltetradecanoate. The relative simplicity of the glycolipid combined with the application of a mild methylation procedure and californium-252 plasma desorption mass spectrometry allowed recognition of three such acyl residues on the 3-, 4-, and 6-hydroxyl positions of the terminal glucosyl residue of the trehalose unit. Thus, the glycolipid is decidedly amphipathic yet is clearly not membranous. This observation leads to speculation about the role of these novel lipooligosaccharides in contributing to the outer segment of the hydrophobic barrier of the cell wall of certain mycobacteria. Images PMID:3680168

  4. cDNA sequence coding for the alpha'-chain of the third complement component in the African lungfish.

    PubMed

    Sato, A; Sültmann, H; Mayer, W E; Figueroa, F; Tichy, H; Klein, J

    1999-04-01

    cDNA clones coding for almost the entire C3 alpha-chain of the African lungfish (Protopterus aethiopicus), a representative of the Sarcopterygii (lobe-finned fishes), were sequenced and characterized. From the sequence it is deduced that the lungfish C3 molecule is probably a disulphide-bonded alpha:beta dimer similar to that of the C3 components of other jawed vertebrates. The deduced sequence contains conserved sites presumably recognized by proteolytic enzymes (e.g. factor I) involved in the activation and inactivation of the component. It also contains the conserved thioester region and the putative site for binding properdin. However, the site for the interaction with complement receptor 2 and factor H are poorly conserved. Either complement receptor 2 and factor H are not present in the lungfish or they bind to different residues at the same or a different site than mammalian complement receptor 2 and factor H. The C3 alpha-chain sequences faithfully reflect the phylogenetic relationships among vertebrate classes and can therefore be used to help to resolve the long-standing controversy concerning the origin of the tetrapods. PMID:10219761

  5. Assessment of reinforcing effects of benztropine analogs and their effects on cocaine self-administration in rats: comparisons with monoamine uptake inhibitors.

    PubMed

    Hiranita, Takato; Soto, Paul L; Newman, Amy H; Katz, Jonathan L

    2009-05-01

    Benztropine (BZT) analogs inhibit dopamine uptake but are less effective than cocaine in producing behavioral effects predicting abuse liability. The present study compared reinforcing effects of intravenous BZT analogs with those of standard monoamine uptake inhibitors and the effects of their oral pretreatment on cocaine self-administration. Responding of rats was maintained by cocaine [0.032-1.0 mg/kg/injection (inj)] or food reinforcement under fixed-ratio five-response schedules. Maximal rates of responding were maintained by 0.32 mg/kg/inj cocaine or substituted methylphenidate, with lower rates maintained at lower and higher doses. The N-methyl BZT analog, AHN 1-055 (3alpha-[bis(4'-fluorophenyl)methoxy]-tropane), also maintained responding (0.1 mg/kg/inj), although maximal rates were less than those with cocaine. Responding was not maintained above vehicle levels by the N-allyl, AHN 2-005 (N-allyl-3alpha-[bis(4'-fluorophenyl)methoxy]-tropane), and N-butyl, JHW 007 [N-(n-butyl)-3alpha-[bis(4'-fluorophenyl)methoxy]-tropane], BZT analogs, and it was not maintained with nisoxetine or citalopram. Presession treatment with methylphenidate (3.2-32 mg/kg) dose-dependently shifted the cocaine self-administration dose-effect curve leftward, whereas nisoxetine and citalopram effects were not significant. An intermediate dose of AHN 1-055 (32 mg/kg) increased responding maintained by low cocaine doses and decreased responding maintained by higher doses. A higher dose of AHN 1-055 completely suppressed cocaine-maintained responding. Both AHN 2-005 and JHW 007 dose-dependently (10-32 mg/kg) decreased cocaine self-administration, shifting its dose-effect curve down. Decreases in cocaine-maintained responding occurred at doses of methylphenidate and BZT analogs that left food-maintained responding unchanged. During a component in which injections were not available, methylphenidate and AHN 1-055, but not AHN 2-005 or JHW 007, increased response rates. These findings further

  6. Molecular cloning and characterization of CIDE-3, a novel member of the cell-death-inducing DNA-fragmentation-factor (DFF45)-like effector family.

    PubMed

    Liang, Liang; Zhao, Mujun; Xu, Zhenhua; Yokoyama, Kazunari K; Li, Tsaiping

    2003-02-15

    DNA fragmentation is one of the critical steps in apoptosis, which is induced by DNA fragmentation factor (DFF). DFF is composed of two subunits, a 40 kDa caspase-activated nuclease (DFF40) and a 45 kDa inhibitor (DFF45). Recently a novel family of cell-death-inducing DFF45-like effectors (CIDEs) has been identified. Among CIDEs, two from human (CIDE-A and CIDE-B) and three from mouse (CIDE-A, CIDE-B and FSP27) have been reported. In this study human CIDE-3, a novel member of CIDEs, was identified upon sequence analysis of a previously unidentified cDNA that encoded a protein of 238 amino acids. It was shown to be a human homologue of mouse FSP27, and shared homology with the CIDE-N and CIDE-C domains of CIDEs. Apoptosis-inducing activity was clearly shown by DNA-fragmentation assay of the nuclear DNA of CIDE-3 transfected 293T cells. The expression pattern of CIDE-3 was different from that of CIDE-B. As shown by Northern-blot analysis, CIDE-3 was expressed mainly in human small intestine, heart, colon and stomach, while CIDE-B showed strong expression in liver and small intestine and at a lower level in colon, kidney and spleen. Green-fluorescent-protein-tagged CIDE-3 was revealed in some cytosolic corpuscles. Alternative splicing of the CIDE-3 gene was also identified by reverse transcription PCR, revealing that two transcripts, CIDE-3 and CIDE-3alpha, were present in HepG2 and A375 cells. CIDE-3 comprised a full-length open reading frame with 238 amino acids; in CIDE-3alpha exon 3 was deleted and it encoded a protein of 164 amino acids. Interestingly the CIDE-3alpha isoform still kept the apoptosis-inducing activity and showed the same pattern of subcellular localization as CIDE-3. Consistent with its chromosome localization at 3p25, a region associated with high frequency loss of heterozygosity in many tumours, CIDE-3 may play an important role in prevention of tumorigenesis.

  7. Analysis of collagen types synthesized by rabbit ear cartilage chondrocytes in vivo and in vitro.

    PubMed Central

    Madsen, K; von der Mark, K; van Menxel, M; Friberg, U

    1984-01-01

    This study compares the collagen types present in rabbit ear cartilage with those synthesized by dissociated chondrocytes in cell culture. The cartilage was first extracted with 4M-guanidinium chloride to remove proteoglycans. This step also extracted type I collagen. After pepsin solubilization of the residue, three additional, genetically distinct collagen types could be separated by fractional salt precipitation. On SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis they were identified as type II collagen, (1 alpha, 2 alpha, 3 alpha) collagen and M-collagen fragments, a collagen pattern identical with that found in hyaline cartilage. Types I, II, (1 alpha, 2 alpha, 3 alpha) and M-collagen fragments represent 20, 75, 3.5, and 1% respectively of the total collagen. In frozen sections of ear cartilage, type II collagen was located by immunofluorescence staining in the extracellular matrix, whereas type I collagen was closely associated with the chondrocytes. Within 24h after release from elastic cartilage by enzymic digestion, auricular chondrocytes began to synthesize type III collagen, in addition to the above-mentioned collagens. This was shown after labelling of freshly dissociated chondrocytes with [3H]proline 1 day after plating, fractionation of the pepsin-treated collagens from medium and cell layer by NaCl precipitation, and analysis of the fractions by CM(carboxymethyl)-cellulose chromatography and SDS/polyacrylamide-gel electrophoresis. The 0.8 M-NaCl precipitate of cell-layer extracts consisted predominantly of type II collagen. The 0.8 M-NaCl precipitate obtained from the medium contained type I, II, and III collagen. In the supernatant of the 0.8 M-NaCl precipitation remained, both in the cell extract and medium, predominantly 1 alpha-, 2 alpha-, and 3 alpha-chains and M-collagen fragments. These results indicate that auricular chondrocytes are similar to chondrocytes from hyaline cartilage in that they produce, with the exception of

  8. Exposure to inhalable dust, wheat flour and alpha-amylase allergens in industrial and traditional bakeries.

    PubMed

    Bulat, Petar; Myny, Katrien; Braeckman, Lutgart; van Sprundel, Marc; Kusters, Edouard; Doekes, Gert; Pössel, Kerstin; Droste, Jos; Vanhoorne, Michel

    2004-01-01

    This study was designed to characterize exposure to inhalable dust, wheat flour and alpha-amylase allergens in industrial and traditional bakeries. The study included 70 bakeries from the northern part of Belgium. Based on the degree of automation and a clear division of individual job tasks, four bakeries were identified as industrial and the remaining 66 were identified as traditional ones. Personal, as well as stationary, samples of inhalable dust were collected during full shift periods, usually 5-7 h. The portable pumps aspirated 2 l/min through Teflon personal dust samplers (Millipore, pore size 1.0 microm) mounted in PAS-6 sampling heads. In the collected samples the inhalable dust, wheat flour and alpha-amylase allergens were determined. Wheat flour allergens were measured using enzyme-linked immunosorbent assay inhibition and an antiwheat IgG4 serum pool. The alpha-amylase allergens were measured using a sandwich enzyme immunoassay with affinity-purified polyclonal rabbit IgG antibodies. In total, 440 samples (300 personal and 140 stationary) were processed. The highest inhalable dust exposure was observed in traditional bakeries among bread [geometric mean (GM) 2.10 mg/m3] and bread and pastry workers (GM 1.80 mg/m3). In industrial bakeries the highest dust exposure was measured in bread-producing workers (GM 1.06 mg/m3). Similar relations were observed for wheat flour and alpha-amylase allergens. Bread baking workers in traditional bakeries had the highest exposure to both allergens (wheat flour GM 22.33 microg/m(3), alpha-amylase GM 0.61 ng/m3). The exposure to wheat flour and alpha-amylase allergens in industrial bakeries was higher in bread baking workers (wheat flour GM 6.15 microg/m3, alpha-amylase GM 0.47 ng/m3) than in bread packing workers (wheat flour GM 2.79 microg/m3, alpha-amylase GM 0.15 ng/m3). The data presented suggest that, on average, exposure in the Belgium bakeries studied-industrial as well as traditional-is lower than or similar to

  9. Alpha Resonances in {sup 13}C Excited by the {sup 9}Be ({sup 6}Li,d) Reaction

    SciTech Connect

    Rodrigues, M. R. D.; Borello-Lewin, T.; Horodynski-Matsushigue, L. B.; Duarte, J. L. M.; Rodrigues, C. L.; Souza, M. A.; Miyake, H.; Cunsolo, A.; Cappuzzello, F.; Ukita, G. M.

    2010-05-21

    The {sup 9}Be({sup 6}Li,d){sup 13}C reaction was used to investigate alpha resonant states in {sup 13}C up to 13 MeV of excitation. The reaction was measured at a bombarding energy of 25.5 MeV employing the Sao Paulo Pelletron-Enge-Spectrograph facility and the nuclear emulsion detection technique. The resolution of 50 keV allowed for the separation of the resonant contributions to the known 7/2{sup -} at 10.753 MeV and (5/2{sup -}) at 10.818 MeV {sup 13}C states. The alpha resonance seen at the (3alpha+n) threshold was not previously reported. The experimental angular distributions are presented in comparison with DWBA predictions.

  10. A stereochemical examination of the equine metabolism of 17alpha-methyltestosterone.

    PubMed

    McKinney, Andrew R; Suann, Craig J; Stenhouse, Allen M

    2007-01-01

    An investigation was conducted into the stereochemistry of the equine urinary metabolites of 17alpha-methyltestosterone observed after oral administration. Standards of the complete range of C3/C5/C16 stereoisomeric 17alpha-methylandrostane-3,17beta-diols, 17alpha-methylandrostane-3,16,17beta-triols and 17alpha-hydroxymethylandrostane-3,17beta-diols were purchased or synthesised, and were used to unequivocally identify the absolute structures of the metabolites. Phase I metabolism was found to involve combinations of Delta(4)-3-ketone reduction with both 5alpha,3beta- and 5beta,3alpha-stereochemistry, hydroxylation at C16 with both 16alpha- and 16beta-stereochemistry and hydroxylation of the 17alpha-methyl substituent. Phase II metabolism involved mainly sulfation with a lesser degree of beta-glucuronidation.

  11. Genetic polymorphism of six plasma proteins in the domestic cat.

    PubMed

    Juneja, R K; Niini, T; Larsson, H E; Sandberg, K

    1991-01-01

    Phenotypes of cat plasma apolipoprotein A4 (APOA4), antithrombin 3 (AT3), alpha 1B-glycoprotein (A1BG), transferrin (TF), vitamin D-binding protein (GC), and an unidentified pretransferrin (PTF) were determined by using simple methods of horizontal, nondenaturing gel electrophoresis followed by protein staining. The cat proteins were identified by immunoblotting using antisera for human plasma proteins. Three alleles were reported for each of TF and PTF, and two alleles were reported for each of GC, APOA4, AT3, and A1BG. The mongrels and Persians showed a high degree of polymorphism at most of the loci whereas the Birmans exhibited much less variation. Genetic evidence indicating the occurrence of a monomeric and a dimeric form of APOA4 in cat plasma was reported. PMID:2013693

  12. Statistical methodology and assessment of seismic event characterization capability. Final report, 2 June 1993-2 September 1995

    SciTech Connect

    Fisk, M.D.; Gray, H.L.; McCartor, G.D.

    1995-10-31

    This project has focused on developing and applying statistical methods to perform seismic event characterization/identification and on quantifying capabilities with regard to monitoring of a Comprehensive Test Ban. An automated procedure is described to categorize seismic events, based on multivariate analysis of features derived from seismic waveforms. Second, preliminary event identification results are presented for a seismic event which occurred on 5 January 1995 in the Southern Ural Mountains region. Third, various statistics are compiled regarding 1786 seismic events which occurred between 11 January 1995 and 12 February 1995 and were detected by a set of 30 GSETT-3 Alpha stations. Fourth, a fundamental problem is addressed of how to utilize multivariate discriminant data from a multistation network in order to optimize the power of the outlier test for fixed false alarm rate.

  13. [Age Effect on Relationship Between Intelligence and EEG Characteristics].

    PubMed

    Belousova, L V; Razumnikova, O M; Volf, N V

    2015-01-01

    Age effect on EEG correlates of psychometrically estimated intelligence (IQ) in the younger (N = 132, age mean = 21.8 ± 3.1) and elder groups (N = 84, age mean = 64.1 ± 6.6) was studied. Regression analysis of individual alpha peak frequency's meanings, total power of biopotentials in eight frequency ranges indicated that a decrease of IQ correlates with age increase, or with decrease of individual alpha peak frequency with positive contribution of the alpha3 power and negative--of the beta1. High meaning of the alpha3 power and low meaning of the beta1 are the predictors of high intelligence in the younger group. High intelligence in the elder group is accompanied by a trend to increase of the individual alpha peak frequency and to decrease of the theta/beta1 power ration together with significant decrease of the alpha3/alpha2 power ratio.

  14. Subpellicular and flagellar microtubules of Trypanosoma brucei brucei contain the same alpha-tubulin isoforms

    PubMed Central

    1987-01-01

    The cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei brucei essentially consists of two microtubule-based structures: a subpellicular layer of singlet microtubules, which are in close contact with the cell membrane, and the flagellar axoneme. In addition, the cells contain a small pool of soluble tubulin. Two-dimensional gel electrophoretic analysis of the tubulins present in these subcellular compartments revealed two distinct electrophoretic isoforms of alpha- tubulin, termed alpha 1 and alpha 3. alpha 1-Tubulin most likely represents the primary translation product, while alpha 3-tubulin is a posttranslationally acetylated derivative of alpha 1-tubulin. In the pool of soluble cytoplasmic tubulin, alpha 1 is the predominant species, while the very stable flagellar microtubules contain almost exclusively the alpha 3-tubulin isoform. The subpellicular microtubules contain both isoforms. Neither of the two alpha-tubulin isoforms is organelle specific, but the alpha 3 isoform is predominantly located in stable microtubules. PMID:3818788

  15. Evidence That GABA Mediates Dopaminergic and Serotonergic Pathways Associated with Locomotor Activity in Juvenile Chinook Salmon (Oncorhynchus tshawytscha)

    USGS Publications Warehouse

    Clements, S.; Schreck, C.B.

    2004-01-01

    The authors examined the control of locomotor activity in juvenile salmon (Oncorhynchus tshawytscha) by manipulating 3 neurotransmitter systems-gamma-amino-n-butyric acid (GABA), dopamine, and serotonin-as well as the neuropeptide corticotropin releasing hormone (CRH). Intracerebroventricular (ICV) injections of CRH and the GABAAagonist muscimol stimulated locomotor activity. The effect of muscimol was attenuated by administration of a dopamine receptor antagonist, haloperidol. Conversely, the administration of a dopamine uptake inhibitor (4???,4??? -difluoro-3-alpha-[diphenylmethoxy] tropane hydrochloride [DUI]) potentiated the effect of muscimol. They found no evidence that CRH-induced hyperactivity is mediated by dopaminergic systems following concurrent injections of haloperidol or DUI with CRH. Administration of muscimol either had no effect or attenuated the locomotor response to concurrent injections of CRH and fluoxetine, whereas the GABAA antagonist bicuculline methiodide potentiated the effect of CRH and fluoxetine.

  16. New (alpha-Hydroxyalkyl)phosphorus Amphiphiles: Synthesis and Dissociation Constants.

    PubMed

    Albouy, Dominique; Brun, Alice; Munoz, Aurelio; Etemad-Moghadam, Guita

    1998-10-16

    Direct synthesis of free (alpha-hydroxyalkyl)phosphinic acid amphiphiles 1 can be readily realized by sonication of the heterogeneous mixture of 50% aqueous hypophosphorous acid and long-chain aldehydes in the presence of catalytic amounts of hydrochloric acid. Oxidation of these phosphinic acids by DMSO in the presence of catalytic amounts of iodine quantitatively leads to the corresponding phosphonic acids 3. IR spectra of the phosphinic acids 1 in the condensed phase and in solution reveal the presence of intra- and intermolecular associations. Dissociation constants of the phosphorus acids 1 and 3 determined by potentiometric and (31)P NMR titrations show a good correlation between the two methods. The phosphinic acid amphiphiles 1 are slightly stronger than the corresponding phosphonic acids 3. (alpha-Hydroxyalkyl)phosphonium chlorides are prepared in good yields from the phosphine PH(3) and long-chain aldehydes in acidic media.

  17. Acidic attack of perfluorinated alkyl ether lubricant molecules by metal oxide surfaces

    NASA Technical Reports Server (NTRS)

    Zehe, Michael J.; Faut, Owen D.

    1989-01-01

    The reactions of linear perfluoropolyalkylether (PFAE) lubricants with alpha-Fe2O3 and Fe2O3-based solid superacids were studied. The reaction with alpha-Fe2O3 proceeds in two stages. The first stage is an initial slow catalytic decomposition of the fluid. This reaction releases reactive gaseous products which attach the metal oxide and convert it to FeF3. The second stage is a more rapid decomposition of the fluid, effected by the surface FeF3. A study of the inital breakdown step was performed using alpha-Fe2O3, alpha-Fe2O3 preconverted to FeF3, and sulfate-promoted alpha-Fe2O3 superacids. The results indicate that the breakdown reaction involves acidic attack at fluorine atoms on acetal carbons in the linear PFAE. Possible approaches to combat the problem are outlined.

  18. Symplectic structure and monopole strength in {sup 12}C

    SciTech Connect

    Yoshida, T.; Itagaki, N.; Kato, K.

    2011-02-15

    The relation between the monopole transition strength and existence of cluster structure in the excited states is discussed based on an algebraic cluster model. The structure of {sup 12}C is studied with a 3{alpha} model, and the wave function for the relative motions between {alpha} clusters are described by the symplectic algebra Sp(2, R){sub z}, which corresponds to the linear combinations of SU(3) states with different multiplicities. Introducing Sp(2,R){sub z} algebra works well for reducing the number of the basis states, and it is also shown that states connected by the strong monopole transition are classified by a quantum number {Lambda} of the Sp(2,R){sub z} algebra.

  19. Magnetic structures of actinide materials by pulsed neutron diffraction

    SciTech Connect

    Lawson, A.C.; Goldstone, J.A.; Huber, J.G.; Giorgi, A.L.; Conant, J.W.; Severing, A.; Cort, B.; Robinson, R.A.

    1990-01-01

    We describe some attempts to observe magnetic structure in various actinide (5f-electron) materials. Our experimental technique is neutron powder diffraction as practiced at a spallation (pulsed) neutron source. We will discuss our investigations of {alpha}-Pu, {delta}-Pu, {alpha}-UD{sub 3} and {beta}-UD{sub 3}. {beta}-UD{sub 3} is a simple ferromagnet: surprisingly, the moments on the two non-equivalent uranium atoms are the same within experimental error. {alpha}-UD{sub 3}, {alpha}-Pu and {delta}-Pu are non-magnetic, within the limits of our observations. Our work with pulsed neutron diffraction shows that it is a useful technique for research on magnetic materials.

  20. Large third-order optical nonlinearity in Au:SiO{sub 2} composite films near the percolation threshold

    SciTech Connect

    Liao, H.B.; Xiao, R.F.; Fu, J.S.; Yu, P.; Wong, G.K.; Sheng, P.

    1997-01-01

    Very large third-order optical nonlinearity, {chi}{sup (3)}{approximately}2.5{times}10{sup {minus}6} esu, measured by a degenerate four wave mixing method using a short pulse (70 picosecond) laser, has been found in the rapid-thermal annealed Au:SiO{sub 2} composite films at concentrations below the Au percolation threshold. The dependence of the {chi}{sup (3)} on Au concentration, p, follows a cubic power law. The maximum figure of merit, {chi}{sup (3)}/{alpha} (with {alpha} being the absorption coefficient) is about 10{sup {minus}11} esu cm. We explain this result as due to local field enhancement arising from the Mie resonance of the Au nanoclusters, with strong interaction between the nanoclusters further promoting the effect. {copyright} {ital 1997 American Institute of Physics.}

  1. Calculation of radiation therapy dose using all particle Monte Carlo transport

    DOEpatents

    Chandler, W.P.; Hartmann-Siantar, C.L.; Rathkopf, J.A.

    1999-02-09

    The actual radiation dose absorbed in the body is calculated using three-dimensional Monte Carlo transport. Neutrons, protons, deuterons, tritons, helium-3, alpha particles, photons, electrons, and positrons are transported in a completely coupled manner, using this Monte Carlo All-Particle Method (MCAPM). The major elements of the invention include: computer hardware, user description of the patient, description of the radiation source, physical databases, Monte Carlo transport, and output of dose distributions. This facilitated the estimation of dose distributions on a Cartesian grid for neutrons, photons, electrons, positrons, and heavy charged-particles incident on any biological target, with resolutions ranging from microns to centimeters. Calculations can be extended to estimate dose distributions on general-geometry (non-Cartesian) grids for biological and/or non-biological media. 57 figs.

  2. Calculation of radiation therapy dose using all particle Monte Carlo transport

    DOEpatents

    Chandler, William P.; Hartmann-Siantar, Christine L.; Rathkopf, James A.

    1999-01-01

    The actual radiation dose absorbed in the body is calculated using three-dimensional Monte Carlo transport. Neutrons, protons, deuterons, tritons, helium-3, alpha particles, photons, electrons, and positrons are transported in a completely coupled manner, using this Monte Carlo All-Particle Method (MCAPM). The major elements of the invention include: computer hardware, user description of the patient, description of the radiation source, physical databases, Monte Carlo transport, and output of dose distributions. This facilitated the estimation of dose distributions on a Cartesian grid for neutrons, photons, electrons, positrons, and heavy charged-particles incident on any biological target, with resolutions ranging from microns to centimeters. Calculations can be extended to estimate dose distributions on general-geometry (non-Cartesian) grids for biological and/or non-biological media.

  3. Hormonal monitoring of age at sexual maturation in female Goeldi's monkeys (Callimico goeldii) in their family groups.

    PubMed

    Dettling, A; Pryce, C R

    1999-01-01

    The aim of this study was to determine whether or not sexual maturation is attained in the family group in captive-born Goeldi's monkey (Callimico goeldii) and if so, at what age and body weight. To monitor ovarian activity in 14 female Goeldi's monkeys, urinary content of pregnanediol-3alpha-glucuronide (PdG) was determined using radioimmunoassay. Urinary samples were collected between the ages of 6 and 70 weeks. Subjects became sexually mature while still housed in their family groups, at a median age of 57 weeks (48-< 70 weeks). Median body weight at the age of sexual maturity was 473 g (N=10; 420-543 g). This corresponded to 90% of the median non-pregnant body weight of breeding females in our colony (526 g, N=8). Therefore, Goeldi's monkey is similar to Leontopithecus but different from Cebuella, Callithrix, and Saguinus, in terms of daughters ovulating in the family group and at a relatively young age.

  4. In vitro metabolism of androstenedione and identification of endogenous steroids in Helix aspersa

    SciTech Connect

    Le Guellec, D.; Thiard, M.C.; Remy-Martin, J.P.; Deray, A.; Gomot, L.; Adessi, G.L.

    1987-06-01

    In vitro metabolism of androstenedione in gonads of juvenile and adult Helix aspersa has been investigated. The conversion of (/sup 3/H)androstenedione into testosterone, 5 alpha-dihydrotestosterone, androsterone, and estriol was demonstrated. In juvenile animals testosterone (59.8%) is the major metabolite whereas in adult animals androsterone (18.8%) is. The following endogenous steroids have been identified by gas chromatography-mass spectrometry in adult gonads: androsterone, dehydroepiandrosterone, androstenedione, 3 alpha-androstanediol, estrone, estradiol-17 beta, and estriol. The levels of testosterone, 5 alpha-dihydrotestosterone, androstenedione, and progesterone have been measured by RIAs in gonads and hemolymph. Their levels vary with the physiological stage: the gonadal and circulating levels of testosterone decrease with the sexual maturation whereas the 5 alpha-dihydrotestosterone increases. These differences observed in metabolism and in level of steroids between the juvenile and the adult snails allow us to suppose that these steroids have a biological role.

  5. Lupane-triterpene glycosides from the leaves of Acanthopanax gracilistylus.

    PubMed

    Yook, Chang-Soo; Liu, Xiang-Qian; Chang, Seung-Yeup; Park, Sang-Yong; Nohara, Toshihiro

    2002-10-01

    A novel lupane-triterpene glycoside, called wujiapioside B (1), was isolated from the leaves of Acanthopanax gracilistylus (Araliaceae) together with three known lupane-triterpene glycosides, acankoreoside C (2), acantrifoside A (3) and 3-epibetulinic acid 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester (4). Based on spectroscopic data, the chemical structure of 1 was determined as 3alpha,23-dihydroxy-lup-20(29)-en-28-oic acid 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester. Compounds 2-3 were obtained for the first time from this plant and compound 4 has not been isolated from Acanthopanax genus yet. PMID:12372869

  6. Development of gluten-free bread using tartary buckwheat and chia flour rich in flavonoids and omega-3 fatty acids as ingredients.

    PubMed

    Costantini, Lara; Lukšič, Lea; Molinari, Romina; Kreft, Ivan; Bonafaccia, Giovanni; Manzi, Laura; Merendino, Nicolò

    2014-12-15

    In this study, chia seed flour, which is rich in omega-3 alpha-linolenic acid, and common and tartary buckwheat flour, which has a high antioxidant activity, were integrated into different types of bread with the aim of improving their nutritional value and healthy features. Our results indicate that bread made with chia and tartary buckwheat flour was more acceptable in many nutritional aspects compared to the control (common wheat bread); it contained a higher amount of protein (20%), insoluble dietary fibres (74%), ash (51%), and alpha-linolenic acid (67.4%). Moreover, this bread possessed lower energy (14%) and carbohydrate contents (24%) compared to the control. Tartary buckwheat also improved the total antioxidant capacity of the bread (about 75%) and provided a considerable amount of flavonoids, which are healthy non-nutritional compounds. Overall, chia and tartary buckwheat represent excellent raw materials for the formulation of gluten-free bread with high nutritional value.

  7. Purification and characterization of a 4-hydroxybiphenyl UDP-glucuronosyltransferase from rat liver microsomes

    SciTech Connect

    Styczynski, B.; Green, M.; Coffman, B.; Puig, J.; Tephly, T. )

    1991-03-11

    A phenobarbital-inducible rat liver microsomal UDP-glucuronosyltransferase (4-HBP UDPGT) which catalyzes the glucuronidation of 4-hydroxybiphenyl has been purified to homogeneity. The apparent subunit molecular weight of this protein is 52,500 as determined by SDS-PAGE. 4-HBP UDPGT was shown to react with 4-hydroxybiphenyl, p-nitrophenol and 4-methylumbelliferone, but did not react with morphine, testosteron or chloramphenicol. Upon treatment with Endoglycosidase H, the 4-HBP UDPGT underwent about a 2,000 dalton decrease in subunit molecular weight, suggesting that this protein in N-glycosylated. Western blot analysis has revealed immunorecognition of 4-HBP UDPGT by antibodies raised in rabbit against rat 3{alpha}- and 17{beta}-hydroxysteroid UDPGTs. Additionally, the authors have obtained the N-terminal amino acid sequence of 4-HBP UDPGT. These data provide evidence which suggests that this protein is different from another UDPGT previously shown to react with 4-hydroxybiphenyl, testosterone and chloramphenicol.

  8. Is there A Role for Alpha-Linolenic Acid in the Fetal Programming of Health?

    PubMed

    Leikin-Frenkel, Alicia I

    2016-03-23

    The role of ω3 alpha linolenic acid (ALA) in the maternal diet during pregnancy and lactation, and its effect on the prevention of disease and programming of health in offspring, is largely unknown. Compared to ALA, ω3 docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids have been more widely researched due to their direct implication in fetal neural development. In this literature search we found that ALA, the essential ω3 fatty acid and metabolic precursor of DHA and EPA has been, paradoxically, almost unexplored. In light of new and evolving findings, this review proposes that ALA may have an intrinsic role, beyond the role as metabolic parent of DHA and EPA, during fetal development as a regulator of gene programming for the prevention of metabolic disease and promotion of health in offspring.

  9. Use of morphology index histograms to quantify populations of the fungal pathogen Paracoccidioides brasiliensis.

    PubMed

    San-Blas, G; Padrón, R; Alamo, L; San-Blas, F

    1997-01-01

    To quantify the dimorphic process in wild and mutant strains of Paracoccidioides brasiliensis, we defined a morphology index (Mi) in terms of the maximum cell length (l), maximum cell diameter (d), and septal diameter (s), according to the equation Mi = 2.13 + 1.13 log10 (ls/d2), whose intercept and slope were such that Mi was around 1 for yeast (spherical) cells or 4 for hyphal (elongated) cells. This discriminatory power was used to quantify morphological population mixtures through Mi histograms. During the temperature-induced dimorphic transition (either way), mean Mi (Mi) varied linearly with time, suggesting a continuity in the process. Also, in wild strains and mutants thereof we found an inverse relationship between Mi and content of both cell wall chitin and 1,3-alpha-glucan.

  10. Alpha and gamma band oscillations index differential processing of acoustically reduced and full forms.

    PubMed

    Drijvers, Linda; Mulder, Kimberley; Ernestus, Mirjam

    2016-02-01

    Reduced forms like yeshay for yesterday often occur in conversations. Previous behavioral research reported a processing advantage for full over reduced forms. The present study investigated whether this processing advantage is reflected in a modulation of alpha (8-12Hz) and gamma (30+Hz) band activity. In three electrophysiological experiments, participants listened to full and reduced forms in isolation (Experiment 1), sentence-final position (Experiment 2), or mid-sentence position (Experiment 3). Alpha power was larger in response to reduced forms than to full forms, but only in Experiments 1 and 2. We interpret these increases in alpha power as reflections of higher auditory cognitive load. In all experiments, gamma power only increased in response to full forms, which we interpret as showing that lexical activation spreads more quickly through the semantic network for full than for reduced forms. These results confirm a processing advantage for full forms, especially in non-medial sentence position. PMID:26878718

  11. Isolation and characterization of Exodus-2, a novel C-C chemokine with a unique 37-amino acid carboxyl-terminal extension.

    PubMed

    Hromas, R; Kim, C H; Klemsz, M; Krathwohl, M; Fife, K; Cooper, S; Schnizlein-Bick, C; Broxmeyer, H E

    1997-09-15

    Chemokines are a group of small, homologous proteins that regulate leukocyte migration, hemopoiesis, and HIV-1 absorption. We report here the cloning and characterization of a novel murine and human C-C chemokine termed Exodus-2 for its similarity to Exodus-1/MIP-3alpha/LARC, and its chemotactic ability. This novel chemokine has a unique 36 or 37 (murine and human, respectively) amino acid carboxyl-terminal extension not seen in any other chemokine family member. Purified recombinant Exodus-2 was found to have two activities classically associated with chemokines: inhibiting hemopoiesis and stimulating chemotaxis. However, Exodus-2 also had unusual characteristics for C-C chemokines. It selectively stimulated the chemotaxis of T-lymphocytes and was preferentially expressed in lymph node tissue. The combination of these characteristics may be a functional correlate for the unique carboxyl-terminal structure of Exodus-2.

  12. Is there A Role for Alpha-Linolenic Acid in the Fetal Programming of Health?

    PubMed Central

    Leikin-Frenkel, Alicia I.

    2016-01-01

    The role of ω3 alpha linolenic acid (ALA) in the maternal diet during pregnancy and lactation, and its effect on the prevention of disease and programming of health in offspring, is largely unknown. Compared to ALA, ω3 docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids have been more widely researched due to their direct implication in fetal neural development. In this literature search we found that ALA, the essential ω3 fatty acid and metabolic precursor of DHA and EPA has been, paradoxically, almost unexplored. In light of new and evolving findings, this review proposes that ALA may have an intrinsic role, beyond the role as metabolic parent of DHA and EPA, during fetal development as a regulator of gene programming for the prevention of metabolic disease and promotion of health in offspring. PMID:27023621

  13. Studies on synthetic pathway of xylose-containing N-linked oligosaccharides deduced from substrate specificities of the processing enzymes in sycamore cells (Acer pseudoplatanus L.).

    PubMed

    Tezuka, K; Hayashi, M; Ishihara, H; Akazawa, T; Takahashi, N

    1992-02-01

    We measured the activities of alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase, alpha-1,6-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase, beta-1,4-mannosyl-glycoprotein beta-1,2-xylosyltransferase and glycoprotein 3-alpha-L-fucosyltransferase in the Golgi fraction of suspension-cultured cells of sycamore (Acer pseudoplatanus L.) using fluorescence-labelled oligosaccharides as acceptor substrates for these transferase reactions. The structures of the pyridylaminated oligosaccharides produced by these reactions were analyzed by two-dimensional sugar mapping using high-performance liquid chromatography. We demonstrated that (formula; see text) was processed to produce by these in vitro reactions. On the basis of these results, we discuss a biosynthetic pathway for xylose containing N-linked oligosaccharides in plant glycoproteins.

  14. Metabolites from the Lichen Ochrolechia parella growing under two different heliotropic conditions.

    PubMed

    Millot, Marion; Tomasi, Sophie; Articus, Kristina; Rouaud, Isabelle; Bernard, Aurélie; Boustie, Joël

    2007-02-01

    A new chloro-depsidone (1) and five known compounds, variolaric acid (2), lecanoric acid (3), alpha-alectoronic acid (4), atranorin (5), and ergosterol peroxide (6), have been isolated from the lichen Ochrolechia parella. The structure of compound 1 was elucidated by spectroscopic analysis. Additionally, the tautomeric equilibrium of compound 4 was investigated. In the present study, two specimens of this lichen, growing under different light conditions, were analyzed. The major compound in both samples was found to be 2, but the amount of this metabolite was significantly higher in the shaded specimen (0.76% w/w). The new compound parellin (1) predominated in the specimen grown under shady conditions, while atranorin (5) was found only in the sunlit specimen. The cytotoxic activities of 2, 4, and 6 against B16 melanoma cells were evaluated. PMID:17256903

  15. Antifungal constituents from the Chinese moss Homalia trichomanoides.

    PubMed

    Wang, Xiao-ning; Yu, Wen-tao; Lou, Hong-xiang

    2005-01-01

    Bioautographic assay on TLC plates was adopted to guide the fractionation of the Et2O extract of Homalia trichomanoides (Hedw.) B. S. G., which led to the isolation of the novel p-terphenyl derivative trichomanin (= 4,4''-dihydroxy-1,1':4',1''-terphenyl-2',3',5',6'-tetrayl tetrakis(phenylacetate); 1), together with five known compounds: 3alpha-methoxyserrat-14-en-21beta-ol (2), 3beta-methoxyserrat-14-en-21beta-ol (3), 3beta-methoxyserrat-14-en-21-one (4), atranorin (5), and methyl 2,4-dihydroxy-3,6-dimethylbenzoate (6). Their structures were determined on the basis of spectral data (1D- and 2D-NMR, MS), X-ray crystallographic analysis, and chemical transformation. Compounds 3, 5, and 6 exhibited antifungal activity against Candida albicans, with minimum inhibitory doses (MID) of 2.0, 2.0, and 0.6 microg, respectively. PMID:17191927

  16. Isolation of glucose-containing high-mannose glycoprotein core oligosaccharides.

    PubMed Central

    Tsai, P K; Ballou, L; Esmon, B; Schekman, R; Ballou, C E

    1984-01-01

    The total cell wall mannoprotein has been isolated from a mutant of Saccharomyces cerevisiae that fails to remove the glucose units of the dolichol-linked precursor after transfer of the oligosaccharide to asparagine units in the protein. The oligosaccharides released from this mannoprotein by endoglucosaminidase H digestion show 1H NMR signals assignable to three alpha-linked glucose units as delta 5.52, 5.27, and 5.17, and a comparison with the chemical shifts of reference compounds shows that these signals are consistent with the structure alpha Glc----2 alpha Glc----3 alpha Man----2. This provides a direct confirmation for the structure previously assigned to the lipid-linked precursor. Analysis of the larger oligosaccharides confirms that the presence of the glucose units does not prevent elongation of the alpha 1----6-linked polymannose backbone or addition of alpha 1----3-linked mannose to the core. Images PMID:6387703

  17. Isolation of glucose-containing high-mannose glycoprotein core oligosaccharides.

    PubMed

    Tsai, P K; Ballou, L; Esmon, B; Schekman, R; Ballou, C E

    1984-10-01

    The total cell wall mannoprotein has been isolated from a mutant of Saccharomyces cerevisiae that fails to remove the glucose units of the dolichol-linked precursor after transfer of the oligosaccharide to asparagine units in the protein. The oligosaccharides released from this mannoprotein by endoglucosaminidase H digestion show 1H NMR signals assignable to three alpha-linked glucose units as delta 5.52, 5.27, and 5.17, and a comparison with the chemical shifts of reference compounds shows that these signals are consistent with the structure alpha Glc----2 alpha Glc----3 alpha Man----2. This provides a direct confirmation for the structure previously assigned to the lipid-linked precursor. Analysis of the larger oligosaccharides confirms that the presence of the glucose units does not prevent elongation of the alpha 1----6-linked polymannose backbone or addition of alpha 1----3-linked mannose to the core. PMID:6387703

  18. Layered cathode materials for lithium ion rechargeable batteries

    DOEpatents

    Kang, Sun-Ho; Amine, Khalil

    2007-04-17

    A number of materials with the composition Li.sub.1+xNi.sub..alpha.Mn.sub..beta.Co.sub..gamma.M'.sub..delta.O.sub.2-- zF.sub.z (M'=Mg,Zn,Al,Ga,B,Zr,Ti) for use with rechargeable batteries, wherein x is between about 0 and 0.3, .alpha. is between about 0.2 and 0.6, .beta. is between about 0.2 and 0.6, .gamma. is between about 0 and 0.3, .delta. is between about 0 and 0.15, and z is between about 0 and 0.2. Adding the above metal and fluorine dopants affects capacity, impedance, and stability of the layered oxide structure during electrochemical cycling.

  19. Isolation of the mouse (MFH-1) and human (FKHL14) mesenchyme fork head-1 genes reveals conservation of their gene and protein structures

    SciTech Connect

    Miura, Naoyuki; Iida, Kiyoshi; Yang, Xiao-Li

    1997-05-01

    The very recently found evolutionarily conserved DNA-binding domain of 100 amino acids, termed the fork head domain, emerged from a sequence comparison of the rat hepatocyte transcription factor HNF-3{alpha} and the homeotic gene fork head of Drosophila. We previously isolated a new member of this family, the mesenchyme fork head-1 (MFH-1) gene, which is expressed in developing mesenchyme. Here we describe the isolation of the mouse (MFH-1) and human (FKHL14) chromosomal MFH-1 genes and the determination of the gene and protein structures of MFH-1. We found that the MFH-1 gene has no introns and that the identity of the amino acid sequences of mouse and human MFH-1 proteins is 94%. We also investigated the transcriptional activity of the mouse and human MFH-1 proteins and found that both proteins act as positive transactivators. 31 refs., 3 figs.

  20. The synthesis and characterization of analogs of the antimicrobial compound squalamine: 6 beta-hydroxy-3-aminosterols synthesized from hyodeoxycholic acid.

    PubMed

    Jones, S R; Kinney, W A; Zhang, X; Jones, L M; Selinsky, B S

    1996-10-01

    Analogs of the aminosterol antimicrobial agent squalamine have been synthesized beginning from hyodeoxycholic acid. After carboxylic acid esterification and oxidation of both alcohol functions to ketones, the A/B ring junction was converted from cis to trans by acid-catalyzed isomerization. Different polyamines were added to the 3-keto group by reductive amination, yielding both the 3 alpha and 3 beta addition products. The synthetic products exhibited potent, broad-spectrum antimicrobial activity similar to that of the parent compound. Changing the identity of the polyamine or the stereochemistry of addition has little effect upon antimicrobial activity but appears to change the selectivity of the agents. The analogs are synthesized with high yield from inexpensive starting materials and are promising alternatives to squalamine as potential antibiotics. PMID:8910969

  1. Design, synthesis and biological evaluation of nonpeptide integrin antagonists.

    PubMed

    Nicolaou, K C; Trujillo, J I; Jandeleit, B; Chibale, K; Rosenfeld, M; Diefenbach, B; Cheresh, D A; Goodman, S L

    1998-08-01

    Recent studies demonstrated that peptide and antibody antagonists of integrin alpha v beta 3 block angiogenesis and tumor growth. In this article, the design, synthesis and biological evaluation of a series of nitroaryl ether-based, nonpeptide mimetics are described. The design of these compounds was based on Merck's arylether/alpha-aminoacid/guanidine framework and incorporates a novel nitroaryl system. The synthesized mimetics were tested against a variety of integrins (alpha v beta 3, alpha IIb beta 3, and alpha v beta 5) in order to determine their binding selectivity and ability to inhibit cell adhesion. Selected compounds were also tested for their ability to inhibit angiogenesis in vivo in the CAM (chick chorioallantoic membrane) assay. From the generated compound library, compounds 16 and 19 proved to be potent and selective inhibitors of alpha IIb beta 3 (IC50 = 14 nM) whereas compound 11 showed excellent in vivo inhibition of angiogenesis (at 30 micrograms/embryo).

  2. GDP-fucose:GM1 alpha 1----2fucosyltransferase is activated in parenchymal cells of rat liver during early stages of N-2-acetylaminofluorene induced hepatocarcinogenesis.

    PubMed

    Holmes, E H

    1990-01-01

    Gangliosides from liver parenchymal and non-parenchymal cells were isolated from Fischer 344 rats that had been fed normal diet or a diet supplemented with 0.03% N-2-acetylaminofluorene (AAF) for 4 weeks. Gangliosides from liver cell fractions were characterized by an induction of both II3NeuAcIV3 alpha GalIV2FucGg4 and GM3 synthesis in the parenchymal cells of AAF-fed animals which were missing in parenchymal cells from animals fed normal diet. In addition, new bands corresponding to GM1 and GD1a were observed in cell fractions of AAF-fed animals. The activity of the GM1-specific alpha 1----2fucosyltransferase induced after AAF feeding was found to be enriched 5- to 6-fold in the parenchymal cell fraction of AAF-fed animals and correlated with the parenchymal cell marker enzyme glucose-6-phosphatase in these cell fractions. Feeding animals the hepatotoxin acetaminophen at 1.87% in the diet for 10 weeks resulted in no increase in the levels of the alpha 1----2fucosyltransferase. Antibodies specific for II3NeuAcIV3 alpha GalIV2FucGg4 were produced and utilized in tissue localization studies. These results indicated little or no staining of normal liver tissue or that after acetaminophen feeding was observed. In contrast, focal areas of staining of liver tissue from animals after 3 weeks of 0.03% AAF feeding were readily apparent. These results indicate that induction of alpha 1----2fucosyltransferase and fucoganglioside synthesis is most probably a property of liver parenchymal cells and is associated with events occurring during early stages of AAF-induced carcinogenesis. PMID:2295130

  3. Determining structure and function of steroid dehydrogenase enzymes by sequence analysis, homology modeling, and rational mutational analysis.

    PubMed

    Duax, William L; Thomas, James; Pletnev, Vladimir; Addlagatta, Anthony; Huether, Robert; Habegger, Lukas; Weeks, Charles M

    2005-12-01

    The short-chain oxidoreductase (SCOR) family of enzymes includes over 6,000 members identified in sequenced genomes. Of these enzymes, approximately 300 have been characterized functionally, and the three-dimensional crystal structures of approximately 40 have been reported. Since some SCOR enzymes are steroid dehydrogenases involved in hypertension, diabetes, breast cancer, and polycystic kidney disease, it is important to characterize the other members of the family for which the biological functions are currently unknown and to determine their three-dimensional structure and mechanism of action. Although the SCOR family appears to have only a single fully conserved residue, it was possible, using bioinformatics methods, to determine characteristic fingerprints composed of 30-40 residues that are conserved at the 70% or greater level in SCOR subgroups. These fingerprints permit reliable prediction of several important structure-function features including cofactor preference, catalytic residues, and substrate specificity. Human type 1 3beta-hydroxysteroid dehydrogenase isomerase (3beta-HSDI) has 30% sequence identity with a human UDP galactose 4-epimerase (UDPGE), a SCOR family enzyme for which an X-ray structure has been reported. Both UDPGE and 3-HSDI appear to trace their origins back to bacterial 3alpha,20beta-HSD. Combining three-dimensional structural information and sequence data on the 3alpha,20beta-HSD, UDPGE, and 3beta-HSDI subfamilies with mutational analysis, we were able to identify the residues critical to the dehydrogenase function of 3-HSDI. We also identified the residues most probably responsible for the isomerase activity of 3beta-HSDI. We test our predictions by specific mutations based on sequence analysis and our structure-based model.

  4. Relationship between in vivo occupancy at the dopamine transporter and behavioral effects of cocaine, GBR 12909 [1-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-4-(3-phenylpropyl)piperazine], and benztropine analogs.

    PubMed

    Desai, Rajeev I; Kopajtic, Theresa A; French, Dawn; Newman, Amy H; Katz, Jonathan L

    2005-10-01

    Analogs of benztropine (BZT) bind to the dopamine (DA) transporter and inhibit DA uptake but often have behavioral effects that differ from those of cocaine and other DA-uptake inhibitors. To better understand these differences, we examined the relationship between locomotor-stimulant effects of cocaine, 1-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-4-(3-phenylpropyl)-piperazine (GBR 12909), and BZT analogs [(3alpha-[bis(4'-fluorophenyl)methoxy]-tropane) (AHN 1-055) and (N-allyl-3alpha-[bis(4'-fluorophenyl)methoxy]-tropane) (AHN 2-005)] and their in vivo displacement of the DA transporter ligand [125I]3beta-(4-iodophenyl)-tropan-2beta-carboxylic acid isopropyl ester hydrochloride (RTI-121) in striatum. Cocaine, GBR 12909, and BZT analogs each displaced [125I]RTI-121 and stimulated locomotor activity in a dose- and time-dependent manner. The time course revealed a slower onset of both effects for AHN 1-055 and AHN 2-005 compared with cocaine and GBR 12909. The BZT analogs were less effective than cocaine and GBR 12909 in stimulating locomotor activity. Locomotor stimulant effects of cocaine were generally greater than predicted by the regression of displacement of [125I]RTI-121 and effect at short times after injection and less than predicted at longer times after injection. This result suggests that the apparent rate of occupancy of the DA transporter, in addition to percentage of sites occupied, contributes to the behavioral effects of cocaine. The present results suggest that among drugs that act at the DA transporter, the slower apparent rates of occupancy with the DA transporter by the BZT analogs may contribute in an important way to differences in their effectiveness.

  5. Applicability of the dopamine and rate hypotheses in explaining the differences in behavioral pharmacology of the chloro-benztropine analogs: studies conducted using intracerebral microdialysis and population pharmacodynamic modeling.

    PubMed

    Othman, Ahmed A; Newman, Amy Hauck; Eddington, Natalie D

    2007-08-01

    Previous studies indicated that the chloro-benztropine analogs differed significantly in their cocaine-like activity, which was not expected based on the similarity in their in vitro binding affinity and functional potency at the dopamine transporter (DAT). The present study was designed to extend the understanding of the involvement of both pharmacokinetic and pharmacodynamic factors in mediating the behavioral differences among these analogs. The pharmacokinetics of 3'-chloro-3alpha-(diphenylmethoxy)tropane (3'-Cl BZT), the analog showing a cocaine-like behavioral profile in rodents, was compared with previously reported pharmacokinetic characteristics of cocaine and 4',4''-dichloro-3alpha-(diphenylmethoxy)tropane (4',4''-diCl BZT), an analog totally devoid of cocaine-like actions. Microdialysis studies in rats were conducted to determine whether 3'-Cl and 4',4''-diCl BZT differed significantly in their effect on nucleus accumbens extracellular dopamine levels, with cocaine serving as a reference. A mechanistic model based on DAT association/dissociation kinetics was used to describe the time delay between the plasma concentrations of the chloro-analogs and their dopaminergic effects. 3'-Cl BZT had plasma elimination half-life of 1.9 h versus 0.5 and 21.1 h for cocaine and 4',4''-diCl BZT, respectively. 4',4''-diCl BZT increased the DA levels at a slower rate and to a significantly lower extent relative to 3'-Cl BZT that were, in turn, lower than cocaine. The duration of dopamine elevation was as follows: 4',4''-diCl BZT > 3'-Cl BZT > cocaine. The model indicated faster association and dissociation with DAT for 3'-Cl BZT relative to 4',4''-diCl BZT. The present results indicate that behavioral differences among the chloro-analogs may be explainable based on both the dopamine and rate hypotheses of drug abuse.

  6. Synergistic neuroprotective effects of lithium and valproic acid or other histone deacetylase inhibitors in neurons: roles of glycogen synthase kinase-3 inhibition.

    PubMed

    Leng, Yan; Liang, Min-Huei; Ren, Ming; Marinova, Zoya; Leeds, Peter; Chuang, De-Maw

    2008-03-01

    Lithium and valproic acid (VPA) are two primary drugs used to treat bipolar mood disorder and have frequently been used in combination to treat bipolar patients resistant to monotherapy with either drug. Lithium, a glycogen synthase kinase-3 (GSK-3) inhibitor, and VPA, a histone deacetylase (HDAC) inhibitor, have neuroprotective effects. The present study was undertaken to demonstrate synergistic neuroprotective effects when both drugs were coadministered. Pretreatment of aging cerebellar granule cells with lithium or VPA alone provided little or no neuroprotection against glutamate-induced cell death. However, copresence of both drugs resulted in complete blockade of glutamate excitotoxicity. Combined treatment with lithium and VPA potentiated serine phosphorylation of GSK-3 alpha and beta isoforms and inhibition of GSK-3 enzyme activity. Transfection with GSK-3alpha small interfering RNA (siRNA) and/or GSK-3beta siRNA mimicked the ability of lithium to induce synergistic protection with VPA. HDAC1 siRNA or other HDAC inhibitors (phenylbutyrate, sodium butyrate or trichostatin A) also caused synergistic neuroprotection together with lithium. Moreover, combination of lithium and HDAC inhibitors potentiated beta-catenin-dependent, Lef/Tcf-mediated transcriptional activity. An additive increase in GSK-3 serine phosphorylation was also observed in mice chronically treated with lithium and VPA. Together, for the first time, our results demonstrate synergistic neuroprotective effects of lithium and HDAC inhibitors and suggest that GSK-3 inhibition is a likely molecular target for the synergistic neuroprotection. Our results may have implications for the combined use of lithium and VPA in treating bipolar disorder. Additionally, combined use of both drugs may be warranted for clinical trials to treat glutamate-related neurodegenerative diseases.

  7. Detection and characterization of a new metabolite of 17alpha-methyltestosterone.

    PubMed

    Pozo, Oscar J; Van Eenoo, Peter; Deventer, Koen; Lootens, Leen; Van Thuyne, Wim; Parr, Maria K; Schänzer, Wilhelm; Sancho, Juan V; Hernández, Felix; Meuleman, Philip; Leroux-Roels, Geert; Delbeke, Frans T

    2009-11-01

    The misuse of the anabolic steroid methyltestosterone is currently routinely monitored in doping control laboratories by gas chromatography-mass spectrometry (GC-MS) of two of its metabolites: 17alpha-methyl-5beta-androstane-3alpha,17beta-diol and 17alpha-methyl-5alpha-androstane-3alpha,17beta-diol. Because of the absence of any easy ionizable moiety, these metabolites are poorly detectable using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI). In this study, the metabolism of methyltestosterone has been reinvestigated by the use of a precursor ion scan method in LC-ESI-MS/MS. Two metabolites have been detected using this method. Both compounds have been confirmed in postadministration urine samples of an urokinase plasminogen activator-severe combined immunodeficiency (uPA-SCID) mouse with humanized liver and were characterized by LC-MS/MS and GC-MS using both quadrupole and time of flight analyzers. From the detailed study of the fragmentation, these metabolites were proposed to be epimethyltestosterone and a dehydrogenated compound. Epimethyltestosterone has previously been described as a minor metabolite, whereas the occurrence of the oxidized metabolite has not been reported. Comparison with the synthesized reference revealed that the structure of the dehydrogenated metabolite is 6-ene-epimethyltestosterone. A selected reaction monitoring method including three transitions for each metabolite has been developed and applied to samples from an excretion study and to samples declared positive after GC-MS analysis. 6-Ene-epimethyltestosterone was found in all samples, showing its applicability in the detection of methyltestosterone misuse.

  8. Comparison of sensitivity between gas chromatography-low-resolution mass spectrometry and gas chromatography-high-resolution mass spectrometry for determining metandienone metabolites in urine.

    PubMed

    Kokkonen, J; Leinonen, A; Tuominen, J; Seppälä, T

    1999-11-12

    In doping control laboratories the misuse of anabolic androgenic steroids is commonly investigated in urine by gas chromatography-low-resolution mass spectrometry with selected ion monitoring (GC-LRMS-SIM). By using high-resolution mass spectrometry (HRMS) detection sensitivity is improved due to reduction of biological background. In our study HRMS and LRMS methods were compared to each other. Two different sets were measured both with HRMS and LRMS. In the first set metandienone (I) metabolites 17alpha-methyl-5beta-androstan-3alpha,17beta-dio l (II), 17-epimetandienone (III), 17beta-methyl-5beta-androst-1-ene-3alpha,17alpha-diol (IV) and 6beta-hydroxymetandienone (V) were spiked in urine extract prepared by solid-phase extraction, hydrolysis with beta-glucuronidase from Escherichia coli and liquid-liquid extraction. In the second set the metabolites were first spiked in blank urine samples of four male persons before pretreatment. Concentration range of the spiked metabolites was 0.1-10 ng/ml in both sets. With HRMS (resolution of 5000) detection limits were 2-10 times lower than with LRMS. However, also with the HRMS method the biological background hampered detection and compounds from matrix were coeluted with some metabolites. For this reason the S/N values of the metabolites spiked had to be first compared to S/N values of coeluted matrix compounds to get any idea of detection limits. At trace concentrations selective isolation procedures should be implemented in order to confirm a positive result. The results suggest that metandienone misuse can be detected by HRMS for a prolonged period after stopping the intake of metandienone. PMID:10595716

  9. Chronic anabolic-androgenic steroid treatment affects brain GABA(A) receptor-gated chloride ion transport.

    PubMed

    Bitran, D; Hilvers, R J; Frye, C A; Erskine, M S

    1996-01-01

    Previous research in this laboratory has shown that chronic treatment of adult male rats with an anabolic-androgenic steroid (AAS) produced anxiolytic behavior and increased the functional response of cortical gamma-aminobutyric acid(A) (GABA(A)) receptors. The experiments reported here were aimed at further characterizing the effect of chronic AAS exposure on cerebral cortical GABA(A) receptors. Adult male rats were injected with dianabol (1,4-androstadien-17alpha-methyl-17beta-ol-3-one; 10 mg/kg/day, SC) for 4 weeks. A significant decrease in ventral prostate gland weight was found after 2 weeks of dianabol, and returned to control levels 3 and 10 days after steroid discontinuation. Testicular weights decreased throughout the treatment period but reached statistical significance only during the withdrawal period. Serum 3alpha-androstanediol level was marginally increased afer 2 weeks of dianabol injection, and was significantly decreased at 3 and 10 days after withdrawal. GABA-stimulated 36chloride (Cl-) influx in cortical synaptoneurosomes was increased in animals treated with dianabol for 2 and 4 weeks, and remained elevated 3 days after dianabol withdrawal, returning to control levels at withdrawal day 10. The increase in receptor efficacy was associated with a transient increase in receptor sensitivity (inverse of EC50), apparent after 2 weeks of AAS treatment and at withdrawal day 3. In a follow-up experiment, metabolites of dianabol were tested for the in vitro efficacy in potentiating GABA-stimulated Cl- transport. Only 3alpha-androstanedial and androsterone were found to have potent stimulatory effects. The 3beta-reduced metabolites were inactive, as were metabolites that contained a methyl group at the 17alpha position. These results point to significant facilitative effects of dianabol treatment on brain GABA(A) receptors via the metabolic formation of neuroactive steroids. PMID:8632710

  10. Extinction of albumin gene expression in a panel of human chromosome 2 microcell hybrids

    SciTech Connect

    Cerosaletti, K.M.; Fournier, R.E.K.

    1996-02-01

    Expression of the serum albumin gene is extinguished in rat hepatoma microcell hybrids that retain mouse chromosome 1. These data define a trans-dominant extinguisher locus, Tse-2, on mouse chromosome 1. To localize the human TSE2 locus, we prepared and characterized rat/human microcell hybrids that contained either human chromosome 1 or chromosome 2, the genetic homologues of mouse chromosome 1. Rat hepatoma microcell hybrids retaining a derivative human chromosome 1 [der 1 t(1;17)(p34.3;q11.2)] expressed their serum albumin genes at levels similar to those of parental hepatoma cells. In contrast, microcell transfer of human chromosome 2 into rat hepatoma recipients produced karyotypically heterogeneous collections of hybrid clones, some of which displayed dramatic albumin extinction phenotypes. For example, albumin mRNA levels in hapatoma x fibroblast whole-cell hybrids. Expression of several other liver genes, including {alpha}1-antitrypsin, aldolase B, alcohol dehydrogenase, and phosphoenolpyruvate carboxykinase, was also affected in some of the microcell hybrids, but expression of these genes was no concordant with expression of albumin. Hybrid segregants were prepared from the albumin-extinguished hybrids, and reexpression of albumin mRNA and protein was observed in sublines that had lost or fragmented human chromosome 2. Finally, expression of mRNAs encoding the liver-enriched transactivators HNF-1, HNF-4, HNF-3{alpha}, and HNF-3{beta} was not affected in any of the chromosome 2-containing hybrids. These data define and map a genetic locus on human chromosome 2 that extinguishes albumin gene expression in trans, and they suggest that TSE2-mediated extinction is independent of HNF-1, -4, -3{alpha}, and -3{beta} expression. 61 refs., 2 figs., 3 tabs.

  11. N-containing sugars from Morus alba and their glycosidase inhibitory activities.

    PubMed

    Asano, N; Oseki, K; Tomioka, E; Kizu, H; Matsui, K

    1994-06-17

    The reexamination of N-containing sugars from the roots of Morus alba by improved purification procedures led to the isolation of eighteen N-containing sugars, including seven that were isolated from the leaves of Morus bombycis. These N-containing sugars are 1-deoxynojirimycin (1), N-methyl-1-deoxynojirimycin (2), fagomine (3), 3-epi-fagomine (4), 1,4-dideoxy-1,4-imino-D-arabinitol (5), 1,4-dideoxy-1,4-imino-D-ribitol (6), calystegin B2 (1 alpha,2 beta,3 alpha,4 beta-tetrahydroxy-nor-tropane, 7), calystegin C1 (1 alpha,2 beta,3 alpha,4 beta,6 alpha-pentahydroxy-nor-tropane, 8), 1,4-dideoxy-1,4-imino-(2-O-beta-D-glucopyranosyl)-D-arabinitol (9), and nine glycosides of 1. These glycosides consist of 2-O- and 6-O-alpha-D-galactopyranosyl-1-deoxynojirimycins (10 and 11, respectively), 2-O-, 3-O- and 4-O-alpha-D-glucopyranosyl-1-deoxynojirimycins (12, 13, and 14, respectively), and 2-O-, 3-O-, 4-O- and 6-O-beta-D-glucopyranosyl-1-deoxynojirimycins (15, 16, 17, and 18, respectively). Compound 4 is a new member of polyhydroxylated piperidine alkaloids, and the isolation of 6 is the first report of its natural occurrence. It has recently been found that the polyhydroxy-nor-tropane alkaloids possess potent glycosidase inhibitory activities. Calystegin A3 is the trihydroxy-nor-tropane, and calystegins B1 and B2 are the tetrahydroxy-nor-tropane. Calystegin C1, a new member of calystegins, is the first naturally occurring pentahydroxy-nor-tropane alkaloid. The inhibitory activities of these compounds were investigated against rat digestive glycosidases and various commercially available glycosidases.

  12. Structure and diversity of the T-cell receptor alpha chain in the Mexican axolotl.

    PubMed

    Fellah, J S; Kerfourn, F; Dumay, A M; Aubet, G; Charlemagne, J

    1997-01-01

    Polymerase chain reaction was used to isolate cDNA clones encoding putative T-cell receptor (TCR) alpha chains in an amphibian, the Mexican axolotl (Ambystoma mexicanum). Five TCRalpha-V chain-encoding segments were identified, each belonging to a separate family. The best identity scores for these axolotl TCRalpha-V segments were all provided by sequences belonging to the human TCRalpha-V1 family and the mouse TCRalpha-V3 and TCRalpha-V8 families. A total of 14 different TCRA-J segments were identified from 44 TCRA-V/TCRA-J regions sequenced, suggesting that a large repertoire of TCRA-J segments is a characteristic of most vertebrates. The structure of the axolotl CDR3 alpha chain loop is in good agreement with that of mammals, including a majority of small hydrophobic residues at position 92 and of charged, hydrophilic, or polar residues at positions 93 and 94, which are highly variable and correspond to the TCRA-V/J junction. This suggests that some positions of the axolotl CDR3 alpha chain loop are positively selected during T-cell differentiation, particularly around residue 93 that could be selected for its ability to makes contacts with major histocompatibility complex-associated antigenic peptides, as in mammals. The axolotl Calpha domain had the typical structure of mammalian and avian Calpha domains, including the charged residues in the TM segment that are thought to interact with other proteins in the membrane, as well as most of the residues forming the conserved antigen receptor transmembrane motif. PMID:9002443

  13. Steroid modulation of the chloride ionophore in rat brain: structure-activity requirements, regional dependence and mechanism of action

    SciTech Connect

    Gee, K.W.; Bolger, M.B.; Brinton, R.E.; Coirini, H.; McEwen, B.S.

    1988-08-01

    Further in vitro studies of steroids active at the gamma-aminobutyric acidA (GABAA) receptor regulated Cl- channel labeled by (35S)-t-butylbicyclophosphorothionate ((35S)TBPS) reveal additional structural requirements necessary for activity. Evaluation of selected steroids for activity against TBPS-induced convulsions show similar requirements for activity. Interestingly, steroids (e.g., 5 alpha-pregnan-3 alpha, 20 alpha-diol) were identified that have high potency but limited efficacy as modulators of (35S)TBPS binding. These characteristics are reminiscent of the clinically useful benzodiazepines (BZs) such as clonazepam. However, interactions between the prototypical anesthetic-barbiturate, sodium pentobarbital, and steroids active at the Cl- channel suggest that they do not share a common site of action as allosteric modulators of (35S)TBPS and BZ receptor binding. The most potent steroid evaluated, 5 alpha-pregnan-3 alpha-ol-20-one, modulates (35S)TBPS binding at low concentrations (IC50 approximately 17 nM) in a regionally dependent manner. All (35S)TBPS binding sites appear to be functionally coupled to a steroid modulatory site. Because several of the active steroids are metabolites of progesterone, their ability to inhibit the binding of (3H)promegestrone to the cytosolic progestin receptor in rat uterus was evaluated. Those steroids showing potent activity at the GABAA receptor-Cl- ionophore were inactive at the intracellular progestin receptor. Such specificity coupled with their high potency provide additional support for the hypothesis that some of these steroids may be involved in the homeostatic regulation of brain excitability via the GABAA-BZ receptor complex.

  14. Time-dependent aldosterone metabolism in toad urinary bladder

    SciTech Connect

    Brem, A.S.; Pacholski, M.; Morris, D.J.

    1988-04-01

    Aldosterone (Aldo) metabolism was examined in the toad bladder. Bladders were incubated with (/sup 3/H)aldosterone (10(-7) M) for 5 h, 1 h, or 10 min. Tissues were analyzed for metabolites using high-pressure liquid chromatography (HPLC). In separate experiments, Na+ transport was assessed by the short-circuit current (SCC) technique. Following a 5-h tissue incubation, about 25% of the (/sup 3/H)-aldosterone was converted into metabolites including a polar monosulfate metabolite, 20 beta-dihydroaldo (20 beta-DHAldo), small quantities of 5 beta-reduced products, and a variety of 5 alpha-reduced Aldo products including 5 alpha-DHAldo, 3 alpha,5 alpha-tetrahydroaldo (3 alpha,5 alpha-THAldo), and 3 beta,5 alpha-THAldo. Tissues metabolized approximately 10% of the labeled hormone into the same compounds by 1 h. Measurable quantities of these metabolites were also synthesized by bladders exposed to Aldo for only 10 min and then incubated in buffer for an additional 50 min without Aldo. Bladders pretreated with the spironolactone, K+-canrenoate (3.5 X 10(-4) M), and stimulated with Aldo (10(-7) M) generated a peak SCC 44 +/- 6% of that observed in matched pairs stimulated with Aldo (P less than 0.001; n = 6). K+-canrenoate also markedly diminished (/sup 3/H)aldosterone metabolism at both 5 and 1 h. Thus, metabolic transformation of Aldo begins prior to hormone-induced increases in Na+ transport. Both the generation of certain metabolites (e.g., 5 alpha-reductase pathway products) and the increase in Na+ transport can be selectively inhibited by K+-canrenoate.

  15. Hierarchical phosphorylation at N-terminal transformation-sensitive sites in c-Myc protein is regulated by mitogens and in mitosis.

    PubMed Central

    Lutterbach, B; Hann, S R

    1994-01-01

    The N-terminal domain of the c-Myc protein has been reported to be critical for both the transactivation and biological functions of the c-Myc proteins. Through detailed phosphopeptide mapping analyses, we demonstrate that there is a cluster of four regulated and complex phosphorylation events on the N-terminal domain of Myc proteins, including Thr-58, Ser-62, and Ser-71. An apparent enhancement of Ser-62 phosphorylation occurs on v-Myc proteins having a mutation at Thr-58 which has previously been correlated with increased transforming ability. In contrast, phosphorylation of Thr-58 in cells is dependent on a prior phosphorylation of Ser-62. Hierarchical phosphorylation of c-Myc is also observed in vitro with a specific glycogen synthase kinase 3 alpha, unlike the promiscuous phosphorylation observed with other glycogen synthase kinase 3 alpha and 3 beta preparations. Although both p42 mitogen-activated protein kinase and cdc2 kinase specifically phosphorylate Ser-62 in vitro and cellular phosphorylation of Thr-58/Ser-62 is stimulated by mitogens, other in vivo experiments do not support a role for these kinases in the phosphorylation of Myc proteins. Unexpectedly, both the Thr-58 and Ser-62 phosphorylation events, but not other N-terminal phosphorylation events, can occur in the cytoplasm, suggesting that translocation of the c-Myc proteins to the nucleus is not required for phosphorylation at these sites. In addition, there appears to be an unusual block to the phosphorylation of Ser-62 during mitosis. Finally, although the enhanced transforming properties of Myc proteins correlates with the loss of phosphorylation at Thr-58 and an enhancement of Ser-62 phosphorylation, these phosphorylation events do not alter the ability of c-Myc to transactivate through the CACGTG Myc/Max binding site. Images PMID:8035827

  16. Rates of ubiquitin conjugation increase when muscles atrophy, largely through activation of the N-end rule pathway

    NASA Technical Reports Server (NTRS)

    Solomon, V.; Baracos, V.; Sarraf, P.; Goldberg, A. L.

    1998-01-01

    The rapid loss of muscle mass that accompanies many disease states, such as cancer or sepsis, is primarily a result of increased protein breakdown in muscle, and several observations have suggested an activation of the ubiquitin-proteasome system. Accordingly, in extracts of atrophying muscles from tumor-bearing or septic rats, rates of 125I-ubiquitin conjugation to endogenous proteins were found to be higher than in control extracts. On the other hand, in extracts of muscles from hypothyroid rats, where overall proteolysis is reduced below normal, the conjugation of 125I-ubiquitin to soluble proteins decreased by 50%, and treatment with triiodothyronine (T3) restored ubiquitination to control levels. Surprisingly, the N-end rule pathway, which selectively degrades proteins with basic or large hydrophobic N-terminal residues, was found to be responsible for most of these changes in ubiquitin conjugation. Competitive inhibitors of this pathway that specifically block the ubiquitin ligase, E3alpha, suppressed most of the increased ubiquitin conjugation in the muscle extracts from tumor-bearing and septic rats. These inhibitors also suppressed ubiquitination in normal extracts toward levels in hypothyroid extracts, which showed little E3alpha-dependent ubiquitination. Thus, the inhibitors eliminated most of the differences in ubiquitination under these different pathological conditions. Moreover, 125I-lysozyme, a model N-end rule substrate, was ubiquitinated more rapidly in extracts from tumor-bearing and septic rats, and more slowly in those from hypothyroid rats, than in controls. Thus, the rate of ubiquitin conjugation increases in atrophying muscles, and these hormone- and cytokine-dependent responses are in large part due to activation of the N-end rule pathway.

  17. Pyroxene structures, cathodoluminescence and the thermal history of the enstatite chondrites

    NASA Technical Reports Server (NTRS)

    Zhang, Yanhong; Huang, Shaoxiong; Schneider, Diann; Benoit, Paul H.; Sears, Derek W. G.; DeHart, John M.; Lofgren, Gary E.

    1996-01-01

    In order to explore the thermal history of enstatite chondrites, we examined the cathodoluminescence (CL) and thermoluminescence (TL) properties of 15 EH chondrites and 21 EL chondrites, including all available petrographic types, both textural types 3-6 and mineralogical types alpha-delta. The CL properties of EL3(alpha) and EH3(alpha) chondrites are similar. Enstatite grains high in Mn and other transition metals display red CL, while enstatite with low concentrations of these elements show blue CL. A few enstatite grains with greater than 5 wt% FeO display no CL. In contrast, the luminescent properties of the metamorphosed EH chondrites are very different from those of metamorphosed EL chondrites. While the enstatites in metamorphosed EH chondrites display predominantly blue CL, the enstatites in metamorphosed EL chondrites display a distinctive magenta CL with blue and red peaks of approximately equal intensity in their spectra. The TL sensitivities of the enstatite chondrites correlate with the intensity of the blue CL and, unlike other meteorite classes, are not simply related to metamorphism. The different luminescent properties of metamorphosed EH and EL chondrites cannot readily be attributed to compositional differences. But x-ray diffraction data suggests that the enstatite in EH5(gamma),(delta) chondrites is predominantly disordered orthopyroxene, while enstatite in EL6(beta) chondrites is predominantly ordered orthopyroxene. The difference in thermal history of metamorphosed EL and EH chondrites is so marked that the use of single 'petrographic' types is misleading, and separate textural and mineralogical types are preferable. Our data confirm earlier suggestions that metamorphosed EH chondrites underwent relatively rapid cooling, and the metamorphosed EL chondrites cooled more slowly and experienced prolonged heating in the orthopyroxene field.

  18. Side chain hydroxylation of C27-steroids and vitamin D3 by a cytochrome P-450 enzyme system isolated from human liver mitochondria

    SciTech Connect

    Oftebro, H.; Saarem, K.; Bjoerkhem, I.; Pedersen, J.I.

    1981-11-01

    The present study was undertaken to obtain information on the involvement of cytochrome P-450 in the 26-hydroxylation on bile acid intermediates and in the 25-hydroxylation of vitamin D3 in human liver mitochondria. Cytochrome P-450 was solubilized from human liver mitochondria and purified two times to a specific content of 0.125 nmol per mg protein. Furthermore, a ferredoxin was isolated from the mitochondria and partly purified. This iron-sulfur protein had properties similar to bovine adrenal ferredoxin. A mitochondrial NADPH-ferredoxin reductase was also isolated and purified to homogeneity. This enzyme was a flavoprotein with properties very similar to the bovine adrenal NADPH-ferredoxin reductase. The cytochrome P-450 preparation catalyzed 26-hydroxylation of C27-steroids and 25-hydroxylation of vitamin D3 when reconstructed with NADPH, the ferredoxin and the ferredoxin reductase. With different substrates the following turnover numbers (nmol product X nmol P-450(-1) X min-1) were found: cholesterol, 8; 5-cholestene-3 beta, 7 alpha-diol, 10; 7 alpha-hydroxy-4-cholesten-3-one, 23; 7 alpha, 12 alpha-dihydroxy-4-cholesten-3-one, 27; 5 beta-cholestane-3 alpha, 7 alpha-diol, 28; 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, 41; and vitamin D3, 0.16. The hydroxylation reactions were inhibited by CO and metyrapone. The human liver mitochondrial ferredoxin and ferredoxin reductase could be replaced by adrenal ferredoxin and adrenal ferredoxin reductase without reduction of activity, but they could not be replaced by microsomal NADPH-cytochrome P-450 reductase. It is concluded that human liver mitochondria contain cytochrome P-450 involved in the oxidation of the side chain of C27-steroids and vitamin D3.

  19. Effect of culture media on the chemical and physical characteristics of polysaccharides isolated from Poria cocos mycelia.

    PubMed

    Jin, Yong; Zhang, Lina; Chen, Li; Chen, Yan; Cheung, Peter Chi Keung; Chen, Liguo

    2003-07-01

    Mycelia of a wild strain Poria cocos were cultured in two media differing in one constituent: bran extract or corn steep liquor, and are designated as wb and wc, respectively. Six polysaccharide fractions were isolated sequentially from the two mycelia by 0.9% NaCl (PCM1), hot water (PCM2), 0.5 M NaOH (PCM3-I and -II) and 88% formic acid (PCM4-I and -II). Their chemical and physical characteristics were determined by infrared spectroscopy (IR), gas chromatography (GC), 13C NMR, light scattering (LS) and viscometry. The results indicated that wb-, wc-PCM1, and PCM2 were heteropolysaccharides mainly composed of alpha-D-glucose, mannose, and galactose, whereas wb-PCM3-I and wc-PCM3-I were mainly (1-->3)-alpha-D-glucans, and wb- and wc-PCM3-II, PCM4-I and PCM4-II were (1-->3)-beta-D-glucans. Interestingly, (1-->3) alpha- and (1-->3)-beta-D-glucans co-existed in the 0.5 M NaOH fraction and were separated individually into the two fractions (PCM3-I and PCM3-II) after neutralizing with acetic acid. The polysaccharides from wc-PCM cultured in media containing corn steep liquor contained relatively more protein. The polysaccharide fractions also existed in conformations including random coil (as in PCM0 and PCM1) and expanded chain (as in PCM3), and differed molecular mass. In addition, two exo-polysaccharides isolated from the two culture media by methanol precipitation (wb- and wc-PCM0) also differed in their monosaccharide composition. PMID:12829396

  20. Structure of the human gene and two rat cDNAs encoding the alpha chain of GTP-binding regulatory protein Go: two different mRNAs are generated by alternative splicing.

    PubMed Central

    Tsukamoto, T; Toyama, R; Itoh, H; Kozasa, T; Matsuoka, M; Kaziro, Y

    1991-01-01

    Go is a specific class ("other") of signal-transducing heterotrimeric GTP-binding proteins (G proteins) that is expressed in high levels in mammalian brain. We have cloned two different rat cDNAs encoding the alpha subunit of Go (Go alpha-1 and Go alpha-2) and a human Go alpha chromosomal gene. The human Go alpha gene spans more than 100 kilobases and contains 11 exons, including one noncoding exon in the 3' flanking region. The 5' flanking region is highly G + C-rich and contains five G.C boxes (Sp1 binding sites) but no TATA box. Exons 7 and 8 coding for amino acid residues 242-354 of Go alpha protein are duplicated (referred to as exons 7A, 7B, 8A, and 8B). It was found that exons 7A and 8A code for Go alpha-1, and 7B and 8B code for Go alpha-2. This indicates that two different Go alpha mRNAs may be generated by alternative splicing of a single Go alpha gene. The splice sites of the Go alpha-1 and Go alpha-2 genes are completely identical with those encoding human inhibitory G protein alpha subunits Gi2 alpha and Gi3 alpha [Itoh, H., Toyama, R., Kozasa, T., Tsukamoto, T., Matsuoka, M. & Kaziro, Y. (1988) J. Biol. Chem. 263, 6656-6664] and also transducin G protein alpha subunit Gt1 alpha [Raport, C. J., Dere, B. & Hurley, J. (1989) J. Biol. Chem. 264, 7122-7128]. Sequence homology and conservation of the exon-intron organization indicate that the genes coding for Go alpha, Gi2 alpha, Gi3 alpha, Gt1 alpha, and probably Gi1 alpha may be evolved from a common progenitor. Like Go alpha-1, Go alpha-2 is expressed mainly in brain. Images PMID:1901650

  1. a Combined Molecular Dynamics and NMR Spectroscopic Protocol for the Conformational Analysis of Oligosaccharides.

    NASA Astrophysics Data System (ADS)

    Varma, Vikram

    A combined experimental and theoretical protocol for the conformational analysis of oligosaccharides is presented. Three disaccharides, methyl alpha - scD-mannopyranosyl-(1 to 3)-alpha- scD-mannopyranoside, methyl beta- scD-galactopyranosyl-(1 to 4)-beta- scD-glucopyranoside, and propyl beta- scD-2-acetamido -2-deoxy glucopyranosyl-(1 to 3)- alpha- scL-rhamnopyranoside, are used to evaluate a protocol for conformational analysis that makes use of molecular dynamics calculations with the CHARMM force field. Dynamics trajectories computed in vacuo and in water are used to calculate time-averaged NMR parameters such as spin-lattice relaxation times (T_1 ), Nuclear Overhauser Enhancements (NOE), and heteronuclear spin-spin coupling constants (^3J _{rm CH}). The calculated NMR parameters are then compared to experimental values and used to evaluate the computational procedure. The energetically accessible conformations are effectively sampled by the simulations. The method has been extended to the conformational analysis of higher-order oligosaccharides corresponding to the cell-wall polysaccharide of the Streptococcus Group A, and the Shigella flexneri Y O-antigen. The Streptococcus Group A cell-wall polysaccharide is comprised of a backbone of rhamnopyranosyl units connected by alternating alpha- scL-(1 to 3) and alpha- scL -(1 to 2) linkages, to which are attached N-acetyl-beta- scD-glucosamine ( beta- scD-GlcpNAc) residues at the 3 positions of the rhamnose backbone.rm A&rm B^'qquad A^'& rm Bqquad Acr[{-alpha}{-}L{-}Rha {it p}{-}(1to2){-alpha }{-}L{-}Rha{it p} {-}(1to3){-alpha}{ -}L{-}Rha{it p}-(1to2) -alpha-L-Rha{it p}{-}(1 to3){-alpha}{-}L{- }Rha{it p}{-}cr&uparrow(1 to3)&uparrow(1to3)crbeta {-}D{-}&rm Glc{it p }NAcqquadbeta{-}D{-}& rm Glc{it p}NAccr&rm C ^'&rm C] A branched trisaccharide (A^' -(C)B), a tetrasaccharide (A^' -(C)B-A), a pentasaccharide (C^' -B^'-A ^'-(C)B), and two hexasaccharides (C^'-B^ '-A^' -(C)B-A) and (A-(C^')B ^'-A^' -(C)B), have been chosen

  2. Fatty acid composition, eicosanoid production and permeability in skin tissues of rainbow trout (Oncorhynchus mykiss) fed a control or an essential fatty acid deficient diet.

    PubMed

    Ghioni, C; Bell, J G; Bell, M V; Sargent, J R

    1997-06-01

    4 (LTC4) and leukotriene C5 (LTC5) in skin cells challenged with the calcium ionophore A23187, and of prostaglandin F2alpha (PGF2alpha), 12-HETE and 12-HEPE in gill cells challenged similarly. Prostaglandin F3alpha (PGF3alpha) production by ionophore stimulated gill cells was significantly reduced in fish fed the EFA-deficient diet. 14-HDHE produced by gill cells was 3.3 fold higher in EFA deficient fish compared to controls.

  3. Compounds exhibiting selective efficacy for different beta subunits of human recombinant gamma-aminobutyric acid A receptors.

    PubMed

    Smith, Alison J; Oxley, Beth; Malpas, Sallie; Pillai, Gopalan V; Simpson, Peter B

    2004-11-01

    Inhibitory GABA(A) receptor modulators are widely used therapeutic agents for a variety of central nervous system disorders. Ltk(-) cells stably expressing human recombinant GABA(A) subunits (alpha1beta1-3gamma2s) were seeded into 96-well plates, loaded with chlorocoumarin-2-dimyristoyl phosphatidylethanolamine and bis(1,3-diethyl-2-thiobarbiturate)trimethineoxonol, and rapid fluorescence resonance energy transfer technique (FRET) measurements were made of GABA-evoked depolarizations in low-Cl(-) buffer using a voltage/ion probe reader. The influence of different betasubunits on the ability of agents to modulate and directly activate the ion channel was examined. GABA evoked concentration-dependent decreases in FRET, increasing fluorescence emission ratio (460/580 nm) at alpha1beta1gamma2, alpha1beta2gamma2, and alpha1beta3gamma2 receptors with similar maximal amplitude (P > 0.05, n = 17) and EC(50) values of 2.4 +/- 0.2, 2.5 +/- 0.2, and 1.3 +/- 0.1 microM, respectively. Piperidine-4-sulfonic acid and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol were less potent, with EC(50) values of 8.7 +/- 0.9, 9.2 +/- 0.5, and 11.7 +/- 1.2, and 43.7 +/- 6.4, 24.8 +/- 1.6, and 26.1 +/- 2.4 microM, respectively. Potency and maximal efficacy of propofol, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, pentobarbital, and steroids, 5alpha-pregnan-3alpha-ol-20-one and 5beta-pregnan-3alpha-ol-20-one, were unaffected by the beta isoform present in the receptor complex. However, several compounds displayed beta2/3 subunit selectivity, notably loreclezole, R(-)-etomidate, and a group of anti-inflammatory agents including mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, niflumic acid, and diflunisal. The anti-inflammatories exhibited varying levels of efficacy at beta2/3 subunits, with micromolar potency, while having antagonist or weak inverse agonist profiles at alpha1beta1gamma2. Diflunisal was the most efficacious compound, eliciting greater

  4. Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

    SciTech Connect

    Soeda, Junpei; Morgan, Maelle; McKee, Chad; Mouralidarane, Angelina; Lin, ChingI; Roskams, Tania; Oben, Jude A.

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Cigarette smoke may induce liver fibrosis via nicotine receptors. Black-Right-Pointing-Pointer Nicotine induces proliferation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Nicotine activates hepatic fibrogenic pathways. Black-Right-Pointing-Pointer Nicotine receptor antagonists attenuate HSC proliferation. Black-Right-Pointing-Pointer Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine - which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells in the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed - RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-{alpha}2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-{beta}1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type ({alpha}1, {beta}1, delta and epsilon) and neuronal type ({alpha}3, {alpha}6, {alpha}7, {beta}2 and {beta}4) nAChR subunits at the mRNA level. Among these subunits, {alpha}3, {alpha}7, {beta}1 and {epsilon} were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-{alpha}2 and TGF-{beta}1 mRNA expression were significantly upregulated by nicotine and inhibited by

  5. Bile acid concentrations in serum and duodenal aspirates of healthy preterm infants: effects of gestational and postnatal age.

    PubMed

    Boehm, G; Braun, W; Moro, G; Minoli, I

    1997-01-01

    In 41 healthy human-milk-fed preterm infants the preprandial total bile acid (BA) concentrations in serum and duodenal juice were simultaneous measured during the first 60 days of life. The infants were subdivided into four groups according to their gestational age: 6 infants with a gestational age of 27 and 28 weeks, 7 infants with a gestational age of 29 and 30 weeks, 21 infants with a gestational age of 31 and 32 weeks and 7 infants with a gestational age of 33 and 34 weeks. The BA levels were enzymatically determined using 3-alpha-hydroxysteroid dehydrogenase. In the duodenal juice, cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid and lithocholic acid were separately quantified by thin-layer chromatography. During the first month of life, the serum BA concentrations increased significantly with postnatal age (p < 0.01) but remained nearly constant during the second month of life. In the duodenal aspirates, the BA concentrations increased continuously up to the end of the observations period (p < 0.001). In the duodenal aspirates, the CA/CDCA ratio was high immediately after birth and decreased significantly with increasing postnatal age (p < 0.001). During the first weeks of life, the BA levels were preferentially conjugated with taurine, but in spite of the taurine-rich diet during the whole observation period the taurine/glycine ratio decreased with postnatal age (p < 0.001). In all samples of duodenal juice, the sum of primary BA was > 98% of total 3-alpha-hydroxy-BA. These data indicate that the establishment of an intestinal microbial flora necessary for intestinal BA transformation and the development of the enterohepatic BA circulation lasts some months of postnatal life. The serum BA concentration reflects hepatic synthesis, intestinal absorption, renal excretion and hepatocellular transport into bile in a very complex way which may limit the diagnostic value of serum BA during this time. Additionally, a duodenal BA concentration below 4

  6. Millisecond autothermal catalytic reforming of carbohydrates for synthetic fuels by reactive flash volatilization

    NASA Astrophysics Data System (ADS)

    Dauenhauer, Paul Jakob

    Carbohydrates including glucose, cellulose, starch and polyols including glycerol, ethylene glycol and methanol produced in large quantities from biomass are considered as a carbon-based feedstock for high temperature catalytic reforming by catalytic partial oxidation. Autothermal catalytic partial oxidation of methanol, ethylene glycol, and glycerol with Rh and Pt-based catalysts with ceria on alumina foam supports at residence times less than ten milliseconds produced equilibrium selectivity to synthesis gas. The addition of steam at S/C>4 produced selectivity to H2 higher than 80% with little or no selectivity to minor products. In a new process referred to as 'reactive flash volatilization,' catalytic partial oxidation was combined with pyrolysis of biomass by directly impinging particles of cellulose, starch, polyethylene, soy oil, or Aspen (Populous Tremuloides) on an operating Rh-based reforming catalyst at 700-800°C. Solid particles endothermically pyrolyzed to volatile organic compounds which mixed with air and reformed on the catalyst exothermically generating heat to drive the overall process. Particles of ˜250 mum microcrystalline cellulose processed at the conditions of C/O=1.0 on a RhCe/gamma-Al2O3/alpha-Al 2O3 at a residence time of ˜70 milliseconds produced a gaseous effluent stream selecting for 50% H2 and 50% CO with no observable side products other than H2O and CO2, and <1% CH4. To obtain a more optimal synthesis gas stream, the reforming of ˜400 mum microcrystalline particles was examined over a fixed bed of RhCe/gamma-Al2O3/alpha-Al2O 3 spheres by varying the feed ratio of N2/O2, the temperature of the feed gas, the total particle feed rate, and the addition of steam permitting cellulose conversion with ˜75% fuel efficiency. Cellulose, sucrose, and glycerol particle conversion was examined with high-speed photography (1000 frames/second) revealing the formation of a liquid intermediate from cellulose permitting extremely high heat flux (

  7. Residues and metabolism of 19-nortestosterone laurate in steers.

    PubMed

    Sauer, M J; Samuels, T P; Howells, L G; Seymour, M A; Nedderman, A; Houghton, E; Bellworthy, S J; Anderson, S; Coldham, N G

    1998-12-01

    The illegal use of 19-nortestosterone (19NT; 4-estren-17 beta-ol-3-one; nandrolone) and its esters in livestock, for growth promotion purposes, has been widely reported in the European Union. The target residues for surveillance of abuse in bovine urine and bile samples are 17 alpha- and 17 beta-19NT, although this choice of target residues is not based on in vivo radiotracer biotransformation data. In this study, four steers were administered [3H2]- and [2H3] 17 beta-19NT laurate (2 mg kg-1 body mass) by intramuscular injection and blood, urine, faeces and bile samples were taken for 30 d until slaughter, after which tissues were sampled for total residue analysis. Total plasma radiolabelled residues reached a maximum of 56.3 +/- 15.9 pmol ml-1 at 36 h and were still appreciable (13.3 +/- 1.6 pmol ml-1) 30 d after treatment. Throughout the study period, total residue concentrations in bile (about 2-16 nmol ml-1), urine and faeces (0.5-3 nmol ml-1 or g-1) were higher than in other tissues sampled at slaughter. At slaughter there was evidence of residue accumulation in pigmented eye tissue (33.1 +/- 6.1 pmol g-1) and in white (13.4 +/- 3.4 pmol g-1) and black hair (28.9 +/- 8.9 pmol g-1). Evaluation of radio-HPLC profiles of urine and bile extracts generally indicated that 19NT and 19NT laurate residues were present in relatively small amounts among a complex mixture of metabolites. GC-MS analysis of glucuronidase-hydrolysed bile extracts indicated that the major metabolites were 5 beta-estrane-3 alpha, 17 alpha-diol, 5 alpha-estrane-3 beta, 17 alpha-diol. 5 alpha-estran-3 alpha-ol-17-one (norandrosterone) and estra-1,3,5(10)-triene-3,17 alpha-diol (17 alpha-estradiol). PMID:10435319

  8. Synthesis of the 5-aminopentyl glycoside of beta-D-Gal p-(1-->4)-beta-D-Glc p NAc-(1-->3)-L-Fuc p and fragments thereof related to glycopeptides of human Christmas factor and the marine sponge Microciona prolifera.

    PubMed

    Ziegler, T

    1994-09-15

    The marine sponge Microciona prolifera and human coagulation factor IX (Christmas factor)-related mono- to tri-saccharide 5-aminopentyl glycosides beta-D-Gal p-R (5), beta-D-Glc pNAc-R (16), beta-D-Gal p-(1-->4)-beta-D-Glc p NAc-R (26), beta-D-Glc p NAc-(1-->3)-beta-L-Fuc p-R (39), beta-D-Glc pNAc-(1-->3)-alpha-L-Fuc p-R (43), beta-D-Gal p-(1-->4)-beta-D- Glc pNAc-(1-->3)-beta-L-Fuc p-R (45), and beta-D-Gal p-(1-->4)-beta-D-Glc p NAc-(1-->3)-alpha-L-Fuc p-R (47), where R is a 5-aminopentyloxy spacer moiety, which allowed the construction of glycoconjugates, were prepared. Thus, 3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2- trichloroethoxycarbonyl-amino)-alpha-D-glucopyranosyl trichloroacetimidate (10) and 1,3,4,6-tetra-O-acetyl-2-chloro-acetamido-2- deoxy-beta-D-glucopyranose (13) were condensed with N-Z-protected 5-amino-pentanol (2) followed by conversion of the coupling products into the corresponding N-acetylglucosamine derivatives, to give compound 16 after deblocking. Similarly, the donors 10 and 13 were coupled to position 3 of suitably protected aminopentyl beta- (32) and alpha- (37) -L-fucopyranosides, to give the disaccharides 39 and 43, respectively. Starting from lactose, O-(2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl)-(1-->4)-3,6-di-O- benzoyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-alpha-D-glu copyranosyl trichloroacetimidate (23) was prepared and used as an efficient disaccharide donor for the construction of ligand 26 from 2 and of the trisaccharide ligands 45 and 47 from fucosides 32 and 37, respectively.

  9. Detection of dehydroepiandrosterone misuse by means of gas chromatography- combustion-isotope ratio mass spectrometry.

    PubMed

    Mareck, Ute; Geyer, Hans; Flenker, Ulrich; Piper, Thomas; Thevis, Mario; Schänzer, Wilhelm

    2007-01-01

    According to World Anti-Doping Agency (WADA) rules (WADA Technical Document-TD2004EAAS) urine samples containing dehydroepiandrosterone (DHEA) concentrations greater than 100 ng ML(-1) shall be submitted to isotope ratio mass spectrometry (IRMS) analysis. The threshold concentration is based on the equivalent to the glucuronide, and the DHEA concentrations have to be adjusted for a specific gravity value of 1.020. In 2006, 11,012 doping control urine samples from national and international federations were analyzed in the Cologne doping control laboratory, 100 (0.9%) of them yielding concentrations of DHEA greater than 100 ng mL(-1). Sixty-eight percent of the specimens showed specific gravity values higher than 1.020, 52% originated from soccer players, 95% were taken in competition, 85% were male urines, 99% of the IRMS results did not indicate an application of testosterone or related prohormones. Only one urine sample was reported as an adverse analytical finding having 319 ng mL(-1) DHEA (screening result), more than 10,000 ng mL(-1) androsterone and depleted carbon isotope ratio values for the testosterone metabolites androsterone and etiocholanolone. Statistical evaluation showed significantly different DHEA concentrations between specimens taken in- and out-of- competition, whereas females showed smaller DHEA values than males for both types of control. Also a strong influence of the DHEA excretion on different sport disciplines was detectable. The highest DHEA values were detected for game sports (soccer, basketball, handball, ice hockey), followed by boxing and wrestling. In 2007, 6622 doping control urine samples were analyzed for 3alpha,5-cyclo-5alpha-androstan-6beta-ol-17-one (3alpha,5-cyclo), a DHEA metabolite which was described as a useful gas chromatography-mass spectrometry (GC-MS) screening marker for DHEA abuse. Nineteen urine specimens showed concentrations higher than the suggested threshold of 140 ng mL(-1), six urine samples yielded

  10. Structural basis of substrate binding in WsaF, a rhamnosyltransferase from Geobacillus stearothermophilus.

    PubMed

    Steiner, Kerstin; Hagelueken, Gregor; Messner, Paul; Schäffer, Christina; Naismith, James H

    2010-03-26

    Carbohydrate polymers are medically and industrially important. The S-layer of many Gram-positive organisms comprises protein and carbohydrate polymers and forms an almost paracrystalline array on the cell surface. Not only is this array important for the bacteria but it has potential application in the manufacture of commercially important polysaccharides and glycoconjugates as well. The S-layer glycoprotein glycan from Geobacillus stearothermophilus NRS 2004/3a is mainly composed of repeating units of three rhamnose sugars linked by alpha-1,3-, alpha-1,2-, and beta-1,2-linkages. The formation of the beta-1,2-linkage is catalysed by the enzyme WsaF. The rational use of this system is hampered by the fact that WsaF and other enzymes in the pathway share very little homology to other enzymes. We report the structural and biochemical characterisation of WsaF, the first such rhamnosyltransferase to be characterised. Structural work was aided by the surface entropy reduction method. The enzyme has two domains, the N-terminal domain, which binds the acceptor (the growing rhamnan chain), and the C-terminal domain, which binds the substrate (dTDP-beta-l-rhamnose). The structure of WsaF bound to dTDP and dTDP-beta-l-rhamnose coupled to biochemical analysis identifies the residues that underlie catalysis and substrate recognition. We have constructed and tested by site-directed mutagenesis a model for acceptor recognition. PMID:20097205

  11. Wide spectrum of steroids serving as substrates for the formation of lipoidal derivatives in ZR-75-1 human breast cancer cells.

    PubMed

    Poulin, R; Poirier, D; Thériault, C; Couture, J; Bélanger, A; Labrie, F

    1990-02-01

    Recently, several natural steroids have been found to be esterified to long-chain fatty acids (FAE) in various mammalian tissues. The purpose of the present study was to determine the ability of a series of 3H-labeled steroids to serve as substrates for the formation and accumulation of such non-polar derivatives in intact cells, using the hormone-responsive ZR-75-1 human breast cancer cell line as model. All 14 steroids tested were found to be converted, directly or following further metabolism, to lipoidal ester derivatives. The percentage of intracellular steroids recovered as FAE derivatives was usually substantial (14-90%), especially in the case of C-19 steroids (75-90%). The composition of the lipoidal steroid fractions recovered from the labeled cell extracts was characterized by chromatographic comparison with synthetic steroid FAEs and by saponification of the steroid FAEs and identification of the released steroidal moieties. Following metabolism, most steroid substrates were converted into multiple lipoidal esters. Furthermore, 5 alpha-androstane-3 alpha, 17 beta-diol, 5 alpha-androstane-3 beta, 17 beta-diol, as well as androst-5-ene-3 beta, 17 beta-diol formed lipoidal diesters in addition to the monoester form. The high level of intracellular steroid FAE accumulation reported in this study suggests that these yet poorly known steroid derivatives may play important functions in the regulation of steroid hormone metabolism and action.

  12. Reading the molecular clock from the decay of internal symmetry of a gene.

    PubMed Central

    Gibbs, P E; Dugaiczyk, A

    1994-01-01

    The closely related serum albumin, alpha-fetoprotein, and vitamin D-binding proteins are derived from a common ancestor, which itself was the result of a triplication of an ancestral gene. We have aligned the sequences of these proteins against themselves to assess the degree to which the ancestral 3-fold symmetry has been retained; in a dot plot, relics of the molecular symmetry appear as a series of alignments parallel to the main diagonal. The decay of internal symmetry reflects the rate of change of a gene in a single line of evolutionary descent. We examined 11 serum albumins, 2 ceruloplasmins, 3 alpha-fetoproteins, and 3 vitamin D-binding proteins. We have found that ceruloplasmin evolves at the same rate in human and rat, whereas albumin, alpha-fetoprotein, and vitamin D-binding protein evolve at different rates. The human genes had the highest alignment scores, indicating the most preserved ancestral features. We conclude that the molecular clock may run at different rates for the same gene in different species. PMID:7512733

  13. Antifungal and molluscicidal saponins from Serjania salzmanniana.

    PubMed

    Ekabo, O A; Farnsworth, N R; Henderson, T O; Mao, G; Mukherjee, R

    1996-04-01

    An investigation of Serjania salzmanniana for biologically active substances has led to the isolation of two novel saponins, salzmannianoside A (3-O-[[beta-D- glucopyranosyl-(1-->4)]-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L- arabinopyranosyl] gypsogenin) [3] and salzmannianoside B (3-O-[[beta-D-glucopyranosyl-(1-->4)]-[alpha-L- arabinopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl-(1-->2)] -alpha-L-arabinopyranosyl] hederagenin) (4). Two known saponins, pulsatilla saponin D (3-O-[[beta-D- glucopyranosyl-(1-->4)]-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L- arabinopyranosyl] hederagenin) (1) and 3-O-[[beta-D-glucopyranosyl-(1-->4)]-[alpha-L-rhamnopyranosyl-(1-->2)]-a lpha-L- arabinopyranosyl] oleanolic acid (2) were also isolated from this plant. The structures of 3 and 4 were elucidated by FABMS and 2D NMR techniques. All these four saponins were mollusicidal, causing 70-100% mortality at 10 ppm against Biomphalaria alexandrina, a vector of Schistosoma mansoni in the Nile Valley. The saponins also showed antifungal activity against Cryptococcus neoformans and Candida albicans at minimal inhibitory concentrations of 8 and 16 micrograms/mL, respectively.

  14. Durangite from the Black Range, New Mexico, and new data on durangite from Durango and Cornwall.

    USGS Publications Warehouse

    Foord, E.E.; Oakman, M.R.; Maxwell, C.H.

    1985-01-01

    Durangite, associated with cassiterite, hematite, quartz, tridymite, cristobalite and clinopyroxene, occurs in small veinlets within flows, ash-flow tuffs and lithic tuffs in a tin mine near Boiler Peak, New Mexico. It is clear to semi-translucent, pale yellow-orange to medium orange-red with a vitreous lustre, pale yellow streak; H. 5-5.5%; irregular to conchoidal fracture and a good (110) cleavage; elongate along c with (110), (010), (021) and (111) the prominent forms; Dmeas 3.90, Dcalc 3.92 g/cm3; alpha medium yellow orange 1.634(3), beta pale yellow orange 1.663(3), gamma colourless 1.685(3); weak to moderate dispersion r < v. The structural formula is: (Na0.93Li0.07)SIGMA 1.00(Al0.89Fe0.07Mn0.06)SIGMA 1.02As0.99O4(F0.90(OH)0.07)SIGMA 0.97. Indexed XRD powder data are tabulated; a 6.574(1), b 8.505(2), c 7.019(1) A, beta 115.34o; space group C2/c; Z = 4. Additional X-ray and chemical data on durangite from Durango and Cornwall are also included.-L.T.T.

  15. Production and characterization of a monoclonal antibody to human Type IV collagen.

    PubMed Central

    Sakai, L. Y.; Engvall, E.; Hollister, D. W.; Burgeson, R. E.

    1982-01-01

    We have produced a monoclonal antibody to human basement membrane Type IV collagen. The antibody reacts with the pepsin-resistant, collagenase-sensitive domain of Type IV collagen isolated from placental membranes, but not with human collagens of Types I, II, III, V, 1alpha, 2alpha, and 3alpha. The antibody precipitates biosynthetically labeled human Type IV procollagen, and the precipitate contains both the alpha1 (IV) and alpha2 (IV) chains, suggesting the occurrence of both of these chains within the same triple-helical molecule. When used in indirect immunofluorescence, the antibody gives brilliant staining of basement membranes from a variety of human tissues but does not stain tissues of bovine, canine, rabbit, rat, or mouse origin. It is suggested that this antibody will be of value in research on the structure of human basement membrane collagen, on the distribution of this collagen in various basement membranes, and particularly for the study of basement membranes in normal human development and pathologic processes. Images Figure 1 Figure 5 PMID:6287846

  16. Role of Triton X-100 in chemiluminescent enzyme immunoassays capable of diagnosing genetic disorders.

    PubMed

    Chong, Richard; Rho, Jee-Eun R; Yoon, Hye-Joo; Park, Paul S; Rho, Tae-Ho D; Park, Jee Y; Park, Lucienne; Kim, Young-Hwan; Lee, Ji Hoon

    2013-11-15

    The use of Triton X surfactants in developing 1,1'-oxalylimidazole chemiluminescent enzyme immunoassays (ODI CEIs) with extended linear response range for the quantification of unconjugated estriol (uE3), alpha-fetoprotein (AFP), and human chorionic gonadotropin (hCG) is reported for the first time. The wider linear dynamic range in ODI CLEIA results from Triton X series (e.g., Triton X-100, -114, -405, -705) acting as an inhibitor in the interaction between Amplex Red (hydrophobic substrate) and horseradish peroxidase (hydrophilic enzyme) to produce resorufin (hydrophobic fluorescent dye). Triton X-100 acts as the appropriate inhibitor in ODI CLEIA. The maximum concentrations of AFP and hCG quantified with sandwich ODI CLEIA in the presence of Triton X-100 were 8 times higher than when analyzed with the same system in the absence of Triton X-100. In addition, the lowest concentration of uE3 determined using competitive ODI CLEIA in the presence of Triton X-100 was 20 times lower than that measured with competitive ODI CLEIA in the absence of Triton X-100. These results indicate that rapid quantification of AFP, uE3, and hCG using cost effective and highly sensitive ODI CLEIAs in the presence of Triton X-100 can be applied as an accurate, precise, and reproducible method to diagnose genetic disorders (e.g., trisomy 18 and trisomy 21) in fetuses.

  17. Population pharmacokinetics, brain distribution, and pharmacodynamics of 2nd generation dopamine transporter selective benztropine analogs developed as potential substitute therapeutics for treatment of cocaine abuse.

    PubMed

    Syed, Shariq A; Newman, Amy H; Othman, Ahmed A; Eddington, Natalie D

    2008-05-01

    A second generation of N-substituted 3alpha-[bis(4'-fluorophenyl)methoxy]-tropanes (GA 1-69, JHW 005 and JHW 013) binds with high affinity to the dopamine transporter (DAT) and are highly selective toward DAT compared to muscarinic receptor binding (M1). The objective of this study was to characterize brain distribution, pharmacokinetics, and pharmacodynamics [extracellular brain dopamine (DA) levels] of three novel N-substituted benztropine (BZT) analogs in male Sprague-Dawley rats. The BZT analogs displayed a higher distribution (Vd = 8.69-34.3 vs. 0.9 L/kg) along with longer elimination (t l/2: 4.1-5.4 vs. 0.5 h) than previously reported for cocaine. Brain-to-plasma partition coefficients were 1.3-2.5 vs. 2.1 for cocaine. The effect of the BZT analogs on extracellular brain (DA) levels ranged from minimal effects (GA 1-69) to several fold elevation (approximately 850% of basal DA for JHW 013) at the highest dose evaluated. PK/PD analysis of exposure-response data resulted in lower IC50 values for the BZT analogs compared to cocaine indicating their higher potency to inhibit DA reuptake (0.1-0.3 vs. 0.7 mg/L). These BZT analogs possess significantly different PK and PD profiles as compared to cocaine suggesting that further evaluation as cocaine abuse therapeutics is warranted.

  18. Implications of the neuroprotective effects of lithium for the treatment of bipolar and neurodegenerative disorders.

    PubMed

    Bauer, M; Alda, M; Priller, J; Young, L T

    2003-11-01

    Bipolar disorder is increasingly recognized as an illness that may progress to impairment in neurocognitive functioning and cell loss in cortical and limbic brain regions. Glutamatergic damage and/or damage due to high glucocorticoid levels that inhibit adult neurogenesis are likely contributing mechanisms. Drug treatments with possible neuroprotective effects are becoming increasingly important both clinically and as research tools. Mood stabilizing drugs and lithium in particular may act to prevent neuronal damage and tissue loss that may occur in the brain of patients with bipolar disorders. Lithium has been shown to exert neuroprotective effects in vitro and to stimulate neurogenesis in the hippocampus. Animal studies have demonstrated pharmacological effects of lithium suggestive of its role in neuroprotection, which range from reducing excitotoxicity through increased glutamate uptake, to regulation of a number of signal transduction intermediates such as myo-inositol, protein kinase C, phosphotidylinositol-3 kinase (PI-3K)/protein kinase B (Akt), ras-mitogen-activated protein kinase (MAPK), glycogen synthase kinase (GSK)-3alpha and -3beta and calcium. It remains to be established whether lithium treatment protects against possible cell damage in the same manner as it protects against recurrences of the illness. We propose to examine the effect of long-term lithium treatment on neurocognitive functioning of bipolar patients and the use of lithium in the treatment of chronic neuropsychiatric disorders.

  19. Roles of Wnt/{beta}-catenin signaling in epithelial differentiation of mesenchymal stem cells

    SciTech Connect

    Wang, Yajing; Sun, Zhaorui; Qiu, Xuefeng; Li, Yan; Qin, Jizheng; Han, Xiaodong

    2009-12-25

    Bone marrow-derived mesenchymal stem cells (MSCs) have been demonstrated to be able to differentiate into epithelial lineage, but the precise mechanisms controlling this process are unclear. Our aim is to explore the roles of Wnt/{beta}-catenin in the epithelial differentiation of MSCs. Using indirect co-culture of rat MSCs with rat airway epithelial cells (RTE), MSCs expressed several airway epithelial markers (cytokeratin 18, tight junction protein occudin, cystic fibrosis transmembrance regulator). The protein levels of some important members in Wnt/{beta}-catenin signaling were determined, suggested down-regulation of Wnt/{beta}-catenin with epithelial differentiation of MSCs. Furthermore, Wnt3{alpha} can inhibit the epithelial differentiation of MSCs. A loss of {beta}-catenin induced by Dickkopf-1 can enhance MSCs differentiation into epithelial cells. Lithium chloride transiently activated {beta}-catenin expression and subsequently decreased {beta}-catenin level and at last inhibited MSCs to differentiate into airway epithelium. Taken together, our study indicated that RTE cells can trigger epithelial differentiation of MSCs. Blocking Wnt/{beta}-catenin signaling may promote MSCs to differentiate towards airway epithelial cells.

  20. Microbial acetyl conjugation of T-2 toxin and its derivatives.

    PubMed Central

    Yoshizawa, T; Onomoto, C; Morooka, N

    1980-01-01

    The acetyl conjugation of T-2 toxin and its derivatives, the 12,13-epoxytrichothecene mycotoxins, was studied by using mycelia of trichothecene-producing strains of Fusarium graminearum, F. nivale, Calonectria nivalis, and F. sporotrichoides, T-2 toxin was efficiently converted into acetyl T-2 toxin by all strains except a T-2 toxin-producing strain of F. sporotrichoides, which hydrolyzed the substrate to HT-2-toxin and neosolaniol. HT-2 toxin was conjugated to 3-acetyl HT-2 toxin as an only product by mycelia of F. graminearum and C. nivalis, but was also resistant to conjugation by both F. nivale and F. sporotrichoides. Neosolaniol was also biotransformed selectively into 3-acetyl neosolaniol by F. graminearum. However, 3-acetyl HT-2 toxin was not acetylated by any of the strains under the conditions employed, but was hydrolyzed to HT-2 toxin by F. graminearum and F. nivale. This is the first report on the biological 3 alpha-O-acetyl conjugation of T-2 toxin and its derivatives. PMID:7396487

  1. Possible mechanisms of fragmentation of {sup 16}O nuclei with a momentum of 4.5 GeV/c per nucleon in track emulsions

    SciTech Connect

    Kotikov, E. A.

    2013-06-15

    The angular distributions of doubly charged fragments of {sup 16}O nuclei having a momentum of 4.5 GeV/c per nucleon and interacting with track-emulsion nuclei were studied. The experimental angular distributions of doubly charged fragments of a {sup 16}O nucleus are not described by the statistical model of the fragmentation of nuclei. The possible channels of fragmentation of {sup 16}O nuclei may include {sup 16}O {yields} 2{sup 8}Be {yields} 4{alpha}, {sup 16}O {yields} {sup 8}Be +{sup 8} Be* {yields} 4{alpha}, {sup 16}O {yields} 2{sup 8}Be* {yields} 4{alpha}, {sup 16}O {yields} {alpha}+{sup 12}C, {sup 16}O {yields} {alpha} +{sup 12}C* {yields} {alpha} + 3{alpha}, {sup 16}O {yields} {alpha} +{sup 12}C* {yields} {alpha} + p{alpha}{sup 7}Li, {sup 16}O {yields} {alpha} +{sup 12}C* {yields} {alpha} + 2{sup 6}Li, {sup 16}O {yields} {alpha} +{sup 12}C* {yields} {alpha} + pt2{alpha}, {sup 16}O {yields} Li + B, and {sup 16}O {yields} Li* + B*.

  2. Regulation of ergosterol biosynthesis and sterol uptake in a sterol-auxotrophic yeast.

    PubMed Central

    Lorenz, R T; Parks, L W

    1987-01-01

    Inhibition of sterol uptake in Saccharomyces cerevisiae sterol auxotroph FY3 (alpha hem1 erg7 ura) by delta-aminolevulinic acid (ALA) is dependent on the ability of the organism to synthesize heme from ALA. Sterol-depleted cells not exposed to ALA or strain PFY3 cells, with a double heme mutation, exposed to ALA did not exhibit inhibition of sterol uptake. Addition of ALA to sterol-depleted FY3 stimulated production of a high endogenous concentration of 2,3-oxidosqualene (25.55 micrograms mg-1 [dry weight]) at 24 h, whereas FY3 not exposed to ALA or PFY3 exposed to ALA did not accumulate 2,3-oxidosqualene. The high concentration of 2,3-oxidosqualene in FY3 with ALA decreased, and 2,3;22,23-dioxidosqualene increased to a very high level. The elevation of 2,3-oxidosqualene by ALA was correlated with a fivefold increase in the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC 1.1.1.34). The enhanced activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase was prevented by cycloheximide but not chloramphenicol and was dependent on a fermentative energy source. Inhibition of sterol uptake could not be attributed to 2,3-oxidosqualene or 2,3;22,23-dioxidosqualene but was due to a nonsaturating level of ergosterol produced as a consequence of heme competency through a leaky erg7 mutation. PMID:3301810

  3. Polymorphism of Alprazolam (Xanax): a review of its crystalline phases and identification, crystallographic characterization, and crystal structure of a new polymorph (form III).

    PubMed

    de Armas, Héctor Novoa; Peeters, Oswald M; Van den Mooter, Guy; Blaton, Norbert

    2007-05-01

    A new polymorphic form of Alprazolam (Xanax), 8-chloro-1-methyl-6-phenyl-4H-[1,2,4]triazolo-[4,3-alpha][1,4]benzodiazepine, C(17)H(13)ClN(4), has been investigated by means of X-ray powder diffraction (XRPD), single crystal X-ray diffraction, and differential scanning calorimetry (DSC). This polymorphic form (form III) was obtained during DSC experiments after the exothermic recrystallization of the melt of form I. The crystal unit cell dimensions for form III were determined from diffractometer methods. The monoclinic unit cell found for this polymorph using XRPD after indexing the powder diffractogram was confirmed by the cell parameters obtained from single crystal X-ray diffractometry on a crystal isolated from the DSC pans. The single crystal unit cell parameters are: a = 28.929(9), b = 13.844(8), c = 7.361(3) angstroms, beta = 92.82(3) degrees , V = 2944(2) angstroms(3), Z = 8, space group P2(1) (No.4), Dx = 1.393 Mg/m(3). The structure obtained from single crystal X-ray diffraction was used as initial model for Rietveld refinement on the powder diffraction data of form III. The temperature phase transformations of alprazolam were also studied using high temperature XRPD. A review of the different phases available in the Powder Diffraction File (PDF) database for this drug is described bringing some clarification and corrections. PMID:17455340

  4. Coagulopathy following major liver resection: the effect of rBPI21 and the role of decreased synthesis of regulating proteins by the liver.

    PubMed

    Meijer, C; Wiezer, M J; Hack, C E; Boelens, P G; Wedel, N I; Meijer, S; Nijveldt, R J; Statius Muller, M G; Wiggers, T; Zoetmulder, F A; Borel Rinkes, I H; Cuesta, M A; Gouma, D J; van de Velde, C J; Tilanus, H W; Scotté, M; Thijs, L G; van Leeuwen, P A

    2001-04-01

    This prospective study investigated the role of reduced hepatic synthesis of regulating proteins in coagulopathy after partial hepatectomy (PH) compared with major abdominal surgery (MAS) without involvement of the liver. Furthermore, we studied the effect of rBPI21, an endotoxin-neutralizing agent, on coagulopathy after PH was studied. Compared with MAS, PH resulted in significantly elevated levels of thrombin-antithrombin-III and plasmin-alpha2-antiplasmin complexes. Levels of antithrombin-3, alpha2-antiplasmin, fibrinogen, plasminogen, alpha2-macroglobulin (alpha2-M), and C1-inhibitor remained lower following PH. Treatment with rBPI21 led to significantly lower levels of tissue-type plasminogen activator (t-PA). Post-operative disseminated intravascular coagulation (DIC) was associated with significantly higher bilirubin and t-PA plasma levels and significantly lower levels of alpha2-M. This study indicates that PH induced hepatic failure results in decreased synthesis of hepatic regulating plasma proteins and subsequent activation of coagulation and fibrinolysis. Prevention of t-PA release by rBPI21 may have important clinical implications. Decreased availability of alpha2-M may be a factor in post-operative DIC.

  5. In vitro metabolism of radioactive progesterone and testosterone by the gonads of the protandrous Rhabdosargus sarba at various sexual phases

    SciTech Connect

    Yeung, W.S.; Chan, S.T.

    1985-08-01

    The in vitro steroidogenic capacity of the gonadal tissue in the protandrous Rhabdosargus sarba was studied. Testicular and ovarian tissues from various sexual phases were used either separately or combined. With progesterone as precursor, high yield of 5 beta-reduced metabolites, and no 11-ketotestosterone or 11 beta-hydroxytestosterone were found. The production of 5 beta-pregnan-3 alpha-ol-20-one and 5 beta-pregnane-3,20-dione was very high in incubations with testicular tissue from intersexes or males and was low in those with ovarian tissue only. The production of 17 alpha-hydroxyprogesterone was high in the female but was low in other sexual phases. With testosterone as precursor, 11-ketotestoterone and 11 beta-hydroxytestosterone were identified. 5 beta-Reductase activity was high in the male and relatively low in the female. 5 alpha-Reduced products, however, were absent. There was an increase in the production of androstenedione as the animal underwent sex reversal. The significance of this change in steroidogenesis in this protandrous fish is at present under investigation. Experimental results also indicated that in the intersexual gonad there may be interaction between the testicular component and the ovarian component in steroidgenesis.

  6. Soluble factor cross-talk between human bone marrow-derived hematopoietic and mesenchymal cells enhances in vitro CFU-F and CFU-O growth and reveals heterogeneity in the mesenchymal progenitor cell compartment.

    PubMed

    Baksh, Dolores; Davies, John E; Zandstra, Peter W

    2005-11-01

    The homeostatic adult bone marrow (BM) is a complex tissue wherein physical and biochemical interactions serve to maintain a balance between the hematopoietic and nonhematopoietic compartments. To focus on soluble factor interactions occurring between mesenchymal and hematopoietic cells, a serum-free adhesion-independent culture system was developed that allows manipulation of the growth of both mesenchymal and hematopoietic human BM-derived progenitors and the balance between these compartments. Factorial experiments demonstrated a role for stem cell factor (SCF) and interleukin 3 (IL-3) in the concomitant growth of hematopoietic (CD45+) and nonhematopoietic (CD45-) cells, as well as their derivatives. Kinetic tracking of IL-3alpha receptor (CD123) and SCF receptor (CD117) expression on a sorted CD45- cell population revealed the emergence of CD45-CD123+ cells capable of osteogenesis. Of the total fibroblast colony-forming units (CFU-Fs) and osteoblast colony-forming units (CFU-O), approximately 24% of CFU-Fs and about 22% of CFU-Os were recovered from this population. Cell-sorting experiments demonstrated that the CD45+ cell population secreted soluble factors that positively affect the survival and proliferation of CFU-Fs and CFU-Os generated from the CD45- cells. Together, our results provide insight into the intercellular cytokine network between hematopoietic and mesenchymal cells and provide a strategy to mutually culture both mesenchymal and hematopoietic cells in a defined scalable bioprocess.

  7. Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV

    NASA Technical Reports Server (NTRS)

    Lim, Kap; Ho, Joseph X.; Keeling, Kim; Gilliland, Gary L.; Ji, Xinhua; Rueker, Florian; Carter, Daniel C.

    1994-01-01

    The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(sub 3)2(sub 1)2 with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.

  8. Lytic enzyme, labiase for a broad range of Gram-positive bacteria and its application to analyze functional DNA/RNA.

    PubMed

    Niwa, Takashi; Kawamura, Yoshiaki; Katagiri, Yoshihiro; Ezaki, Takayuki

    2005-05-01

    The lytic activity of labiase and achromopeptidase for bacterial DNA/RNA extraction were compared. Rapid lysis of many bacterial strains was observed with labiase followed by SDS treatment. Both labiase and achromopeptidase showed high lytic activity against bacterial strains with the A1alpha chemotype (e.g., Aerococcus viridans) and the A3alpha chemotype (e.g., Staphylococcus epidermidis) for cell wall peptidoglycan structures. The lytic activity of labiase was higher than that of achromopeptidase against strains with the A1gamma chemotype (e.g., Bacillus subtilis). The activity of labiase was not detrimentally affected with increasing NaCl concentration. Labiase lysates were successfully used for rapid extraction of DNA and RNA, whereas achromopeptidase lysates degraded RNA. The DNA and RNA obtained were successfully used for 16S rRNA amplification and real-time RT-PCR detection. It is concluded that labiase is useful for rapid lysis of a wide variety of Gram-positive bacteria and can be used for DNA/RNA isolation protocols.

  9. Terpenoid composition of the wound-induced bark exudate of Commiphora tenuis from Ethiopia.

    PubMed

    Asres, K; Tei, A; Moges, G; Sporer, F; Wink, M

    1998-06-01

    The bark of Commiphora tenuis Vollensen exudes a translucent, free-flowing odoriferous liquid upon wounding which was analysed by capillary GLC and GLC-MS. 42 mono- and sesquiterpenes were detected and 37 identified. The main components of the monoterpenoid fraction were alpha-pinene (60.8%), beta-pinene (8.8%), sabinene (6.3%), alpha-thujene (8.9%), limonene (5.5%), 3-carene (3.7%), beta-myrcene (1.8%), and beta-elemene (1.1%) constituting 97% of the oil. Identified sesquiterpenoid components constituted approximately 1.6% of the oil. Oleanolic acid acetate was isolated and identified as the main triterpene from the resin by 1H- and 13C-NMR. Three other triterpenes of the olean-12-ene group were also detected using GC-MS. The essential oil exhibited antibacterial activities against Staphylococcus aureus. Proteus mirabilis and E. coli with MIC between 0.5 and 1%. PMID:9690355

  10. Estrogen receptor ligands counteract cognitive deficits caused by androgen deprivation in male rats.

    PubMed

    Lagunas, Natalia; Calmarza-Font, Isabel; Grassi, Daniela; Garcia-Segura, Luis M

    2011-04-01

    Androgen deprivation causes impairment of cognitive tasks in rodents and humans, and this deficit can be reverted by androgen replacement therapy. Part of the effects of androgens in the male may be mediated by their local metabolism to estradiol or 3-alpha androstanediol within the brain and the consequent activation of estrogen receptors. In this study we have assessed whether the administration of estradiol benzoate, the estrogen receptor β selective agonist diarylpropionitrile or the estrogen receptor α selective agonist propyl pyrazole triol affect performance of androgen-deprived male Wistar rats in the cross-maze test. In addition, we tested the effect of raloxifene and tamoxifen, two selective estrogen receptor modulators used in clinical practice. The behavior of the rats was assessed 2 weeks after orchidectomy or sham surgery. Orchidectomy impaired acquisition in the cross-maze test. Estradiol benzoate and the selective estrogen receptor β agonist significantly improved acquisition in the cross-maze test compared to orchidectomized animals injected with vehicle. Raloxifene and tamoxifen at a dose of 1mg/kg, but not at doses of 0.5 or 2mg/kg, also improved acquisition of orchidectomized animals. Our findings suggest that estrogenic compounds with affinity for estrogen receptor β and selective estrogen receptor modulators, such as raloxifene and tamoxifen, may represent good candidates to promote cognitive performance in androgen-deprived males.

  11. Acidic metabolites. VI. 20 alpha-isosteroids as intermediates in 20 alpha-dihydrosteroid formation

    SciTech Connect

    Monder, C.; Bradlow, H.L.; Han, C.A.; Zumoff, B.

    1985-03-01

    We have previously shown that human subjects metabolize the 20 beta-epimer of isocortisol (11 beta, 17,20 beta-trihydroxy-3-oxo-pregn-4-en-21-al) to both 20 alpha- and 20 beta-hydroxy steroid end products. In this paper we describe the synthesis of tritium labeled 20 alpha-epimers of isocortisol and isoTHF (3 alpha, 11 beta, 17,20 alpha-tetrahydroxy-5 beta-pregnan-21-al) and their metabolic fate in humans. Both steroids yielded 20 alpha-hydroxy urinary neutral end-products (cortols and cortolones) and no 20 beta-hydroxy epimers. Regeneration of 17-ketols from aldols occurred to a small extent with isoTHF, but not with isocortisol. Isocortisol and isoTHF yielded less cortoic acids than did the corresponding ketols. The results provide further evidence that in man the stereochemistry at C-20 of the end-products of corticosteroid metabolism is determined by the configuration of the aldol at C-20 prior to subsequent metabolic events.

  12. Purification, characterization, and crystallization of single molecular species of beta-conglycinin from soybean seeds.

    PubMed

    Morita, S; Fukase, M; Yamaguchi, M; Fukuda, Y; Morita, Y

    1996-05-01

    Four major molecular species of beta-conglycinin, alpha 3, alpha 2 beta, alpha beta 2, and beta 3, were isolated and purified from seeds of an alpha' subunit-deficient strain of soybeans (Glycine max). All components were found to be homogeneous by high pressure liquid chromatography, SDS-polyacrylamide gel electrophoresis, and amino acid and amino terminal sequence analyses. The amino acid compositions of the alpha 3 and beta 3 components agreed fairly well with the compositions deduced from the cDNA sequences, and all of the components were highly glycosylated. The alpha 3 and beta 3 components were compared regarding their secondary structures. The secondary structure of the alpha 3 component deduced from CD measurements showed a higher alpha-helix content than that of the beta 3 component. The beta 3 component was crystallized by decreasing the ionic strength from 0.5 to 0.14 in phosphate buffer, pH 7.3, and the crystals grew to a size (1.0 mm x 0.2 mm x 0.2 mm) suitable for X-ray crystallographic analysis. A preliminary X-ray analysis showed that the crystal belonged to an orthorhombic crystal system having the space group P2(1)2(1)2(1) and unit cell dimensions of a = 185.1 A, b = 107.9 A, and c = 97.6 A.

  13. Balancing omega-6 and omega-3 fatty acids in ready-to-use therapeutic foods (RUTF).

    PubMed

    Brenna, J Thomas; Akomo, Peter; Bahwere, Paluku; Berkley, James A; Calder, Philip C; Jones, Kelsey D; Liu, Lei; Manary, Mark; Trehan, Indi; Briend, André

    2015-05-15

    Ready-to-use therapeutic foods (RUTFs) are a key component of a life-saving treatment for young children who present with uncomplicated severe acute malnutrition in resource limited settings. Increasing recognition of the role of balanced dietary omega-6 and omega-3 polyunsaturated fatty acids (PUFA) in neurocognitive and immune development led two independent groups to evaluate RUTFs. Jones et al. (BMC Med 13:93, 2015), in a study in BMC Medicine, and Hsieh et al. (J Pediatr Gastroenterol Nutr 2015), in a study in the Journal of Pediatric Gastroenterology and Nutrition, reformulated RUTFs with altered PUFA content and looked at the effects on circulating omega-3 docosahexaenoic acid (DHA) status as a measure of overall omega-3 status. Supplemental oral administration of omega-3 DHA or reduction of RUTF omega-6 linoleic acid using high oleic peanuts improved DHA status, whereas increasing omega-3 alpha-linolenic acid in RUTF did not. The results of these two small studies are consistent with well-established effects in animal studies and highlight the need for basic and operational research to improve fat composition in support of omega-3-specific development in young children as RUTF use expands.

  14. Parametric Evaluation of Thin, Transonic Circulation-Control Airfoils

    NASA Technical Reports Server (NTRS)

    Schlecht, Robin; Anders, Scott

    2007-01-01

    Wind-tunnel tests were conducted in the NASA Langley Transonic Dynamics Tunnel on a 6 percent-thick, elliptical circulation-control airfoil with upper-surface and lower-surface blowing capability. Results for elliptical Coanda trailing-edge geometries, biconvex Coanda trailing-edge geometries, and leading-edge geometries are reported. Results are presented at subsonic and transonic Mach numbers of 0.3 and 0.8, respectively. When considering one fixed trailing-edge geometry, for both the subsonic and transonic conditions it was found that the [3.0:1] ratio elliptical Coanda surface with the most rounded leading-edge [03] performed favorably and was determined to be the best compromise between comparable configurations that took advantage of the Coanda effect. This configuration generated a maximum. (Delta)C(sub 1) = 0.625 at a C(sub mu) = 0.06 at M = 0.3, alpha = 6deg. This same configuration generated a maximum (Delta)C(sub 1) = 0.275 at a C(sub mu) = 0.0085 at M = 0.8, alpha = 3deg.

  15. Vitamin E delivery to human skin: studies using deuterated alpha-tocopherol measured by APCI LC-MS.

    PubMed

    Vaule, Heather; Leonard, Scott W; Traber, Maret G

    2004-02-15

    Enrichment of skin surface lipids with deuterium-labeled alpha-tocopherol was compared with plasma enrichment to evaluate kinetics of the delivery of vitamin E to skin surface lipids. For 7 d, subjects consumed 75 mg each of RRR-alpha-[5-(C2H3)]- (d3) and all rac-alpha-[5,7-(C2H3)2]- (d6) tocopheryl acetates with breakfast. Blood was drawn and skin lipids were collected daily for 2 weeks, then every other day for 2 weeks. A liquid chromatography-mass spectrometry atmospheric pressure chemical ionization method for quantification of deuterium labeled (d3, d6, d9-alpha-tocopherols) and unlabeled (d0-) alpha- and gamma-tocopherols was developed. Tocopherols were quantified at their m/z [M-1] using single ion recording. alpha-Tocopherol detection was linear from 1 to 100 pmol with a detection limit of 40 pg (93 fmol). Detection of gamma-tocopherol was twice as sensitive due to greater ionization efficiency. Though d3- and d6-alpha-tocopherols appeared in plasma within 24 h of the first dose, d3-alpha-tocopherol was not detected in skin surface lipids until approximately 1 week. Plasma percentage d3 peaked at day 8, while skin surface lipid percentage d3 increased on average until day 19. Apparently skin employs a mechanism to deliver alpha-tocopherol into skin via lipid secretions.

  16. Purification and properties of extracellular glucosyltransferases from Streptococcus mutans serotype a.

    PubMed

    Tsumori, H; Shimamura, A; Mukasa, H

    1983-10-01

    Extracellular glucosyltransferases (sucrose: 1,6-alpha-D-glucan 3-alpha- and 6-alpha-glucosyltransferase) of Streptococcus mutans HS6 (serotype a) were purified from the culture supernatant by DEAE-Sepharose chromatography, ConA-Sepharose chromatography and chromatofocusing. The enzymes I and II with specific activities of 6.20 and 5.86 i.u. mg-1, respectively, exhibited slightly different isoelectric points (pI 4.5 and 4.2) and the molecular weights were estimated to be 161000 and 174000, respectively, by SDS-PAGE. The enzymes had the same optimum pH of 5.5 and the same Km values of 1.3 mM for sucrose and of 83 microM-glucose equivalent for dextran T10. By double immunodiffusion test on agar, these enzymes were immunologically identical to each other. Analysis by GLC of the glucans synthesized de novo from sucrose by the enzymes (I and II) established that they were 1,6-alpha-D-glucans with 20 and 24.5 mol% 1,3,6-branch points, respectively. Both are therefore bifunctional enzymes. PMID:6228638

  17. Isolation and characterization of poly- and oligosaccharides from the red microalga Porphyridium sp.

    PubMed

    Geresh, Shimona; Arad, Shoshana Malis; Levy-Ontman, Oshrat; Zhang, Wang; Tekoah, Yoram; Glaser, Robert

    2009-02-17

    The current study forms part of an ongoing research effort focusing on the elucidation of the chemical structure of the sulfated extracellular polysaccharide of the red microalga Porphyridium sp. (UTEX 637). We report here on the chemical structure of a fraction separated from an acidic crude extract of the polysaccharide, as investigated by methylation analysis, carboxyl reduction-methylation analysis, desulfation-methylation analysis, partial acid hydrolysis, Smith degradation, together with 1D and 2D (1)H and (13)C NMR spectroscopy. This fraction with a molar mass of 2.39x10(5)g mol(-1) comprised D- and L-Gal, D-Glc, D-Xyl, D-GlcA, and sulfate groups in a molar ratio of 1.0:1.1:2.1:0.2:0.7. The almost linear backbone of the fraction is composed of (1-->2)- or (1-->4)-linked d-xylopyranosyl, (1-->3)-linked L-galactopyranosyl, (1-->3)-linked D-glucopyranosyl, and (1-->3)-linked D-glucopyranosyluronic acid and comprises a possible acidic building unit: [(2 or 4)-beta-D-Xylp-(L-->3)]m-alpha-D-Glcp-(1-->3)-alpha-D-GLCPA-(1-->3)-L-Galp(l-->. Attached to the backbone are sulfate groups and nonreducing terminal D-xylopyranosyl and galactopyranosyl residues, which occur at the O-6 positions of Glc-derived moieties in the main chain.

  18. Effect of musical modelling on late auditory evoked potentials.

    PubMed

    Paulus, W

    1988-01-01

    Late auditory evoked potentials were recorded in four subjects during musical tasks. A PDP 12 computer synchronized stimuli, which were produced by an integrated circuit, and recording with the help of a quartz time basis. The content of each experiment was different modelling of an ambiguous identical acoustic stimulus. In experiment 1, subjects had to model a 6-note melody according to the classic metric foot. In experiment 2, segmentation of an 8-note melody into 5- and 3- versus 3- and 5-tone motifs had to be performed. In experiment 1 an intra-individually reliable, but inter-individually variable neurophysiological correlate was detected during the heavy tone: (1) positivity, (2) negativity, (3) alpha blocking and (4) DC shift. Experiment 2 yielded an intra- and inter-individually reliable positive DC shift of about 4 microV between the two motifs. Myogenic, ocular, dermal, respiratory or electrocardiographic artefacts were excluded in each case. The results indicate that conclusions from evoked potentials to musical perception might be possible and that possible modelling mechanisms with subsequent undesirable influence on recordings have to be considered in any kind of evoked potential experimental design. PMID:3169066

  19. Experimental determination of ampicillin adsorption to nanometer-size Al2O3 in water.

    PubMed

    Peterson, Jonathan W; Burkhart, Rachel S; Shaw, Drew C; Schuiling, Amanda B; Haserodt, Megan J; Seymour, Michael D

    2010-09-01

    Transport of antibiotics in soil-water systems is controlled in part by adsorption to nanometer-size (10(-9)m) particles. Batch adsorption experiments were performed with ampicillin, a common amphoteric antibiotic, and 50 nm-Al(2)O(3) (alpha-alumina) at different pH conditions. Sorption to Al(2)O(3) can be described by linear isotherms for 2.9 microM-2.9 mM ampicillin concentrations. Distribution coefficients (K(d)) are 11.1 (+/-0.32)L kg(-1) at pH 2, 0.55 (+/-.04) L kg(-1) at pH 4, 21.9 (+/-0.9) L kg(-1) at pH 6, and 39.5 (+/-2.2) L kg(-1) at pH 8. At pH 2, approximately 47% of the initially adsorbed drug was removable by rinsing, at pH 4-56% was removed. Only 7% of the drug could be removed by rinsing at pH 6, and 3% at pH 8. Weak electrostatic forces dominate at pH<4, and stronger attachment mechanisms at higher pH. Low yields in rinsing (desorption) experiments at pH6 indicate strong attachment mechanisms, either electrostatic or possibly surface complexation. PMID:20638098

  20. Lysophospholipid presentation by CD1d and recognition by a human Natural Killer T-cell receptor

    SciTech Connect

    López-Sagaseta, Jacinto; Sibener, Leah V.; Kung, Jennifer E.; Gumperz, Jenny; Adams, Erin J.

    2014-10-02

    Invariant Natural Killer T (iNKT) cells use highly restricted {alpha}{beta} T cell receptors (TCRs) to probe the repertoire of lipids presented by CD1d molecules. Here, we describe our studies of lysophosphatidylcholine (LPC) presentation by human CD1d and its recognition by a native, LPC-specific iNKT TCR. Human CD1d presenting LPC adopts an altered conformation from that of CD1d presenting glycolipid antigens, with a shifted {alpha}1 helix resulting in an open A pocket. Binding of the iNKT TCR requires a 7-{angstrom} displacement of the LPC headgroup but stabilizes the CD1d-LPC complex in a closed conformation. The iNKT TCR CDR loop footprint on CD1d-LPC is anchored by the conserved positioning of the CDR3{alpha} loop, whereas the remaining CDR loops are shifted, due in part to amino-acid differences in the CDR3{beta} and J{beta} segment used by this iNKT TCR. These findings provide insight into how lysophospholipids are presented by human CD1d molecules and how this complex is recognized by some, but not all, human iNKT cells.

  1. Lipid content and fatty acid composition of green algae Scenedesmus obliquus grown in a constant cell density apparatus

    NASA Technical Reports Server (NTRS)

    Choi, K. J.; Nakhost, Z.; Barzana, E.; Karel, M.

    1987-01-01

    The lipids of alga Scenedesmus obliquus grown under controlled conditions were separated and fractionated by column and thin-layer chromatography, and fatty acid composition of each lipid component was studied by gas-liquid chromatography (GLC). Total lipids were 11.17%, and neutral lipid, glycolipid and phospholipid fractions were 7.24%, 2.45% and 1.48% on a dry weight basis, respectively. The major neutral lipids were diglycerides, triglycerides, free sterols, hydrocarbons and sterol esters. The glycolipids were: monogalactosyl diglyceride, digalactosyl diglyceride, esterified sterol glycoside, and sterol glycoside. The phospholipids included: phosphatidyl choline, phosphatidyl glycerol and phosphatidyl ethanolamine. Fourteen fatty acids were identified in the four lipid fractions by GLC. The main fatty acids were C18:2, C16:0, C18:3(alpha), C18:1, C16:3, C16:1, and C16:4. Total unsaturated fatty acid and essential fatty acid compositions of the total algal lipids were 80% and 38%, respectively.

  2. Fluorescence measurements reveal stoichiometry of K+ channels formed by modulatory and delayed rectifier alpha-subunits.

    PubMed

    Kerschensteiner, Daniel; Soto, Florentina; Stocker, Martin

    2005-04-26

    Modulatory alpha-subunits, which comprise one-fourth of all voltagegated K(+) channel (Kv) alpha-subunits, do not assemble into homomeric channels, but selectively associate with delayed rectifier Kv2 subunits to form heteromeric channels of unknown stoichiometry. Their distinct expression patterns and unique functional properties have made these channels candidate molecular correlates for a broad set of native K(+) currents. Here, we combine FRET and electrophysiological measurements to determine the stoichiometry and geometry of heteromeric channels composed of the delayed rectifier Kv2.1 subunit and the modulatory Kv9.3 alpha-subunit. Kv channel alpha-subunits were fused with GFP variants, and heteromerization of different combinations of tagged and untagged alpha-subunits was studied. FRET, evaluated by acceptor photobleaching, was only observed upon formation of functional channels. Our results, obtained from two independent experimental paradigms, suggest the formation of heteromeric Kv2.1/Kv9.3 channels of fixed stoichiometry consisting of three Kv2.1 subunits and one Kv9.3 subunit. Strikingly, despite this uneven stoichiometry, we find that heteromeric Kv2.1/Kv9.3 channels maintain a pseudosymmetric arrangement of subunits around the central pore. PMID:15827117

  3. Exploration of cone cyclic nucleotide-gated channel-interacting proteins using affinity purification and mass spectrometry.

    PubMed

    Ding, Xi-Qin; Matveev, Alexander; Singh, Anil; Komori, Naoka; Matsumoto, Hiroyuki

    2014-01-01

    Photopic (cone) vision essential for color sensation, central vision, and visual acuity is mediated by the activation of photoreceptor cyclic nucleotide-gated (CNG) channels. Naturally occurring mutations in the cone channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. This work investigated the functional modulation of cone CNG channel by exploring the channel-interacting proteins. Retinal protein extracts prepared from cone-dominant Nrl (- / -) mice were used in CNGA3 antibody affinity purification, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. The peptide mass fingerprinting of the tryptic digests and database search identified a number of proteins including spectrin alpha-2, ATPase (Na(+)/K(+) transporting) alpha-3, alpha and beta subunits of ATP synthase (H(+) transporting, mitochondrial F1 complex), and alpha-2 subunit of the guanine nucleotide-binding protein. In addition, the affinity-binding assays demonstrated an interaction between cone CNG channel and calmodulin but not cone Na(+)/Ca(2+)-K(+) exchanger in the mouse retina. Results of this study provide insight into our understanding of cone CNG channel-interacting proteins and the functional modulations.

  4. Chemical composition, larvicidal action, and adult repellency of Thymus magnus against Aedes albopictus.

    PubMed

    Park, Young-Uk; Koo, Hyun-Na; Kim, Gil-Hah

    2012-09-01

    Thymus magnus, an endemic species, is found in the Republic of Korea. The volatile compounds extracted by SPME from T. magnus were investigated for their chemical composition and electrophysiological response against the Asian tiger mosquito, Aedes albopictus. The volatile compounds of T. magnus as determined by gas chromatography mass spectrometry were gamma-terpinene (33.0%), thymol (29.9%), beta-bisabolene (8.9%), p-cymene (8.3%), alpha-terpinene (5.0%), myrcene (4.7%), beta-caryophyllene (4.0%), alpha-thujene (2.7%), camphene (1.3%), carvacrol (1.2%), and alpha-pinene (1.1%). Among these candidates, thymol exhibited complete (100%) repellent activity against female Ae. albopictus, an effect that was confirmed through evaluating the electrophysiological response on the antenna of Ae. albopictus. The effectiveness of a binary 1:2 mixture of thymol and vanillin (0.05:0.1 microl per cm2) was found to be significantly more effective than thymol alone for a period of 120 min. In addition, thymol, alpha-terpinene, and carvacrol showed high larvicidal activity against on the third-stage larvae with LC50 values of 0.9 microl per 100 ml. PMID:23833899

  5. Accumulation of 5 alpha-reduced androgen glucosiduronates associated with impaired removal in young male hemodialysis patients.

    PubMed

    Boudou, P; Naret, C; Fiet, J; Bonete, R; Tritto, G; Le Duc, A; Poignet, J L; Man, N K

    1995-12-01

    Hypothalamic-pituitary gonadal function is commonly altered in dialysis patients. Even though an improvement in general status and well-being has been noted after recombinant human erythropoietin supplementation, no significant changes were observed in the sex hormone profile. Pituitary gonadal axis as well as 5 alpha-reduced androgen glucosiduronates (i.e. 5 alpha-androstane,3 alpha,17 beta-diol and androsterone) profiles were studied in 23 young male stable dialyzed patients and compared to an age-matched group of healthy subjects. 5 alpha-Reduced androgen glucosiduronates are products of peripheral testosterone (T) metabolism and seem to be a useful tool in assessment of the male androgen status. Their polarity facilitates their urinary excretion, and their clearance is similar to the glomerular filtration rate in healthy men. We observed 1) a pituitary-Leydig cell dysfunction supported by normal serum estradiol and T levels, low free T, and increased LH levels; 2) an alteration of the dehydroepiandrosterone (DHEA) sulfate-DHEA interconversion, reflected by a dramatic decrease in DHEA while DHEA sulfate levels remained in the normal range; 3) an accumulation of 5 alpha-reduced androgen glucosiduronates, whose removal was impaired as shown by their very low sieving coefficients (< 0.012). Taken together, the above observations are consistent with alteration of spermatogenesis with respect to dialysis duration in which earlier elevated baseline serum LH levels indicate a primary defect in Leydig cell function.

  6. Hierarchy and positive/negative interplays of the hepatocyte nuclear factors HNF-1, -3 and -4 in the liver-specific enhancer for the human alpha-1-microglobulin/bikunin precursor.

    PubMed Central

    Rouet, P; Raguenez, G; Tronche, F; Mfou'ou, V; Salier, J P

    1995-01-01

    Alpha-1-microglobulin and bikunin are two plasma glycoproteins encoded by an alpha-1-microglobulin/bikunin precursor (AMBP) gene. The strict liver-specific expression of the AMBP gene is controlled by a potent enhancer made of six clustered boxes numbered 1-6 that have been reported to be proven or potential binding sites for the hepatocyte-enriched nuclear factors HNF-1, -4, -3, -1, -3, -4, respectively. In the present study, electromobility shift assays of wild-type or mutated probes demonstrated that the boxes 1-5 have a binding capacity for their cognate HNF protein. Box 5 is also a target for another, as yet unidentified, factor. A functional analysis of the wild-type or mutated enhancer, driving its homologous promoter and a reporter CAT gene in the HepG2 hepatoma cell line, demonstrated that all six boxes participate in the enhancer activity, with the primary influence of box 4 (HNF-1) and box 2 (HNF-4). A similar analysis in the HNF-free CHO cell line co-transfected with one or several HNF factors further demonstrated various interplays between boxes: box 3 (HNF-3 alpha and beta) has a negative influence over the major HNF-4 box 2 as well as a positive influence over the major HNF-1 box 4. Images PMID:7533900

  7. Polymorphism of Alprazolam (Xanax): a review of its crystalline phases and identification, crystallographic characterization, and crystal structure of a new polymorph (form III).

    PubMed

    de Armas, Héctor Novoa; Peeters, Oswald M; Van den Mooter, Guy; Blaton, Norbert

    2007-05-01

    A new polymorphic form of Alprazolam (Xanax), 8-chloro-1-methyl-6-phenyl-4H-[1,2,4]triazolo-[4,3-alpha][1,4]benzodiazepine, C(17)H(13)ClN(4), has been investigated by means of X-ray powder diffraction (XRPD), single crystal X-ray diffraction, and differential scanning calorimetry (DSC). This polymorphic form (form III) was obtained during DSC experiments after the exothermic recrystallization of the melt of form I. The crystal unit cell dimensions for form III were determined from diffractometer methods. The monoclinic unit cell found for this polymorph using XRPD after indexing the powder diffractogram was confirmed by the cell parameters obtained from single crystal X-ray diffractometry on a crystal isolated from the DSC pans. The single crystal unit cell parameters are: a = 28.929(9), b = 13.844(8), c = 7.361(3) angstroms, beta = 92.82(3) degrees , V = 2944(2) angstroms(3), Z = 8, space group P2(1) (No.4), Dx = 1.393 Mg/m(3). The structure obtained from single crystal X-ray diffraction was used as initial model for Rietveld refinement on the powder diffraction data of form III. The temperature phase transformations of alprazolam were also studied using high temperature XRPD. A review of the different phases available in the Powder Diffraction File (PDF) database for this drug is described bringing some clarification and corrections.

  8. {alpha}-cluster structure and density waves in oblate nuclei

    SciTech Connect

    Kanada-En'yo, Yoshiko; Hidaka, Yoshimasa

    2011-07-15

    Pentagon and triangle shapes in {sup 28}Si and {sup 12}C are discussed in relation to nuclear density waves. In the antisymmetrized molecular dynamics calculations, the K{sup {pi}=}5{sup -} band in {sup 28}Si and the K{sup {pi}=}3{sup -} band in {sup 12}C are described by the pentagon and triangle shapes, respectively. These negative-parity bands can be interpreted as the parity partners of the K{sup {pi}=}0{sup +} ground bands and they are constructed from the parity-asymmetric-intrinsic states. The pentagon and the triangle shapes originate in 7{alpha}- and 3{alpha}-cluster structures, respectively. In a mean-field picture, they are described also by the static one-dimensional density waves at the edge of the oblate states. In analyses with ideal {alpha}-cluster models using Brink-Bloch cluster wave functions and that with a simplified model, we show that the static edge density waves for the pentagon and triangle shapes can be understood by spontaneous breaking of axial symmetry, i.e., the instability of the oblate states with respect to the edge density waves. The density wave is enhanced in the Z=N nuclei due to the proton-neutron coherent density waves, while it is suppressed in Z{ne}N nuclei.

  9. Identification of a major cytokinin in coconut milk.

    PubMed

    Kobayashi, H; Morisaki, N; Tago, Y; Hashimoto, Y; Iwasaki, S; Kawachi, E; Nagata, R; Shudo, K

    1995-11-15

    A major cytokinin found in coconut milk was isolated by using the tobacco callus growth-promoting assay as a guide during purification. The structure of the factor was determined to be 14-O-(3-O-[beta-D-galactopyranosyl-(1-->2)-alpha-D-galactopyranosyl-(1-- >3)- alpha-L-arabinofuranosyl]-4-O-(alpha-L-arabinofuranosyl)-beta-D- galactopyranosyl)-trans-zeatin riboside [G3A2-ZR] by various NMR techniques, including heteronuclear multiple bond connectivity by 2D multiple quantum NMR (HMBC), as well as mass spectroscopy and sugar analysis. The optimum concentration of G3A2-ZR for cytokinin activity in the tobacco callus assay was estimated to be 5 x 10(-6) M, so that G3A2-ZR is one order of magnitude more potent than 1,3-diphenylurea and one order less potent than zeatin riboside. At least 20% of the cytokinin activity of coconut milk could be attributed to G3A2-ZR. PMID:7498450

  10. Fatty acid profile, tocopherol, squalene and phytosterol content of walnuts, almonds, peanuts, hazelnuts and the macadamia nut.

    PubMed

    Maguire, L S; O'Sullivan, S M; Galvin, K; O'Connor, T P; O'Brien, N M

    2004-05-01

    Nuts are high in fat but have a fatty acid profile that may be beneficial in relation to risk of coronary heart disease. Nuts also contain other potentially cardioprotective constituents including phytosterols, tocopherols and squalene. In the present study, the total oil content, peroxide value, composition of fatty acids, tocopherols, phytosterols and squalene content were determined in the oil extracted from freshly ground walnuts, almonds, peanuts, hazelnuts and the macadamia nut. The total oil content of the nuts ranged from 37.9 to 59.2%, while the peroxide values ranged from 0.19 to 0.43 meq O2/kg oil. The main monounsaturated fatty acid was oleic acid (C18:1) with substantial levels of palmitoleic acid (C16:1) present in the macadamia nut. The main polyunsaturated fatty acids present were linoleic acid (C18:2) and linolenic acid (C18:3). alpha-Tocopherol was the most prevalent tocopherol except in walnuts. The levels of squalene detected ranged from 9.4 to 186.4 microg/g. beta-Sitosterol was the most abundant sterol, ranging in concentration from 991.2 to 2071.7 microg/g oil. Campesterol and stigmasterol were also present in significant concentrations. Our data indicate that all five nuts are a good source of monounsaturated fatty acid, tocopherols, squalene and phytosterols.

  11. A salicylic acid-based analogue discovered from virtual screening as a potent inhibitor of human 20alpha-hydroxysteroid dehydrogenase.

    PubMed

    Dhagat, Urmi; Carbone, Vincenzo; Chung, Roland P-T; Matsunaga, Toshihiro; Endo, Satoshi; Hara, Akira; El-Kabbani, Ossama

    2007-11-01

    20alpha-hydroxysteroid dehydrogenase (AKR1C1) plays a key role in the metabolism of progesterone and other steroid hormones, thereby regulating their action at the pre-receptor level. AKR1C1 is implicated in neurological and psychiatric conditions such as catamenial epilepsy and depressive disorders. Increased activity of AKR1C1 is associated with termination of pregnancy and the development of breast cancer, endometriosis and endometrial cancer. Inhibition of the undesired activity of AKR1C1 will help reduce risks of premature birth, neurological disorders and the development of cancer. In order to identify potential leads for new inhibitors of AKR1C1 we adopted a virtual screening-based approach using the automated DOCK program. Approximately 250,000 compounds from the NCI database were screened for potential ligands based on their chemical complementarity and steric fit within the active site of AKR1C1. Kinetic analysis revealed 3,5-diiodosalicylic acid, an analogue of salicylic acid, as a potent competitive inhibitor with respect to the substrate 5beta-pregnane-3alpha,20alpha-diol with a K(i) of 9 nM. Aspirin, which is a well known salicylic acid-based drug, was also found to inhibit AKR1C1 activity. This is the first report to show aspirin (IC(50)=21 microM) and its metabolite salicylic acid (IC(50)=7.8 microM) as inhibitors of AKR1C1.

  12. Dietary fat composition modifies the effect of boron on bone characteristics and plasma lipids in rats.

    PubMed

    Nielsen, Forrest H

    2004-01-01

    Female and male rats weighing about 170 g and 200 g, respectively, were fed diets (approximately 70 microg boron/kg) in a factorial arrangement with supplemental boron at 0 (deficient) and 3 (adequate) mg/kg and canola oil or palm oil at 75 g/kg of diet as variables. After 5 weeks, six females in each treatment were bred. Dams and pups continued on their respective dietary treatments through gestation, lactation and post-weaning. Thirteen weeks after weaning, plasma and bones were collected from 12 male and 12 female offspring in each treatment. Boron supplementation increased femur strength measured by the breaking variable bending moment; tibial calcium and phosphorus concentrations; and plasma alkaline phosphatase. Femur breaking stress was greatest in boron-supplemented rats fed canola oil, and lowest in boron-deprived females fed canola oil; this group also exhibited the lowest femur bending moment. Minerals associated with bone organic matrix, zinc and potassium, were increased by boron supplementation in tibia. Plasma phospholipids were decreased by boron deprivation in females, but not males. Plasma cholesterol was decreased in boron-supplemented males by replacing canola oil with palm oil. The findings suggest that a diet high in omega-3 alpha-linolenic acid promotes femur strength best when the dietary boron is adequate.

  13. Development of a spectroscopic assay for bifunctional ligand-protein conjugates based on copper.

    PubMed

    Brady, Erik D; Chong, Hyun-Soon; Milenic, Diane E; Brechbiel, Martin W

    2004-08-01

    A simple, non-radioactive method for the determination of ligand-to-protein ratio (L/P) for novel ligand-antibody conjugates has been developed based on an exchange equilibrium with the purple Cu(II) complex of arsenazo III. The method requires a UV/Vis spectrometer and has been verified for monoclonal antibody Herceptin conjugates of a variety of ligand modalities, including common macrocyclic compounds NOTA and TETA, and with a new bifunctional tachpyridine (1H-Pyrrole-1-butanamide,N-[4-[[(1alpha,3alpha,5alpha)-3,5-bis[(2-pyridinylmethyl)amino]cyclohexyl](2-pyridinylmethyl)amino]butyl]-2,5-dihydro-2,5-dioxo-(9CI)). The spectroscopically derived values for L/P were verified by titration of the ligand-antibody conjugate with 64Cu. In each case, the value obtained by UV/Vis spectroscopy matches that found by radiolabeling. The method is rapid, taking less than 30 minutes with each ligand in this study.

  14. High risk groups in oil shale workforce

    SciTech Connect

    Gratt, L.B.; Perry, B.W.; Marine, W.M.; Savitz, D.A.

    1984-04-01

    The workforce risks of a hypothetical one million barrels-per-day oil shale industry were estimated. The risks for the different workforce segments were compared and high risk groups were identified. Accidents and injuries were statistically described by rates for fatalities, for accidents with days lost from work, and for accidents with no days lost from work. Workforce diseases analyzed were cancers, silicosia, pneumoconiosis, chronic bronchitis, chronic airway obstruction, and high frequency hearing loss. A comparison of the workforce groups under different risk measures (occurrence, fatality, and life-loss expectancy) was performed. The miners represented the group with the largest fatality and the most serious accident rate, although the estimated rates were below the average industry-wide underground mining experience. Lung disease from inhalation exposure of about the nuisance dust threshold limit value presents a significant risk for future concerns. If future environmental dust exposure is at the 100 ..mu..g/m/sup 3/ alpha-quartz level, safety improvements in the mining sector are of prime importance to reduce the oil shale worker's life-loss expectancy. 11 references, 1 figure, 11 tables.

  15. A Highly Tilted Binding Mode by a Self-Reactive T Cell Receptor Results in Altered Engagement of Peptide and MHC

    SciTech Connect

    D Sethi; D Schubert; A Anders; A Heroux; D Bonsor; C Thomas; E Sundberg; J Pyrdol; K Wucherpfennig

    2011-12-31

    Self-reactive T cells that escape elimination in the thymus can cause autoimmune pathology, and it is therefore important to understand the structural mechanisms of self-antigen recognition. We report the crystal structure of a T cell receptor (TCR) from a patient with relapsing-remitting multiple sclerosis that engages its self-peptide-major histocompatibility complex (pMHC) ligand in an unusual manner. The TCR is bound in a highly tilted orientation that prevents interaction of the TCR-{alpha} chain with the MHC class II {beta} chain helix. In this structure, only a single germline-encoded TCR loop engages the MHC protein, whereas in most other TCR-pMHC structures all four germline-encoded TCR loops bind to the MHC helices. The tilted binding mode also prevents peptide contacts by the short complementarity-determining region (CDR) 3{beta} loop, and interactions that contribute to peptide side chain specificity are focused on the CDR3{alpha} loop. This structure is the first example in which only a single germline-encoded TCR loop contacts the MHC helices. Furthermore, the reduced interaction surface with the peptide may facilitate TCR cross-reactivity. The structural alterations in the trimolecular complex are distinct from previously characterized self-reactive TCRs, indicating that there are multiple unusual ways for self-reactive TCRs to bind their pMHC ligand.

  16. A highly tilted binding mode by a self-reactive T cell receptor results in altered engagement of peptide and MHC

    SciTech Connect

    Sethi, D.K.; Heroux, A.; Schubert, D. A.; Anders, A.-K.; Bonsor, D. A.; Thomas, C. P.; Sundberg, E. J.; Pyrdol, J.; Wucherpfennig, K. W.

    2011-01-17

    Self-reactive T cells that escape elimination in the thymus can cause autoimmune pathology, and it is therefore important to understand the structural mechanisms of self-antigen recognition. We report the crystal structure of a T cell receptor (TCR) from a patient with relapsing-remitting multiple sclerosis that engages its self-peptide-major histocompatibility complex (pMHC) ligand in an unusual manner. The TCR is bound in a highly tilted orientation that prevents interaction of the TCR-{alpha} chain with the MHC class II {beta} chain helix. In this structure, only a single germline-encoded TCR loop engages the MHC protein, whereas in most other TCR-pMHC structures all four germline-encoded TCR loops bind to the MHC helices. The tilted binding mode also prevents peptide contacts by the short complementarity-determining region (CDR) 3{beta} loop, and interactions that contribute to peptide side chain specificity are focused on the CDR3{alpha} loop. This structure is the first example in which only a single germline-encoded TCR loop contacts the MHC helices. Furthermore, the reduced interaction surface with the peptide may facilitate TCR cross-reactivity. The structural alterations in the trimolecular complex are distinct from previously characterized self-reactive TCRs, indicating that there are multiple unusual ways for self-reactive TCRs to bind their pMHC ligand.

  17. Darwin's evolution theory, brain oscillations, and complex brain function in a new "Cartesian view".

    PubMed

    Başar, Erol; Güntekin, Bahar

    2009-01-01

    Comparatively analyses of electrophysiological correlates across species during evolution, alpha activity during brain maturation, and alpha activity in complex cognitive processes are presented to illustrate a new multidimensional "Cartesian System" brain function. The main features are: (1) The growth of the alpha activity during evolution, increase of alpha during cognitive processes, and decrease of the alpha entropy during evolution provide an indicator for evolution of brain cognitive performance. (2) Human children younger than 3 years are unable to produce higher cognitive processes and do not show alpha activity till the age of 3 years. The mature brain can perform higher cognitive processes and demonstrates regular alpha activity. (3) Alpha activity also is significantly associated with highly complex cognitive processes, such as the recognition of facial expressions. The neural activity reflected by these brain oscillations can be considered as constituent "building blocks" for a great number of functions. An overarching statement on the alpha function is presented by extended analyzes with multiple dimensions that constitute a "Cartesian Hyperspace" as the basis for oscillatory function. Theoretical implications are considered. PMID:18805445

  18. Use of isotope ratio mass spectrometry to detect doping with oral testosterone undecanoate: inter-individual variability of 13C/12C ratio.

    PubMed

    Baume, Norbert; Saudan, Christophe; Desmarchelier, Aurélien; Strahm, Emmanuel; Sottas, Pierre-Edouard; Bagutti, Carlo; Cauderay, Michel; Schumacher, Yorck Olaf; Mangin, Patrice; Saugy, Martial

    2006-05-01

    The metabolic effect of multiple oral testosterone undecanoate (TU) doses over 4 weeks was assessed in seven voluntary men. The protocol was designed to detect accumulation of the substance by choosing the appropriate spot urines collections time and to study the urinary clearance of the substance after weeks of treatment. Urines were analysed by a new GC/C/isotope ratio mass spectrometry (IRMS) method to establish the delta(13)C-values of testosterone metabolites (androsterone and etiocholanolone) together with an endogenous reference compound (16(5alpha)-androsten-3alpha-ol). The significant differences in inter-individual metabolism following TU intake was illustrated by large variations in delta(13)C-values of both T metabolites (maximum Deltadelta(13)C-values = 5.5 per thousand), as well as by very stable longitudinal T/E profiles and carbon isotopic ratios in the first hours following administration. According to T/E ratios and delta(13)C-values, the washout period after 80 mg TU intake was less than 48 h for all subjects and no accumulation phenomenon was observed upon chronic oral administration. PMID:16438998

  19. Application of factorial design in optimization of preconcentration procedure for copper determination in soft drink by flame atomic absorption spectrometry.

    PubMed

    Castro, Martha T P O; Baccan, Nivaldo

    2005-03-15

    In the present paper, a procedure for preconcentration and determination of copper in soft drink using flame atomic absorption spectrometry (FAAS) is proposed, which is based on solid-phase extraction of copper(II) ions as its ion pair of 1,10-phenanthroline complexes with the anionic surfactant sodium dodecil sulphate (SDS), by Amberlite XAD-2 resin. The optimization process was carried out using 2(4-1) factorial and 2(2) factorial with a center point designs. Four variables (XAD-2 mass, copper mass, sample flow rate and elution flow rate) were regarded as factors in the optimization. Student's t-test on the results of the 2(4-1) factorial design with eight runs for copper extraction, demonstrated that the factors XAD-2 mass and sample flow rate in the levels studied are statistically significant. The 2(2) factorial with a center point design was applied in order to determine the optimum conditions for extraction. The procedure proposed allowed the determination of copper with detection limits (3alpha/S) of 3.9mugl(-1). The precision, calculated as relative standard deviation (R.S.D.) was 1.8% for 20.0mugl(-1) of copper. The preconcentration factor was 100. The robustness of this procedure is demonstrated by the recovery achieved for determination of copper in the presence of several cations. This procedure was applied to the determination of copper in soft drink samples collected in Campinas, SP, Brazil. PMID:18969940

  20. Potentiation of quantitative electroencephalograms following prefrontal repetitive transcranial magnetic stimulation in patients with major depression.

    PubMed

    Noda, Yoshihiro; Nakamura, Motoaki; Saeki, Takashi; Inoue, Misa; Iwanari, Hideo; Kasai, Kiyoto

    2013-01-01

    The long-lasting effects of repetitive transcranial magnetic stimulation (rTMS) on electroencephalogram (EEG) activity are not clear. We aimed to investigate the cumulative rTMS effects on EEG and clinical outcomes in patients with major depression. Twenty-five patients with medication-resistant depression underwent 10 daily rTMS sessions over the left dorsolateral prefrontal cortex. We measured resting EEG and spectrum-power before and after the rTMS course. Clinical efficacy was evaluated with the Hamilton's Depression Rating Scale (HAM-D) and Wisconsin Card Sorting Test (WCST). In an ANOVA model, including all prefrontal electrodes, post hoc analyses revealed significant time effects on the theta (F1,24 = 7.89, P = 0.010; +43%), delta (F1,24 = 6.58, P = 0.017; +26%), and alpha (F1,24 = 4.64, P = 0.042; 31%) bands without site specificity. Clinical correlations were observed between F4 alpha power increases and improvements in HAM-D retardation, F3 alpha power increases and improvements of the absolute changes in perseveration and error number on the WCST, and C3 and C4 theta power increases and improvements of the percent change in perseveration and error number on the WCST following rTMS. Consecutive prefrontal rTMS could induce long-lasting EEG potentiations beyond the aftereffects, resulting in improved cognitive and depressive symptoms.

  1. Potentiation of quantitative electroencephalograms following prefrontal repetitive transcranial magnetic stimulation in patients with major depression.

    PubMed

    Noda, Yoshihiro; Nakamura, Motoaki; Saeki, Takashi; Inoue, Misa; Iwanari, Hideo; Kasai, Kiyoto

    2013-01-01

    The long-lasting effects of repetitive transcranial magnetic stimulation (rTMS) on electroencephalogram (EEG) activity are not clear. We aimed to investigate the cumulative rTMS effects on EEG and clinical outcomes in patients with major depression. Twenty-five patients with medication-resistant depression underwent 10 daily rTMS sessions over the left dorsolateral prefrontal cortex. We measured resting EEG and spectrum-power before and after the rTMS course. Clinical efficacy was evaluated with the Hamilton's Depression Rating Scale (HAM-D) and Wisconsin Card Sorting Test (WCST). In an ANOVA model, including all prefrontal electrodes, post hoc analyses revealed significant time effects on the theta (F1,24 = 7.89, P = 0.010; +43%), delta (F1,24 = 6.58, P = 0.017; +26%), and alpha (F1,24 = 4.64, P = 0.042; 31%) bands without site specificity. Clinical correlations were observed between F4 alpha power increases and improvements in HAM-D retardation, F3 alpha power increases and improvements of the absolute changes in perseveration and error number on the WCST, and C3 and C4 theta power increases and improvements of the percent change in perseveration and error number on the WCST following rTMS. Consecutive prefrontal rTMS could induce long-lasting EEG potentiations beyond the aftereffects, resulting in improved cognitive and depressive symptoms. PMID:23827366

  2. Dystonin/Bpag1 is a necessary endoplasmic reticulum/nuclear envelope protein in sensory neurons

    SciTech Connect

    Young, Kevin G.; Kothary, Rashmi

    2008-09-10

    Dystonin/Bpag1 proteins are cytoskeletal linkers whose loss of function in mice results in a hereditary sensory neuropathy with a progressive loss of limb coordination starting in the second week of life. These mice, named dystonia musculorum (dt), succumb to the disease and die of unknown causes prior to sexual maturity. Previous evidence indicated that cytoskeletal defects in the axon are a primary cause of dt neurodegeneration. However, more recent data suggests that other factors may be equally important contributors to the disease process. In the present study, we demonstrate perikaryal defects in dorsal root ganglion (DRG) neurons at stages preceding the onset of loss of limb coordination in dt mice. Abnormalities include alterations in endoplasmic reticulum (ER) chaperone protein expression, indicative of an ER stress response. Dystonin in sensory neurons localized in association with the ER and nuclear envelope (NE). A fusion protein ofthe dystonin-a2 isoform, which harbors an N-terminal transmembrane domain, associated with and reorganized the ER in cell culture. This isoform also interacts with the NE protein nesprin-3{alpha}, but not nesprin-3{beta}. Defects in dt mice, as demonstrated here, may ultimately result in pathogenesis involving ER dysfunction and contribute significantly to the dt phenotype.

  3. [Clinical findings and differential diagnosis in ketosis and hypocalcemia in sheep].

    PubMed

    Bickhardt, K; Henze, P; Ganter, M

    1998-11-01

    Clinical and laboratory examinations were carried out in a prospective study from 1978 to 1998 on 454 spontaneously diseased sheep of different breeds aged 2-8 years. Three groups of sheep were defined by plasma concentrations of 3-hydroxybutyrate (3-OHB) and calcium. 214 ewes suffered from ketosis (pregnancy toxaemia, 3-OHB > 1.6 mmol/l), 195 ewes and 5 rams suffered from hypocalcaemia (Ca < 2.0 mmol/l), and 40 ewes were sick with booth diseases simultaneously. Clinical findings differ only slightly in all three groups. In ketosis the time until the onset of clinical signs and the duration of the disease is somewhat longer, while locomotion disturbances are less severe than in hypocalcaemia and in the combination of both. In individual cases a differential diagnosis is not possible on the basis of clinical findings, and also acetonuria is not a reliable criterion. Therefore, a therapy is suggested to treat all three possible forms of disease: daily oral application of Na-propionate, Ca-lactate and K-chloride, and additional subcutanous injection of Ca-borogluconate, vitamin D3, alpha-Tocopherol and selenium at the beginning of the disease. Pathogenesis of both diseases are discussed, recommendations for feeding management are given.

  4. The new species Lecanora bicinctoidea, its position and considerations about phenotypic evolution in the Lecanora rupicola group.

    PubMed

    Blaha, Juliane; Grube, Martin

    2007-01-01

    A phylogenetic analysis of the Lecanora rupicola group based on combined nITS rDNA and beta-tubulin sequences and a combined dataset of ITS, beta-tubulin and partial sequences of polyketide synthase genes reveals a previously unrecognized species, which here is introduced under the name Lecanora bicinctoidea. The new species is a sister group of the L. swartzii complex (including L. swartzii and L. lojkaeana), which is characterized by eucorticate ascomata, and a morphological diversity that includes also a dwarf-fruticose lineage. The preferential occurrence on vertical to overhanging siliceous rocks corresponds more closely to L. swartzii. A detailed investigation of phenotypic characters reveals that the new species differs from the superficially similar morphospecies L. bicincta in several ways, such as a thallus of comparatively small areoles and broadly sessile ascomata and the development of an amphithecial cortex devoid of algal remnants (i.e. an eucortex). L. bicinctoidea contains methyl 3alpha-hydroxy-4-O-demethylbarbatate, a chemical compound not known from other members of the L. rupicola group. We also discuss the importance of eucortex formation as one of several factors that are involved in the evolution of substrate-detached thallus structures.

  5. Identification of a major cytokinin in coconut milk.

    PubMed

    Kobayashi, H; Morisaki, N; Tago, Y; Hashimoto, Y; Iwasaki, S; Kawachi, E; Nagata, R; Shudo, K

    1995-11-15

    A major cytokinin found in coconut milk was isolated by using the tobacco callus growth-promoting assay as a guide during purification. The structure of the factor was determined to be 14-O-(3-O-[beta-D-galactopyranosyl-(1-->2)-alpha-D-galactopyranosyl-(1-- >3)- alpha-L-arabinofuranosyl]-4-O-(alpha-L-arabinofuranosyl)-beta-D- galactopyranosyl)-trans-zeatin riboside [G3A2-ZR] by various NMR techniques, including heteronuclear multiple bond connectivity by 2D multiple quantum NMR (HMBC), as well as mass spectroscopy and sugar analysis. The optimum concentration of G3A2-ZR for cytokinin activity in the tobacco callus assay was estimated to be 5 x 10(-6) M, so that G3A2-ZR is one order of magnitude more potent than 1,3-diphenylurea and one order less potent than zeatin riboside. At least 20% of the cytokinin activity of coconut milk could be attributed to G3A2-ZR.

  6. Biotransformation of norgestimate in women

    SciTech Connect

    Alton, K.B.; Hetyei, N.S.; Shaw, C.; Patrick, J.E.

    1984-01-01

    The metabolism of norgestimate (ORF-10131; /sup 14/C-d-13-ethyl-17-acetoxy-18,19-dinor-17 alpha-pregn-4-en-20- yn -3-oxime) was studied in humans. Compound labeled with carbon-14 in the 17 alpha-ethynyl group was administered to four female subjects. An average of 46.8 percent of the administered radioactivity was excreted in the urine and 36.8 percent in the feces over a two-week collection period. About 12 percent of the urinary radioactivity represented freely extractable metabolites and another 57 percent consisted of extractable material released by enzyme hydrolysis. The ethynylated metabolites of norgestimate were separated from endogenous compounds and non- ethynylated metabolites by silver- sulfoethyl cellulose column chromatography. Metabolites were subsequently isolated by high performance liquid chromatography and thin layer chromatography. The identification of five urinary metabolites was accomplished by combined gas-liquid chromatography/mass spectrometry. These metabolites include norgestrel, 16 beta- hydroxynorgestrel, 2 alpha- hydroxynorgestrel, 3 alpha, 5 beta- tetrahydronorgestrel, and a fifth trihydroxylated metabolite of undetermined stereochemical configuration; 3,16-dihydroxy-5- tetrahydronorgestrel .

  7. Effect of the bile-acid sequestrant colestipol on postprandial serum bile-acid concentration: evaluation by bioluminescent enzymic analysis.

    PubMed

    Rossi, S S; Wayne, M L; Smith, R B; Wright, C E; Andreadis, N A; Hofmann, A F

    1989-02-01

    Chronic ingestion of bile-acid sequestrants has been shown to decrease the serum cholesterol concentration and coronary events in hypercholesterolaemic patients. To develop improved sequestrants, a rapid, convenient method for testing the bile-acid binding efficacy of sequestrants is needed. Serum bile-acid concentrations could be used to detect bile-acid binding by an administered sequestrant, since the serum bile-acid concentration is determined largely by the rate of intestinal absorption in healthy individuals. To test this, serum bile-acid concentrations were measured at frequent intervals over 24 h in five otherwise healthy hypercholesterolaemic subjects during the ingestion of three standard meals, with or without the addition of 5 g colestipol granules administered 30 min before each meal. Total serum bile-acid concentration was measured with a previously reported bioluminescent enzymic assay, that uses a 3 alpha-hydroxysteroid dehydrogenase, an oxido-reductase, and a bacterial luciferase co-immobilized on to Sepharose beads. Bile acids in 1 ml of serum were isolated by solid-phase extraction chromatography with reversed-phase C18 cartridges. Colestipol lowered the postprandial elevation of serum bile acids by one half, with a subsequent decrease in the cumulative area under the curve. The data suggest that measurement of serum bile-acid concentrations by bioluminescence is a rapid, simple way to document the efficacy of bile-acid sequestrants.

  8. Achromatopsia as a potential candidate for gene therapy.

    PubMed

    Pang, Ji-Jing; Alexander, John; Lei, Bo; Deng, Wentao; Zhang, Keqing; Li, Qiuhong; Chang, Bo; Hauswirth, William W

    2010-01-01

    Achromatopsia is an autosomal recessive retinal disease involving loss of cone function that afflicts approximately 1 in 30,000 individuals. Patients with achromatopsia usually have visual acuities lower than 20/200 because of the central vision loss, photophobia, complete color blindness and reduced cone-mediated electroretinographic (ERG) amplitudes. Mutations in three genes have been found to be the primary causes of achromatopsia, including CNGB3 (beta subunit of the cone cyclic nucleotide-gated cation channel), CNGA3 (alpha subunit of the cone cyclic nucleotide-gated cation channel), and GNAT2 (cone specific alpha subunit of transducin). Naturally occurring mouse models with mutations in Cnga3 (cpfl5 mice) and Gnat2 (cpfl3 mice) were discovered at The Jackson Laboratory. A natural occurring canine model with CNGB3 mutations has also been found. These animal models have many of the central phenotypic features of the corresponding human diseases. Using adeno-associated virus (AAV)-mediated gene therapy, we and others show that cone function can be restored in all three models. These data suggest that human achromatopsia may be a good candidate for corrective gene therapy. PMID:20238068

  9. Modulation of cerebral microvascular permeability by endothelial nicotinic acetylcholine receptors.

    PubMed

    Hawkins, Brian T; Egleton, Richard D; Davis, Thomas P

    2005-07-01

    Nicotine increases the permeability of the blood-brain barrier in vivo. This implies a possible role for nicotinic acetylcholine receptors in the regulation of cerebral microvascular permeability. Expression of nicotinic acetylcholine receptor subunits in cerebral microvessels was investigated with immunofluorescence microscopy. Positive immunoreactivity was found for receptor subunits alpha3, alpha5, alpha7, and beta2, but not subunits alpha4, beta3, or beta4. Blood-brain barrier permeability was assessed via in situ brain perfusion with [14C]sucrose. Nicotine increased the rate of sucrose entry into the brain from 0.3 +/- 0.1 to 1.1 +/- 0.2 microl.g(-1).min(-1), as previously described. This nicotine-induced increase in blood-brain barrier permeability was significantly attenuated by both the blood-brain barrier-permeant nicotinic antagonist mecamylamine and the blood-brain barrier-impermeant nicotinic antagonist hexamethonium to 0.5 +/- 0.2 and 0.3 +/- 0.2 microl.g(-1).min(-1), respectively. These data suggest that nicotinic acetylcholine receptors expressed on the cerebral microvascular endothelium mediate nicotine-induced changes in blood-brain barrier permeability.

  10. Altered profile of gene expression in rat hearts induced by chronic nicotine consumption.

    PubMed

    Hu, Dahai; Cao, Kun; Peterson-Wakeman, Robert; Wang, Rui

    2002-10-01

    Using a cDNA microarray technique, we analyzed the expression profile of 1081 genes in the whole heart tissue of rats. The expressions of three classes of genes encoding cellular energy metabolism enzymes, transmembrane receptors, and intracellular kinase network members were reduced by more than 2.5-fold in cardiac tissues from the rats fed with nicotine (3mg/kg/day) for 3 months. The down-regulated 11 genes included mitochondrial ATP synthase beta subunit, mitochondrial H(+) transporting ATP synthase F1 complex alpha subunit isoform 1, liver mitochondrial aldehyde dehydrogenase 2, glutathione-S-transferase mu type 2, corticotropin-releasing factor receptor 2, metabotropic glutamate receptor 2, N-methyl-D-aspartate receptor subtype 2B, muscarinic acetylcholine receptor M3, transmembrane receptor Unc5H1, glycogen synthase kinase 3alpha, and Ca(2+)/calmodulin-dependent protein kinase II beta subunit. It appears that chronic nicotine treatment affects cardiac function by modulating the expressions of genes involved in energy metabolism and signal transduction.

  11. Levels of progesterone, testosterone, and estradiol, and androstenedione metabolism in the gonads of Lytechinus variegatus (Echinodermata:echinoidea).

    PubMed

    Wasson, K M; Gower, B A; Hines, G A; Watts, S A

    2000-06-01

    Levels of progesterone (P4), testosterone (T) and estradiol (E2) indicated significant variation among individual echinoids during the annual cycle, reflecting generally the variation in gamete development that can be observed among individuals. Testosterone and E2 levels in both the ovaries and testes were higher during the period of gonadal growth. Levels of all steroids were greatly reduced compared to those levels reported for asteroids. Differences in the levels of P4, T, and estrogens between asteroids and Lytechinus variegatus may be related to differences in gonad morphology and nutrient storage capacity between asteroids and echinoids. It was hypothesized that the low levels of steroids detected in L. variegatus reflect paracrine-like mechanisms in cell signaling as compared to endocrine-like mechanisms proposed to be involved in regulating gonad function in asteroids. Both the ovaries and testes of L. variegatus had the capacity to synthesize T and a variety of 5alpha-reduced androgens including 5alpha-androstane-3beta,17beta-diol and 5alpha-androstane-3alpha,17beta-diol (5alpha-adiols) from androstenedione (AD) in 8 h. Estrogen synthesis was not detected. The sex-specific pattern of accumulation of 5alpha-adiols in the ovaries and testes suggests that the 5alpha-adiols may affect processes related to reproduction in L. variegatus. PMID:11050687

  12. Proscar (Finasteride) inhibits 5 alpha-reductase activity in the ovaries and testes of Lytechinus variegatus Lamarck (Echinodermata: Echinoidea).

    PubMed

    Wasson, K M; Watts, S A

    1998-10-01

    Recent investigations into the steroid metabolic pathway in the echinoid Lytechinus variegatus demonstrated the capacity of the gonads to convert androstenedione, the classical mammalian precursor to bioactive androgens, into testosterone and a variety of 5 alpha-reduced androgens including 5 alpha-androstane-3 beta, 17 beta-diol and 5 alpha-androstane-3 alpha, 17 beta-diol. The synthesis of these steroids, which requires 5 alpha-reductase activity, varies with sex and reproductive state in L. variegatus, suggesting that these steroids may be involved in reproductive processes. The classical method of castration followed by steroid replacement therapy to determine the biological role of steroids in the gonads of higher vertebrates is not possible in echinoids. Therefore, this study was designed to determine the efficacy of finasteride, a selective 5 alpha-reductase inhibitor in the mammalian prostate gland, on 5 alpha-reductase activity in the gonads of L. variegatus. Finasteride inhibits echinoid 5 alpha-reductase in a dose-dependent manner with IC50 approximately 2.7 microM for both ovaries and testes. These echinoid IC50s are significantly higher than those reported for humans and rats. In addition, oral administration of finasteride to the echinoids appeared to inhibit 5 alpha-reductase with no apparent stress (no spine loss) to the animals. These data suggest that finasteride may be used to selectively and chemically ablate 5 alpha-reduced androgen synthesis in the gonads of L. variegatus. PMID:9827060

  13. Overview of the magnetic properties experiments on the Mars Exploration Rovers

    USGS Publications Warehouse

    Madsen, M.B.; Goetz, W.; Bertelsen, P.; Binau, C.S.; Folkmann, F.; Gunnlaugsson, H.P.; Hjollum, J.I.; Hviid, S.F.; Jensen, J.; Kinch, K.M.; Leer, K.; Madsen, D.E.; Merrison, J.; Olsen, M.; Arneson, H.M.; Bell, J.F.; Gellert, Ralf; Herkenhoff, K. E.; Johnson, J. R.; Johnson, M.J.; Klingelhofer, G.; McCartney, E.; Ming, D. W.; Morris, R.V.; Proton, J.B.; Rodionov, D.; Sims, M.; Squyres, S. W.; Wdowiak, T.; Yen, A. S.

    2009-01-01

    The Mars Exploration Rovers have accumulated airborne dust on different types of permanent magnets. Images of these magnets document the dynamics of dust capture and removal over time. The strongly magnetic subset of airborne dust appears dark brown to black in Panoramic Camera (Pancam) images, while the weakly magnetic one is bright red. Images returned by the Microscopic Imager reveal the formation of magnetic chains diagnostic of magnetite-rich grains with substantial magnetization (>8 Am2 kg-1). On the basis of M??ssbauer spectra the dust contains magnetite, olivine, pyroxene, and nanophase oxides in varying proportions, depending on wind regime and landing site. The dust contains a larger amount of ferric iron (Fe3+/Fe tot ??? 0.6) than rocks in the Gusev plains (???0.1-0.2) or average Gusev soil (???0.3). Alpha Particle X-Ray Spectrometer data of the dust show that some of the iron in magnetite is substituted by titanium and chromium. The good correlation of the amount of calcium and sulfur in the dust may be caused by the presence of a calcium sulfate related phase. The overall mineralogical composition points to a basaltic origin of the airborne dust, although some alteration has taken place as indicated by the large degree of oxidation. Copyright 2009 by the American Geophysical Union.

  14. The structure of the carbohydrate backbone of the lipopolysaccharide of Pectinatus frisingensis strain VTT E-79104.

    PubMed

    Vinogradov, Evgeny; Li, Jianjun; Sadovskaya, Irina; Jabbouri, Said; Helander, Ilkka M

    2004-06-22

    The structure of the carbohydrate backbone of the lipopolysaccharide from Pectinatus frisingensis strain VTT E-79104 was analyzed using chemical degradations, NMR spectroscopy, mass spectrometry, and chemical methods. The LPS contains two major structural variants, differing in the presence or absence of an octasaccharide fragment. The largest structure of the carbohydrate backbone of the LPS, that could be deduced from experimental results, consists of 20 monosaccharides arranged in a nonrepetitive sequence: [carbohydrate structure: see text] where R is H or 4-O-Me-alpha-L-Fuc-(1-2)-4-O-Me-beta-Hep-(1-3)-alpha-GlcNAc-(1-2)-beta-Man-(1-3)-beta-ManNAc-(1-4)-alpha-Gal-(1-4)-beta-Hep-(1-3)-beta-GalNAc-(1- where Hep is a residue of D-glycero-D-galacto-heptose; all monosaccharides have the D-configuration except for 4-O-Me-L-Fuc and L-Ara4N. This structure is architecturally similar to the oligosaccharide system reported previously in P. frisingensis VTT E-82164 LPS, but differs from the latter in composition and also in the size of the outer region.

  15. Triple tracks in CR-39 as the result of Pd-D Co-deposition: evidence of energetic neutrons.

    PubMed

    Mosier-Boss, Pamela A; Szpak, Stanislaw; Gordon, Frank E; Forsley, Lawrence P G

    2009-01-01

    Since the announcement by Fleischmann and Pons that the excess enthalpy generated in the negatively polarized Pd-D-D(2)O system was attributable to nuclear reactions occurring inside the Pd lattice, there have been reports of other manifestations of nuclear activities in this system. In particular, there have been reports of tritium and helium-4 production; emission of energetic particles, gamma or X-rays, and neutrons; as well as the transmutation of elements. In this communication, the results of Pd-D co-deposition experiments conducted with the cathode in close contact with CR-39, a solid-state nuclear etch detector, are reported. Among the solitary tracks due to individual energetic particles, triple tracks are observed. Microscopic examination of the bottom of the triple track pit shows that the three lobes of the track are splitting apart from a center point. The presence of three alpha-particle tracks outgoing from a single point is diagnostic of the (12)C(n,n')3alpha carbon breakup reaction and suggests that DT reactions that produce > or = 9.6 MeV neutrons are occurring inside the Pd lattice. To our knowledge, this is the first report of the production of energetic (> or = 9.6 MeV) neutrons in the Pd-D system.

  16. Mechanisms of stem cell osteogenic differentiation on TiO2 nanotubes.

    PubMed

    Yu, Weiqiang; Qian, Chao; Jiang, Xinquan; Zhang, Fuqiang; Weng, Weimin

    2015-12-01

    TiO2 nanotubes could stimulate osteogenic differentiation of stem cells, but the molecular mechanisms underlying the interactions between nanotubes and stem cells remain unclear. In this study, we investigated the response of bone marrow stromal cells to nanotubes of different diameters using microarray-based bioinformatics approach. Gene ontology (GO) and GO enrichment network analysis indicated that larger TiO2 nanotubes were more potent than smaller nanotubes in inducing the expression of genes involved in cell proliferation, differentiation, and immune responses, and inhibiting that of genes responsible for cell adhesion. The analysis of the signaling network containing significantly affected genes suggested that Na(+)/K(+) transporting ATPases ATP1A2 (alpha 2 polypeptide) and ATP1A3 (alpha 3 polypeptide), and MAP3K11 (mitogen-activated protein kinase kinase kinase 11) were important for inducing osteogenic differentiation of bone marrow stromal cells without additional osteogenic stimuli. The upregulation of the ATP1A2 and MAP3K11 genes confirmed by real-time PCR indicates that the response of bone marrow stromal cells to nanotube cues may be mediated by the pathways previously implicated in transducing mechanical stress signals. Our results revealed some molecular mechanisms by which TiO2 nanotubes may direct osteogenic differentiation of stem cells.

  17. Endothelium-dependent induction of vasorelaxation by the butanol extract of Phellinus igniarius in isolated rat aorta.

    PubMed

    Kang, Dae Gill; Cao, Li Hua; Lee, Jun Kyoung; Choi, Deok Ho; Kim, Seung Ju; Lee, Hyuck; Kim, Jin Sook; Lee, Ho Sub

    2006-01-01

    The butanol extract of Phellinus igniarius (BPI) induced relaxation of the phenylephrin e-precontracted rat aorta in a dose-dependent manner, and its effect was abolished by the removal of functional endothelium. Pretreatment of the aortic tissues with N(G)-nitro-L-arginine methyl ester (L-NAME), methylene blue, or 1H-[1,2,4]-oxadiazole-[4,3-alpha]-quinoxalin1-one (ODQ) inhibited the vascular relaxation induced by BPI. BPI-induced vascular relaxations were also markedly attenuated by the addition of verapamil or diltiazem, while the relaxant effect of BPI was not blocked by pretreatment with indomethacine, glibenclamide, tetraethylammonium (TEA), atropine, or propranolol. Incubation of endothelium-intact rat aorta with BPI increased the production of cGMP in a dose-dependent manner. These results suggest that BPI dilates vascular smooth muscle via endothelium-dependent nitric oxide-cGMP signaling pathway, with the possible involvement of L-type Ca(2+) channels. PMID:16883636

  18. Chia (Salvia hispanica L.) seed as an n-3 fatty acid source for finishing pigs: effects on fatty acid composition and fat stability of the meat and internal fat, growth performance, and meat sensory characteristics.

    PubMed

    Coates, W; Ayerza, R

    2009-11-01

    Coronary heart disease is caused by arteriosclerosis, which is triggered by an unbalanced fatty acid profile in the body. Today, Western diets are typically low in n-3 fatty acids and high in SFA and n-6 fatty acids; consequently, healthier foods are needed. Chia seed (Salvia hispanica L.), which contains the greatest known plant source of n-3 alpha-linolenic acid, was fed at the rate of 10 and 20% to finishing pigs, with the goal to determine if this new crop would increase the n-3 content of the meat as has been reported for other n-3 fatty acid-rich crops. The effects of chia on fatty acid composition of the meat, internal fats, growth performance, and meat sensory characteristics were determined. Productive performance was unaffected by dietary treatment. Chia seed modified the fatty acid composition of the meat fat, but not of the internal fat. Significantly (P < 0.05) less palmitic, stearic, and arachidic acids were found with both chia treatments. This is different than trials in which flaxseed, another plant based source of omega-3 fatty acid, has been fed. Alpha-linolenic acid content increased with increasing chia content of the diet; however, only the effect of the 20% ration was significantly (P < 0.05) different from that of the control. Chia seed increased panel member preferences for aroma and flavor of the meat. This study tends to show that chia seems to be a viable feed that can produce healthier pork for human consumption.

  19. Alcohol absorption inhibitors from bay leaf (Laurus nobilis): structure-requirements of sesquiterpenes for the activity.

    PubMed

    Yoshikawa, M; Shimoda, H; Uemura, T; Morikawa, T; Kawahara, Y; Matsuda, H

    2000-08-01

    Through a bioassay-guided separation using inhibitory activity on blood ethanol elevation in oral ethanol-loaded rat, various sesquiterpenes having an alpha-methylene-gamma-butyrolactone moiety, costunolide (1), dehydrocostus lactone (2), zaluzanin D (3), reynosin (4), santamarine (5), 3alpha-acetoxyeudesma-1,4(15),11(13)-trien-12,6alpha-+ ++olide (6) and 3-oxoeudesma-1,4,11(13)-trien-12,6alpha-olide (7), were isolated as the active principle from the leaves of Laurus nobilis (bay leaf, laurel). In order to characterize the structure requirement for the activity, several reduction products (2a-2d) and amino acid adducts (2e, 2f) of the alpha-methylene-gamma-butyrolactone moiety were synthesized from 2 and the inhibitory activities of these sesquiterpenes, together with alpha-methylene-gamma-butyrolactone (12) and its related compounds (13-16), were examined. These results indicated that the gamma-butyrolactone or gamma-butyrolactol moiety having alpha-methylene or alpha-methyl group was essential for the inhibitory activity on ethanol absorption. Since 1, 2 and 12 showed no significant effect on glucose absorption, these sesquiterpenes appeared to selectively inhibit ethanol absorption. In addition, the acute toxicities of 1 and 2 in a single oral administration were found to be lower than that of 12.

  20. Termination of pseudopregnancy in the rat produces an anxiogenic-like response that is associated with an increase in benzodiazepine receptor binding density and a decrease in GABA-stimulated chloride influx in the hippocampus.

    PubMed

    Bitran, Daniel; Smith, Sheryl S

    2005-01-30

    The neurosteroid, 3alpha-OH-5alpha-pregnan-20-one (allopregnanolone) is a potent positive modulator of the GABA(A) receptor complex. Its pharmacological spectrum of action is shared by the benzodiazepines and alcohol, and includes anxiolytic, anticonvulsant, ataxic, and hypnotic effects. Discontinuation from chronic exposure to allopregnanolone or other neuroactive steroids has been shown to elicit behavioral effects that are typically seen in benzodiazepine or alcohol withdrawal. In this series of experiments, the effects of an endogenous elevation of ovarian steroids on brain GABA(A) receptor function was examined by inducing pseudopregnancy. In female rats, pseudopregnancy did not affect behavior in the elevated plus-maze, despite a persistent increase in circulating levels of allopregnanolone. Pseudopregnancy was associated with a decrease in the maximal binding density of 3H-flunitrazepam in the cerebral cortex and cerebellum; however, GABA-stimulated chloride influx in cerebral cortical, hippocampal, and cerebellar synaptoneurosomes remained unaffected during pseudopregnancy. Termination of pseudopregnancy by ovariectomy precipitated an anxiogenic-like effect in the elevated plus-maze. The withdrawal from elevated ovarian steroid levels also increased the number of benzodiazepine receptors and decreased GABA-stimulated chloride influx in the hippocampus.

  1. Synthesis of 8-thiabicyclo[3.2.1]octanes and their binding affinity for the dopamine and serotonin transporters.

    PubMed

    Pham-Huu, Duy-Phong; Deschamps, Jeffrey R; Liu, Shanghao; Madras, Bertha K; Meltzer, Peter C

    2007-01-15

    Cocaine is a potent stimulant of the central nervous system. Its reinforcing and stimulant properties have been associated with inhibition of the dopamine transporter (DAT) on presynaptic neurons. In the search for medications for cocaine abuse, we have prepared 2-carbomethoxy-3-aryl-8-thiabicyclo[3.2.1]octane analogues of cocaine. We report that this class of compounds provides potent and selective inhibitors of the DAT and SERT. The selectivity resulted from reduced activity at the SERT. The 3beta-(3,4-dichlorophenyl) analogue inhibits the DAT and SERT with a potency of IC(50)=5.7 nM and 8.0 nM, respectively. The 3-(3,4-dichlorophenyl)-2,3-unsaturated analogue inhibits the DAT potently (IC(50)=4.5 nM) and selectively (>800-fold vs SERT). Biological enantioselectivity of DAT inhibition was limited for both the 3-aryl-2,3-unsaturated and the 3alpha-aryl analogues (2-fold), but more robust (>10-fold) for the 3beta-aryl analogues. The (1R)-configuration provided the eutomers. PMID:17070057

  2. Brassinosteroid stimulation of hypocotyl elongation and wall relaxation in pakchoi (Brassica chinensis cv Lei-Choi)

    SciTech Connect

    Tzannwei Wang; Cosgrove, D.J.; Arteca, R.N. )

    1993-03-01

    Hypocotyl elongation of pakchoi (Brassica chinensis cv Lei-Choi) was stimulated by applying 300 ng of brassinosteroid (2[alpha],3[alpha],22[beta],23[beta]-tetrahydroxy-24[beta]-methyl-B-homo-7-oxa-5[alpha]-cholestan-6-one, BR) in 1 [mu]L of 50% ethanol to the apex of hypocotyls. BR had its greatest effect on elongation of the apical 3-mm region below the cotyledonary node (75% stimulation) between 6 and 18 h after treatment. Stress/strain (Instron) analysis of this 3-mm region revealed that plastic and elastic components of extension were not significantly different between BR-treated and control seedlings. In pressure-block experiments, the initial rate of relaxation was 2-fold faster in BR-treated plants as compared with controls, whereas after 125 min the total amount of relaxation and the relaxation rate were the same for the two treatments. Osmotic pressure of cell sap expressed from this 3-mm region showed a large decrease (28%) in BR-treated seedlings compared to the controls. The authors conclude that BR stimulates growth in pakchoi by accelerating the biochemical processes that cause wall relaxation, without inducing a large change in wall mechanical properties. 19 refs., 3 figs., 1 tab.

  3. 17 beta-estradiol modulates GABAergic synaptic transmission and tonic currents during development in vitro.

    PubMed

    Pytel, Maria; Wójtowicz, Tomasz; Mercik, Katarzyna; Sarto-Jackson, Isabella; Sieghart, Werner; Ikonomidou, Chrysanthy; Mozrzymas, Jerzy W

    2007-05-01

    Estrogens exert a variety of modulatory effects on the structure and function of the nervous system. In particular, 17 beta-estradiol was found to affect GABAergic inhibition in adult animals but its action on GABAergic currents during development has not been elucidated. In the present study, we investigated the effect of 17 beta-estradiol on hippocampal neurons developing in vitro. In this model, mIPSC kinetics showed acceleration with age along with increased alpha1 subunit expression, similarly as in vivo. Long-term treatment with 17 beta-estradiol increased mIPSC amplitudes in neurons cultured for 6-8 and 9-11DIV and prolonged the mIPSC decaying phase only in the 9-11DIV group. The time needed for the onset of 17 beta-estradiol effect on mIPSC amplitude was approximately 48 h. In the period of 9-11DIV, treatment with 17 beta-estradiol strongly reduced the tonic conductance activated by low GABA concentrations. The effects of 17 beta-estradiol on mIPSCs and tonic conductance were not correlated with any change in expression of considered GABAAR subunits (alpha1-3, alpha5-6, gamma2) while alpha4 and delta subunits were at the detection limit. In conclusion, we provide evidence that 17 beta-estradiol differentially affects the phasic and tonic components of GABAergic currents in neurons developing in vitro.

  4. Photolysis of alpha-xylyl chlorides: an efficient deep-UV photoinitiating system for radical and cationic polymerization.

    PubMed

    Ponce, Patricia; Catalani, Luiz Henrique

    2004-07-01

    Photoacid generators (PAG) are chemical systems where light absorption renders strong acid formation, typically with quantum yields greater than one. Many compounds bearing halogen atoms are reported to produce hydrogen halides upon photolysis. Here, alpha-chloroxylene derivatives (ortho, meta and para) were subject of a photolysis study in order to: (i) determine the operative mechanism, (ii) identify the products formed and (iii) quantify the amount of HCl formed. Product structure and quantum yields of HCl formation where determined for the photolysis of alpha-chloro-o-xylene (1), alpha-chloro-m-xylene (2), alpha-chloro-p-xylene (3), alpha, alpha'-dichloro-o-xylene (4), alpha, alpha'-dichloro-m-xylene (5) and alpha, alpha'-dichloro-p-xylene (6) in apolar (benzene, cumene, ethylbenzene, toluene and isooctane) and polar (methanol, n-propanol, isopropyl alcohol) solvents. Some of these compounds were analysed by laser flash photolysis in argon-purged isooctane as solvent to examine the possible reaction intermediates involved. The observed products are derived from typical radical reactions like recombination, dimerization and hydrogen abstraction from the starting compound or from solvents. The formation of HCl is expected as the result of C-Cl homolysis followed by hydrogen abstraction by chlorine atom. The results showed yields ranging from 1.2 to 18, depending on the conditions used. These numbers indicate the potential use of these compounds as PAG systems for the deep UV region.

  5. What do we really know about the ubiquitin-proteasome pathway in muscle atrophy?

    NASA Technical Reports Server (NTRS)

    Jagoe, R. T.; Goldberg, A. L.

    2001-01-01

    Studies of many different rodent models of muscle wasting have indicated that accelerated proteolysis via the ubiquitin-proteasome pathway is the principal cause of muscle atrophy induced by fasting, cancer cachexia, metabolic acidosis, denervation, disuse, diabetes, sepsis, burns, hyperthyroidism and excess glucocorticoids. However, our understanding about how muscle proteins are degraded, and how the ubiquitin-proteasome pathway is activated in muscle under these conditions, is still very limited. The identities of the important ubiquitin-protein ligases in skeletal muscle, and the ways in which they recognize substrates are still largely unknown. Recent in-vitro studies have suggested that one set of ubquitination enzymes, E2(14K) and E3(alpha), which are responsible for the 'N-end rule' system of ubiquitination, plays an important role in muscle, especially in catabolic states. However, their functional significance in degrading different muscle proteins is still unclear. This review focuses on the many gaps in our understanding of the functioning of the ubiquitin-proteasome pathway in muscle atrophy, and highlights the strengths and limitations of the different experimental approaches used in such studies.

  6. Substitutional and Interstitial Diffusion in alpha2-Ti3Al(O)

    NASA Technical Reports Server (NTRS)

    Copland, Evan; Young, David J.; Gleeson, Brian; Jacobson, Nathan

    2007-01-01

    The reaction between Al2O3 and alpha2-Ti3Al was studied with a series of Al2O3/alpha2-Ti3Al multiphase diffusion couples annealed at 900, 1000 and 1100 C. The diffusion-paths were found to strongly depend on alpha2- Ti3Al(O) composition. For alloys with low oxygen concentrations the reaction involved the reduction of Al2O3, the formation of a gamma-TiAl reaction-layer and diffusion of Al and O into the alpha2-Ti3Al substrate. Measured concentration profiles across the interaction-zone showed "up-hill" diffusion of O in alpha2-Ti3Al(O) indicating a significant thermodynamic interaction between O and Al, Ti or both. Diffusion coefficients for the interstitial O in alpha2-Ti3Al(O) were determined independently from the interdiffusion of Ti and Al on the substitutional lattice. Diffusion coefficients are reported for alpha2-Ti3Al(O) as well as gamma-TiAl. Interpretation of the results were aided with the subsequent measurement of the activities of Al, Ti and O in alpha 2-Ti3Al(O) by Knudsen effusion-cell mass spectrometry.

  7. Comparison of different progestagen assays for measuring progesterone metabolites in faeces of the bitch.

    PubMed

    Möstl, E; Brunner, I

    1997-12-01

    From 12 bitches of various breeds with fertile oestrus cycles faecal samples were collected daily from the onset of pro-oestrus till 20 days after the start of vulval bleeding, then once per week till about 1 week before term. Immunoreactive progesterone metabolites were extracted from the samples using methanol and measured using immunoassays. In a first experiment four different assays were compared in regard to the amounts of immunoreactive substances measured: the enzyme immunoassay against 20-oxo-3-hydroxypregnanes showed twice to four times higher values of immunoreactive material than another using an antibody against 6 beta-hydroxyprogesterone. An enzyme immunoassay for pregnanediol measured only low concentrations of immunoreactive material. Also a radio immunoassay using an antibody against 11 beta-hydroxyprogesterone detected only small amounts of reacting material. High performance liquid chromatography showed that in faeces of bitches the immunoreactive progesterone metabolites were present in unconjugated form, mainly as 3 alpha/beta hydroxylated progestagens with a 20-oxo group. In the second experiment the samples were measured with the assay system using the 20-oxo-3-hydroxypregnane antibody. A few days before mating the concentration of progesterone metabolites increased, reaching 5.77 mumol/kg faeces (median) at the day of mating. High levels (10.45 mumol/kg faeces) were measured till the end of the first month after mating. Thereafter, the concentrations decreased, reaching 2.68 mumol/kg (median) at the end of the second month.

  8. Variation in total polyphenolics contents of aerial parts of Potentilla species and their anticariogenic activity.

    PubMed

    Tomczyk, Michał; Pleszczyńska, Małgorzata; Wiater, Adrian

    2010-07-01

    The aerial parts of selected Potentilla species (P. anserina, P. argentea, P. erecta, P. fruticosa, P. grandiflora, P. nepalensis, P. norvegica, P. pensylvanica, P. crantzii and P. thuringiaca) were investigated in order to determine their contents of polyphenolic compounds. The results showed that P. fruticosa has relatively high concentrations of tannins (167.3 +/- 2.0 mg/g dw), proanthocyanidins (4.6 +/- 0.2 mg/g dw) and phenolic acids (16.4 +/- 0.8 mg/g dw), as well as flavonoids (7.0 +/- 1.1 mg/g dw), calculated as quercetin. Furthermore, we investigated the in vitro inhibitory effects of aqueous extracts from these species against cariogenic Streptococcus spp. strains. It was found that the tested samples moderately inhibit the growth of oral streptococci. However, all the preparations exhibited inhibitory effects on water-insoluble alpha-(1-->3)-, alpha-(1-->6)-linked glucan (mutan) and artificial dental plaque formation. The extract from P. fruticosa showed the highest anti-biofilm activities, with minimum mutan and biofilm inhibition concentrations of 6.25-25 and 50-100 microg/mL, respectively. The results indicate that the studied Potentilla species could be a potential plant material for extracting biologically active compounds, and could become a useful supplement for pharmaceutical products as a new anticariogenic agent in a wide range of oral care products. PMID:20657382

  9. Stereoselective recognition of monolayers of cholesterol, ent-cholesterol, and epicholesterol by an antibody.

    PubMed

    Geva, M; Izhaky, D; Mickus, D E; Rychnovsky, S D; Addadi, L

    2001-04-01

    The interaction between a monoclonal antibody and four distinct monolayers with varying degrees of structural, chemical, and stereochemical similarity were studied and quantified. The antibody, raised and selected against cholesterol monohydrate crystals, interacts with cholesterol monolayers stereospecifically, but not enantiospecifically. Monolayers of ent-cholesterol molecules, which are chemically identical to cholesterol and whose structure is the exact mirror image of the cholesterol monolayer, interact with the antibody to the same extent as the cholesterol monolayers. The affinity of the antibody for both enantiomeric monolayers is extremely high. However, the antibody does not interact with monolayers of epicholesterol, which is an epimer of cholesterol: The hydroxy group in epicholesterol is in the 3alpha position rather than in the 3beta position, imposing a different angle between the hydroxy group and the rigid steroid backbone, and a different packing of the molecules. Monolayers of triacontanol, a long-chain primary aliphatic alcohol, interact with the antibody to a lesser extent than the cholesterol and ent-cholesterol monolayers, presumably due to the structural flexibility of the triacontanol molecule. The lack of chiral discrimination by the antibody is thus correlated to the level at which the chirality is exposed at the surface of the monolayers.

  10. Chemical composition and biological activity of Nepeta parnassica oils and isolated nepetalactones.

    PubMed

    Gkinis, Giorgos; Tzakou, Olga; Iliopoulou, Dimitra; Roussis, Vassilios

    2003-01-01

    Essential oils of Nepeta parnassica, collected at different developmental stages, were analyzed by means of GC/MS. From the fifty-five identified constituents in samples A and B, representing 94.8% and 98.7% of the oils respectively, 4a(alpha),7alpha,7alpha(beta)-nepetalactone (22.0%), 1,8-cineole (21.1%), alpha-pinene (9.5%) and 4a(alpha),7,beta,7alpha(beta)-nepetalactone (7.9%) were the major components of sample A (vegetative stage), whereas in sample B (flowering stage) the main contributors were 1,8-cineole (34.6%), 4a(alpha),7alpha,7a(alpha)-nepetalactone (17.3%), alpha-pinene (11.4%) and 4a(alpha),7alpha,7alpha(beta)-nepetalactone (8.9%). The oils were tested on human health important insects such as the Pogonomyrmex sp. ants and the Culex pipiens molestus mosquitoes with promising results on insect repellency/toxicity.

  11. Ubiquitin-protein ligases in muscle wasting: multiple parallel pathways?

    NASA Technical Reports Server (NTRS)

    Lecker, Stewart H.; Goldberg, A. L. (Principal Investigator)

    2003-01-01

    PURPOSE OF REVIEW: Studies in a wide variety of animal models of muscle wasting have led to the concept that increased protein breakdown via the ubiquitin-proteasome pathway is responsible for the loss of muscle mass seen as muscle atrophy. The complexity of the ubiquitination apparatus has hampered our understanding of how this pathway is activated in atrophying muscles and which ubiquitin-conjugating enzymes in muscle are responsible. RECENT FINDINGS: Recent experiments have shown that two newly identified ubiquitin-protein ligases (E3s), atrogin-1/MAFbx and MURF-1, are critical in the development of muscle atrophy. Other in-vitro studies also implicated E2(14k) and E3alpha, of the N-end rule pathway, as playing an important role in the process. SUMMARY: It seems likely that multiple pathways of ubiquitin conjugation are activated in parallel in atrophying muscle, perhaps to target for degradation specific classes of muscle proteins. The emerging challenge will be to define the protein targets for, as well as inhibitors of, these E3s.

  12. GSK3A Is Redundant with GSK3B in Modulating Drug Resistance and Chemotherapy-Induced Necroptosis

    PubMed Central

    Grassilli, Emanuela; Ianzano, Leonarda; Bonomo, Sara; Missaglia, Carola; Cerrito, Maria Grazia; Giovannoni, Roberto; Masiero, Laura; Lavitrano, Marialuisa

    2014-01-01

    Glycogen Synthase Kinase-3 alpha (GSK3A) and beta (GSK3B) isoforms are encoded by distinct genes, are 98% identical within their kinase domain and perform similar functions in several settings; however, they are not completely redundant and, depending on the cell type and differentiative status, they also play unique roles. We recently identified a role for GSK3B in drug resistance by demonstrating that its inhibition enables necroptosis in response to chemotherapy in p53-null drug-resistant colon carcinoma cells. We report here that, similarly to GSK3B, also GSK3A silencing/inhibition does not affect cell proliferation or cell cycle but only abolishes growth after treatment with DNA-damaging chemotherapy. In particular, blocking GSK3A impairs DNA repair upon exposure to DNA-damaging drugs. As a consequence, p53-null cells overcome their inability to undergo apoptosis and mount a necroptotic response, characterized by absence of caspase activation and RIP1-independent, PARP-dependent AIF nuclear re-localization. We therefore conclude that GSK3A is redundant with GSK3B in regulating drug-resistance and chemotherapy-induced necroptosis and suggest that inhibition of only one isoform, or rather partial inhibition of overall cellular GSK3 activity, is enough to re-sensitize drug-resistant cells to chemotherapy. PMID:24984063

  13. Feeding Glycyrrhiza glabra (liquorice) and Astragalus membranaceus (AM) alters innate immune and physiological responses in yellow perch (Perca flavescens).

    PubMed

    Elabd, Hiam; Wang, Han-Ping; Shaheen, Adel; Yao, Hong; Abbass, Amany

    2016-07-01

    The current work assessed the potential immunomodulatory and growth-promoting effects of Astragalus membranaceus (AM) and Glycyrrhiza glabra (liquorice) in Yellow perch (Perca flavescens). In this regard, fish with an average weight of 31 ± 1.0 g were divided into five groups, and fed daily with an additive-free basal diet (control); 1, 2, and 3% (w/w) Glycyrrhiza glabra, and the fifth diet was incorporated with a combination of 1% G. glabra-AM for a four-week period. Immunological, biochemical and growth parameters were measured; and sub-groups of fish were exposed to 1-week starvation. The results showed that incorporating AM and liquorice in the diet significantly improved Immunological [superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), Lipid peroxidase (LPx) and lysozyme activities], biochemical [Aspartate Aminotransferase (AST) and Alanine Transaminase (ALT) activities; and glucose and cortisol concentrations] and growth performance parameters [body mass gain (BMG), specific growth rate (SGR), length, condition factor (K) and feed conversion ratio (FCR)]. In addition, markedly up-regulated the expression of related genes [Insulin-Like Growth Factor-1 (IGF-1), Serum amyloid A (SAA), Complement Component C3 (CCC3), Alpha 2 Macroglobulin (A2M), SOD and GPx] in treated fish groups compared to the control. Conclusively, feeding AM and liquorice diets significantly increased (P < 0.05) growth performance, antioxidant and immune response profiles throughout the entire experiment, suggesting their beneficial rule as natural anti-stress agents.

  14. Lytic enzyme, labiase for a broad range of Gram-positive bacteria and its application to analyze functional DNA/RNA.

    PubMed

    Niwa, Takashi; Kawamura, Yoshiaki; Katagiri, Yoshihiro; Ezaki, Takayuki

    2005-05-01

    The lytic activity of labiase and achromopeptidase for bacterial DNA/RNA extraction were compared. Rapid lysis of many bacterial strains was observed with labiase followed by SDS treatment. Both labiase and achromopeptidase showed high lytic activity against bacterial strains with the A1alpha chemotype (e.g., Aerococcus viridans) and the A3alpha chemotype (e.g., Staphylococcus epidermidis) for cell wall peptidoglycan structures. The lytic activity of labiase was higher than that of achromopeptidase against strains with the A1gamma chemotype (e.g., Bacillus subtilis). The activity of labiase was not detrimentally affected with increasing NaCl concentration. Labiase lysates were successfully used for rapid extraction of DNA and RNA, whereas achromopeptidase lysates degraded RNA. The DNA and RNA obtained were successfully used for 16S rRNA amplification and real-time RT-PCR detection. It is concluded that labiase is useful for rapid lysis of a wide variety of Gram-positive bacteria and can be used for DNA/RNA isolation protocols. PMID:15722152

  15. Dietary source of stearidonic acid promotes higher muscle DHA concentrations than linolenic acid in hybrid striped bass.

    PubMed

    Bharadwaj, Anant S; Hart, Steven D; Brown, Billie J; Li, Yong; Watkins, Bruce A; Brown, Paul B

    2010-01-01

    Rapid expansion of aquacultural production is placing increasing demand on fish oil supplies and intensified the search for alternative lipid sources. Many of the potential alternative sources contain low concentrations of long chain n-3 fatty acids and the conversion of dietary linolenic acid to longer chain highly unsaturated fatty acids is a relatively inefficient process in some species. A 6-week study was conducted to compare tissue fatty acid (FA) concentrations in hybrid striped bass fed either 18:3n-3 (alpha-linolenic acid; ALA) or 18:4n-3 (stearidonic acid; SDA). Hybrid striped bass were fed either a control diet containing fish oil, or diets containing ALA or SDA at three different levels (0.5, 1 and 2% of the diet). There were no significant differences in whole animal responses between fish fed ALA or SDA. Liver and muscle concentrations of ALA and SDA were responsive to dosages fed. However, only 22:6n-3 concentrations in muscle were significantly affected by dietary source of 18 carbon precursors. Muscle 22:6n-3 concentrations were significantly higher in fish fed SDA compared to fish fed ALA. Based on these data, it appears that feeding SDA can increase long chain n-3 fatty acid concentrations in fish muscle.

  16. Nonexpression of cartilage type II collagen in a case of Langer-Saldino achondrogenesis.

    PubMed

    Eyre, D R; Upton, M P; Shapiro, F D; Wilkinson, R H; Vawter, G F

    1986-07-01

    A lethal short-limbed dwarfism was diagnosed at autopsy as the Langer-Saldino variant of achondrogenesis by radiological, histological, and gross pathological criteria. Cartilage was obtained for biochemical and ultrastructural analyses from the ends of long bones, from ribs and from a scapula of the newborn infant. At all sites, it had an abnormal gelatinous texture and translucent appearance. Biochemical analyses of the cartilages to identify pepsin-solubilized collagen alpha-chains and collagen-specific CNBr-peptides failed to detect type II collagen at any site where it would normally be the main constituent. Instead, type I was the predominant collagen present. However, three cartilage-specific minor collagen chains identified as 1 alpha, 2 alpha, and 3 alpha chains by their electrophoretic mobility were present at about 10% of the total collagen. Cartilage-specific proteoglycans also appeared to be abundant in the tissue judging by its high hexosamine content and high ratio of galactosamine to glucosamine. The findings indicate that a chondrocyte phenotype had differentiated but without the expression of type II collagen. In addition to the skeletal abnormalities, the severe pulmonary hypoplasia was also felt to be directly related to the underlying pathology in collagen expression. The term chondrogenesis imperfecta rather than achondrogenesis should be considered a more accurate description of this and related conditions. PMID:3752081

  17. Compact prisms for polarisation splitting of fibre laser beams

    SciTech Connect

    Davydov, B L; Yagodkin, D I

    2005-11-30

    Simple compact monoprisms for spatial splitting of polarised laser beams with relatively small diameters (no more than 1 mm) are considered. Prisms can be made of optically inactive CaCO{sub 3}, {alpha}-BaB{sub 2}O{sub 4} ({alpha}-BBO), LiIO{sub 3}, LiNbO{sub 3}, YVO{sub 4}, and TiO{sub 2} crystals known in polarisation optics. The exact solution of the Snell equation for the extraordinary wave reflected from a surface arbitrarily tilted to its wave vector is obtained. The analysis of variants of the solution allows the fabrication of prisms with any deviation angles of the extraordinary wave by preserving the propagation direction of the ordinary wave. Three variants of prisms are considered: with minimised dimensions, with the Brewster output of the extraordinary beam, and with the deviation of the extraordinary wave by 90{sup 0}. Calcite prisms with the deviation angles for the extraordinary beam {approx}19{sup 0} and 90{sup 0} are tested experimentally. (control of laser radiation parameters)

  18. A role for Src kinase in progestin facilitation of estrous behavior in estradiol-primed female rats.

    PubMed

    González-Flores, Oscar; Beyer, Carlos; Gómora-Arrati, Porfirio; García-Juárez, Marcos; Lima-Hernández, Francisco J; Soto-Sánchez, Alfonso; Etgen, Anne M

    2010-07-01

    This study tested the hypothesis that the Src/Raf/MAPK signaling pathway is involved in the facilitation of the lordosis and proceptive behaviors induced by progesterone (P) and its ring A-reduced metabolites in ovariectomized, estradiol-primed rats. Intraventricular (icv) infusion of PP2 (7.5, 15 and 30 microg), a Src kinase inhibitor, significantly depressed P-dependent estrous behavior (lordosis and proceptivity) in estradiol-primed rats. Icv infusion of 30 microg of PP2 also significantly attenuated estrous behavior induced by the ring A-reduced P metabolites 5 alpha-dihydroprogesterone (5 alpha-DHP) and 5 alpha-pregnan-3alpha-ol-20-one (allopregnanolone). PP2 did not inhibit estrous behavior induced by administration of high doses of estradiol alone to ovariectomized rats. We also assessed if the ventromedial hypothalamus (VMH) is one of the neural sites at which progestins activate Src signaling to facilitate estrous behavior. Bilateral administration of 15 microg of PP2 into the VMH inhibited the stimulation of both lordosis and proceptive behaviors elicited by subcutaneous P administration to estradiol-primed rats. These results suggest that progestins act through Src/Raf/MAPK signaling to initiate estrous behaviors in estrogen-primed rats. This event is one component of the cellular pathways leading to the display of estrous behaviors induced by P and its ring A-reduced metabolites in female rats.

  19. Synthesis of 8-thiabicyclo[3.2.1]octanes and their binding affinity for the dopamine and serotonin transporters.

    PubMed

    Pham-Huu, Duy-Phong; Deschamps, Jeffrey R; Liu, Shanghao; Madras, Bertha K; Meltzer, Peter C

    2007-01-15

    Cocaine is a potent stimulant of the central nervous system. Its reinforcing and stimulant properties have been associated with inhibition of the dopamine transporter (DAT) on presynaptic neurons. In the search for medications for cocaine abuse, we have prepared 2-carbomethoxy-3-aryl-8-thiabicyclo[3.2.1]octane analogues of cocaine. We report that this class of compounds provides potent and selective inhibitors of the DAT and SERT. The selectivity resulted from reduced activity at the SERT. The 3beta-(3,4-dichlorophenyl) analogue inhibits the DAT and SERT with a potency of IC(50)=5.7 nM and 8.0 nM, respectively. The 3-(3,4-dichlorophenyl)-2,3-unsaturated analogue inhibits the DAT potently (IC(50)=4.5 nM) and selectively (>800-fold vs SERT). Biological enantioselectivity of DAT inhibition was limited for both the 3-aryl-2,3-unsaturated and the 3alpha-aryl analogues (2-fold), but more robust (>10-fold) for the 3beta-aryl analogues. The (1R)-configuration provided the eutomers.

  20. Liver specific transcription factors of the HNF3-, C/EBP- and LFB1-families interact with the A-activator binding site.

    PubMed Central

    Drewes, T; Klein-Hitpass, L; Ryffel, G U

    1991-01-01

    The A-activator binding site (AABS), present in the Xenopus A2 vitellogenin gene and several mammalian liver specifically expressed genes, interacts with different liver specific transcription factors including LFB1- and C/EBP-isobinders. We have now isolated some additional proteins interacting with AABS and show that they are HNF3-isobinders. The interactions between AABS and members of the HNF3 family are confirmed by binding studies using bacterially made HNF3-alpha protein. Thus a short DNA module of 24 bp is able to bind proteins of three different families of liver specific transcription factors. Competition experiments in the cell free in vitro transcription show that AABS dependent transcriptional activation is mediated by transcription factors belonging to at least two different families, the C/EBP- and the HNF3-isobinders. Being able to mediate the action of several distinct transactivators, AABS may thus be a prototype for a novel kind of tissue specific promoter modules with unique regulatory capacities. Images PMID:1754374

  1. Regulation of naïve fetal T-cell migration by the chemokines Exodus-2 and Exodus-3.

    PubMed

    Christopherson, K; Brahmi, Z; Hromas, R

    1999-08-01

    We and other workers have recently isolated three novel CC chemokines termed Exodus-1/LARC/Mip-3alpha, Exodus-2/6Ckine/SLC/TCA4, and Exodus-3/Mip-3beta/CKbeta11/ELC. These chemokines share an amino terminal Asp-Cys-Cys-Leu sequence, unique among all chemokines. They also selectively regulate migration of adult T cells. Indeed, there is evidence that Exodus-2 and -3 are critical for adult T-cell adhesion to high endothelial venules in lymph nodes, a rate-limiting step for T-cell trafficking through nodal tissue. Less is known of the factors controlling migration of naïve human fetal T cells. We tested whether these chemokines could regulate chemotaxis in cord blood T-cell populations, and compared that efficacy with normal peripheral blood adult T cells. The findings indicated that naive CD45RA+ cord blood T-cell migration is stimulated by Exodus-2 and -3, and CD4+ cord blood T cells are attracted preferentially by Exodus-2 or -3 as compared with CD8+. Exodus-2 and -3 are likely to be critical in regulating the flux of naive CD4 + fetal T-cell population of secondary lymphoid tissue.

  2. The exodus subfamily of CC chemokines inhibits the proliferation of chronic myelogenous leukemia progenitors.

    PubMed

    Hromas, R; Cripe, L; Hangoc, G; Cooper, S; Broxmeyer, H E

    2000-02-15

    Chemokines are a family of related proteins that regulate leukocyte infiltration into inflamed tissue and play important roles in disease processes. Among the biologic activities of chemokines is inhibition of proliferation of normal hematopoietic progenitors. However, chemokines that inhibit normal progenitors rarely inhibit proliferation of hematopoietic progenitors from patients with chronic myelogenous leukemia (CML). We and others recently cloned a subfamily of CC chemokines that share similar amino-terminal peptide sequences and a remarkable ability to chemoattract T cells. These chemokines, Exodus-1/LARC/MIP-3alpha, Exodus-2/SLC/6Ckine/TCA4, and Exodus-3/CKbeta11/MIP-3beta, were found to inhibit proliferation of normal human marrow progenitors. The study described here found that these chemokines also inhibited the proliferation of progenitors in every sample of marrow from patients with CML that was tested. This demonstration of consistent inhibition of CML progenitor proliferation makes the 3 Exodus chemokines unique among chemokines. (Blood. 2000;95:1506-1508)

  3. G-protein. alpha. -subunit expression, myristoylation, and membrane association in COS cells

    SciTech Connect

    Mumby, S.M.; Gilman, A.G. ); Heukeroth, R.O.; Gordon, J.I. )

    1990-01-01

    Myristolyation of seven different {alpha} subunits of guanine nucleotide-binding regulatory proteins (G proteins) was examined by expressing these proteins in monkey kidney COS cells. Metabolic labeling studies of cells transfected with cytomegalovirus-based expression vectors indicated that ({sup 3}H)myristate was incorporated into {alpha}{sub i1}, {alpha}{sub i2}, {alpha}{sub i3}, {alpha}{sub 0}, and {alpha}{sub 1}, and {alpha}{sub z} but not {alpha}{sub s} subunits. The role of myristoylation in the association of {alpha} subunits with membranes was analyzed by site-directed mutagenesis and by substitution of myristate with a less hydrophobic analog, 10-(propoxy)decanoate (11-oxamyristate). Myristoylation of {alpha}{sub 0} was blocked when an alanine residue was substituted for its amino-terminal glycine, as was association of the protein with membranes. Substitution of the myristoyl group with 11-oxamyristate affected the cellular distribution of a subset of acylated {alpha} subunits. The results are consistent with a model wherein the hydrophobic interaction of myristate with the bilayer permits continued association of the protein with the plasma membrane when G-protein {alpha} subunits dissociated from {beta}{gamma}.

  4. Darwin's evolution theory, brain oscillations, and complex brain function in a new "Cartesian view".

    PubMed

    Başar, Erol; Güntekin, Bahar

    2009-01-01

    Comparatively analyses of electrophysiological correlates across species during evolution, alpha activity during brain maturation, and alpha activity in complex cognitive processes are presented to illustrate a new multidimensional "Cartesian System" brain function. The main features are: (1) The growth of the alpha activity during evolution, increase of alpha during cognitive processes, and decrease of the alpha entropy during evolution provide an indicator for evolution of brain cognitive performance. (2) Human children younger than 3 years are unable to produce higher cognitive processes and do not show alpha activity till the age of 3 years. The mature brain can perform higher cognitive processes and demonstrates regular alpha activity. (3) Alpha activity also is significantly associated with highly complex cognitive processes, such as the recognition of facial expressions. The neural activity reflected by these brain oscillations can be considered as constituent "building blocks" for a great number of functions. An overarching statement on the alpha function is presented by extended analyzes with multiple dimensions that constitute a "Cartesian Hyperspace" as the basis for oscillatory function. Theoretical implications are considered.

  5. Conversion of cassava starch to biomass, carbohydrates, and acids by Aspergillus niger.

    PubMed

    Tan, K H; Ferguson, L B; Carlton, C

    1984-01-01

    The filamentous fungus, Aspergillus niger, efficiently converted cassava polysaccharides to mycelial mass, simple sugars, and acids during the course of its growth. A typical 70-ml culture broth containing 2% cassava polysaccharides yielded 0.38 g dry mycelial mass, 1.14 mmol reducing sugars, and 1.17 meq acids at the end of 42 h. About 70% of the initial total carbohydrate in the medium was degraded during the same period. The sugars and acids in the culture broths were analyzed by HPLC on a single Aminex HPX-87 column at 55 degrees C, using 0.013 N H2SO4 as the eluting solvent. Cassava polysaccharides were degraded to oligosaccharides, maltotriose, maltose, and glucose beyond the 20-h growth periods, with maltotriose emerging as the major simple sugar. The appearance of citric, malic, gluconic, succinic, and fumaric acids accounted mostly for the decreasing pH in the growth media. Formation of carbohydrate species in the culture broths was closely related to the biosynthesis and secretion of several carbohydrases by A. niger. The extracellular carbohydrases were separated and identified by chromatofocusing and polyacrylamide gel electrophoresis to be amyloglucosidase (EC 3.1.2.3), alpha-amylase (EC 3.2.1.1), and alpha-glucosidase (EC 3.2.1.20), respectively.

  6. Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating Occludin, Claudin-1 and E-cadherin expression

    SciTech Connect

    Severson, Eric A.; Kwon, Mike; Hilgarth, Roland S.; Parkos, Charles A.; Nusrat, Asma

    2010-07-02

    The Apical Junctional Complex (AJC) encompassing the tight junction (TJ) and adherens junction (AJ) plays a pivotal role in regulating epithelial barrier function and epithelial cell proliferative processes through signaling events that remain poorly characterized. A potential regulator of AJC protein expression is Glycogen Synthase Kinase-3 (GSK-3). GSK-3 is a constitutively active kinase that is repressed during epithelial-mesenchymal transition (EMT). In the present study, we report that GSK-3 activity regulates the structure and function of the AJC in polarized model intestinal (SK-CO15) and kidney (Madin-Darby Canine Kidney (MDCK)) epithelial cells. Reduction of GSK-3 activity, either by small molecule inhibitors or siRNA targeting GSK-3 alpha and beta mRNA, resulted in increased permeability to both ions and bulk solutes. Immunofluorescence labeling and immunoblot analyses revealed that the barrier defects correlated with decreased protein expression of AJC transmembrane proteins Occludin, Claudin-1 and E-cadherin without influencing other TJ proteins, Zonula Occludens-1 (ZO-1) and Junctional Adhesion Molecule A (JAM-A). The decrease in Occludin and E-cadherin protein expression correlated with downregulation of the corresponding mRNA levels for these respective proteins following GSK-3 inhibition. These observations implicate an important role of GSK-3 in the regulation of the structure and function of the AJC that is mediated by differential modulation of mRNA transcription of key AJC proteins, Occludin, Claudin-1 and E-cadherin.

  7. Galectin-4 expression is down-regulated in response to autophagy during differentiation of rat trophoblast cells.

    PubMed

    Arikawa, Tomohiro; Liao, Shengjun; Shimada, Hiroki; Inoue, Tomoki; Sakata-Haga, Hiromi; Nakamura, Takanori; Hatta, Toshihisa; Shoji, Hiroki

    2016-01-01

    Placental development and trophoblast invasion of the maternal endometrium establish the maternal-fetal interface, which is critical for the developing embryo and fetus. Herein we show that overexpression of Galectin-4 (Gal-4) during trophoblast differentiation inhibited the enlargement of Rcho-1 cells (a model for rat trophoblast differentiation) and promoted cell-cell adhesion, whereas trophoblast specific markers and MMP-9 activity were not affected. In the rat placenta, microtubule associated protein 1 light chain 3 alpha (LC3) protein, an autophagy marker, is highly expressed on the maternal side of the decidua where Gal-4 expression is weak. In vitro assays showed that the expression of trophoblast-specific differentiation markers was reduced by 3-Methyladenine (3-MA) and Bafilomycin A1, known as autophagy inhibitors, compared to control cells. Furthermore, Gal-4 expression in Rcho-1 cells, which is normally down-regulated during differentiation, was not attenuated in the presence of autophagy inhibitors, suggesting that autophagy is upstream of Gal-4 expression. We herein describe a possible mechanism by which autophagy regulates trophoblast differentiation via regulation of Gal-4 expression in order to establish the maternal-fetal interface. PMID:27572741

  8. NASA Tech Briefs, April 2008

    NASA Technical Reports Server (NTRS)

    2008-01-01

    Topics covered include: Gas Sensors Based on Coated and Doped Carbon Nanotubes; Tactile Robotic Topographical Mapping Without Force or Contact Sensors; Thin-Film Magnetic-Field-Response Fluid-Level Sensor for Non-Viscous Fluids; Progress in Development of Improved Ion-Channel Biosensors; Simulating Operation of a Complex Sensor Network; Using Transponders on the Moon to Increase Accuracy of GPS; Controller for Driving a Piezoelectric Actuator at Resonance; Coaxial Electric Heaters; Dual-Input AND Gate From Single-Channel Thin-Film FET; High-Density, High-Bandwidth, Multilevel Holographic Memory; Fabrication of Gate-Electrode Integrated Carbon-Nanotube Bundle Field Emitters; Hydroxide-Assisted Bonding of Ultra-Low-Expansion Glass; Photochemically Synthesized Polyimides; Optimized Carbonate and Ester-Based Li-Ion Electrolytes; Compact 6-DOF Stage for Optical Adjustments; Ultrasonic/Sonic Impacting Penetrators; Miniature, Lightweight, One-Time-Opening Valve; Supplier Management System; Improved CLARAty Functional-Layer/Decision-Layer Interface; JAVA Stereo Display Toolkit; Remote-Sensing Time Series Analysis, a Vegetation Monitoring Tool; PyPele Rewritten To Use MPI; Data Assimilation Cycling for Weather Analysis; Hydrocyclone/Filter for Concentrating Biomarkers from Soil; Activating STAT3 Alpha for Promoting Healing of Neurons; and Probing a Spray Using Frequency-Analyzed Light Scattering.

  9. Structure and binding kinetics of three different human CD1d-alpha-galactosylceramide-specific T cell receptors.

    PubMed

    Gadola, Stephan D; Koch, Michael; Marles-Wright, Jon; Lissin, Nikolai M; Shepherd, Dawn; Matulis, Gediminas; Harlos, Karl; Villiger, Peter M; Stuart, David I; Jakobsen, Bent K; Cerundolo, Vincenzo; Jones, E Yvonne

    2006-03-20

    Invariant human TCR Valpha24-Jalpha18+/Vbeta11+ NKT cells (iNKT) are restricted by CD1d-alpha-glycosylceramides. We analyzed crystal structures and binding characteristics for an iNKT TCR plus two CD1d-alpha-GalCer-specific Vbeta11+ TCRs that use different TCR Valpha chains. The results were similar to those previously reported for MHC-peptide-specific TCRs, illustrating the versatility of the TCR platform. Docking TCR and CD1d-alpha-GalCer structures provided plausible insights into their interaction. The model supports a diagonal orientation of TCR on CD1d and suggests that complementarity determining region (CDR)3alpha, CDR3beta, and CDR1beta interact with ligands presented by CD1d, whereas CDR2beta binds to the CD1d alpha1 helix. This docking provides an explanation for the dominant usage of Vbeta11 and Vbeta8.2 chains by human and mouse iNKT cells, respectively, for recognition of CD1d-alpha-GalCer.

  10. Tertiary oil recovery processes research at the University of Texas. Annual report, October 1981-September 1982

    SciTech Connect

    Schechter, R.S.; Wade, W.H.

    1985-01-01

    During the past year we have continued three major projects: (I) further delineation of surfactant adsorption mechanisms, (II) the systematics of chromatographic separation processes, and (III) the design and evaluation of surfactants. In I we have: (a) explored surface condensed structures and plateau adsorption levels; and (b) expanded absorption theory to encompass heterogeneities, studied the critical admicelle concentration and multicomponent adsorption. In II we have traced the composition path for several pure alkyl benzene sulfonates in the grid diagram and examined surfactant wave fronts. In III we have developed two and quantitative surfactant synthesis schemes and finished studies on ..cap alpha..-olefin sulfonates, ethoxylated oleyl sulfonates and alkane sulfonates. The following 5 papers in III have been processed for inclusion in the Energy Data Base: (1) synthesis and performance of isomer-free secondary alkane sulfonate surfactants; (2) synthesis and performance of linear monoisomeric ethylene oxide sulfonate surfactants; (3) alpha-olefin sulfonates for enhanced oil recovery; (4) ethoxylated oleyl sulfonates as model compounds for enhanced oil recovery; (5) phase behavior of simple salt tolerant sulfonates.

  11. Probing transmembrane mechanical coupling and cytomechanics using magnetic twisting cytometry

    NASA Technical Reports Server (NTRS)

    Wang, N.; Ingber, D. E.

    1995-01-01

    We recently developed a magnetic twisting cytometry technique that allows us to apply controlled mechanical stresses to specific cell surface receptors using ligand-coated ferromagnetic microbeads and to simultaneously measure the mechanical response in living cells. Using this technique, we have previously shown the following: (i) beta 1 integrin receptors mediate mechanical force transfer across the cell surface and to the cytoskeleton, whereas other transmembrane receptors (e.g., scavenger receptors) do not; (ii) cytoskeletal stiffness increases in direct proportion to the level of stress applied to integrins; and (iii) the slope of this linear stiffening response differs depending on the shape of the cell. We now show that different integrins (beta 1, alpha V beta 3, alpha V, alpha 5, alpha 2) and other transmembrane receptors (scavenger receptor, platelet endothelial cell adhesion molecule) differ in their ability to mediate force transfer across the cell surface. In addition, the linear stiffening behavior previously observed in endothelial cells was found to be shared by other cell types. Finally, we demonstrate that dynamic changes in cell shape that occur during both cell spreading and retraction are accompanied by coordinate changes in cytoskeletal stiffness. Taken together, these results suggest that the magnetic twisting cytometry technique may be a powerful and versatile tool for studies analyzing the molecular basis of transmembrane mechanical coupling to the cytoskeleton as well as dynamic relations between changes in cytoskeletal structure and alterations in cell form and function.

  12. Molecular cloning and characterization of a Candida tsukubaensis alpha-glucosidase gene in the yeast Saccharomyces cerevisiae.

    PubMed

    Kinsella, B T; Larkin, A; Bolton, M; Cantwell, B A

    1991-07-01

    The molecular cloning of an alpha-glucosidase gene isolated from a Candida tsukubaensis (CBS 6389) genomic library in Saccharomyces cervisiae is reported. The cloned gene is contained within a 6.2 kb Sau3A DNA fragment and directs the synthesis and secretion of an amylolytic enzyme into the extracellular medium of the recombinant host, S. cerevisiae. The cloned enzyme was found to have an unusually broad substrate specificity and is capable of hydrolysing alpha-1,2, alpha-1,3, alpha-1,4 and alpha-1,6 linked, as well as aryl and alkyl, D-glucosides. On the basis of its substrate specificity profile, the cloned enzyme was classified as an alpha-glucosidase (E.C. 3.2.1.20). It has a pH optimum in the range 4.2-4.6, a temperature optimum of 58 degrees C and is readily inactivated at pasteurization temperature (60 degrees C). Southern blot analysis failed to reveal any homology between the cloned gene and genomic DNA isolated from other well characterized amylolytic yeasts. A rapid plate-assay, based on the utilization of a chromogenic substrate X-alpha-D-glucoside to detect the expression of the cloned alpha-glucosidase in S. cerevisiae transformants, was developed. PMID:1934116

  13. Immediate postnatal rise in whole body androgen content in male rats: correlation with increased testicular content and reduced body clearance of testosterone.

    PubMed

    Baum, M J; Brand, T; Ooms, M; Vreeburg, J T; Slob, A K

    1988-06-01

    Whole body content of androgen (testosterone + 5 alpha-dihydrotestosterone) was invariably higher in male than in female rat pups killed 1 or 3 h after natural delivery, whereas androgen content was equivalent in males and females killed immediately or 6, 12, and 24 h after birth. Testicular content of androgen was significantly elevated in males killed 1 and 24 h after birth, compared with levels in males killed immediately, or 3, 6, and 12 h after birth. Thus, heightened testicular androgen content was only initially associated with increased systemic levels of androgen in males during the immediate postpartum period. A second study assessed the possibility that the body's clearance (i.e., metabolism plus excretion) of testosterone is lower in newborn rats upon separation from the placental circulation than in slightly older pups. Rats of both sexes killed 1 and 3 h after s.c. injection of [3H] testosterone had significantly higher plasma concentrations of [3H] testosterone as well as several 5 alpha-reduced androgens (5 alpha-dihydrotestosterone, 3 alpha-androstanediol, and androsterone) when injections were given within minutes as opposed to 24 h after birth. This suggests that in both sexes the clearance of testosterone is slower immediately after birth than at later ages. This phenomenon together with a brief postnatal elevation in the testicular synthesis and secretion of testosterone may explain the temporary rise in circulating androgen concentrations that occurs in the newborn male rat.

  14. Diamond detector for high rate monitors of fast neutrons beams

    SciTech Connect

    Giacomelli, L.; Rebai, M.; Cippo, E. Perelli; Tardocchi, M.; Fazzi, A.; Andreani, C.; Pietropaolo, A.; Frost, C. D.; Rhodes, N.; Schooneveld, E.; Gorini, G.

    2012-06-19

    A fast neutron detection system suitable for high rate measurements is presented. The detector is based on a commercial high purity single crystal diamond (SDD) coupled to a fast digital data acquisition system. The detector was tested at the ISIS pulsed spallation neutron source. The SDD event signal was digitized at 1 GHz to reconstruct the deposited energy (pulse amplitude) and neutron arrival time; the event time of flight (ToF) was obtained relative to the recorded proton beam signal t{sub 0}. Fast acquisition is needed since the peak count rate is very high ({approx}800 kHz) due to the pulsed structure of the neutron beam. Measurements at ISIS indicate that three characteristics regions exist in the biparametric spectrum: i) background gamma events of low pulse amplitudes; ii) low pulse amplitude neutron events in the energy range E{sub dep}= 1.5-7 MeV ascribed to neutron elastic scattering on {sup 12}C; iii) large pulse amplitude neutron events with E{sub n} < 7 MeV ascribed to {sup 12}C(n,{alpha}){sup 9}Be and 12C(n,n')3{alpha}.

  15. Staphylococcus aureus exotoxins are present in vivo in tampons.

    PubMed

    Schlievert, Patrick M; Nemeth, Kimberly A; Davis, Catherine C; Peterson, Marnie L; Jones, Bruce E

    2010-05-01

    Staphylococcal toxic shock syndrome toxin 1 (TSST-1) is the cause of menstrual toxic shock syndrome (mTSS) associated with vaginal colonization by Staphylococcus aureus. In this pilot study, we measured TSST-1 and alpha-toxin, another exotoxin, on used tampons from four healthy women with S. aureus on tampons and from two women with tampon-associated mTSS. Tampons from all six women were sectioned into approximately 0.5-cm(3) pieces, some containing menstrual blood and some lacking menstrual blood. The pH of tampon sections with or without menstrual blood was neutral. S. aureus CFU were present in tampon sections at approximately equivalent counts (total counts were 1 x 10(8) to 2 x 10(9) CFU/tampon). TSST-1 (2 to 80 microg/tampon) and alpha-toxin (28 to 30 microg/tampon) were present only in the sections containing little or no menstrual blood (low hemoglobin density). In the tampons from TSS patients, the cytokine gamma interferon (IFN-gamma) was detected only in menstrual-blood-containing sections, whereas the chemokines macrophage inflammatory protein 3alpha and interleukin-8 were detected in all sections. Thus, IFN-gamma was being produced systemically, whereas the chemokines were being produced both locally by epithelial cells and systemically. The data show that S. aureus exotoxins can be identified in tampons ex vivo in sites with low hemoglobin density.

  16. Pharmacological properties of rat alpha 7 nicotinic receptors expressed in native and recombinant cell systems.

    PubMed

    Virginio, Caterina; Giacometti, Angelo; Aldegheri, Laura; Rimland, Joseph M; Terstappen, Georg C

    2002-06-12

    The pharmacological properties of the rat alpha7 nicotinic acetylcholine receptor endogenously expressed in PC12 cells and recombinantly expressed in GH4C1 cells (alpha7-GH4C1 cells) were characterized and compared. Patch-clamp recordings demonstrated that activation by choline and block by methyllycaconitine and dihydro-beta-erythroidine were similar, but block by mecamylamine was different. Whereas in alpha7-GH4C1 cells the inhibition curve for mecamylamine was monophasic (IC(50) of 1.6 microM), it was biphasic in PC12 cells (IC(50) values of 341 nM and 9.6 microM). The same rank order of potency was obtained for various nicotinic agonists, while acetylcholine was 3.7-fold less potent and 1.5-fold more effective in PC12 cells. Dihydro-beta-erythroidine differentially blocked acetylcholine-evoked currents in both systems. Since reverse transcriptase polymerase chain reaction (RT-PCR) experiments revealed expression of alpha3, alpha4, alpha5, alpha7 and beta4 subunits in PC12 cells, whereas GH4C1 cells express only the beta4 subunit, our results suggest that more than one form of alpha7 containing heteromeric nicotinic receptors might be functionally expressed in PC12 cells.

  17. Roles of extracellular matrix in follicular development.

    PubMed

    Rodgers, R J; van Wezel, I L; Irving-Rodgers, H F; Lavranos, T C; Irvine, C M; Krupa, M

    1999-01-01

    The cellular biology and changes in the extracellular matrix of ovarian follicles during their development are reviewed. During growth of the bovine ovarian follicle the follicular basal lamina doubles 19 times in surface area. It changes in composition, having collagen IV alpha 1-26 and laminin alpha 1, beta 2 and gamma 1 at the primordial stage, and collagen IV alpha 1 and alpha 2, reduced amounts of alpha 3-alpha 5, and a higher content of laminin alpha 1, beta 2 and gamma 1 at the antral stage. In atretic antral follicles laminin alpha 2 was also detected. The follicular epithelium also changes from one layer to many layers during follicular growth. It is clear that not all granulosal cells have equal potential to divide, and we have evidence that the granulosal cells arise from a population of stem cells. This finding has important ramifications and supports the concept that different follicular growth factors can act on different subsets of granulosal cells. In antral follicles, the replication of cells occurs in the middle layers of the membrana granulosa, with older granulosal cells towards the antrum and towards the basal lamina. The basal cells in the membrana granulosa have also been observed to vary in shape between follicies. In smaller antral follicles, they were either columnar or rounded, and in follicles > 5 mm the cells were all rounded. The reasons for these changes in matrix and cell shapes are discussed in relation to follicular development. PMID:10692866

  18. Isolation of a bile salt sulfatase-producing Clostridium strain from rat intestinal microflora.

    PubMed Central

    Huijghebaert, S M; Mertens, J A; Eyssen, H J

    1982-01-01

    Bile acid sulfates, formed in human and rat livers, are desulfated by the intestinal microflora. In our study we first isolated from conventional rat feces an unnamed bacterium, termed strain S1, which desulfated the 5 beta-bile salt 3 alpha-sulfates in vitro and in vivo after association with gnotobiotic rats. Strain S1 also possessed 12 alpha-hydroxysteroid dehydrogenase and bile salt-deconjugating activities. The strain was a strict anaerobic, CO2-requiring, gram-negative, sporeforming rod and was designated as belonging to the genus Clostridium. Growth was scarce in culture media, unless in the presence of 0.1% taurine, a sulfur-containing amino acid. Addition of this substance raised the number of bacteria in thioglycolate and peptone yeast media from 10(4) per ml to 10(6) to 10(7) per ml and increased the colony diameter on agar medium from 0.2 mm to 0.5 to 0.9 mm. Sulfatase activity was specific for the 5 beta-bile salt sulfates, leaving the 5 alpha-bile salt sulfates unchanged. In addition, the sulfatase activity was cell bound, and its production was dependent on the composition of the culture medium, although no minimal sulfur medium was required for sulfatase activity. Images PMID:7055372

  19. Bismuth Oxide Nanoparticles in the Stratosphere

    NASA Technical Reports Server (NTRS)

    Rietmeijer, Frans J. M.; Mackinnon, Ian D. R.

    1997-01-01

    Platey grains of cubic Bi2O3, alpha-Bi2O3, and Bi2O(2.75), nanograins were associated with chondritic porous interplanetary dust particles W7029C1, W7029E5, and 2011C2 that were collected in the stratosphere at 17-19 km altitude. Similar Bi oxide nanograins were present in the upper stratosphere during May 1985. These grains are linked to the plumes of several major volcanic eruptions during the early 1980s that injected material into the stratosphere. The mass of sulfur from these eruptions is a proxy for the mass of stratospheric Bi from which we derive the particle number densities (p/cu m) for "average Bi2O3 nanograins" due to this volcanic activity and those necessary to contaminate the extraterrestrial chondritic porous interplanetary dust particles via collisional sticking. The match between both values supports the idea that Bi2O3 nanograins of volcanic origin could contaminate interplanetary dust particles in the Earth's stratosphere.

  20. Direct determination of anabolic steroids in pig urine by a new SPME-GC-MS method.

    PubMed

    Zhang, Zhuomin; Duan, Hongbin; Zhang, Lan; Chen, Xi; Liu, Wei; Chen, Guonan

    2009-05-15

    A new solid phase microextraction (SPME) method coupled with gas chromatography-mass spectrometry (GC-MS) was developed for rapid determination of four anabolic steroids such as 3alpha-hydroxy-5alpha-androstane-17-one (HA), dihydrotestosterone (DHT), androstenedione (AD) and methyltestosterone (MT) in pig urine. SPME was used to extract the four anabolic compounds directly without derivatization. The optimum SPME sampling conditions were based on the home-made carbowax-divinylbenzene (CW-DVB) fiber coating during extraction at 40 degrees C for 50 min with 0.18 g/mL NaCl solution and 750 rpm stirring speed. The linear ranges of the proposed method were in the range of 8-640 pg/mL for HA and DHT and 16-510 pg/mL for AD and MT, respectively. The detection limits (S/N=3) were from 2 to 8 pg/mL for the four anabolic steroids. This SPME method provided very high enrichment factors for the four anabolic steroids, which were 1063-fold and 965-fold for HA and DHT at the concentration of 8 pg/mL and 207-fold and 451-fold for AD and MT at the concentration of 16 pg/mL, respectively. The recoveries ranged from 71.3 to 121%, and the RSDs were lower than 12.9%. The method was sensitive and reliable for determination of trace anabolic steroids in biological samples.

  1. Association of HLA-DR3 with human immune response to Lol p I and Lol p II allergens in allergic subjects.

    PubMed

    Freidhoff, L R; Ehrlich-Kautzky, E; Meyers, D A; Ansari, A A; Bias, W B; Marsh, D G

    1988-04-01

    Associations between HLA type and IgE or IgG antibody (Ab) responses to two well-characterized, antigenetically non-crossreactive components of Lolium perenne (rye grass) pollen extract, Lol p I (Rye I) and Lol p II (Rye II) were studied in two groups of skin-test positive (ST+) Caucasoid adults. By both nonparametric and parametric statistical methods, significant associations were found between Ab responses to both Lol I and Lol II and the possession of HLA-DR3. In view of the well-known associations of both DR3 and B8 (which are in linkage disequilibrium) with many autoimmune diseases, differences in anti-Lol I and anti-Lol II mean log[Ab] levels between B8+, DR3- vs B8-, DR3- subjects and B8+, DR3+ vs B8-, DR3+ subjects were investigated. No differences were found. Our data, along with recent RFLP and DNA sequence studies, suggest that an Ia molecule involved in immune recognition of a similar major Ia recognition site of both the Lol molecules may consist of a DR3 alpha-beta I pair. Abbreviations used: Ab: Antibody. HLA: Human leukocyte antigen. Lol p I, Lol I: Group I allergen from Lolium perenne pollen (Rye I). Lol p II, Lol II: Group II allergen from Lolium perenne pollen (Rye II). Mr: Relative molecular mass. Rx: Immunotherapy with grass pollen extracts. ST: Skin test.

  2. Rhizobium fredii and Rhizobium meliloti produce 3-deoxy-D-manno-2-octulosonic acid-containing polysaccharides that are structurally analogous to group II K antigens (capsular polysaccharides) found in Escherichia coli.

    PubMed

    Reuhs, B L; Carlson, R W; Kim, J S

    1993-06-01

    The polysaccharide components from cultured cells of Rhizobium fredii USDA205 and Rhizobium meliloti AK631 were extracted with hot phenol-water and separated by repetitive gel filtration chromatography. Polyacrylamide gel electrophoresis, nuclear magnetic resonance spectrometry, and gas chromatography analyses showed that both of these bacterial species produce unique polysaccharides that contain a high proportion of 3-deoxy-D-manno-2-octulosonic acid (Kdo). These polysaccharides, which constituted a major portion of the extracted carbohydrate, are not excreted into the growth media (i.e., they are not extracellular polysaccharides) and are structurally distinct from the lipopolysaccharides. The primary structure of the preponderant polysaccharide from R. fredii USDA205 was determined by high-performance anion-exchange liquid chromatography, nuclear magnetic resonance spectrometry, fast atom bombardment-mass spectrometry, and gas chromatography-mass spectrometry; it consists of repeating units of [-->3)-alpha-D-Galp-(1-->5)-beta-D-Kdop-(2-->]n. This molecule is structurally analogous to the constituents of one subgroup of K antigens (capsular polysaccharides) produced by Escherichia coli. Polysaccharides of this type have not previously been identified as components of rhizobial cells. The Kdo-containing polysaccharide from R. meliloti, which has not been completely characterized, appears to be structurally related to that of R. fredii.

  3. Chemical composition and antioxidant, antimicrobial, and larvicidal activities of the essential oils of Annona salzmannii and A. pickelii (Annonaceae).

    PubMed

    Costa, Emmanoel Vilaça; Dutra, Lívia Macedo; de Jesus, Hugo César Ramos; Nogueira, Paulo Cesar de Lima; Moraes, Valéria Regina de Souza; Salvador, Marcos José; Cavalcanti, Sócrates Cabral de Holanda; dos Santos, Roseli La Corte; Prata, Ana Paula do Nacimento

    2011-06-01

    The essential oils from the leaves of Annona salzmannii and A. pickelii (Annonaceae) growing in Sergipe, northeastern region of Brazil, were obtained by hydrodistillation using a Clevenger-type apparatus, and analyzed by GC/MS and GC/FID. Thirty-four compounds were identified in the essential oil of A. salzmannii and twenty-seven in that of A. pickelii; sesquiterpenes predominated in both essential oils. Bicyclogermacrene (20.3%), (E)-caryophyllene (19.9%), delta-cadinene (15.3%), alpha-copaene (10.0%), and allo-aromadendrene (5.7%) were the main components of A. salzmannii, and bicyclogermacrene (45.4%), (E)-caryophyllene (14.6%), and alpha-copaene (10.6%) of A. pickelii. The essential oils showed significant antioxidant capacity in the ORAC(FL) and DPPH assays. The antimicrobial activity of these essential oils was also evaluated against bacteria and fungi, as well as the larvicidal activity against Aedes aegypti larvae.

  4. Incorporation of radiolabeled whey proteins into casein micelles by heat processing

    SciTech Connect

    Noh, B.; Richardson, T. )

    1989-07-01

    Skim milk was heated at .70, 95, and 140{degree}C to simulate the processes of pasteurization, forewarming, and UHT sterilization, and the specific interactions between {alpha}-lactalbumin or {beta}-lactoglobulin and the caseins studied using tracer amounts of added {sup 14}C-labeled whey protein. Radioactivities of the whey and of the washed casein pellets from renneted skim milk were measured and the extent of the interaction estimated. Upon heating skim milk at 70{degree}C for 45 s, less than 2% {beta}-lactoglobulin and less than .3% {alpha}-lactalbumin were incorporated into the curd. Heating at 95{degree}C for .5 to 20 min resulted in 58 to 85% of the {beta}-lactoglobulin and 8 to 55% of the {alpha}-lactalbumin becoming associated with the curd. Heating at 140{degree}C for 2 and 4 s caused 43 and 54% of the {beta}-lactoglobulin and 9 and 12% of the {alpha}-lactalbumin, respectively, to be bound to the curd fraction. The radiolabeling technique is very sensitive and useful for tracing low levels of interaction between whey proteins and casein in heated milk systems.

  5. Impact of climate change on communities: revealing species' contribution.

    PubMed

    Davey, Catherine M; Devictor, Vincent; Jonzén, Niclas; Lindström, Ake; Smith, Henrik G

    2013-05-01

    1. Although climate is known to play an important role in structuring biological communities, high-resolution analyses of recent climatic impacts on multiple components of diversity are still sparse. Additionally, there is a lack of knowledge about which species drive community response to environmental change. 2. We used a long-term breeding bird data set that encompasses a large latitudinal and altitudinal range to model the effect of temperature on spatial and temporal patterns in alpha and beta diversity. We also established a novel framework for identifying species-specific contributions to these macroecological patterns, hence combining two different approaches for identifying climatic impacts. 3. Alpha diversity increased over time, whilst beta diversity declined; both diversity metrics showed a significant relationship with recent temperature anomalies. By partitioning beta diversity, we showed that the decline was predominately driven by changes in species turnover rather than nestedness suggesting a process of replacement by more common species. 4. Using jackknife analyses we identified how individual species influenced the modelled relationships of diversity with temperature and time. Influential species tended to be habitat generalists with moderate to large distributions. 5. We demonstrate that different facets of avian diversity can respond rapidly to temperature anomalies and as a result have undergone significant changes in the last decade. In general, it appears that warming temperatures are driving compositional homogenization of temperate bird communities via range expansion of common generalist species.

  6. Galectin-4 expression is down-regulated in response to autophagy during differentiation of rat trophoblast cells

    PubMed Central

    Arikawa, Tomohiro; Liao, Shengjun; Shimada, Hiroki; Inoue, Tomoki; Sakata-Haga, Hiromi; Nakamura, Takanori; Hatta, Toshihisa; Shoji, Hiroki

    2016-01-01

    Placental development and trophoblast invasion of the maternal endometrium establish the maternal-fetal interface, which is critical for the developing embryo and fetus. Herein we show that overexpression of Galectin-4 (Gal-4) during trophoblast differentiation inhibited the enlargement of Rcho-1 cells (a model for rat trophoblast differentiation) and promoted cell-cell adhesion, whereas trophoblast specific markers and MMP-9 activity were not affected. In the rat placenta, microtubule associated protein 1 light chain 3 alpha (LC3) protein, an autophagy marker, is highly expressed on the maternal side of the decidua where Gal-4 expression is weak. In vitro assays showed that the expression of trophoblast-specific differentiation markers was reduced by 3-Methyladenine (3-MA) and Bafilomycin A1, known as autophagy inhibitors, compared to control cells. Furthermore, Gal-4 expression in Rcho-1 cells, which is normally down-regulated during differentiation, was not attenuated in the presence of autophagy inhibitors, suggesting that autophagy is upstream of Gal-4 expression. We herein describe a possible mechanism by which autophagy regulates trophoblast differentiation via regulation of Gal-4 expression in order to establish the maternal-fetal interface. PMID:27572741

  7. The pharmacology of spontaneously open alpha 1 beta 3 epsilon GABA A receptor-ionophores.

    PubMed

    Maksay, Gábor; Thompson, Sally A; Wafford, Keith A

    2003-06-01

    Human alpha(1)beta(3) epsilon GABA(A) receptors were expressed in Xenopus oocytes and examined using the conventional two-electrode voltage-clamp technique and compared to alpha(1)beta(3)gamma(2) receptors. The effects of several GABA(A) agonists were studied, and the allosteric modulation of the channel by a number of GABAergic modulators investigated. The presence of the epsilon subunit increased the potency and efficacy of direct activation by partial GABA(A) agonists (piperidine-4-sulphonic acid and thio-4-PIOL), pentobarbital and neuro-steroids. Direct activation by 3-hydroxylated neurosteroids was restricted to 3alpha epimers, while chirality at C5 was indifferent. The 3beta-sulfate esters of pregnenolone and dehydroepiandrosterone inhibited the spontaneous currents with efficacies higher, while bicuculline methiodide and SR 95531 did so lower than picrotoxin and TBPS. Furosemide, fipronil, triphenylcyanoborate and Zn(2+) blocked the spontaneous currents of alpha(1)beta(3) epsilon receptors with different efficacies. Flunitrazepam and 4'-chlorodiazepam inhibited the spontaneous currents with micromolar potencies. In conclusion, spontaneously active alpha(1)beta(3) epsilon GABA(A) receptors can be potentiated and blocked by GABAergic agents within a broad range of efficacy.

  8. Lipoprotein and apolipoprotein levels among Mexican-Americans in Starr County, Texas.

    PubMed

    Hanis, C L; Hewett-Emmett, D; Douglas, T C; Schull, W J

    1991-01-01

    Mexican-Americans represent the single largest component of the US Hispanic population and have been shown to bear a disproportionate burden of chronic disease. A representative sample of 1,004 Mexican-Americans aged 15-74 years from Starr County, Tex., was recruited for this study. Each subject was provided a detailed physical evaluation that included measurement of fasting levels of cholesterol, triglycerides, high density lipoprotein (HDL) cholesterol and its subfractions (HDL2 and HDL3) alpha- and beta-lipoprotein cholesterol, and low density lipoprotein cholesterol. Apolipoproteins A-I, A-II, B, C-II, C-III, and E were determined for approximately 550 of these individuals. Age- and sex-specific mean levels and percentile cut points are presented. The distributions of lipoproteins are quite similar to those of the general population except for consistently higher triglycerides in males and females and lower HDL cholesterol levels in females. These findings are consistent with the high frequency of obesity. Comparative age- and sex-specific data for the apolipoproteins are not widely available. Where such data exist, apolipoprotein levels observed in the Mexican-American population tend to be similar to or lower than the comparative data. PMID:1987989

  9. An Alpha Motif at Tas3C Terminus Mediates RITS Cis Spreading and Promotes Heterochromatic Gene Silencing

    SciTech Connect

    Li, H.; Motamedi, M; Yip, C; Wang, Z; Walz, T; Patel, D; Moazed, D

    2009-01-01

    RNA interference (RNAi) plays a pivotal role in the formation of heterochromatin at the fission yeast centromeres. The RNA-induced transcriptional silencing (RITS) complex, composed of heterochromatic small interfering RNAs (siRNAs), the siRNA-binding protein Ago1, the chromodomain protein Chp1, and the Ago1/Chp1-interacting protein Tas3, provides a physical tether between the RNAi and heterochromatin assembly pathways. Here, we report the structural and functional characterization of a C-terminal Tas3 {alpha}-helical motif (TAM), which self-associates into a helical polymer and is required for cis spreading of RITS in centromeric DNA regions. Site-directed mutations of key residues within the hydrophobic monomer-monomer interface disrupt Tas3-TAM polymeric self-association in vitro and result in loss of gene silencing, spreading of RITS, and a dramatic reduction in centromeric siRNAs in vivo. These results demonstrate that, in addition to the chromodomain of Chp1 and siRNA-loaded Ago1, Tas3 self-association is required for RITS spreading and efficient heterochromatic gene silencing at centromeric repeat regions.

  10. Stellar evolution of high mass based on the Ledoux criterion for convection

    NASA Technical Reports Server (NTRS)

    Stothers, R.; Chin, C.

    1972-01-01

    Theoretical evolutionary sequences of models for stars of 15 and 30 solar masses were computed from the zero-age main sequence to the end of core helium burning. During the earliest stages of core helium depletion, the envelope rapidly expands into the red-supergiant configuration. At 15 solar mass, a blue loop on the H-R diagram ensues if the initial metals abundance, initial helium abundance, or C-12 + alpha particle reaction rate is sufficiently large, or if the 3-alpha reaction rate is sufficiently small. These quantities affect the opacity of the base of the outer convection zone, the mass of the core, and the thermal properties of the core. The blue loop occurs abruptly and fully developed when the critical value of any of these quantities is exceeded, and the effective temperature range and fraction of the lifetime of core helium burning during the slow phase of the blue loop vary surprisingly little. At 30 solar mass no blue loop occurs for any reasonable set of input parameters.

  11. Inhibition of cell-to-cell transmission of human T-cell lymphotropic virus type 1 in vitro by carbohydrate-binding agents.

    PubMed

    Balestrieri, Emanuela; Ascolani, Arianna; Igarashi, Yasuhiro; Oki, Toshikazu; Mastino, Antonio; Balzarini, Jan; Macchi, Beatrice

    2008-08-01

    Peripheral blood mononuclear cells (PBMCs) from healthy individuals can be infected by human T-lymphotropic virus type 1 (HTLV-1) upon cocultivation of the PBMCs with irradiated HTLV-1-transformed human MT-2 cells. This model system closely mimics HTLV-1 transmission through cell-to-cell contact. Carbohydrate-binding agents (CBAs) such as the alpha(1,3)/alpha(1,6)mannose-specific Hippeastrum hybrid agglutinin and the GlcNAc-specific Urtica dioica agglutinin, and also the small, nonpeptidic alpha(1,2)-mannose-specific antibiotic pradimicin A, were able to efficiently prevent cell-to-cell HTLV-1 transmission at nontoxic concentrations, as evidenced by the lack of appearance of virus-specific mRNA and of the viral protein Tax in the acceptor cells. Consistently, antivirally active doses of CBAs fully prevented HTLV-1-induced stimulation of PBMC growth. The inhibitory effects of CBAs on HTLV-1 transmission were also evident when HTLV-1-infected C5MJ cells were used in place of MT-2 cells as a virus donor cell line. The anti-HTLV-1 properties of the CBAs highlight the importance of the envelope glycans in events underlying HTLV-1 passage from cell to cell and indicate that CBAs should be further investigated for their potential to prevent HTLV-1 infection, including mother-to-child virus transmission by cell-to-cell contact through breast milk feeding. PMID:18505856

  12. Method and apparatus for preparation of spherical metal carbonates and lithium metal oxides for lithium rechargeable batteries

    DOEpatents

    Kang, Sun-Ho; Amine, Khalil

    2008-10-14

    A number of materials with the composition Li.sub.1+xNi.sub..alpha.Mn.sub..beta.Co.sub..gamma.M'.sub..delta.O.sub.2-- zF.sub.z (M'=Mg,Zn,Al,Ga,B,Zr,Ti) for use with rechargeable batteries, wherein x is between about 0 and 0.3, .alpha. is between about 0.2 and 0.6, .beta. is between about 0.2 and 0.6, .gamma. is between about 0 and 0.3, .delta. is between about 0 and 0.15, and z is between about 0 and 0.2. Adding the above metal and fluorine dopants affects capacity, impedance, and stability of the layered oxide structure during electrochemical cycling. Another aspect of the invention includes materials with the composition Li.sub.1+xNi.sub..alpha.Co.sub..beta.Mn.sub..gamma.M'.sub..delta.O.sub.yF- .sub.z (M'=Mg,Zn,Al,Ga,B,Zr,Ti), where the x is between 0 and 0.2, the .alpha. between 0 and 1, the .beta. between 0 and 1, the .gamma. between 0 and 2, the .delta. between about 0 and about 0.2, the y is between 2 and 4, and the z is between 0 and 0.5.

  13. De novo prediction of polypeptide conformations using dihedral probability grid Monte Carlo methodology.

    PubMed Central

    Evans, J. S.; Mathiowetz, A. M.; Chan, S. I.; Goddard, W. A.

    1995-01-01

    We tested the dihedral probability grid Monte Carlo (DPG-MC) methodology to determine optimal conformations of polypeptides by applying it to predict the low energy ensemble for two peptides whose solution NMR structures are known: integrin receptor peptide (YGRGDSP, Type II beta-turn) and S3 alpha-helical peptide (YMSEDEL KAAEAAFKRHGPT). DPG-MC involves importance sampling, local random stepping in the vicinity of a current local minima, and Metropolis sampling criteria for acceptance or rejection of new structures. Internal coordinate values are based on side-chain-specific dihedral angle probability distributions (from analysis of high-resolution protein crystal structures). Important features of DPG-MC are: (1) Each DPG-MC step selects the torsion angles (phi, psi, chi) from a discrete grid that are then applied directly to the structure. The torsion angle increments can be taken as S = 60, 30, 15, 10, or 5 degrees, depending on the application. (2) DPG-MC utilizes a temperature-dependent probability function (P) in conjunction with Metropolis sampling to accept or reject new structures. For each peptide, we found close agreement with the known structure for the low energy conformational ensemble located with DPG-MC. This suggests that DPG-MC will be useful for predicting conformations of other polypeptides. PMID:7549884

  14. Identification of 7(8) and 8(9) unsaturated adrenal steroid metabolites produced by patients with 7-dehydrosterol-delta7-reductase deficiency (Smith-Lemli-Opitz syndrome).

    PubMed

    Shackleton, Cedric; Roitman, Esther; Guo, Li Wei; Wilson, William K; Porter, Forbes D

    2002-10-01

    Patients with Smith-Lemli-Opitz syndrome have impaired ability to synthesize cholesterol due to attenuated activity of 7-dehydrosterol-delta(7)-reductase which catalyses the final step in cholesterol synthesis. Accumulation of 7- and 8-dehydrocholesterol is a result of the disorder and potentially these sterols could be used as precursors of a novel class of delta(7) and delta(8) unsaturated adrenal steroids and their metabolites. In this study, we have analyzed urine from SLOS patients in the anticipation of characterizing such metabolites. Gas chromatography/mass spectrometry (GC/MS) was used in the identification of two major metabolites as 7- and 8-dehydroversions of the well-known steroid pregnanetriol. Other steroids, such as 8-dehydro dehydroepiandrosterone (8-dehydro DHEA) and 7- or 8-dehydroandrostenediol were also identified, and several more steroids are present in urine but remain uncharacterized. As yet, the study provides no evidence for the production of ring-B unsaturated metabolites of complex steroids, such as cortisol. We believe that the following transformations can utilize ring-B dehydroprecursors: StAR transport of cholesterol, p450 side chain cleavage, 17-hydroxylase/17,20-lyase, 3beta-hydroxysteroid dehydrogenase, 3alpha-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase, 20alpha-hydroxysteroid dehydrogenase and 5beta-reductase. We have yet to prove the activity of adrenal 21-hydroxylase, 11beta-hydroxylase or 5alpha-reductase towards 7- or 8-dehydroprecursors.

  15. Identification of specific protein markers in microdissected hepatocellular carcinoma.

    PubMed

    Melle, Christian; Ernst, Günther; Scheibner, Olaf; Kaufmann, Roland; Schimmel, Bettina; Bleul, Annett; Settmacher, Utz; Hommann, Merten; Claussen, Uwe; von Eggeling, Ferdinand

    2007-01-01

    At present, the molecular mechanisms of hepatocellular carcinogenesis are not well-understood, and hepatocellular carcinoma (HCC) stays one of the most frequent and high-risk metastatic visceral neoplasms worldwide. For the identification of tumor-relevant proteins, we analyzed microdissected cells from nontumorous liver tissue (n = 28) and tissue derived from hepatic tumor center (n = 25), as well as tumor margin (n = 23). We unequivocally identified 53 proteins from hepatic tumor tissues by peptide fingerprint mapping and SELDI mass spectrometry that were separated using two-dimensional gel electrophoresis. Among a number of signals that were detected as significantly different in the protein profiling analysis, we identified for the first time ferritin light subunit (FLS) and adenylate kinase 3 alpha-like 1 (AK3), showing decreased expressions in hepatic tumor, as well as biliverdin reductase B (BVRB) that was upregulated in HCC. The use of ProteinChip technology in combination with tissue microdissection gives insight of the complex changes occurring at the protein level in hepatocellular cancer associated with tumor development and progression and resulted in three new potential diagnostically useful markers. PMID:17203974

  16. A serendipitous discovery of antifreeze protein-specific activity in C-linked antifreeze glycoprotein analogs.

    PubMed

    Eniade, Adewale; Purushotham, Madhusudhan; Ben, Robert N; Wang, J B; Horwath, Kathleen

    2003-01-01

    Structurally diverse carbon-linked (C-linked) analogs of antifreeze glycoprotein (AFGP) have been prepared via linear or convergent solid phase synthesis. These analogs range in molecular weight from approx 1.5-4.1 KDa and do not possess the beta-D-galactose-1,3-alpha-D-N-acetylgalactosamine carbohydrate moiety or the L-threonine-L-alanine-L-alanine polypeptide backbone native to the AFGP wild-type. Despite these dramatic structural modifications, the 2.7-KDa and 4.1-KDa analogs possess antifreeze protein-specific activity as determined by recrystallization-inhibition (RI) and thermal hysteresis (TH) assays. These analogs are weaker than the wild-type in their activity, but nanoliter osmometry indicates that these compounds are binding to ice and affecting a localized freezing point depression. This is the first example of a C-linked AFGP analog that possesses TH and RI activity and suggests that the rational design and synthesis of chemically and biologically stable AFGP analogs is a feasible and worthwhile endeavor. Given the low degree of TH activity, these compounds may prove useful for the protection of cells during freezing and thawing cycles. PMID:12777711

  17. Fatty acid profile, tocopherol, squalene and phytosterol content of walnuts, almonds, peanuts, hazelnuts and the macadamia nut.

    PubMed

    Maguire, L S; O'Sullivan, S M; Galvin, K; O'Connor, T P; O'Brien, N M

    2004-05-01

    Nuts are high in fat but have a fatty acid profile that may be beneficial in relation to risk of coronary heart disease. Nuts also contain other potentially cardioprotective constituents including phytosterols, tocopherols and squalene. In the present study, the total oil content, peroxide value, composition of fatty acids, tocopherols, phytosterols and squalene content were determined in the oil extracted from freshly ground walnuts, almonds, peanuts, hazelnuts and the macadamia nut. The total oil content of the nuts ranged from 37.9 to 59.2%, while the peroxide values ranged from 0.19 to 0.43 meq O2/kg oil. The main monounsaturated fatty acid was oleic acid (C18:1) with substantial levels of palmitoleic acid (C16:1) present in the macadamia nut. The main polyunsaturated fatty acids present were linoleic acid (C18:2) and linolenic acid (C18:3). alpha-Tocopherol was the most prevalent tocopherol except in walnuts. The levels of squalene detected ranged from 9.4 to 186.4 microg/g. beta-Sitosterol was the most abundant sterol, ranging in concentration from 991.2 to 2071.7 microg/g oil. Campesterol and stigmasterol were also present in significant concentrations. Our data indicate that all five nuts are a good source of monounsaturated fatty acid, tocopherols, squalene and phytosterols. PMID:15223592

  18. Self-similar collapse and accretion of radiative gas

    NASA Astrophysics Data System (ADS)

    Boily, C. M.; Lynden-Bell, D.

    1995-09-01

    origin, these flows have free-fall density and velocity profiles, provided that delta<~-1/2, where delta=MAX¾(alpha-2)/(beta+3), -1/2}. When delta=-1/2, and provided that gamma=5/3, pressure gradients scale with GM/r^2 all the way to r=0. The temperature has a power-law radial dependence, T~r^2delta, and diverges near the mass, provided that delta<0. The equations of motion allow for a continuum of accretion solutions for each value of alpha, beta. These are classified in terms of the ratio of gravitational force to pressure gradient, W, and the Mach number, which are constant at large radii. Solutions exist that have arbitrarily large values of the Mach number and inward velocities. Accretion by a mass surrounded by a hydrostatic envelope is found in the limit W-->1 and zero velocity; solutions are given for 18 different pairs of alpha and beta. Outflowing envelopes cause the appearance of a `ring' in the luminosity profile, which expands about the central mass. When the outflow is such that the gas leaves the central region, we find no solution except when beta=1-3alpha and with 4/3<alpha<3/2, in which cases outflows lead to holes expanding self-similarly. The specific case of a dusty emission law is considered in detail.

  19. Mycobacterium tuberculosis acyl carrier protein synthase adopts two different pH-dependent structural conformations

    SciTech Connect

    Gokulan, Kuppan; Aggarwal, Anup; Shipman, Lance; Besra, Gurdyal S.; Sacchettini, James C.

    2011-09-20

    The crystal structures of acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) and Corynebacterium ammoniagenes determined at pH 5.3 and pH 6.5, respectively, are reported. Comparison of the Mtb apo-AcpS structure with the recently reported structure of the Mtb AcpS-ADP complex revealed that AcpS adopts two different conformations: the orthorhombic and trigonal space-group structures show structural differences in the {alpha}2 helix and in the conformation of the {alpha}3-{alpha}4 connecting loop, which is in a closed conformation. The apo-AcpS structure shows electron density for the entire model and was obtained at lower pH values (4.4-6.0). In contrast, at a higher pH value (6.5) AcpS undergoes significant conformational changes, resulting in disordered regions that show no electron density in the AcpS model. The solved structures also reveal that C. ammoniagenes AcpS undergoes structural rearrangement in two regions, similar to the recently reported Mtb AcpS-ADP complex structure. In vitro reconstitution experiments show that AcpS has a higher post-translational modification activity between pH 4.4 and 6.0 than at pH values above 6.5, where the activity drops owing to the change in conformation. The results show that apo-AcpS and AcpS-ADP adopt different conformations depending upon the pH conditions of the crystallization solution.

  20. Release of arginine, glutamate and glutamine in the hippocampus of freely moving rats: Involvement of nitric oxide.

    PubMed

    Watts, Jo; Fowler, Leslie; Whitton, Peter S; Pearce, Brian

    2005-05-30

    Using in vivo microdialysis, we have monitored the release of three amino acids (arginine, glutamate and glutamine) in the hippocampus of freely moving rats in response to various drugs. In response to N-methyl-d-aspartate (NMDA) infusion, extracellular glutamate was increased, glutamine was decreased and arginine remained unchanged. By contrast, alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) elicited an increase in arginine release but had no effect on either glutamate or glutamine. When S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide (NO) donor, was infused into the hippocampus, an increase in glutamate, a decrease in glutamine and no change in arginine were recorded. The effect of SNAP on extracellular glutamine levels was reversed by prior infusion of the guanylate cyclase inhibitor oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), however its effect on glutamate release was unchanged. Interestingly, SNAP was found to promote the release of arginine in the presence of ODQ. We also assessed the effect of two nitric oxide synthase inhibitors, N-nitro-l-arginine methylester (l-NAME) and 7-nitroindazole (7-NI), on the release of these amino acids. l-NAME was found to increase arginine and glutamate levels but decrease those of glutamine. In contrast, 7-NI reduced the release of all three amino acids. The results presented here confirm some but not all of the findings previously obtained using in vitro preparations. In addition, they suggest that complex relationships exist between the release of these amino acids, and that endogenous NO plays an important role in regulating their release.

  1. Unexpected Inflammatory Effects of Intravaginal Gels (Universal Placebo Gel and Nonoxynol-9) on the Upper Female Reproductive Tract: A Randomized Crossover Study

    PubMed Central

    Smith-McCune, Karen; Chen, Joseph C.; Greenblatt, Ruth M.; Shanmugasundaram, Uma; Shacklett, Barbara L.; Hilton, Joan F.; Johnson, Brittni; Irwin, Juan C.; Giudice, Linda C

    2015-01-01

    Intravaginal anti-HIV microbicides could provide women with a self-controlled means for HIV prevention, but results from clinical trials have been largely disappointing. We postulated that unrecognized effects of intravaginal gels on the upper female reproductive tract might contribute to the lower-than-expected efficacy of HIV microbicides. Our objective was to study the effects of intravaginal gels on the immune microenvironment of the cervix and uterus. In this randomized crossover study, 27 healthy female volunteers used a nightly application of intravaginal nonoxynol-9 (N9) gel as a “failed” microbicide or the universal placebo gel (UPG) as a “safe” gel (intervention cycles), or nothing (control cycle) from the end of menses to the mid-luteal phase. At a specific time-point following ovulation, all participants underwent sample collection for measurements of T-cell phenotypes, gene expression, and cytokine/chemokine protein concentrations from 3 anatomic sites above the vagina: the cervical transformation zone, the endocervix and the endometrium. We used hierarchical statistical models to estimate mean (95% CI) intervention effects, for N9 and UPG relative to control. Exposure to N9 gel and UPG generated a common “harm signal” that included transcriptional up-regulation of inflammatory genes chemokine (C-C motif) ligand 20 (macrophage inflammatory factor-3alpha) and interleukin 8 in the cervix, decreased protein concentrations of secretory leukocyte protease inhibitor, and transcriptional up-regulation of inflammatory mediators glycodelin-A and osteopontin in the endometrium. These results need to be replicated with a larger sample, but underscore the need to consider the effects of microbicide agents and gel excipients on the upper female reproductive tract in studies of vaginal microbicides. PMID:26177352

  2. Allopregnanolone modulates spontaneous GABA release via presynaptic Cl- permeability in rat preoptic nerve terminals.

    PubMed

    Haage, David; Druzin, Michael; Johansson, Staffan

    2002-12-27

    The endogenous neurosteroid 3alpha-hydroxy-5alpha-pregnane-20-one (allopregnanolone) affects presynaptic nerve terminals and thereby increases the frequency of spontaneous GABA release. The present study aimed at clarifying the mechanisms underlying this presynaptic neurosteroid action, by recording the frequency of spontaneous GABA-mediated inhibitory postsynaptic currents (sIPSCs) in neurons from the medial preoptic nucleus (MPN) of rat. Acutely dissociated neurons with functional adhering nerve terminals were studied by perforated-patch recording under voltage-clamp conditions. It was shown that the sIPSC frequency increased with the external K(+) concentration ([K(+)](o)). Further, the effect of allopregnanolone on the sIPSC frequency was strongly dependent on [K(+)](o). In a [K(+)](o) of 5 mM, 2.0 microM allopregnanolone caused a clear increase in sIPSC frequency. However, the effect declined rapidly with increased [K(+)](o) and at high [K(+)](o) allopregnanolone reduced the sIPSC frequency. The effect of allopregnanolone was also strongly dependent on the external Cl(-) concentration ([Cl(-)](o)). In a reduced [Cl(-)](o) (40 mM, but with a standard [K(+)](o) of 5 mM), the effect on sIPSC frequency was larger than that in the standard [Cl(-)](o) of 146 mM. The dependence of the effect of allopregnanolone on [K(+)](o) and on estimated presynaptic membrane potential was also altered by the reduction in [Cl(-)](o). As in standard [Cl(-)](o), the effect in low [Cl(-)](o) declined when [K(+)](o) was raised, but reversed at a higher [K(+)](o). The GABA(A) receptor agonist muscimol also potentiated the sIPSC frequency. Altogether, the results suggest that allopregnanolone exerts its presynaptic effect by increasing the presynaptic Cl(-) permeability, most likely via GABA(A) receptors. PMID:12470877

  3. Markedly inhibited 7-dehydrocholesterol-delta 7-reductase activity in liver microsomes from Smith-Lemli-Opitz homozygotes.

    PubMed Central

    Shefer, S; Salen, G; Batta, A K; Honda, A; Tint, G S; Irons, M; Elias, E R; Chen, T C; Holick, M F

    1995-01-01

    We investigated the enzyme defect in late cholesterol biosynthesis in the Smith-Lemli-Opitz syndrome, a recessively inherited developmental disorder characterized by facial dysmorphism, mental retardation, and multiple organ congenital anomalies. Reduced plasma and tissue cholesterol with increased 7-dehydrocholesterol concentrations are biochemical features diagnostic of the inherited enzyme defect. Using isotope incorporation assays, we measured the transformation of the precursors, [3 alpha- 3H]lathosterol and [1,2-3H]7-dehydrocholesterol into cholesterol by liver microsomes from seven controls and four Smith-Lemli-Opitz homozygous subjects. The introduction of the double bond in lathosterol at C-5[6] to form 7-dehydrocholesterol that is catalyzed by lathosterol-5-dehydrogenase was equally rapid in controls and homozygotes liver microsomes (120 +/- 8 vs 100 +/- 7 pmol/mg protein per min, P = NS). In distinction, the reduction of the double bond at C-7 [8] in 7-dehydrocholesterol to yield cholesterol catalyzed by 7-dehydrocholesterol-delta 7-reductase was nine times greater in controls than homozygotes microsomes (365 +/- 23 vs 40 +/- 4 pmol/mg protein per min, P < 0.0001). These results demonstrate that the pathway of lathosterol to cholesterol in human liver includes 7-dehydrocholesterol as a key intermediate. In Smith-Lemli-Opitz homozygotes, the transformation of 7-dehydrocholesterol to cholesterol by hepatic microsomes was blocked although 7-dehydrocholesterol was produced abundantly from lathosterol. Thus, lathosterol 5-dehydrogenase is equally active which indicates that homozygotes liver microsomes are viable. Accordingly, microsomal 7-dehydrocholesterol-delta 7-reductase is inherited abnormally in Smith-Lemli-Opitz homozygotes. PMID:7560069

  4. Purification, structural characterization, and antioxidant activity of antioxidant substance from the red seaweed Gloiopeltis tenax.

    PubMed

    Lim, Byung-Lak; Ryu, Il-Hwan

    2009-04-01

    An antioxidant substance (AOS) obtained from an enzymatic extract of the red seaweed Gloiopeltis tenax was purified by DEAE-Sephadex CL-6B and Sephadex G-100 column chromatography. The purification yield of AOS was 14.3%. The AOS predominantly contained fucose, mannose, and galactose but also contained a sulfate group. The structure of AOS was investigated by periodate oxidation, desulfation, Fourier transform-infrared spectroscopy, and nuclear magnetic resonance spectroscopy. AOS was mainly composed of alternating units of beta-D-Glc(1 --> 2)alpha-D-Man(1 --> 4)beta-D-Gal(1 --> 4)alpha-D-Man(1 --> 4)beta-D-Gal alpha-D-Man (1 --> 4) beta-D-Glc (or Xyl)- and branched linkage of alpha-D-Man(1 --> 3) alpha-D-Fuc. In addition, the fucose residues were shown to be 2-O- and 4-O-sulfated and, therefore, were either terminal or 3-linked. The antioxidative activity of AOS was measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and the beta-carotene-linoleate assay systems and was compared with those of butylated hydroxytoluene and ascorbic acid (AscA). The results showed that AOS exhibited higher antioxidative activity than AscA in the DPPH assay model and in the beta-carotene-linoleate assay system at all of the four concentration levels tested (from 50 to 200 microg/mL). These results suggested that AOS from the red seaweed G. tenax is an efficient novel antioxidant. PMID:19459750

  5. Purification, characterization and gene cloning of two alpha-L-arabinofuranosidases from streptomyces chartreusis GS901.

    PubMed

    Matsuo, N; Kaneko, S; Kuno, A; Kobayashi, H; Kusakabe, I

    2000-02-15

    alpha-L-Arabinofuranosidases I and II were purified from the culture filtrate of Streptomyces chartreusis GS901 and were found to have molecular masses of 80 and 37 kDa and pI values of 6.6 and 7.5 respectively. Both enzymes demonstrated slight reactivity towards arabinoxylan and arabinogalactan as substrates but did not hydrolyse gum arabic or arabinoxylo-oligosaccharides. alpha-L-Arabinofuranosidase I hydrolysed all of the alpha-linkage types that normally occur between two alpha-L-arabinofuranosyl residues, with the following decreasing order of reactivity being observed for the respective disaccharide linkages: alpha-(1-->2) alpha-(1-->3) alpha-(1-->5). This enzyme cleaved the (1-->3) linkages of the arabinosyl side-chains of methyl 3, 5-di-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside in preference to the (1-->5) linkages. alpha-L-Arabinofuranosidase I hydrolysed approx. 30% of the arabinan but hydrolysed hardly any linear arabinan. In contrast, alpha-L-Arabinofuranosidase II hydrolysed only (1-->5)-arabinofuranobioside among the regioisomeric methyl arabinobiosides and did not hydrolyse the arabinotrioside. Linear 1-->5-linked arabinan was a good substrate for this enzyme, but it hydrolysed hardly any of the arabinan. Synergism between the two enzymes was observed in the conversion of arabinan and debranched arabinan into arabinose. Complete amino acid sequencing of alpha-L-arabinofuranosidase I indicated that the enzyme consists of a central catalytic domain that belongs to family 51 of the glycoside hydrolases and additionally that unknown functional domains exist in the N-terminal and C-terminal regions. The amino acid sequence of alpha-L-arabinofuranosidase II indicated that this enzyme belongs to family 43 of the glycoside hydrolase family and, as this is the first report of an exo-1, 5-alpha-L-arabinofuranosidase, it represents a novel type of enzyme.

  6. The high affinity dopamine uptake inhibitor, JHW 007, blocks cocaine-induced reward, locomotor stimulation and sensitization.

    PubMed

    Velázquez-Sánchez, C; Ferragud, A; Murga, J; Cardá, M; Canales, J J

    2010-07-01

    The discovery and evaluation of high affinity dopamine transport inhibitors with low abuse liability is an important step toward the development of efficacious medications for cocaine addiction. We examined in mice the behavioural effects of (N-(n-butyl)-3alpha-[bis(4'-fluorophenyl)methoxy]-tropane) (JHW 007), a benztropine (BZT) analogue that blocks dopamine uptake, and assessed its potential to influence the actions of cocaine in clinically-relevant models of cocaine addiction. In the conditioned place preference (CPP) paradigm, JHW 007 exposure did not produce place conditioning within an ample dose range but effectively blocked the CPP induced by cocaine administration. Similarly, in the CPP apparatus JHW 007 treatment failed to stimulate locomotor activity at any dose but dose-dependently suppressed the hyperactivity evoked by cocaine treatment. In locomotor sensitization assays performed in the open field, JHW 007 did not produce sensitized locomotor behaviour when given alone, but it prevented the sensitized component of the locomotor response elicited by subchronic (8-day) cocaine exposure. In the elevated plus maze (EPM), acute treatment with JHW 007, cocaine and combinations of the BZT analogue and cocaine produced an anxiogenic-like profile. Re-test in the EPM following subchronic (8-day) exposure enhanced the anxiogenic-like effect of the same drug treatments. The present findings indicate that JHW 007 exposure counteracts some critical behavioural correlates of cocaine treatment, including conditioned reward, locomotor stimulation and sensitization, and lend support to the further development of BZT analogues as potential replacement medications in cocaine addiction.

  7. Evaluation of the blood-brain barrier transport, population pharmacokinetics, and brain distribution of benztropine analogs and cocaine using in vitro and in vivo techniques.

    PubMed

    Raje, Sangeeta; Cao, Jianjing; Newman, Amy Hauck; Gao, Huanling; Eddington, Natalie D

    2003-11-01

    The N-substituted 3alpha-[bis(4'-fluorophenyl)methoxy]tropanes (AHN 2-003, AHN 1-055, AHN 2-005, and JHW 007) bind with high affinity to the dopamine transporter and inhibit dopamine uptake more potently than cocaine, but they demonstrate behavioral profiles in animal models of psychostimulant abuse that are unlike that of cocaine. The objective of this study was to characterize the in vitro permeability, brain distribution, and pharmacokinetics of the benztropine (BZT) analogs. Transport studies of cocaine and the BZT analogs (10-4 M) were conducted across bovine brain microvessel endothelial cells. Male Sprague-Dawley rats (approximately 300 g) were administered BZT analogs (10 mg/kg) or cocaine (5 mg/kg) via the tail vein. Blood and brain samples were collected over 36 h and assayed using UV-high-performance liquid chromatography. Transport of both AHN 1-055 (2.15 x 10-4 cm/s) and JHW 007 (2.83 x 10-4 cm/s) was higher (p < 0.05) than that of cocaine (1.63 x 10-4 cm/s). The volume of distribution (12.3-30.5 l/kg) of the analogs was significantly higher than cocaine (0.9 l/kg). The BZT analogs displayed a > or =8-fold higher elimination half-life (4.12-16.49 h) compared with cocaine (0.49 h). The brain-to-plasma partition coefficients were at least two-fold higher for the BZTs versus cocaine, except for AHN 2-003. The BZT analogs are highly permeable across the blood-brain barrier and possess a pharmacokinetic profile different from that of cocaine. These characteristics, in addition to their distinctive behavioral profiles, suggest that the BZT analogs may be promising candidates for the treatment of cocaine abuse.

  8. GSK3 inactivation is involved in mitochondrial complex IV defect in transforming growth factor (TGF) {beta}1-induced senescence

    SciTech Connect

    Byun, Hae-Ok; Jung, Hyun-Jung; Seo, Yong-Hak; Lee, Young-Kyoung; Hwang, Sung-Chul; Seong Hwang, Eun; Yoon, Gyesoon

    2012-09-10

    Transforming growth factor {beta}1 (TGF {beta}1) induces Mv1Lu cell senescence by persistently producing mitochondrial reactive oxygen species (ROS) through decreased complex IV activity. Here, we investigated the molecular mechanism underlying the effect of TGF {beta}1 on mitochondrial complex IV activity. TGF {beta}1 progressively phosphorylated the negative regulatory sites of both glycogen synthase kinase 3 (GSK3) {alpha} and {beta}, corresponding well to the intracellular ROS generation profile. Pre-treatment of N-acetyl cysteine, an antioxidant, did not alter this GSK3 phosphorylation (inactivation), whereas pharmacological inhibition of GSK3 by SB415286 significantly increased mitochondrial ROS, implying that GSK3 phosphorylation is an upstream event of the ROS generation. GSK3 inhibition by SB415286 decreased complex IV activity and cellular O{sub 2} consumption rate and eventually induced senescence of Mv1Lu cell. Similar results were obtained with siRNA-mediated knockdown of GSK3. Moreover, we found that GSK3 not only exists in cytosol but also in mitochondria of Mv1Lu cell and the mitochondrial GSK3 binds complex IV subunit 6b which has no electron carrier and is topologically located in the mitochondrial intermembrane space. Involvement of subunit 6b in controlling complex IV activity and overall respiration rate was proved with siRNA-mediated knockdown of subunit 6b. Finally, TGF {beta}1 treatment decreased the binding of the subunit 6b to GSK3 and subunit 6b phosphorylation. Taken together, our results suggest that GSK3 inactivation is importantly involved in TGF {beta}1-induced complex IV defects through decreasing phosphorylation of the subunit 6b, thereby contributing to senescence-associated mitochondrial ROS generation.

  9. Detection and identification of plasma progesterone metabolites in the female Florida manatee (Trichechus manatus latirostris) using GC/MS/MS.

    PubMed

    Tripp, K M; Dubois, M; Delahaut, P; Verstegen, J P

    2009-08-01

    Florida manatees (Trichechus manatus latirostris) have relatively low peripheral concentrations of progesterone (P4). The objective of this study was to determine if these relatively low P4 concentrations are associated with a high ratio of progestin metabolites and to document metabolite concentrations from individual blood samples obtained from manatees during diestrus or pregnancy. Metabolites known to exist in elephants-terrestrial manatee relatives-were targeted. These included 5alpha-reduced progestins (5alpha-pregnane-3,20-dione [5alpha-DHP] and 3alpha-hydroxy-5alpha-pregnan-20-one [5alpha-P3-OH]) and 17alpha-hydroxyprogesterone (17alpha-OHP), which occurs in Asian elephants. An additional, inactive metabolite, 20alpha-hydroxyprogesterone (20alpha-OHP), indicative of P4 overproduction, was also targeted. Progesterone itself was the predominant progestin detected in pregnant and nonpregnant manatee plasma (n = 10) using gas chromatography-mass spectrometry with tandem quadrupole detectors (GC/MS/MS). Progesterone concentrations in pregnant females varied from early (moderate to high) through mid and late (low) pregnancy. Progesterone concentrations ranged from low to high in nonpregnant, nonlactating females. The most commonly detected metabolite was 5alpha-P3-OH (n = 7), which occurred in pregnant (lower limit of detection [LLOD] to high) and nonpregnant (trace to high) females. The 5alpha-DHP metabolite was also detected in pregnant (LLOD to moderate) and nonpregnant (low) females. The 17alpha-OHP metabolite was not detected in any tested female. The 20alpha-OHP metabolite was detected in one nonpregnant, nonlactating, captive female (LLOD). Metabolites were most prevalent during early pregnancy, concurrent with maximum P4 concentrations. Based on their concentrations in peripheral circulation, we inferred that these metabolites may have, opposite to elephants, a limited physiologic role during luteal, pregnant, and nonpregnant phases in the manatee.

  10. Crystallographic refinement and structure analysis of the complex of wheat germ agglutinin with a bivalent sialoglycopeptide from glycophorin A.

    PubMed

    Wright, C S; Jaeger, J

    1993-07-20

    Wheat germ agglutinin (WGA) elicits a number of biological effects in erythrocytes as a result of specific binding to the transmembrane protein glycophorin A. The structure of co-crystals of WGA (isolectin 1: WGA1) with a bivalent sialoglycopeptide fragment of glycophorin A (T5), determined at 2.0 A resolution, has been further refined and analyzed with respect to ligand-induced changes in the tertiary structure, mobility, solvation, saccharide conformation and protein/saccharide interactions at three independent N-acetyl-D-neuraminic (NeuNAc) binding sites. The final model, which includes the two independent WGA1 monomers (composed of domains A, B, C and D), two positions for bound T5 sialo-tetrasaccharide (NeuNAc-alpha 2,3-Gal-beta 1,3-(alpha 2,6-NeuNAc)GalNAc) and 386 water molecules, refined to a crystallographic R-factor of 17.1% (Fo > 1.0 sigma) and an average temperature factor of 31.99 A2. Comparisons between the tertiary structures of the liganded and unliganded WGA1 dimers indicate that the largest deviations from 2-fold symmetry are localized in domains engaged in sugar binding (B1 and C2) and at the C-terminal domain of monomer II (D2), forming a strong lattice contact. Interactions of the tetrasaccharide with amino acid ligands in the three binding sites and with water were carefully analyzed and compared. Bound conformations of terminal NeuNAc match to within a root-mean-square delta r of 0.3 A. The specificity-determining N-acetyl group superimposes best in comparison with other substituents of the sugar ring. Of the five domain binding sites that are not occupied in this dimeric crosslinked complex, only one is accessible to the NeuNAc monosaccharide as determined from a difference Fourier map at 3.0 A resolution.

  11. Chemical analysis and biological activity of the essential oils of two endemic Soqotri Commiphora species.

    PubMed

    Mothana, Ramzi A; Al-Rehaily, Adnan J; Schultze, Wulf

    2010-02-01

    The barks of two endemic Commiphora species namely, Commiphora ornifolia (Balf.f.) Gillett and Commiphora parvifolia Engl., were collected from Soqotra Island in Yemen and their essential oils were obtained by hydrodistillation. The chemical composition of both oils was investigated by GC and GC-MS. Moreover, the essential oils were evaluated for their antimicrobial activity against two Gram-positive bacteria, two Gram-negative bacteria and one yeast species by using a broth micro-dilution assay for minimum inhibitory concentrations (MIC) and for their antioxidant activity by measuring the DPPH radical scavenging activity. A total of 45 constituents of C. ornifolia (85.6%) and 44 constituents of C. parvifolia (87.1%) were identified. The oil of C. ornifolia was characterized by a high content of oxygenated monoterpenes (56.3%), of which camphor (27.3%), alpha-fenchol (15.5%), fenchone (4.4%) and borneol (2.9%) were identified as the main components. High contents of oxygenated sesquiterpenes (36.1%) and aliphatic acids (22.8%) were found in C. parvifolia oil, in which caryophyllene oxide (14.2%), beta-eudesmol (7.7%), bulnesol (5.7%), T-cadinol (3.7%) and hexadecanoic acid (18.4%) predominated. The results of the antimicrobial assay showed that both oils exhibited moderate to high antibacterial activity especially against Gram-positive bacteria. C. ornifolia oil was the most active. In addition, the DPPH-radical scavenging assay exhibited only weak antioxidant activities for both oils at the high concentration tested. PMID:20335939

  12. Evidence for a role of glycogen synthase kinase-3 beta in rodent spermatogenesis.

    PubMed

    Guo, Taylor B; Chan, Kam C; Hakovirta, Harri; Xiao, Yang; Toppari, Jorma; Mitchell, Aaron P; Salameh, Wael A

    2003-01-01

    Glycogen synthase kinase-3 beta (GSK-3 beta) regulates cell metabolism, cell cycle, and cell fate through the phosphorylation of a diverse array of substrates. Herein, we provide evidence that supports a role for GSK-3 in mammalian meiosis and spermatogenesis. Immunostaining of testis sections showed that while GSK-3 alpha was ubiquitous in the seminiferous tubules, GSK-3 beta was expressed in premeiotic type B spermatogonia, in both meiotic preleptotene and leptotene spermatocytes, as well as in Sertoli cells in both the mouse and rat. Thus, GSK-3 beta is expressed in germ cells entering meiosis. In addition, intense immunoreactivity was detected in rat step 6 though 11 spermatids. In situ hybridization (ISH) in rat testis confirmed the immunostaining pattern in leptotene and spermatids and showed a GSK-3 beta messenger RNA (mRNA) signal in some pachytene spermatocytes. The restricted pattern of expression suggests cell-specific regulation of Gsk-3 beta mRNA. To determine whether GSK-3 is required for meiosis entry, rat stage VIIa seminiferous tubule segments were cultured with selective small-molecule GSK-3 inhibitors. These compounds markedly and dose-dependently suppressed meiotic synthesis (S)-phase DNA. Since a yeast GSK-3 homolog, Rim11p (regulator of inducer of meiosis), is pivotal to meiosis entry, we tested whether GSK-3 beta complements Rim11p function in meiosis. Rim11p phosphorylates transcription factors Ume6p (unscheduled meiotic gene expression) and Ime1p (inducer of meiosis) to induce meiosis entry. Overexpression of murine GSK-3 beta in a rim11 mutant yeast failed to rescue the sporulation defect. Our finding that GSK-3 beta interacted only with Ume6p but not with IME1 in a yeast 2-hybrid assay suggests that noncomplementation reflects partial divergence in substrate specificity. This work provides the basis for future studies of GSK-3 beta signaling in mammalian meiosis and spermatogenesis.

  13. Non-targeted Metabolite Profiling and Scavenging Activity Unveil the Nutraceutical Potential of Psyllium (Plantago ovata Forsk)

    PubMed Central

    Patel, Manish K.; Mishra, Avinash; Jha, Bhavanath

    2016-01-01

    Non-targeted metabolomics implies that psyllium (Plantago ovata) is a rich source of natural antioxidants, PUFAs (ω-3 and ω-6 fatty acids) and essential and sulfur-rich amino acids, as recommended by the FAO for human health. Psyllium contains phenolics and flavonoids that possess reducing capacity and reactive oxygen species (ROS) scavenging activities. In leaves, seeds, and husks, about 76, 78, 58% polyunsaturated, 21, 15, 20% saturated, and 3, 7, 22% monounsaturated fatty acids were found, respectively. A range of FAs (C12 to C24) was detected in psyllium and among different plant parts, a high content of the nutritive indicators ω-3 alpha-linolenic acid (57%) and ω-6 linoleic acid (18%) was detected in leaves. Similarly, total content of phenolics and the essential amino acid valine were also detected utmost in leaves followed by sulfur-rich amino acids and flavonoids. In total, 36 different metabolites were identified in psyllium, out of which 26 (13 each) metabolites were detected in leaves and seeds, whereas the remaining 10 were found in the husk. Most of the metabolites are natural antioxidants, phenolics, flavonoids, or alkaloids and can be used as nutrient supplements. Moreover, these metabolites have been reported to have several pharmaceutical applications, including anti-cancer activity. Natural plant ROS scavengers, saponins, were also detected. Based on metabolomic data, the probable presence of a flavonoid biosynthesis pathway was inferred, which provides useful insight for metabolic engineering in the future. Non-targeted metabolomics, antioxidants and scavenging activities reveal the nutraceutical potential of the plant and also suggest that psyllium leaves can be used as a green salad as a dietary supplement to daily food. PMID:27092153

  14. Genetic Epidemiology of Pelvic Organ Prolapse: A Systematic Review

    PubMed Central

    WARD, Renée M.; VELEZ EDWARDS, Digna R.; EDWARDS, Todd; GIRI, Ayush; JEROME, Rebecca N.; WU, Jennifer M.

    2014-01-01

    Given current evidence supporting a genetic predisposition for pelvic organ prolapse (POP), we conducted a systematic review of published literature on the genetic epidemiology of POP. Inclusion criteria were linkage studies, candidate gene association and genome-wide association studies (GWAS) in adult women published in English and indexed in PubMed through December 2012, with no limit on date of publication. Methodology adhered to the PRISMA guidelines. Data were systematically extracted by two reviewers and graded by the Venice criteria for studies of genetic associations. A meta-analysis was performed on all single nucleotide polymorphisms (SNPs) evaluated by two or more studies with similar methodology. The meta-analysis suggests that collagen type 3 alpha 1 (COL3A1) rs1800255 genotype AA is associated with POP, OR 4.79 (95% CI 1.91 to 11.98, p= 0.001) compared to the reference genotype GG in populations of Asian and Dutch women. There was little evidence of heterogeneity for rs1800255 (p-value for heterogeneity= 0.94; proportion of variance due to heterogeneity, I2= 0.00%). There was insufficient evidence to determine whether other SNPs evaluated by two or more papers were associated with POP. An association with POP was seen in individual studies for estrogen receptor alpha (ER-α) rs2228480 GA, COL3A1 exon 31, chromosome 9q21 (HLOD score 3.41) as well as six SNPs identified by a GWAS. Overall, individual studies were of small sample size and often of poor quality. Future studies would benefit from more rigorous study design as outlined in the Venice recommendations. PMID:24721264

  15. A profile of sickle cell disease in Nigeria.

    PubMed

    Akinyanju, O O

    1989-01-01

    Nigeria has a population of 112 million with an annual growth rate of 3.2%. About 25% of adults throughout the country have the sickle cell trait, AS, while the Hb C trait is largely confined to the Yoruba people of southwestern Nigeria in whom it occurs in about 6%. Other variant hemoglobins including beta thalassemia are rare, but alpha thalassemia occurs in 39% (32% with 3 alpha-globin genes; 7% with 2 alpha-globin genes). Of a total of 5.4 million expected live births in 1988, about 90,000 will have SCD and 1.1 million the trait, AS. The clinical phenotype of sickle cell anemia is severe with manifestations occurring very early in childhood and mean Hb level 7.6 g/dl with HbF 5.9%. A very high infant mortality due to infections occurs especially in rural areas. Gallstones, leg ulcerations, and stroke appear less common than in American sicklers, and aplastic crises have not been described. Poor availability of resources to the public health and welfare sectors and economic inflation are severely curtailing access to appropriate medical and social services. This situation is frustrating to the families of a growing number of surviving patients in urban or middle to upper income groups. Efforts to create more awareness of SCD are paradoxically increasing frustration and stigmatization in the absence of a commensurate improvement of services. Any measures aimed at enhancing the sensitization of health professionals, policy makers, and resource allocators to the pertinent issues in the control of SCD would seem to be at this stage an important step in the right direction.

  16. Training-induced changes in the expression of GABAA-associated genes in the amygdala after the acquisition and extinction of Pavlovian fear.

    PubMed

    Heldt, Scott A; Ressler, Kerry J

    2007-12-01

    Previous work suggests the gamma-aminobutyric acid (GABA)ergic system may be dynamically regulated during emotional learning. In the current study we examined training-induced changes in the expression of GABA(A)-related genes and the binding of GABA receptor radioligands in the amygdala after the acquisition and extinction of Pavlovian fear. Using in situ hybridization, we examined the expression pattern changes of mRNAs for GABAergic markers in the lateral, basolateral and central subdivisions of the amygdala in C57Bl/6J mice. These markers included GABA-synthesizing enzymes (GAD67 and GAD65), major GABA(A) receptor subunits (alpha1, alpha2, alpha3, alpha5, beta2 and gamma2) and the expression of mRNAs that are involved in a variety of GABA-related intracellular processes, including GABA transporter-1 (GAT1), GABA(A) receptor-associated protein and the GABA(A) clustering protein, gephyrin. With fear conditioning, we found decreased mRNA levels of alpha1, alpha5 and GAD67, as well as deceased benzodiazepine binding in the amygdala. Fear extinction induced an increase in mRNA levels of alpha2, beta2, GAD67 and gephyrin, as well as a decrease in GAT1. Together, these findings indicate that the acquisition of fear induced a downregulation of mRNA markers related to a decrease in amygdala GABAergic function, whereas the acquisition of fear extinction produced an upregulation of GABAergic markers related to enhanced GABAergic transmission.

  17. The role of Schizosaccharomyces pombe DNA repair enzymes Apn1p and Uve1p in the base excision repair of apurinic/apyrimidinic sites

    SciTech Connect

    Tanihigashi, Haruna; Yamada, Ayako; Igawa, Emi; Ikeda, Shogo . E-mail: ikeda@dbc.ous.ac.jp

    2006-09-08

    In Schizosaccharomyces pombe the repair of apurinic/apyrimidinic (AP) sites is mainly initiated by AP lyase activity of DNA glycosylase Nth1p. In contrast, the major AP endonuclease Apn2p functions by removing 3'-{alpha},{beta}-unsaturated aldehyde ends induced by Nth1p, rather than by incising the AP sites. S. pombe possesses other minor AP endonuclease activities derived from Apn1p and Uve1p. In this study, we investigated the function of these two enzymes in base excision repair (BER) for methyl methanesulfonate (MMS) damage using the nth1 and apn2 mutants. Deletion of apn1 or uve1 from nth1{delta} cells did not affect sensitivity to MMS. Exogenous expression of Apn1p failed to suppress the MMS sensitivity of nth1{delta} cells. Although Apn1p and Uve1p incised the oligonucleotide containing an AP site analogue, these enzymes could not initiate repair of the AP sites in vivo. Despite this, expression of Apn1p partially restored the MMS sensitivity of apn2{delta} cells, indicating that the enzyme functions as a 3'-phosphodiesterase to remove 3'-blocked ends. Localization of Apn1p in the nucleus and cytoplasm hints at an additional function of the enzyme other than nuclear DNA repair. Heterologous expression of Saccharomyces cerevisiae homologue of Apn1p completely restored the MMS resistance of the nth1{delta} and apn2{delta} cells. This result confirms a difference in the major pathway for processing the AP site between S. pombe and S. cerevisiae cells.

  18. Paleomagnetic evidence for Late Miocene counterclockwise rotation of north coast carbonate sequence, Puerto Rico

    SciTech Connect

    Reid, J.A.; Plumley, P.W. ); Schellekens, J.H. )

    1991-03-01

    A paleomagnetic study of the essentially undeformed middle Tertiary carbonate sequence along the north coast of Puerto Rico reveals statistically significant pre-Pliocene discordance of characteristic component directions against those expected from cratonic North America for much of the section. Despite generally weak to moderately weak magnetic intensities, confirmation of the magnetization as primary in origin comes from the presence of two distinct components of magnetization, intrasite bipolarity, and/or the reproducibility of measurements. The mean geographic direction for the upper Oligocene to middle Miocene strata is 335.2{degree}/32.9{degree} and the corrected mean paleomagnetic pole is 207.6{degree}/66.5{degree}, (N = 3, {alpha}95 = 4.3{degree}). This suggests a counter-clockwise (CCW) block rotation of Puerto Rico and its microplate of 24.5{degrees} ({plus minus} 5.8{degrees}) during the late Miocene. Using a width of 250 km for the Northern Caribbean Plate Boundary Zone (NCPBZ) between the North American Plate and Caribbean Plate, the mean left lateral displacement implied is 1.8 to 2.4 cm/yr, which agrees fairly well with published relative motion rates for the two plates. Average rotation rate for 50 Ma to 20 Ma was 0.7{degree}/my but perhaps as great as 4{degree}/my in the Miocene. Resolution of mean paleolatitude indicates northward motion of a degree or less during the period of rotation. Causes of this short-lived rotation may include (1) tectonic escape from the inhibiting presence of the Bahama Banks and Beata Ridge during eastward motion of Puerto Rico along the sinistral transpressive Puerto Rico Trench and Muertos Trough fault systems or (2) changes in relative plate motions of the Caribbean and North American Plate during the late Miocene.

  19. The MUSES-C, mission description and its status

    NASA Astrophysics Data System (ADS)

    Kawaguchi, Jun'ichiro; Uesugi, Kuninori T.; Fujiwara, Akira; Saitoh, Hirobumi

    1999-11-01

    The MUSES-C mission is the world's first sample and return attempt to/from the near Earth asteroid Nereus (4660). It is the ISAS (The Institute of Space and Astronautical Science, Ministry of Education) which manages the mission that started in 1996 scheduling to be launched in January of 2002. The mission is built as a kind of technology demonstration, however, it is aiming at not only the in-situ observation but also the touch-down sampling of the surface fragments. The sample collected is returned to the Earth in January of 2006. The mission is a four year journey. The major purpose of it originally consists of the following four subjects: 1) The Ion thruster propulsion performed in interplanetary field as a primary means, 2) Autonomous guidance, navigation and control during the rendezvous and touch down phase, 3) The sample collection mechanism and 4) The hyperbolic reentry capsule with the asteroid sample contained inside it. The current primary objective is extended to carry the joint small rover with NASA/JPL, which is supposed to be placed on the surface and to look into the crater created by the sampling shot of the projectile. The rover is designated as the Small Science Vehicle (SSV) that weighs about 1 kg carrying three kinds of in-situ instruments: 1) A Visible Camera, 2) Near Infra-Red Spectrometer and potentially 3) Alpha-Proton X-ray Spectrometer (APXS) similar to that delivered on the Mars Path Finder. During the fiscal 1998, the spacecraft undergoes the PM tests and the FM fabrication starts from next year, 1999. The paper presents the latest mission description around the asteroid and shows the current status of the spacecraft as well as the instruments and so on. The mission will be the good example of an international collaboration in the small interplanetary exploration.

  20. Oxidative stress enhances IL-8 and inhibits CCL20 production from intestinal epithelial cells in response to bacterial flagellin.

    PubMed

    Ivison, Sabine M; Wang, Ce; Himmel, Megan E; Sheridan, Jared; Delano, Jonathan; Mayer, Matt L; Yao, Yu; Kifayet, Arnawaz; Steiner, Theodore S

    2010-09-01

    Intestinal epithelial cells act as innate immune sentinels, as the first cells that encounter diarrheal pathogens. They use pattern recognition molecules such as the Toll-like receptors (TLRs) to identify molecular signals found on microbes but not host cells or food components. TLRs cannot generally distinguish the molecular signals on pathogenic bacteria from those found in commensals, yet under healthy conditions epithelial immune responses are kept in check. We hypothesized that, in the setting of tissue damage or stress, intestinal epithelial cells would upregulate their responses to TLR ligands to reflect the greater need for immediate protection against pathogens. We treated Caco-2 cells with the TLR5 agonist flagellin in the presence or absence of H(2)O(2) and measured chemokine production and intracellular signaling pathways. H(2)O(2) increased flagellin-induced IL-8 (CXCL8) production in a dose-dependent manner. This was associated with synergistic phosphorylation of p38 MAP kinase and with prolonged I-kappaB degradation and NF-kappaB activation. The H(2)O(2)-mediated potentiation of IL-8 production required the activity of p38, tyrosine kinases, phospholipase Cgamma, and intracellular calcium, but not protein kinase C or protein kinase D. H(2)O(2) prolonged and augmented NF-kappaB activation by flagellin. In contrast to IL-8, CCL20 (MIP3alpha) production by flagellin was reduced by H(2)O(2), and this effect was not calcium dependent. Oxidative stress biases intestinal epithelial responses to flagellin, leading to increased production of IL-8 and decreased production of CCL20. This suggests that epithelial cells are capable of sensing the extracellular environment and adjusting their antimicrobial responses accordingly.

  1. The neuroactive steroid allopregnanolone suppresses hypothalamic gonadotropin-releasing hormone release through a mechanism mediated by the gamma-aminobutyric acidA receptor.

    PubMed

    Calogero, A E; Palumbo, M A; Bosboom, A M; Burrello, N; Ferrara, E; Palumbo, G; Petraglia, F; D'Agata, R

    1998-07-01

    The central nervous system (CNS) is able to synthesize and/or metabolize steroid hormones. These neuroactive steroids are capable of modulating several brain functions and, among these, they seem to regulate the hypothalamic-pituitary-gonadal (HPG) axis. Indeed, recent observations have shown that 5 alpha-pregnane-3 alpha-ol-20-one (allopregnanolone), one of the most abundant naturally occurring neuroactive steroids, suppresses ovulation and sexual behaviour when administered within the CNS. The present study was undertaken to evaluate the effects of allopregnanolone and its inactive stereoisomer, 5 alpha-pregnane-3 beta-ol-20-one, upon the release of gonadotropin-releasing hormone (GnRH) from individually-incubated hemihypothalami. Allopregnanolone suppressed GnRH release in a concentration-dependent manner with maximal activity in the nanomolar range, a range at which this neurosteroid is capable of playing a biological action. The specificity of allopregnanolone suppression of GnRH release was provided by the lack of effect of its known inactive stereoisomer. To evaluate the involvement of gamma-aminobutyric acidA (GABAA) receptor, we examined the effects of two neurosteroids with GABA-antagonistic properties, pregnanolone sulfate (PREG-S) and dehydroepiandrosterone sulfate (DHEAS), and of bicuculline, a selective antagonist of the GABA binding site on the GABAA receptor, on allopregnanolone (10 nM)-suppressed GnRH release. Both PREG-S and bicuculline overcame the inhibitory effects of allopregnanolone on GnRH release, whereas DHEAS did not. To substantiate the involvement of the GABAA receptor further, we tested the effects of muscimol, a selective agonist for this receptor, which suppressed GnRH release. In conclusion, allopregnanolone suppressed hypothalamic GnRH release in vitro and this effect appeared to be mediated by an interaction with the GABAA receptor. We speculate that the inhibitory effect of allopregnanolone on the HPG axis may also be caused by

  2. Detection and identification of plasma progesterone metabolites in the female Florida manatee (Trichechus manatus latirostris) using GC/MS/MS.

    PubMed

    Tripp, K M; Dubois, M; Delahaut, P; Verstegen, J P

    2009-08-01

    Florida manatees (Trichechus manatus latirostris) have relatively low peripheral concentrations of progesterone (P4). The objective of this study was to determine if these relatively low P4 concentrations are associated with a high ratio of progestin metabolites and to document metabolite concentrations from individual blood samples obtained from manatees during diestrus or pregnancy. Metabolites known to exist in elephants-terrestrial manatee relatives-were targeted. These included 5alpha-reduced progestins (5alpha-pregnane-3,20-dione [5alpha-DHP] and 3alpha-hydroxy-5alpha-pregnan-20-one [5alpha-P3-OH]) and 17alpha-hydroxyprogesterone (17alpha-OHP), which occurs in Asian elephants. An additional, inactive metabolite, 20alpha-hydroxyprogesterone (20alpha-OHP), indicative of P4 overproduction, was also targeted. Progesterone itself was the predominant progestin detected in pregnant and nonpregnant manatee plasma (n = 10) using gas chromatography-mass spectrometry with tandem quadrupole detectors (GC/MS/MS). Progesterone concentrations in pregnant females varied from early (moderate to high) through mid and late (low) pregnancy. Progesterone concentrations ranged from low to high in nonpregnant, nonlactating females. The most commonly detected metabolite was 5alpha-P3-OH (n = 7), which occurred in pregnant (lower limit of detection [LLOD] to high) and nonpregnant (trace to high) females. The 5alpha-DHP metabolite was also detected in pregnant (LLOD to moderate) and nonpregnant (low) females. The 17alpha-OHP metabolite was not detected in any tested female. The 20alpha-OHP metabolite was detected in one nonpregnant, nonlactating, captive female (LLOD). Metabolites were most prevalent during early pregnancy, concurrent with maximum P4 concentrations. Based on their concentrations in peripheral circulation, we inferred that these metabolites may have, opposite to elephants, a limited physiologic role during luteal, pregnant, and nonpregnant phases in the manatee. PMID

  3. Spectroscopic Study of HD 179821 (IRAS 19114+0002): Proto-Planetary Nebula or Supergiant?

    NASA Technical Reports Server (NTRS)

    Reddy, B. E.; Hrivnak, Bruce J.

    1999-01-01

    A detailed chemical composition analysis of the bright post-AGB candidate HD 179821 (IRAS 19114 + 0002) is presented. The LTE analysis, based on high-resolution (R approximately equal 50,000) and high-quality (S/N approximately equal 300) spectra, yields atmospheric parameters T(sub eff) = 6750 K, log g = 0.5, and xi(sub t) = 5.25 km/s. The elemental abundance results of HD 179821 are found to be [Fe/H] = -0.1, [C/Fe] = +0.2, [N/Fe] = +1.3, [O/Fe] = +0.2, [alpha-process/Fe] = +0.5, and [s-process/Fe] = +0.4. These values clearly differ from the elemental abundances of Population I F supergiants. The C, N, and O abundances and the total CNO abundance value relative to Fe, [C+N+O/Fe] = +0.5, indicate that the photosphere of HD 179821 is contaminated with both the H- and He-burning products of the AGB phase. The evidence for He burning through the 3.alpha process and deep AGB mixing also comes from the observed overabundances of s-process elements. Remarkably, the abundance of the element Na is found to be very large, [Na/Fe] = +0.9. The ratio O/C = 2.6 indicates that the atmosphere is oxygen rich. The results of this abundance study support the argument that HD 179821 is a proto-planetary nebula,. probably with an intermediate-mass progenitor. However, the strength of the O I triplet lines at 7774 A and the distance derived from the interstellar Na I D1 and D2 components imply that the star is a luminous object (M(sub bol) approximately -8.9 +/- 1) and thus a massive supergiant. Thus, while this study contributes important new observational results for this star, an unambiguous determination of its evolutionary status has yet to be achieved.

  4. Differential expression of guanosine triphosphate binding proteins in men at high and low risk for the future development of alcoholism.

    PubMed Central

    Wand, G S; Waltman, C; Martin, C S; McCaul, M E; Levine, M A; Wolfgang, D

    1994-01-01

    We evaluated G-proteins that are components of adenylyl cyclase (AC) signal transduction in erythrocyte and lymphocyte membranes from 26 family history positive (FHP) non-alcoholic and 26 family history negative (FHN) nonalcoholic subjects. Subjects were classified as FHP if their father met criteria for alcohol dependence; as FHN, if there was no history of alcoholism in any first or second degree relatives. Immunoblot analysis indicated that levels of erythrocyte membrane Gs alpha from FHP subjects were greater than levels in FHN subjects (171 +/- 11 vs 100 +/- 6, P < 0.001). To confirm the results of the immunoblot analysis, Gs alpha was quantitated by cholera toxin-dependent [32P]ADP-ribosylation. Levels of erythrocyte [32P]ADP-ribose-Gs alpha from FHP subjects were greater than levels in FHN subjects (236 +/- 28 vs 100 +/- 14, P < 0.001). Gs alpha levels did not correlate with age or alcohol consumption. By contrast to differences in Gs alpha, immunoblot analysis showed similar levels of Gi(2)alpha and Gi(3)alpha in erythrocyte membranes of FHP and FHN subjects. Pertussis toxin-catalyzed [32P]ADP-ribosylation of Gi-like G-proteins confirmed the immunoblot observations. Lastly, compared to FHN subjects, FHP subjects had enhanced Gs alpha expression in lymphocyte membranes as well (138 +/- 11 vs 100 +/- 5.5; P < 0.02). In summary, compared to FHN nonalcoholic men, FHP nonalcoholic men had greater levels of the stimulatory G-protein, Gs alpha, in erythrocyte and lymphocyte membranes. Enhanced expression of Gs alpha may be a marker of increased risk for the future development of alcoholism. Images PMID:8083341

  5. Forms of n-3 (ALA, C18:3n-3 or DHA, C22:6n-3) Fatty Acids Affect Carcass Yield, Blood Lipids, Muscle n-3 Fatty Acids and Liver Gene Expression in Lambs.

    PubMed

    Ponnampalam, Eric N; Lewandowski, Paul A; Fahri, Fahri T; Burnett, Viv F; Dunshea, Frank R; Plozza, Tim; Jacobs, Joe L

    2015-11-01

    The effects of supplementing diets with n-3 alpha-linolenic acid (ALA) and docosahexaenoic acid (DHA) on plasma metabolites, carcass yield, muscle n-3 fatty acids and liver messenger RNA (mRNA) in lambs were investigated. Lambs (n = 120) were stratified to 12 groups based on body weight (35 ± 3.1 kg), and within groups randomly allocated to four dietary treatments: basal diet (BAS), BAS with 10.7 % flaxseed supplement (Flax), BAS with 1.8 % algae supplement (DHA), BAS with Flax and DHA (FlaxDHA). Lambs were fed for 56 days. Blood samples were collected on day 0 and day 56, and plasma analysed for insulin and lipids. Lambs were slaughtered, and carcass traits measured. At 30 min and 24 h, liver and muscle samples, respectively, were collected for determination of mRNA (FADS1, FADS2, CPT1A, ACOX1) and fatty acid composition. Lambs fed Flax had higher plasma triacylglycerol, body weight, body fat and carcass yield compared with the BAS group (P < 0.001). DHA supplementation increased carcass yield and muscle DHA while lowering plasma insulin compared with the BAS diet (P < 0.01). Flax treatment increased (P < 0.001) muscle ALA concentration, while DHA treatment increased (P < 0.001) muscle DHA concentration. Liver mRNA FADS2 was higher and CPT1A lower in the DHA group (P < 0.05). The FlaxDHA diet had additive effects, including higher FADS1 and ACOX1 mRNA than for the Flax or DHA diet. In summary, supplementation with ALA or DHA modulated plasma metabolites, muscle DHA, body fat and liver gene expression differently.

  6. Early diagenetic transformation of higher-plant triterpenoids in deep-sea sediments from Baffin Bay

    SciTech Connect

    Ten Haven, H.L. Inst. Francais du Petrole, Rueil-Malmaison ); Peakman, T.M. ); Rullkoetter, J. )

    1992-05-01

    A sediment sequence of early Miocene to Holocene from Ocean Drilling Program Site 645 in central Baffin Bay was found to contain abundant triterpenoids of higher-plant origin including mono-desmethyl derivatives and other oxidative degradation products. 24-nor-triterpenoids of the oleanene, ursene, and lupane series were found in the nonaromatic hydrocarbon and alcohol fractions of the sediment extracts. Other diagenetic transformation products included 24,28-dinor-olean-17-ene and 24-nor-urs-12-en-11-one as well as their tentatively identified des-A counterparts. The identification of these novel degradation products was confirmed by synthesis of a suite of reference compounds. The simple reaction sequence applied to obtain 24-nor-urs-12-ene-from 3{alpha}-acetoxyurs-12-en-24-oic acid is likely to happen in a similar manner during early diagenesis of organic matter in nature. These results have implications for the understanding of the diagenetic reaction sequence leading to the formation of other demethylated triterpenoids of terrigenous origin (e.g., 24,28-dinor-lupanes). 28,30-dinot-17{alpha}-hopane is sometimes the dominant hydrocarbon in the most deeply buried samples. Its co-occurrence with higher plant triterpenoids and the low abundance of other regular hopanoids is not considered an indication, however, that 28,20-dinor-17{alpha}-hopane is derived from a higher plant precursor molecule. It is suggested that two independent sedimentary processes are responsible for the presence of the two types of demethylated triterpenoids observed in Baffin Bay sediments.

  7. Crystal Structure of TDP-Fucosamine Acetyl Transferase (WECD) from Escherichia Coli, an Enzyme Required for Enterobacterial Common Antigen Synthesis

    SciTech Connect

    Hung,M.; Rangarajan, E.; Munger, C.; Nadeau, G.; Sulea, T.; Matte, A.

    2006-01-01

    Enterobacterial common antigen (ECA) is a polysaccharide found on the outer membrane of virtually all gram-negative enteric bacteria and consists of three sugars, N-acetyl-D-glucosamine, N-acetyl-D-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-D-galactose, organized into trisaccharide repeating units having the sequence {yields}(3)-{alpha}-D-Fuc4NAc-(1{yields}4)-{beta}-D-ManNAcA-(1{yields}4)-{alpha}-D-GlcNAc-(1{yields}). While the precise function of ECA is unknown, it has been linked to the resistance of Shiga-toxin-producing Escherichia coli (STEC) O157:H7 to organic acids and the resistance of Salmonella enterica to bile salts. The final step in the synthesis of 4-acetamido-4,6-dideoxy-D-galactose, the acetyl-coenzyme A (CoA)-dependent acetylation of the 4-amino group, is carried out by TDP-fucosamine acetyltransferase (WecD). We have determined the crystal structure of WecD in apo form at a 1.95-Angstroms resolution and bound to acetyl-CoA at a 1.66-Angstroms resolution. WecD is a dimeric enzyme, with each monomer adopting the GNAT N-acetyltransferase fold, common to a number of enzymes involved in acetylation of histones, aminoglycoside antibiotics, serotonin, and sugars. The crystal structure of WecD, however, represents the first structure of a GNAT family member that acts on nucleotide sugars. Based on this cocrystal structure, we have used flexible docking to generate a WecD-bound model of the acetyl-CoA-TDP-fucosamine tetrahedral intermediate, representing the structure during acetyl transfer. Our structural data show that WecD does not possess a residue that directly functions as a catalytic base, although Tyr208 is well positioned to function as a general acid by protonating the thiolate anion of coenzyme A.

  8. Towards improved fat intake and nutrition for Malaysians.

    PubMed

    Ng, T K

    1995-03-01

    An examination of the fat composition of the diet of a Malaysian urban hostel population obtained by chemical analysis of representative meals prepared by a 7-day rotation menu, revealed both nutritional attributes and limitations when compared against the dietary messages contained in the American Heart Association (AHA) and World Health Organisation (WHO) models. The Malaysian diet supplies 26% kcal i.e. 66 g total fat (51 g vegetable fats, 15 g animal fats) and contains <300 mg cholesterol, which are below the upper limits for these dietary constituents in the AHA and WHO models and conflicts with the perception that Malaysians in general, may be consuming too much fat and cholesterol. The supply of essential fatty acids (EFA), however, appears sub-optimal at 3.2% kcal mainly due to the comparatively low content of both the omega-6 (linoleic acid) and omega-3 [alpha-linolenic, eicosapentaenoate (EPA) and docosahexaenoate (DHA)] fatty acids in the Malaysian diet. The estimated omega-6/omega-3 fatty acid ratio of 10 further reflects an imbalance of these two families of polyunsaturated fatty acids (PUFA), which can be corrected to a ratio of 5 to 7 by moderate increases in the consumption of fish, soyabean-based foods, and pulses and nuts. Considering the current status of knowledge on the health effects of the different families of fatty acids, the ratio of 2:3:1 for the saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and PUFA in the diet is judged to improve fat intake and nutrition in Malaysians. Such a dietary fatty acids ratio can be satisfied by the use of a cooking oil containing 28% SFA, 53% MUFA, and 19% PUFA, which may obtained by the judicious blending of palm olein with MUFA-rich and PUFA-rich vegetable oils. Alternatively, moderate increases in the consumption of marine fish, pulses, nuts, soybean-based foods and their products would also serve the same end.

  9. Towards a synthetic glycoconjugate vaccine against Neisseria meningitidis A.

    PubMed

    Berkin, Ali; Coxon, Bruce; Pozsgay, Vince

    2002-10-01

    Albumin conjugates of synthetic fragments of the capsular polysaccharide of the Gram-negative bacterium Neisseria meningitidis serogroup A were prepared. The fragments include monosaccharides 1 [alpha-D-ManpNAc-(1-->O)-(CH(2))(2)NH(2)] and 2 [6-O-P(O)(O(-))(2)-alpha-D-ManpNAc-(1-->O)-(CH(2))(2)NH(2)], disaccharide 3 [alpha-D-ManpNAc-[1-->O-P(O)(O(-))-->6]-alpha-D-ManpNAc-(1-->O)-(CH(2))(2)NH(2)], and trisaccharide 4 [alpha-D-ManpNAc-[1-->O-P(O)(O(-))-->6]-alpha-D-ManpNAc-[1-->O-P(O)(O(-))-->6]-alpha-D-ManpNAc-(1-->O)-(CH(2))(2)NH(2)]. Two monosaccharide blocks were employed as key intermediates. The reducing-end mannose unit featured the NHAc group at C-2, and contained the aminoethyl spacer as the aglycon for the final bioconjugation. The interresidual phosphodiester linkages were fashioned from an anomerically positioned H-phosphonate group in a 2-azido-mannose building block. The spacer-linked saccharides 1-4 were N-acylated with hepta-4,6-dienoic acid and the resulting conjugated diene-equipped saccharides were subjected to Diels-Alder-type addition with maleimidobutyryl-group functionalized human serum albumin to form covalent conjugates containing up to 26 saccharide haptens per albumin molecule. Complete (1)H, (13)C, and (31)P NMR assignments for 1-4 are given. Antigenicity of the neoglycoconjugates containing 1-4 was demonstrated by a double immunodiffusion assay which indicated that a fragment as small as a monosaccharide is recognized by a polyclonal meningococcus group A antiserum and that the O-acetyl group(s) present in the natural capsular material is not essential for antigenicity. PMID:12355530

  10. KDN-containing glycoprotein from loach skin mucus.

    PubMed

    Nakagawa, H; Hama, Y; Sumi, T; Li, S C; Li, Y T

    2001-01-01

    It has been widely recognized that the mucus coat of fish plays a variety of important physical, chemical, and physiological functions. One of the major constituents of the mucus coat is mucus glycoprotein. We found that sialic acids in the skin mucus of the loach, Misgurnus anguillicaudatus, consisted predominantly of KDN. Subsequently, we isolated KDN-containing glycoprotein from loach skin mucus and characterized its chemical nature and structure. Loach mucus glycoprotein was purified from the Tris-HCl buffer extract of loach skin mucus by DEAE-cellulose chromatography, Nuclease P1 treatment, and Sepharose CL-6B gel filtration. The purified mucus glycoprotein was found to contain 38.5 KDN, 0.5% NeuAc, 25.0% GalNAc, 3.5% Gal, 0.5% GlcNAc and 28% amino acids. Exhaustive Actinase digestion of the glycoprotein yielded a glycopeptide with a higher sugar content and higher Thr and Ser contents. The molecular size of this glycopeptide was approximately 1/12 of the intact glycoprotein. These results suggest that approximately 11 highly glycosylated polypeptide units are linked in tandem through nonglycosylated peptides to form the glycoporotein molecule. The oligosaccharide alditols liberated from the loach mucus glycoprotein by alkaline borohydride treatment were separated by Sephadex G-25 gel filtration and HPLC. The purified sugar chains were analyzed b --> 6GalNAc-ol, KDNalpha2 --> 3(GalNAcbeta1 --> 14)GalNAc-ol, KDNalpha2 --> 6(GalNAcalpha1 --> 3)GalNAc-ol, KDNalpha2 --> 6(Gal3alpha1--> 3)GalNAc-ol, and NeuAcalpha2 --> 6Gal NAc-ol. It is estimated that one loach mucus glycoprotein molecule contains more than 500 KDN-containing sugar chains that are linked to Thr and Ser residues of the protein core through GalNAc. PMID:14533798

  11. Use of secondary structural information and C alpha-C alpha distance restraints to model protein structures with MODELLER.

    PubMed

    Reddy, Boojala V B; Kaznessis, Yiannis N

    2007-08-01

    Protein secondary structure predictions and amino acid long range contact map predictions from primary sequence of proteins have been explored to aid in modelling protein tertiary structures. In order to evaluate the usefulness of secondary structure and 3D-residue contact prediction methods to model protein structures we have used the known Q3 (alpha-helix,beta-strands and irregular turns/loops) secondary structure information, along with residue-residue contact information as restraints for MODELLER. We present here results of our modelling studies on 30 best resolved single domain protein structures of varied lengths. The results shows that it is very difficult to obtain useful models even with 100% accurate secondary structure predictions and accurate residue contact predictions for up to 30% of residues in a sequence. The best models that we obtained for proteins of lengths 37, 70, 118, 136 and 193 amino acid residues are of RMSDs 4.17, 5.27, 9.12, 7.89 and 9.69,respectively. The results show that one can obtain better models for the proteins which have high percent of alpha-helix content. This analysis further shows that MODELLER restrain optimization program can be useful only if we have truly homologous structure(s) as a template where it derives numerous restraints, almost identical to the templates used. This analysis also clearly indicates that even if we satisfy several true residue-residue contact distances, up to 30%of their sequence length with fully known secondary structural information, we end up predicting model structures much distant from their corresponding native structures.

  12. Characterization of rapeseed (Brassica napus) oils by bulk C, O, H, and fatty acid C stable isotope analyses.

    PubMed

    Richter, Eva Katharina; Spangenberg, Jorge E; Kreuzer, Michael; Leiber, Florian

    2010-07-14

    Rapeseed ( Brassica napus ) oils differing in cultivar, sites of growth, and harvest year were characterized by fatty acid concentrations and carbon, hydrogen, and oxygen stable isotope analyses of bulk oils (delta(13)C(bulk), delta(2)H(bulk), delta(18)O(bulk) values) and individual fatty acids (delta(13)C(FA)). The delta(13)C(bulk), delta(2)H(bulk), and delta(18)O(bulk) values were determined by continuous flow combustion and high-temperature conversion elemental analyzer-isotope ratio mass spectrometry (EA/IRMS, TC-EA/IRMS). The delta(13)C(FA) values were determined using gas chromatography--combustion-isotope ratio mass spectrometry (GC/C/IRMS). For comparison, other C(3) vegetable oils rich in linolenic acid (flax and false flax oils) and rich in linoleic acid (poppy, sunflower, and safflower oils) were submitted to the same chemical and isotopic analyses. The bulk and molecular delta(13)C values were typical for C(3) plants. The delta(13)C value of palmitic acid (delta(13)C(16:0)) and n-3 alpha-linolenic acid (delta(13)C(18:3n-3)) differed (p < 0.001) between rape, flax, and poppy oils. Also within species, significant differences of delta(13)C(FA) were observed (p < 0.01). The hydrogen and oxygen isotope compositions of rape oil differed between cultivars (p < 0.05). Major differences in the individual delta(13)C(FA) values were found. A plant-specific carbon isotope fractionation occurs during the biosynthesis of the fatty acids and particularly during desaturation of C(18) acids in rape and flax. Bulk oil and specific fatty acid stable isotope analysis might be useful in tracing dietary lipids differing in their origin.

  13. Systematic determination of the peptide acceptor preferences for the human UDP-Gal:glycoprotein-alpha-GalNAc beta 3 galactosyltransferase (T-synthase).

    PubMed

    Perrine, Cynthia; Ju, Tongzhong; Cummings, Richard D; Gerken, Thomas A

    2009-03-01

    Mucin-type protein O-glycosylation is initiated by the addition of alpha-GalNAc to Ser/Thr residues of a polypeptide chain. The addition of beta-Gal to GalNAc by the UDP-Gal:glycoprotein-alpha-GalNAc beta 3 galactosyltransferase (T-synthase), forming the Core 1 structure (beta-Gal(1-3)-alpha-GalNAc-O-Ser/Thr), is a common and biologically significant subsequent step in O-glycan biosynthesis. What dictates the sites of Core 1 glycosylation is poorly understood; however, the peptide sequence and neighboring glycosylation effects have been implicated. To systematically address the role of the peptide sequence on the specificity of T-synthase, we used the oriented random glycopeptide: GAGAXXXX(T-O-GalNAc)XXXXAGAG (where X = G, A, P, V, I, F, Y, S, N, D, E, H, R, and K) as a substrate. The Core 1 glycosylated product was isolated on immobilized PNA (Arachis hypogaea) lectin and its composition determined by Edman amino acid sequencing for comparison with the initial substrate composition, from which transferase preferences were obtained. From these studies, elevated preferences for Gly at the +1 position with moderately high preferences for Phe and Tyr in the +3 position relative to the acceptor Thr-O-GalNAc were found. A number of smaller Pro enhancements were also observed. Basic residues, i.e., Lys, Arg, and His, in any position were disfavored, suggesting electrostatic interactions as an additional important component modulating transferase specificity. This work suggests that there are indeed subtle specific and nonspecific protein-targeting sequence motifs for this transferase.

  14. Cr(VI)-stimulated STAT3 tyrosine phosphorylation and nuclear translocation in human airway epithelial cells requires Lck.

    PubMed

    O'Hara, Kimberley A; Vaghjiani, Rasilaben J; Nemec, Antonia A; Klei, Linda R; Barchowsky, Aaron

    2007-03-01

    Chronic inhalation of low amounts of Cr(VI) promotes pulmonary diseases and cancers through poorly defined mechanisms. SFKs (Src family kinases) in pulmonary airway cells may mediate Cr(VI) signalling for lung injury, although the downstream effectors of Cr(VI)-stimulated SFKs and how they relate to pathogenic gene induction are unknown. Therefore SFK-dependent activation of transcription factors by non-cytotoxic exposure of human bronchial epithelial cells to Cr(VI) was determined. Protein-DNA binding arrays demonstrated that exposing BEAS 2B cells to 5 microM Cr(VI) for 4 and 24 h resulted in increased protein binding to 25 and 43 cis-elements respectively, while binding to 12 and 16 cis-elements decreased. Of note, Cr(VI) increased protein binding to several STAT (signal transducer and activator of transcription) cis-elements. Cr(VI) stimulated acute tyrosine phosphorylation and nuclear translocation of STAT1 over a 4 h period and a prolonged activation of STAT3 that reached a peak between 48 and 72 h. This prolonged activation was observed for both STAT3alpha and STAT3beta. Immunofluorescent confocal microscopy confirmed that Cr(VI) increased nuclear localization of phosphorylated STAT3 for more than 72 h in both primary and BEAS 2B human airway cells. Cr(VI) induced transactivation of both a STAT3-driven luciferase reporter construct and the endogenous inflammatory gene IL-6 (interleukin-6). Inhibition with siRNA (small interfering RNA) targeting the SFK Lck, but not dominant-negative JAK (Janus kinase), prevented Cr(VI)-stimulated phosphorylation of both STAT3 isoforms and induction of IL-6. The results suggest that Cr(VI) activates epithelial cell Lck to signal for prolonged STAT3 activation and transactivation of IL-6, an important immunomodulator of lung disease progression.

  15. Tumor necrosis factor (TNF)-alpha-induced IL-8 expression in gastric epithelial cells: role of reactive oxygen species and AP endonuclease-1/redox factor (Ref)-1.

    PubMed

    O'Hara, Ann M; Bhattacharyya, Asima; Bai, Jie; Mifflin, Randy C; Ernst, Peter B; Mitra, Sankar; Crowe, Sheila E

    2009-06-01

    TNF-alpha contributes to oxidative stress via induction of reactive oxygen species (ROS) and pro-inflammatory cytokines. The molecular basis of this is not well understood but it is partly mediated through the inducible expression of IL-8. As redox factor-1 (Ref-1), is an important mediator of redox-regulated gene expression we investigated whether ROS and Ref-1 modulate TNF-alpha-induced IL-8 expression in human gastric epithelial cells. We found that TNF-alpha treatment of AGS cells enhanced nuclear expression of Ref-1 and potently induced IL-8 expression. Overexpression of Ref-1 enhanced IL-8 gene transcription at baseline and after TNF-alpha treatment whereas Ref-1 suppression and antioxidant treatment inhibited TNF-alpha-stimulated IL-8 expression. TNF-alpha-mediated enhancement of other pro-inflammatory chemokines like MIP-3 alpha and Gro-alpha was also regulated by Ref-1. Although TNF-alpha increased DNA binding activity of Ref-1-regulated transcription factors, AP-1 and NF-kappaB, to the IL-8 promoter, promoter activity was mainly mediated by NF-kappaB binding. Silencing of Ref-1 in AGS cells inhibited basal and TNF-alpha-induced AP-1 and NF-kappaB DNA binding activity, but not their nuclear accumulation. Collectively, we provide the first mechanistic evidence of Ref-1 involvement in TNF-alpha-mediated, redox-sensitive induction of IL-8 and other chemokines in human gastric mucosa. This has implications for understanding the pathogenesis of gastrointestinal inflammatory disorders.

  16. Toxicological evaluation of polyphenol extract from Acerola (Malpighia emarginata DC.) fruit.

    PubMed

    Hanamura, T; Aoki, H

    2008-05-01

    Acerola (Malpighia emarginata DC.) is known to be one of the best fruits rich in ascorbic acid and polyphenols. Acerola polyphenols (APs) were found to have radical scavenging activities and inhibitory effects on both alpha-glucosidase and advanced glycation endproducts (AGEs) production. While polyphenols from several foodstuffs have been shown to be safe, APs, which differ from those of other foodstuffs, have not been evaluated for their toxicological properties. Here, we report preliminary toxicological and safety evaluations of crude APs (C-AP), which were obtained by eluting an XAD7HP column-adsorbed fraction of APs with 70% ethanol containing malic acid. The total polyphenol content of C-AP was 57.7% with the main polyphenols being proanthocyanidin and cyanidin-3-alpha-O-rhamnoside. For toxicological evaluations, C-AP was administered orally to rats at doses of 2000 mg/kg body weight (acute) or 100, 300, and 1000 mg/kg body weight/d for 28 (subacute) and 90 (subchronic) d. In the acute oral toxicological test, no deaths or abnormalities at necropsy on day 14 were observed, confirming that the minimum fatal dose of C-AP is greater than 2000 mg/kg body weight. In both subacute and subchronic toxicological tests, no death was recorded and the body weights and food intakes of the rats did not differ significantly from the control groups. Besides, there were no abnormal clinical signs related to administration of C-AP in any of the experimental animals. These results provide an important reference for the safety of APs as a food supplement for human consumption.

  17. Susceptibility of piglets to enterotoxigenic Escherichia coli is not related to the expression of MUC13 and MUC20.

    PubMed

    Schroyen, M; Stinckens, A; Verhelst, R; Geens, M; Cox, E; Niewold, T; Buys, N

    2012-06-01

    Enterotoxigenic Escherichia coli (ETEC) is one of the most frequently isolated enteropathogens in production animals, especially pigs and calves. Economically, the swine industry is by far the most affected by infections with ETEC because of mortality, morbidity and decreased growth rate of newborn and early-weaned piglets. After ingestion by the animal, these bacteria attach themselves to specific receptors on the small intestinal epithelium by means of proteinaceous surface appendages, the fimbriae. The F4 fimbriae, which attach to the F4 receptor, are the most studied. The aim of our study was to investigate gene expression in the small intestine of piglets of MUC13 and MUC20 in relation to animals with a different treatment towards or a different reaction on ETEC-F4ac by means of quantitative reverse transcription chain reaction (qRT/PCR). MUC13 and MUC20 are positional candidate genes for this F4ac receptor and are located in the region on SSC13q41 that segregates with the susceptibility to ETEC-F4ac. The condition of the small intestine is crucial when examining expression differences between different samples. Therefore, the expression of two genes, fatty-acid binding protein 2, intestinal (FABP2) and pancreatitis-associated protein (PAP), now known as regenerating islet-derived 3 alpha (REG3A) in the small intestine was simultaneously checked. FABP2, a standard for epithelial content, reflects the state of damage, whereas REG3A is a measure for inflammation in the small intestine. The four different substudies presented here suggest that expression of MUC13 and MUC20 is not related to the susceptibility of piglets to ETEC-F4ac. PMID:22486505

  18. Selective down-regulation of the alpha6-integrin subunit in melanocytes by UVB light.

    PubMed

    Krengel, Sven; Stark, Imke; Geuchen, Christian; Knoppe, Bettina; Scheel, Gabriele; Schlenke, Peter; Gebert, Andreas; Wünsch, Lutz; Brinckmann, Jürgen; Tronnier, Michael

    2005-06-01

    In vivo, melanocytes bind to laminin (LM) molecules of the basement membrane (BM) via the integrins alpha3beta1 and alpha6beta1, and they adhere to neighbouring keratinocytes via E-cadherin. Only few studies have addressed the impact of ultraviolet (UV) light on the interaction of melanocytes with their microenvironment. In this report, we examined the influence of UVB irradiation on the expression of the most important melanocyte-adhesion molecules (E-, N-cadherin, alpha2-, alpha3-, alpha5-, alpha6-, alphaV-, beta1-, beta3-integrins and ICAM-1) in vitro by flow cytometry. We were able to demonstrate that the alpha6-integrin subunit is selectively and reversibly down-regulated by UVB in a dwzm 150ose-dependent manner. In comparison, keratinocytes lacked UVB-inducible alterations in the expression of alpha6-integrin. In the presence of LM-1, the UVB-induced down-regulation of alpha6-integrin in melanocytes was significantly reduced. Moreover, LM-1 increased the resistance of melanocytes to UVB-induced cell death, as measured by annexinV-binding analysis. This effect was reversed by preincubation with an alpha6-integrin-blocking antibody. By immunofluorescence, we could demonstrate that UVB leads to a dose-dependent internalization of alpha6-integrin, providing an obvious explanation for the down-regulation on the outer cell surface observed by flow cytometry. We suggest that adhesion to LM-1 through alpha6-integrin represents a protective mechanism for melanocytes to withstand UVB damage. Through alpha6-integrin internalization, sunburns might alter the interaction between melanocytes and the BM, resulting in apoptosis induced by loss of anchorage (anoikis). Repeated sunburns may then lead to the selection of a population of melanocytes which are capable of anchorage-independent survival, culminating in solar nevogenesis and melanoma development.

  19. Flavonol tetraglycosides from fruits of Styphnolobium japonicum (Leguminosae) and the authentication of Fructus Sophorae and Flos Sophorae.

    PubMed

    Kite, Geoffrey C; Veitch, Nigel C; Boalch, Martha E; Lewis, Gwilym P; Leon, Christine J; Simmonds, Monique S J

    2009-04-01

    The dried fruits and seeds of Styphnolobium japonicum (L.) Schott (syn. Sophora japonica L.) are used in traditional Chinese medicine and known as Fructus Sophorae or Huai Jiao. The major flavonoids in these fruits and seeds were studied by LC-MS and other spectroscopic techniques to aid the chemical authentication of Fructus Sophorae. Among the flavonoids were two previously unreported kaempferol glycosides: kaempferol 3-O-beta-glucopyranosyl(1-->2)-beta-galactopyranoside-7-O-alpha-rhamnopyranoside and kaempferol 3-O-beta-xylopyranosyl(1-->3)-alpha-rhamnopyranosyl(1-->6)[beta-glucopyranosyl(1-->2)]-beta-glucopyranoside, the structures of which were determined by NMR. Two further tetraglycosides were identified for the first time in S. japonicum as kaempferol 3-O-beta-glucopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-glucopyranoside-7-O-alpha-rhamnopyranoside and kaempferol 3-O-beta-glucopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-galactopyranoside-7-O-alpha-rhamnopyranoside; the latter was the main flavonoid in mature seeds. The chromatographic profiles of 27 recorded flavonoids were relatively consistent among fruits of similar ages collected from five trees of S. japonicum, and those of maturing unripe and ripe fruits were similar to a market sample of Fructus Sophorae, and thus provide useful markers for authentication of this herbal ingredient. The flower buds (Huai Mi) and flowers (Huai Hua) of S. japonicum (collectively Flos Sophorae) contained rutin as the main flavonoid and lacked the flavone glycosides that were present in flower buds and flowers of Sophora flavescens Ait., reported to be occasional substitutes for Flos Sophorae. The single major flavonoid in fruits of S. flavescens was determined as 3'-hydroxydaidzein. PMID:19447452

  20. Degradation of the proto-oncogene product c-Fos by the ubiquitin proteolytic system in vivo and in vitro: identification and characterization of the conjugating enzymes.

    PubMed Central

    Stancovski, I; Gonen, H; Orian, A; Schwartz, A L; Ciechanover, A

    1995-01-01

    The transcription factor c-Fos is a short-lived cellular protein. The levels of the protein fluctuate significantly and abruptly during changing pathophysiological conditions. Thus, it is clear that degradation of the protein plays an important role in its tightly regulated activity. We examined the involvement of the ubiquitin pathway in c-Fos breakdown. Using a mutant cell line, ts20, that harbors a thermolabile ubiquitin-activating enzyme, E1, we demonstrate that impaired function of the ubiquitin system stabilizes c-Fos in vivo. In vitro, we reconstituted a cell-free system and demonstrated that the protein is multiply ubiquitinated. The adducts serve as essential intermediates for degradation by the 26S proteasome. We show that both conjugation and degradation are significantly stimulated by c-Jun, with which c-Fos forms the active heterodimeric transcriptional activator AP-1. Analysis of the enzymatic cascade involved in the conjugation process reveals that the ubiquitin-carrier protein E2-F1 and its human homolog UbcH5, which target the tumor suppressor p53 for degradation, are also involved in c-Fos recognition. The E2 enzyme acts along with a novel species of ubiquitin-protein ligase, E3. This enzyme is distinct from other known E3s, including E3 alpha/UBR1, E3 beta, and E6-AP. We have purified the novel enzyme approximately 350-fold and demonstrated that it is a homodimer with an apparent molecular mass of approximately 280 kDa. It contains a sulfhydryl group that is essential for its activity, presumably for anchoring activated ubiquitin as an intermediate thioester prior to its transfer to the substrate. Taken together, our in vivo and in vitro studies strongly suggest that c-Fos is degraded in the cell by the ubiquitin-proteasome proteolytic pathway in a process that requires a novel recognition enzyme. PMID:8524278

  1. Artificial rearing of infant mice leads to n-3 fatty acid deficiency in cardiac, neural and peripheral tissues.

    PubMed

    Hussein, Nahed; Fedorova, Irina; Moriguchi, Toru; Hamazaki, Kei; Kim, Hee-Yong; Hoshiba, Junji; Salem, Norman

    2009-08-01

    The ability to control the fatty acid content of the diet during early development is a crucial requirement for a one-generation model of docosahexaenoic acid (DHA; 22:6n3) deficiency. A hand feeding method using artificial rearing (AR) together with sterile, artificial milk was employed for feeding mice from postnatal day 2-15. The pups were fed an n-3 fatty acid adequate (3% alpha-linolenic acid (LNA; 18:3n3) + 1% 22:6n3) or a deficient diet (0.06% 18:3n3) with linoleic acid (LA; 18:2n6) as the only dietary source of essential fatty acids by AR along with a dam-reared control group (3.1% 18:3n3). The results indicate that restriction of n-3 fatty acid intake during postnatal development leads to markedly lower levels of brain, retinal, liver, plasma and heart 22:6n3 at 20 weeks of age with replacement by docosapentaenoic acid (DPAn6; 22:5n6), arachidonic acid (ARA; 20:4n6) and docosatetraenoic acid (DTA; 22:4n6). A detailed analysis of phospholipid classes of heart tissue indicated that phosphatidylethanolamine, phosphatidylcholine and cardiolipin were the major repositories of 22:6n3, reaching 40, 29 and 15%, respectively. A novel heart cardiolipin species containing four 22:6n3 moieties is described. This is the first report of the application of artificially rearing to mouse pup nutrition; this technique will facilitate dietary studies of knockout animals as well as the study of essential fatty acid (EFA) functions in the cardiovascular, neural and other organ systems.

  2. Nicotine activates and up-regulates nicotinic acetylcholine receptors in bronchial epithelial cells.

    PubMed

    Fu, Xiao Wen; Lindstrom, Jon; Spindel, Eliot R

    2009-07-01

    Prenatal nicotine exposure impairs normal lung development and leads to diminished pulmonary function after birth. Previous work from our laboratory has demonstrated that nicotine alters lung development by affecting a nonneuronal cholinergic autocrine loop that is expressed in lung. Bronchial epithelial cells (BECs) express choline acetyltransferase, the choline high-affinity transporter and nicotinic acetylcholine (ACh) receptor (nAChR) subunits. We now demonstrate through a combination of morphological and electrophysiological techniques that nicotine affects this autocrine loop by up-regulating and activating cholinergic signaling. RT-PCR showed the expression of alpha 3, alpha 4, alpha 7, alpha 9, alpha 10, beta2, and beta 4 nAChR mRNAs in rhesus monkey lung and cultured BECs. The expression of alpha 7, alpha 4, and beta2 nAChR was confirmed by immunofluorescence in the cultured BECs and lung. The electrophysiological characteristics of nAChR in BECs were determined using whole-cell patch-clamp on cultured BECs. Both ACh and nicotine evoked an inward current, with a rapid desensitizing current. Nicotine induced inward currents in a concentration-dependent manner, with an EC(50) of 26.7 microM. Nicotine-induced currents were reversibly blocked by the nicotinic antagonists, mecamylamine, dihydro-beta-erythroidine, and methyllcaconitine. Incubation of BECs with 1 microM nicotine for 48 hours enhanced nicotine-induced currents by roughly 26%. The protein tyrosine phosphorylation inhibitor, genistein, increased nicotine-induced currents by 58% and enhanced methyllcaconitine-sensitive currents (alpha 7 nAChR activities) 2.3-fold, whereas the protein tyrosine phosphatase inhibitor, pervanadate, decreased the effects of nicotine. These results demonstrate that chronic nicotine exposure up-regulates nAChR activity in developing lung, and that nAChR activity can be further modified by tyrosine phosphorylation.

  3. High resolution infrared spectroscopy of CN and NH lines: nitrogen abundance in oxygen-rich giants through K to late M

    NASA Astrophysics Data System (ADS)

    Aoki, W.; Tsuji, T.

    1997-12-01

    The analyses of high resolution infrared spectra have been done for CN lines in oxygen-rich cool evolved stars including 2 K giants, 20 M giants and 1 S-type star. Since CN lines analyzed in the present work are weak and resolved well, they are appropriate for quantitative analyses. CN lines of Delta v=-2 and -1 sequences (red system) which are in the K- and the H-window regions, respectively, give the consistent nitrogen abundance for each star. The analyses of NH lines in the L-window region have been done for 5 late M giants for which CN lines have been also analyzed. Although the triplet structure of NH lines cannot be fully resolved, they are preferable because determination of nitrogen abundance is almost independent of other elemental abundances while nitrogen abundance based on CN depends on carbon abundance. The nitrogen abundances derived from NH for late M giants agree well with those from CN for which we adopt 7.75eV as the dissociation energy in the analysis. The results show that the nitrogen abundances in late M giants are larger than those in early M giants while decrease of the carbon abundance was found in late M giants by our previous work (Tsuji 1991). These variations of abundances can not be explained by the first dredge-up model but require additional processing by the CN cycle and mixing after the first dredge-up. However, there is no obvious evidence of other processes such as the 3alpha -process and subsequent hot bottom burning in our program stars. Such variation of the carbon and nitrogen abundances is not well understood by the present evolutionary models of low-mass and intermediate-mass stars. Table~10 is only available in electronic form at the CDS (Strasbourg) via anonymous ftp to cdsarc.u-strasbg.fr (130.79.128.5) or via http://cdsweb.u-strasbg.fr/Abstract.html

  4. Profiles of circulating steroid hormones, gonadotropins, immunoreactive inhibin and prolactin during pregnancy in goats and immunolocalization of inhibin subunits, steroidogenic enzymes and prolactin in the corpus luteum and placenta.

    PubMed

    Kandiel, Mohamed M M; Watanabe, Gen; Sosa, Gamal A; Abou El-Roos, Mahmoud E A; Abdel-Ghaffar, Alaa E; Li, Jun Y; Manabe, Noboru; El Azab, Abd El Salam I; Taya, Kazuyoshi

    2010-04-01

    The current study was performed to follow up the circulating hormonal changes and to correlate the findings with the physiological activity of the corpus luteum (CL) and placenta during pregnancy in goats. Blood samples were collected weekly from five goats during pregnancy for measuring steroid and protein hormones. A gradual increase was observed in immunoreactive (ir-) inhibin, with maximal levels at the 17th week. The plasma concentrations of estradiol and prolactin (PRL) showed nearly similar patterns during pregnancy, where they declined to basal levels during the first 4 weeks post-breeding and then increased significantly, with the maximal concentration during late pregnancy. The plasma FSH and LH concentrations were maintained at basal levels throughout the gestation period. The plasma progesterone concentration abruptly increased in the first week post-breeding and remained at high values throughout the pregnancy period. Immunohistochemical localization of inhibin alpha, beta(A), beta(B) and steroidogenic enzymes cytochrome P450 aromatase, 3alpha-hydroxysteroid dehydrogenase (3betaHSD), cytochrome 17alpha-hydroxylase P450 and cholesterol side-chain cleavage cytochrome P450 in the cyclic and pregnant goat CL revealed positive immunoreactivity without affinity differences between the luteal and pregnancy stages. The placental syncytiotrophoblasts also showed positive staining, except for inhibin beta(A) and 3betaHSD. The giant binucleate cells of the placenta showed positive immunoreactions to PRL. These results suggest that the high concentrations of ir-inhibin, estradiol and PRL during late pregnancy are of placental origin and that the placenta may have a vital role in the maintenance of pregnancy, regulation of mammary growth and preparation for kidding and lactation in goats.

  5. Forms of n-3 (ALA, C18:3n-3 or DHA, C22:6n-3) Fatty Acids Affect Carcass Yield, Blood Lipids, Muscle n-3 Fatty Acids and Liver Gene Expression in Lambs.

    PubMed

    Ponnampalam, Eric N; Lewandowski, Paul A; Fahri, Fahri T; Burnett, Viv F; Dunshea, Frank R; Plozza, Tim; Jacobs, Joe L

    2015-11-01

    The effects of supplementing diets with n-3 alpha-linolenic acid (ALA) and docosahexaenoic acid (DHA) on plasma metabolites, carcass yield, muscle n-3 fatty acids and liver messenger RNA (mRNA) in lambs were investigated. Lambs (n = 120) were stratified to 12 groups based on body weight (35 ± 3.1 kg), and within groups randomly allocated to four dietary treatments: basal diet (BAS), BAS with 10.7 % flaxseed supplement (Flax), BAS with 1.8 % algae supplement (DHA), BAS with Flax and DHA (FlaxDHA). Lambs were fed for 56 days. Blood samples were collected on day 0 and day 56, and plasma analysed for insulin and lipids. Lambs were slaughtered, and carcass traits measured. At 30 min and 24 h, liver and muscle samples, respectively, were collected for determination of mRNA (FADS1, FADS2, CPT1A, ACOX1) and fatty acid composition. Lambs fed Flax had higher plasma triacylglycerol, body weight, body fat and carcass yield compared with the BAS group (P < 0.001). DHA supplementation increased carcass yield and muscle DHA while lowering plasma insulin compared with the BAS diet (P < 0.01). Flax treatment increased (P < 0.001) muscle ALA concentration, while DHA treatment increased (P < 0.001) muscle DHA concentration. Liver mRNA FADS2 was higher and CPT1A lower in the DHA group (P < 0.05). The FlaxDHA diet had additive effects, including higher FADS1 and ACOX1 mRNA than for the Flax or DHA diet. In summary, supplementation with ALA or DHA modulated plasma metabolites, muscle DHA, body fat and liver gene expression differently. PMID:26395388

  6. Autophagy in periodontitis patients and gingival fibroblasts: unraveling the link between chronic diseases and inflammation

    PubMed Central

    2012-01-01

    Background Periodontitis, the most prevalent chronic inflammatory disease, has been related to cardiovascular diseases. Autophagy provides a mechanism for the turnover of cellular organelles and proteins through a lysosome-dependent degradation pathway. The aim of this research was to study the role of autophagy in peripheral blood mononuclear cells from patients with periodontitis and gingival fibroblasts treated with a lipopolysaccharide of Porphyromonas gingivalis. Autophagy-dependent mechanisms have been proposed in the pathogenesis of inflammatory disorders and in other diseases related to periodontitis, such as cardiovascular disease and diabetes. Thus it is important to study the role of autophagy in the pathophysiology of periodontitis. Methods Peripheral blood mononuclear cells from patients with periodontitis (n = 38) and without periodontitis (n = 20) were used to study autophagy. To investigate the mechanism of autophagy, we evaluated the influence of a lipopolysaccharide from P. gingivalis in human gingival fibroblasts, and autophagy was monitored morphologically and biochemically. Autophagosomes were observed by immunofluorescence and electron microscopy. Results We found increased levels of autophagy gene expression and high levels of mitochondrial reactive oxygen species production in peripheral blood mononuclear cells from patients with periodontitis compared with controls. A significantly positive correlation between both was observed. In human gingival fibroblasts treated with lipopolysaccharide from P. gingivalis, there was an increase of protein and transcript of autophagy-related protein 12 (ATG12) and microtubule-associated protein 1 light chain 3 alpha LC3. A reduction of mitochondrial reactive oxygen species induced a decrease in autophagy whereas inhibition of autophagy in infected cells increased apoptosis, showing the protective role of autophagy. Conclusion Results from the present study suggest that autophagy is an important and shared

  7. Development of Valpha4+ NK T cells in the early stages of embryogenesis.

    PubMed Central

    Makino, Y; Kanno, R; Koseki, H; Taniguchi, M

    1996-01-01

    The majority of T lymphocytes start to develop at around day 15 of gestation (d15)-d17 in the thymus and comprise the peripheral repertoire characterized by the expression of polymorphic T-cell antigen receptors (TCRs). Contrary to these conventional T cells, a subset of T cells, called natural killer (NK) T cells (most of them expressing an invariant TCR encoded by the Valpha14Jalpha281 gene with a 1-nt N-region), preferentially differentiates extrathymically and dominates the peripheral T-cell population at a high frequency (5% in splenic T cells and 40% in bone marrow T cells). Here, we investigated the development of NK T cells and found that the invariant Valpha14+ TCR transcripts and the circular DNA created by Valpha14 and Jalpha281 gene rearrangements can be detected in the embryo body at d9.5 of gestation and in the yolk sac and the fetal liver at d11.5-d13.5 of gestation, but not in the thymus, whereas T cells with Valpha1+ TCR expression, a major population in the thymus, were not observed at these early stages of gestation. Fluorescence-activated cell sorter analysis also demonstrated that there exist CD3+ alpha beta+ T cells, almost all of which are Valpha14/Vbeta8+ NK+ T cells, during early embryogenesis. To our knowledge, this demonstrates for the first time that a T lymphocyte subset develops in extrathymic tissues during the early stages of embryogenesis. Images Fig. 1 Fig. 4 PMID:8692847

  8. Protection against TGF-β1-induced fibrosis effects of IL-10 on dermal fibroblasts and its potential therapeutics for the reduction of skin scarring.

    PubMed

    Shi, Ji-Hong; Guan, Hao; Shi, Shan; Cai, Wei-Xia; Bai, Xiao-Zhi; Hu, Xiao-Long; Fang, Xiao-Bin; Liu, Jia-Qi; Tao, Ke; Zhu, Xiong-Xiang; Tang, Chao-Wu; Hu, Da-Hai

    2013-05-01

    Scarring, tightly associated with fibrosis, is a significant symptomatic clinical problem. Interleukin 10 (IL-10) has been identified as a candidate scar-improving therapy based on preclinical studies. However, the molecular mechanism of IL-10 in scar improvement is still uncertain. In this study, human dermal fibroblasts stimulated with TGF-β1 were treated with IL-10 to analyze the mRNA and some of proteins' expression levels of type I collagen (Col1), type III collagen (Col3), alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-1 (MMP1), MMP2, MMP8 and tissue inhibitor of metalloproteinase 1 (TIMP1), TIMP2 by real-time PCR and Western blot, to observe α-SMA-positive fibroblasts by immunocytochemistry. The contracture and improvement of fibroblast-populated collagen lattice (FPCL) and a murine model of wound healing were used to evaluate the scar-improving effects by histological staining. The results showed that IL-10 can significantly down-regulate the mRNA and protein expression levels of Col1, Col3, α-SMA, and up-regulate the mRNA expression levels of MMP1 and MMP8, and decrease α-SMA-positive fibroblasts. FPCL analysis showed that the IL-10 (20 ng/ml) can significantly inhibit the contracture, improve the architecture of FPCL. Wounds injected with IL-10 demonstrated that the appearance of scar was improved, the wound margin of scarring was narrow, and the deposition of collagens (Col1 and Col3) in regenerated tissue was relieved. These results provide direct evidences that IL-10 has the inhibitory effects on the excessive deposition of extracellular matrix components and fibroblast-to-myofibroblast transition, and show that IL-10 has the potential therapy in prevention and reduction of skin scarring.

  9. Feeding Glycyrrhiza glabra (liquorice) and Astragalus membranaceus (AM) alters innate immune and physiological responses in yellow perch (Perca flavescens).

    PubMed

    Elabd, Hiam; Wang, Han-Ping; Shaheen, Adel; Yao, Hong; Abbass, Amany

    2016-07-01

    The current work assessed the potential immunomodulatory and growth-promoting effects of Astragalus membranaceus (AM) and Glycyrrhiza glabra (liquorice) in Yellow perch (Perca flavescens). In this regard, fish with an average weight of 31 ± 1.0 g were divided into five groups, and fed daily with an additive-free basal diet (control); 1, 2, and 3% (w/w) Glycyrrhiza glabra, and the fifth diet was incorporated with a combination of 1% G. glabra-AM for a four-week period. Immunological, biochemical and growth parameters were measured; and sub-groups of fish were exposed to 1-week starvation. The results showed that incorporating AM and liquorice in the diet significantly improved Immunological [superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), Lipid peroxidase (LPx) and lysozyme activities], biochemical [Aspartate Aminotransferase (AST) and Alanine Transaminase (ALT) activities; and glucose and cortisol concentrations] and growth performance parameters [body mass gain (BMG), specific growth rate (SGR), length, condition factor (K) and feed conversion ratio (FCR)]. In addition, markedly up-regulated the expression of related genes [Insulin-Like Growth Factor-1 (IGF-1), Serum amyloid A (SAA), Complement Component C3 (CCC3), Alpha 2 Macroglobulin (A2M), SOD and GPx] in treated fish groups compared to the control. Conclusively, feeding AM and liquorice diets significantly increased (P < 0.05) growth performance, antioxidant and immune response profiles throughout the entire experiment, suggesting their beneficial rule as natural anti-stress agents. PMID:27129627

  10. Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV

    NASA Technical Reports Server (NTRS)

    Lim, K.; Ho, J. X.; Keeling, K.; Gilliland, G. L.; Ji, X.; Ruker, F.; Carter, D. C.

    1994-01-01

    The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) (Muster T et al., 1993, J Virol 67:6642-6647) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169-10184) was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(3)2(1)2, with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.

  11. Loci of Mycobacterium avium ser2 gene cluster and their functions.

    PubMed Central

    Mills, J A; McNeil, M R; Belisle, J T; Jacobs, W R; Brennan, P J

    1994-01-01

    The highly antigenic glycopeptidolipids present on the surface of members of the Mycobacterium avium complex serve to distinguish these bacteria from all others and to define the various serovars that compose this complex. Previously, the genes responsible for the biosynthesis of the disaccharide hapten [2,3-di-O-methyl-alpha-L-fucopyranosyl-(1-->3)-alpha-L-rhamnopyranose] of serovar 2 of the M. avium complex were isolated, localized to a contiguous 22- to 27-kb fragment of the M. avium genome, and designated the ser2 gene cluster (J. T. Belisle, L. Pascopella, J. M. Inamine, P. J. Brennan, and W. R. Jacobs, Jr., J. Bacteriol. 173:6991-6997, 1991). In the present study, transposon saturation mutagenesis was used to map the specific genetic loci within the ser2 gene cluster required for expression of this disaccharide. Four essential loci, termed ser2A, -B, -C, and -D, constituting a total of 5.7 kb within the ser2 gene cluster, were defined. The ser2B and ser2D loci encode the methyltransferases required to methylate the fucose at the 3 and 2 positions, respectively. The rhamnosyltransferase was encoded by ser2A, whereas either ser2C or ser2D encoded the fucosyltransferase. The ser2C and ser2D loci are also apparently involved in the de novo synthesis of fucose. Isolation of the truncated versions of the hapten induced by the transposon insertions provides genetic evidence that the glycopeptidolipids of M. avium serovar 2 are synthesized by an initial transfer of the rhamnose unit to the peptide core followed by fucose and finally O methylation of the fucosyl unit. PMID:8050992

  12. Loci of Mycobacterium avium ser2 gene cluster and their functions.

    PubMed

    Mills, J A; McNeil, M R; Belisle, J T; Jacobs, W R; Brennan, P J

    1994-08-01

    The highly antigenic glycopeptidolipids present on the surface of members of the Mycobacterium avium complex serve to distinguish these bacteria from all others and to define the various serovars that compose this complex. Previously, the genes responsible for the biosynthesis of the disaccharide hapten [2,3-di-O-methyl-alpha-L-fucopyranosyl-(1-->3)-alpha-L-rhamnopyranose] of serovar 2 of the M. avium complex were isolated, localized to a contiguous 22- to 27-kb fragment of the M. avium genome, and designated the ser2 gene cluster (J. T. Belisle, L. Pascopella, J. M. Inamine, P. J. Brennan, and W. R. Jacobs, Jr., J. Bacteriol. 173:6991-6997, 1991). In the present study, transposon saturation mutagenesis was used to map the specific genetic loci within the ser2 gene cluster required for expression of this disaccharide. Four essential loci, termed ser2A, -B, -C, and -D, constituting a total of 5.7 kb within the ser2 gene cluster, were defined. The ser2B and ser2D loci encode the methyltransferases required to methylate the fucose at the 3 and 2 positions, respectively. The rhamnosyltransferase was encoded by ser2A, whereas either ser2C or ser2D encoded the fucosyltransferase. The ser2C and ser2D loci are also apparently involved in the de novo synthesis of fucose. Isolation of the truncated versions of the hapten induced by the transposon insertions provides genetic evidence that the glycopeptidolipids of M. avium serovar 2 are synthesized by an initial transfer of the rhamnose unit to the peptide core followed by fucose and finally O methylation of the fucosyl unit. PMID:8050992

  13. A 28,000-dalton protein of normal mouse serum binds specifically to the inner core region of bacterial lipopolysaccharide.

    PubMed

    Brade, L; Brade, H

    1985-12-01

    Normal mouse serum was found to contain a protein, referred to here as factor, which binds to the inner core region of lipopolysaccharides (LPSs) of various bacterial families. Since factor-LPS interactions resulted in activation of guinea pig complement, factor activity could be assayed by a passive hemolysis test with sheep erythrocytes coated with LPS or lipid A from Acinetobacter calcoaceticus (which was found earlier to bind particularly well to factor). Factor was purified by G-50 and hydroxyapatite chromatography whereby the specific hemolytic activity was enriched 1,675-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed the presence of a 28,000-dalton protein as the main band. The identity of this band was determined by absorption experiments with LPS-coated sheep erythrocytes or latex beads, whereby the 28,000-dalton band disappeared after specific absorption and could be recovered from the absorbent. The binding specificity of factor was determined in a passive hemolysis inhibition assay with defined oligosaccharides representative for the inner core region of LPS. Thus, the di- and trisaccharides alpha-D-mannoheptopyranosyl-(1----5)-2-keto-3-deoxy-D-mannoocto nic acid and alpha-D-mannoheptopyranosyl-(1----3)-alpha-D-mannoheptopyranosy l-(1----5)-2- keto-3-deoxy-D-mannooctonic acid, respectively, were able to inhibit binding of factor to LPS. The results are in accordance with our earlier observation that the heptose-2-keto-3-deoxy-D-mannooctonic acid region represents a common antigen of bacterial LPS. Rabbit hyperimmune serum directed against this common antigen and purified factor was found to exhibit the same specificity for LPS. Factor activity was followed in mice in vivo after injection of LPS; it disappeared completely 15 min after the injection of LPS and reappeared within 1 h.

  14. [Chromatographic determination of bile acids in biological fluids with sensitive and selective detection].

    PubMed

    Goto, J

    1990-11-01

    Separation and determination of bile acids in biological fluids by high-performance liquid chromatography are reviewed. The capacity ratios of bile acids on an ODS column were affected by the number, position and configuration of the hydroxyl group on the steroid nucleus, and the chromatographic behavior was markedly influenced by the pH of a mobile phase according to the conjugated form at C-24. A new pre-column derivatization reagent, 1-anthroyl nitrile, was developed and applied to the analysis of bile acids in biological fluids. Bile acids were derivatized through the 3 alpha-hydroxyl group into the corresponding esters, separated on an ODS column, and monitored by a fluorescence detector with detection limit of 20 fmol. The sensitive method for the determination of bile acids in biological materials by gas chromatography (GC) in combination with negative ion chemical ionization (NICI) mass spectrometry is also described. Of various derivatives for the carboxyl group, the pentafluorobenzyl (PFB) ester provided the highest value of the ratio of the negative to positive ion current. A characteristic carboxylate anion [M-181]- was produced as the most abundant ion by the loss of the PFB group in NICI. PFB esters of bile acids were further derivatized into the dimethylethylsilyl ethers and then separated by GC. The detection limit was 2 fg when the characteristic anion was monitored in the NICI mode. The preparation of 18O-labelled bile acids, as the internal standard for the trace analysis or the tracer for the metabolic study, was developed. Finally, the clean-up procedure for bile acids in biological fluids was investigated. The combined use of solid-phase extraction with a Sep-pak C18 or Bond Elut cartridge and group separation on a lipophilic ion-exchange gel, piperidinohydroxypropyl Sephadex LH-20, was found most effective for this purpose. PMID:2082011

  15. Binding of collagens to an enterotoxigenic strain of Escherichia coli

    SciTech Connect

    Visai, L.; Speziale, P.; Bozzini, S. )

    1990-02-01

    An enterotoxigenic strain of Escherichia coli, B34289c, has been shown to bind the N-terminal region of fibronectin with high affinity. We now report that this strain also binds collagen. The binding of 125I-labeled type II collagen to bacteria was time dependent and reversible. Bacteria expressed a limited number of collagen receptors (2.2 x 10(4) per cell) and bound collagen with a Kd of 20 nM. All collagen types tested (I to V) as well as all tested cyanogen bromide-generated peptides (alpha 1(I)CB2, alpha 1(I)CB3, alpha 1(I)CB7, alpha 1(I)CB8, and alpha 2(I)CB4) were recognized by bacterial receptors, as demonstrated by the ability of these proteins to inhibit the binding of 125I-labeled collagen to bacteria. Of several unlabeled proteins tested in competition experiments, fibronectin and its N-terminal region strongly inhibited binding of the radiolabeled collagen to E. coli cells. Conversely, collagen competed with an 125I-labeled 28-kilodalton fibronectin fragment for bacterial binding. Collagen bound to bacteria could be displaced by excess amounts of either unlabeled fibronectin or its N-terminal fragment. Similarly, collagen could displace 125I-labeled N-terminal peptide of fibronectin bound to the bacterial cell surface. Bacteria grown at 41 degrees C or in the presence of glucose did not express collagen or fibronectin receptors. These results indicate the presence of specific binding sites for collagen on the surface of E. coli cells and furthermore that the collagen and fibronectin binding sites are located in close proximity, possibly on the same structure.

  16. ACCELERATION IN PERPENDICULAR RELATIVISTIC SHOCKS FOR PLASMAS CONSISTING OF LEPTONS AND HADRONS

    SciTech Connect

    Stockem, A.; Fiuza, F.; Fonseca, R. A.; Silva, L. O.

    2012-08-10

    We investigate the acceleration of light particles in perpendicular shocks for plasmas consisting of a mixture of leptonic and hadronic particles. Starting from the full set of conservation equations for the mixed plasma constituents, we generalize the magnetohydrodynamical jump conditions for a multi-component plasma, including information about the specific adiabatic constants for the different species. The impact of deviations from the standard model of an ideal gas is compared in theory and particle-in-cell simulations, showing that the standard MHD model is a good approximation. The simulations of shocks in electron-positron-ion plasmas are for the first time multi-dimensional, transverse effects are small in this configuration, and one-dimensional (1D) simulations are a good representation if the initial magnetization is chosen high. 1D runs with a mass ratio of 1836 are performed, which identify the Larmor frequency {omega}{sub ci} as the dominant frequency that determines the shock physics in mixed component plasmas. The maximum energy in the non-thermal tail of the particle spectra evolves in time according to a power law {proportional_to}t{sup {alpha}} with {alpha} in the range 1/3 < {alpha} < 1, depending on the initial parameters. A connection is made with transport theoretical models by Drury and Gargate and Spitkovsky, which predict an acceleration time {proportional_to}{gamma} and the theory for small wavelength scattering by Kirk and Reville, which predicts a behavior rather as {proportional_to}{gamma}{sup 2}. Furthermore, we compare different magnetic field orientations with B{sub 0} inside and out of the plane, observing qualitatively different particle spectra than in pure electron-ion shocks.

  17. Effect of side chain length on bile acid conjugation: glucuronidation, sulfation and coenzyme A formation of nor-bile acids and their natural C24 homologs by human and rat liver fractions.

    PubMed

    Kirkpatrick, R B; Green, M D; Hagey, L R; Hofmann, A F; Tephly, T R

    1988-01-01

    The effect of side chain length on bile acid conjugation by human and rat liver fractions was examined. The rate of conjugation with glucuronic acid, sulfate and coenzyme A of several natural (C24) bile acids was compared with that of their corresponding nor-bile acids. The rate of coenzyme A ester formation by nor-bile acids was much lower than that of the natural bile acids. In human liver microsomes, the rate of coenzyme A formation was less than 8% of the rate for the corresponding C24 bile acid. Rat liver microsomes formed the coenzyme A ester of nor-bile acids less than 20% of the rate of their corresponding C24 homologs. Glucuronidation rates were greater than sulfation rates in both species. With human liver microsomes, nor-bile acids were glucuronidated more rapidly than their corresponding C24 homologs, whereas with rat liver microsomes the reverse was true. Purified 3 alpha-OH androgen UDP-glucuronyltransferase catalyzed the glucuronidation of both nor-bile acids and bile acids. Human liver cytosol sulfated nor-bile acids more slowly than the corresponding bile acids. Rat liver cytosol, however, sulfated nor-bile acids more rapidly than the corresponding bile acids. The highest rate was seen with lithocholylglycine. The results indicate that the novel biotransformation of nor-bile acids seen in vivo--sulfation and glucuronidation rather than amidation--is most likely explained as a consequent of defective amidation, to which the rate of coenzyme A formation contributes. Thus, side chain and nuclear structures as well as species differences in conjugating enzyme activity are determinants of the pattern of bile acid biotransformation by the mammalian liver.

  18. Ursodeoxycholic acid in the Ursidae: biliary bile acids of bears, pandas, and related carnivores.

    PubMed

    Hagey, L R; Crombie, D L; Espinosa, E; Carey, M C; Igimi, H; Hofmann, A F

    1993-11-01

    The biliary bile acid composition of gallbladder bile obtained from six species of bears (Ursidae), the Giant panda, the Red panda, and 11 related carnivores were determined by reversed phase liquid chromatography and gas chromatography-mass spectrometry. Bile acids were conjugated solely with taurine (in N-acyl linkage) in all species. Ursodeoxycholic acid (3 alpha, 7 beta-dihydroxy-5 beta-cholan-24-oic acid) was present in all Ursidae, averaging 1-39% of biliary bile acids depending on the species; it was not detected or present as a trace constituent (< 0.5%) in all other species, including the Giant panda. Ursodeoxycholic acid was present in 73 of 75 American Black bears, and its proportion averaged 34% (range 0-62%). Ursodeoxycholic acid averaged 17% of biliary bile acids in the Polar bear (n = 4) and 18% in the Brown bear (n = 6). Lower proportions (1-8%) were present in the Sun bear (n = 2), Ceylon Sloth bear (n = 1), and the Spectacled bear (n = 1). Bile of all species contained taurine-conjugated chenodeoxycholic acid and cholic acid. In some related carnivores, deoxycholic acid, the 7-dehydroxylation product of cholic acid, was also present. To determine whether the 7 beta hydroxy group of ursodeoxycholic acid was formed by hepatic or bacterial enzymes, bile acids were determined in hepatic bile obtained from bears with chronic biliary fistulae. Fistula bile samples contained ursodeoxycholic acid, chenodeoxycholic acid, and a trace amount of cholic acid, all as taurine conjugates, indicating that ursodeoxycholic acid is a primary bile acid formed in the liver in Ursidae.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Site-directed mutagenesis of the substrate-binding cleft of human estrogen sulfotransferase.

    PubMed

    Hempel, N; Barnett, A C; Bolton-Grob, R M; Liyou, N E; McManus, M E

    2000-09-16

    The sulfonation of estrogens by human estrogen sulfotransferase (humSULT1E1) plays a vital role in controlling the active levels of these hormones in the body. To understand more fully the structural and functional characteristics of humSULT1E1, we have carried out site-directed mutagenesis of critical amino acids found in the substrate-binding cleft. Three single amino acid mutations of humSULT1E1 (V145E, H107A, and K85A) were created in this study. Kinetic studies were used to provide information about the importance of these residues in substrate specificity and catalysis, using a variety of substrates. Lysine at position 85 has been proposed to be within hydrogen bonding distance to the 3alpha-phenol group of beta-estradiol, thereby stabilising the substrate in the active site. However, substitution to a neutral alanine at this position improved substrate specificity of humSULT1E1 for beta-estradiol, estrone, and dehydroepiandrosterone (DHEA). The exchange of valine 145 for negatively charged glutamic acid markedly improved the ability of humSULT1E1 to sulfonate dopamine, but caused a reduction in specificity constants toward steroids tested, in particular DHEA. The presence of a histidine residue at position 107 was shown to be essential for the production of a functional protein, as substitution of this amino acid to alanine resulted in complete loss of activity of humSULT1E1 towards all substrates tested. PMID:11006110

  20. Unexpected Inflammatory Effects of Intravaginal Gels (Universal Placebo Gel and Nonoxynol-9) on the Upper Female Reproductive Tract: A Randomized Crossover Study.

    PubMed

    Smith-McCune, Karen; Chen, Joseph C; Greenblatt, Ruth M; Shanmugasundaram, Uma; Shacklett, Barbara L; Hilton, Joan F; Johnson, Brittni; Irwin, Juan C; Giudice, Linda C

    2015-01-01

    Intravaginal anti-HIV microbicides could provide women with a self-controlled means for HIV prevention, but results from clinical trials have been largely disappointing. We postulated that unrecognized effects of intravaginal gels on the upper female reproductive tract might contribute to the lower-than-expected efficacy of HIV microbicides. Our objective was to study the effects of intravaginal gels on the immune microenvironment of the cervix and uterus. In this randomized crossover study, 27 healthy female volunteers used a nightly application of intravaginal nonoxynol-9 (N9) gel as a "failed" microbicide or the universal placebo gel (UPG) as a "safe" gel (intervention cycles), or nothing (control cycle) from the end of menses to the mid-luteal phase. At a specific time-point following ovulation, all participants underwent sample collection for measurements of T-cell phenotypes, gene expression, and cytokine/chemokine protein concentrations from 3 anatomic sites above the vagina: the cervical transformation zone, the endocervix and the endometrium. We used hierarchical statistical models to estimate mean (95% CI) intervention effects, for N9 and UPG relative to control. Exposure to N9 gel and UPG generated a common "harm signal" that included transcriptional up-regulation of inflammatory genes chemokine (C-C motif) ligand 20 (macrophage inflammatory factor-3alpha) and interleukin 8 in the cervix, decreased protein concentrations of secretory leukocyte protease inhibitor, and transcriptional up-regulation of inflammatory mediators glycodelin-A and osteopontin in the endometrium. These results need to be replicated with a larger sample, but underscore the need to consider the effects of microbicide agents and gel excipients on the upper female reproductive tract in studies of vaginal microbicides. PMID:26177352

  1. Photoaffinity labeling of rat liver microsomal morphine UDP-glucuronosyltransferase by ( sup 3 H)flunitrazepam

    SciTech Connect

    Thomassin, J.; Tephly, T.R. )

    1990-09-01

    Benzodiazepines have been shown to competitively inhibit morphine glucuronidation in rat and human hepatic microsomes. Flunitrazepam exerted a potent competitive inhibition of rat hepatic morphine UDP-glucuronosyltransferase (UDPGT) activity (Ki = 130 microM). It has no effect on the activity of p-nitrophenol, 17 beta-hydroxysteroid, 3 alpha-hydroxysteroid, or 4-hydroxybiphenyl UDPGTs. Because flunitrazepam is an effective photoaffinity label for benzodiazepine receptors, studied were performed in solubilized rat hepatic microsomes and with partially purified preparations of morphine UDPGT to determine the enhancement of flunitrazepam inhibition and binding to morphine UDPGT promoted by exposure to UV light. Under UV light, flunitrazepam inhibition was markedly enhanced. UV light exposure also led to a marked increase in binding of (3H)flunitrazepam to microsomal protein, which was protected substantially by preincubation with morphine. Testosterone, androsterone, and UDP-glucuronic acid did not protect against UV-enhanced flunitrazepam binding, and morphine did not reverse flunitrazepam binding once binding had occurred. As morphine UDPGT was purified, a good correlation was found between the increases in specific activity of morphine UDPGT and flunitrazepam binding to protein. Chromatofocusing chromatography showed that flunitrazepam bound only to fractions containing active morphine UDPGT, and no binding to 4-hydroxybiphenyl UDPGT was observed. Fluorography of a sodium dodecyl sulfate-polyacrylamide electrophoresis gel of solubilized hepatic microsomes that had been treated with (3H) flunitrazepam under UV light revealed a band with a monomeric molecular weight between 54,000 and 58,000. This monomeric molecular weight compares favorably with the reported monomeric molecular weight of homogeneous morphine UDPGT (56,000).

  2. Spatial and compositional variation in the fungal communities of organic and conventionally grown apple fruit at the consumer point-of-purchase

    PubMed Central

    Abdelfattah, Ahmed; Wisniewski, Michael; Droby, Samir; Schena, Leonardo

    2016-01-01

    The fungal diversity in harvested apples from organic or conventional management practices was analyzed in different fruit locations (stem end, calyx end, peel, and wounded flesh) shortly after fruit purchase (T1) and after 2 weeks of storage (T5). A total of 5,760,162 high-quality fungal sequences were recovered and assigned to 8,504 Operational Taxonomic Units. Members of the phylum Ascomycota were dominant in all samples and accounted for 91.6% of the total number of detected sequences. This was followed by Basidiomycota (8%), Chytridiomycota (0.1%), and unidentified fungi (0.3%). Alpha and beta diversity analyses revealed the presence of significantly different fungal populations in the investigated fruit parts. Among detected fungi, the genus Penicillium prevailed in the peel and in the wounded flesh while Alternaria spp. prevailed in the calyx and stem end samples that included apple core tissues. Several taxonomic units that appear to be closely related to pathogenic fungi associated with secondary human infections were present in peel and wounds. Moreover, significantly different populations were revealed in organic and conventional apples and this result was consistent in all investigated fruit parts (calyx end, peel, stem end, and wounded flesh). Several unique taxa were exclusively detected in organic apples suggesting that management practices may have been a contributing factor in determining the taxa present. In contrast, little differences were revealed in the two assessment times (T1 and T5). Results of the present study represent an advancement of the current knowledge on the fungal microbiota in collected fruit tissues of apple. PMID:27766161

  3. Nitric oxide mediates N-methyl-D-aspartate receptor-induced activation of p21ras.

    PubMed

    Yun, H Y; Gonzalez-Zulueta, M; Dawson, V L; Dawson, T M

    1998-05-12

    N-methyl-D-aspartate (NMDA) glutamate receptor-mediated increases in intracellular calcium are thought to play a critical role in synaptic plasticity. The mechanisms by which changes in cytoplasmic calcium transmit the glutamate signal to the nucleus, which is ultimately important for long-lasting neuronal responses, are poorly understood. We show that NMDA receptor stimulation leads to activation of p21(ras) (Ras) through generation of nitric oxide (NO) via neuronal NO synthase. The competitive NO synthase inhibitor, L-nitroarginine methyl ester, prevents Ras activation elicited by NMDA and this effect is competitively reversed by the NO synthase substrate, L-arginine. NMDA receptor stimulation fails to activate Ras in neuronal cultures from mice lacking neuronal NO synthase. NMDA-induced Ras activation occurs through a cGMP-independent pathway as 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), a potent and selective inhibitor of guanylyl cyclase, has no effect on NMDA receptor-induced activation of Ras, and the cell-permeable cGMP analog, 8Br-cGMP, does not activate Ras. Furthermore, NO directly activates immunoprecipitated Ras from neurons. NMDA also elicits tyrosine phosphorylation of extracellular signal-regulated kinases, a downstream effector pathway of Ras, through a NO/non-cGMP dependent mechanism, thus supporting the physiologic relevance of endogenous NO regulation of Ras. These results suggest that Ras is a physiologic target of endogenously produced NO and indicates a signaling pathway for NMDA receptor activation that may be important for long-lasting neuronal responses.

  4. The biochemical and phenotypic characterization of females homozygous for 5 alpha-reductase-2 deficiency.

    PubMed

    Katz, M D; Cai, L Q; Zhu, Y S; Herrera, C; DeFillo-Ricart, M; Shackleton, C H; Imperato-McGinley, J

    1995-11-01

    The biochemical and physiologic manifestations of decreased 5 alpha-dihydrotestosterone (DHT) in females are characterized. Three females from the large Dominican kindred with 5 alpha-reductase-2 deficiency were identified as homozygous for a point mutation (R246W, C-->T) on exon 5 of the 5 alpha-reductase-2 gene by single strand DNA conformational polymorphism analysis and DNA sequence analysis. Body hair was decreased; there was no history of acne. Despite delayed menarche, all were fertile, and two had twins. Urinary 5 beta/5 alpha C19 and C21 steroid metabolite ratios were elevated. Plasma testosterone was normal to elevated, with low DHT, resulting in an increased testosterone/DHT ratio. 3 alpha,5 alpha-Androstanediol glucuronide was low. Menstrual cycle profiling performed in two subjects showed ovulatory gonadotropin peaks. Sebum production was normal. 5 alpha-Reductase-2-deficient homozygotic females demonstrate the importance of DHT in the physiology and pathophysiology of body hair growth. Normal sebum implies regulation by the 5 alpha-reductase-1 isoenzyme. Delayed puberty suggests involvement of 5 alpha-reductase-2 in menarche at the hypothalamic/pituitary and/or ovarian level. As two had nonidentical twins, DHT and/or the DHT/estradiol ratio may regulate follicular development, with lower levels permitting more than one dominant follicle per cycle and higher levels impairing follicular development and ovulation. Thus, females with 5 alpha-reductase-2 deficiency highlight a role for DHT in hirsutism and/or menstrual disorders. PMID:7593420

  5. Effects of a pure antiandrogen on gonadotropin secretion in normal women and in polycystic ovarian disease.

    PubMed

    Couzinet, B; Thomas, G; Thalabard, J C; Brailly, S; Schaison, G

    1989-07-01

    To assess the role of androgens in gonadotropin regulation in women, we studied the effects of a pure nonsteroidal antiandrogen, Anandron (Cassenne, Paris, France). Nine normally cycling women (group 1) with acne and/or seborrhoea and nine patients with polycystic ovarian disease (PCOD) (group 2) received Anandron (100 mg twice a day) and a placebo. Both treatments were administered orally, in a cross-over randomized design, for two consecutive cycles (group 1) or months (group 2) separated by one cycle or 1 month. Luteinizing hormone (LH) pulse frequency and amplitude (cluster analysis), basal and gonadotropin-releasing hormone (GnRH)-stimulated plasma LH/follicle-stimulating hormone (FSH) levels were determined on day 5 of each treatment or placebo cycle. On days 5, 10, 20, and 24 of each cycle or month, plasma estradiol (E2), estrone (E1), testosterone (T), dihydrotestosterone (DHT), androstenedione (A), dehydroepiandrosterone sulfate (DHAS), sex hormone-binding globulin (SHBG) levels, and urinary androstanediol glucuronide (3 alpha-diol G) were measured. Plasma progesterone (P) levels were determined on days 20 and 24 of each cycle (group 1) and on days 5, 10, 20, and 24 (group 2). In both groups, seborrhea and acne decreased markedly within the first month and practically disappeared after 2 months of Anandron treatment. No adverse side effects were reported. None of the normal patients had any disturbance of menstrual cycles as assessed by basal body temperature shift, ultrasonography, and plasma P levels. In PCOD patients, cycles remained anovulatory.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2744186

  6. Antihormonal activities of 5 alpha-reductase and aromatase inhibitors.

    PubMed

    Zoppi, S; Cocconi, M; Lechuga, M J; Messi, E; Zanisi, M; Motta, M

    1988-10-01

    The problem of developing androgen antagonists has been tackled so far only by synthesizing steroids able to displace testosterone and other androgens from their specific receptor sites. The observation that testosterone has to be converted intracellularly either to 5 alpha-reduced metabolites (DHT, 3 alpha-diol, etc.) or to estrogens, in order to become fully active on androgen-dependent structures (both central and peripheral), has opened the possibility of creating molecules which prevent these conversions, and which could then block the actions of testosterone. The availability of these new compounds has allowed a better understanding of the selective physiological role of each of the metabolites of testosterone, and to provide the basis for the development of new hormone antagonists to be used in those clinical conditions for which an inhibition of the actions of testosterone is foreseen. The usefulness of these enzyme inhibitors is underlined by some examples described in this paper. The results obtained may permit the formulation of the following conclusions: (1) The conversion of testosterone to its 5 alpha-reduced metabolites occurring in the neuroendocrine structures may represent an essential step for the appearance of the inhibitory feedback effect testosterone exerts on LH secretion; (2) Testosterone exhibits its negative feedback effect on FSH secretion as such and not following the local aromatization to estrogens; (3) Testosterone exerts its effect on the intrahypothalamic stores of LHRH acting as such and not following its local conversion either to 5 alpha-reduced metabolites or to estrogenic molecules; (4) Some of the new enzyme inhibitors (e.g. 4-OH-A) may represent an interesting tool for the treatment and/or the prevention of BPH and possibly of other androgen-dependent diseases (prostate carcinoma, acne etc.), as shown by their ability to prevent the in vitro conversion of testosterone to its 5 alpha-reduced metabolites both in the normal

  7. Identification and characterization of genes required for compensatory growth in Drosophila.

    PubMed

    Gerhold, Abigail R; Richter, Daniel J; Yu, Albert S; Hariharan, Iswar K

    2011-12-01

    To maintain tissue homeostasis, some organs are able to replace dying cells with additional proliferation of surviving cells. Such proliferation can be localized (e.g., a regeneration blastema) or diffuse (compensatory growth). The relationship between such growth and the growth that occurs during development has not been characterized in detail. Drosophila melanogaster larval imaginal discs can recover from extensive damage, producing normally sized adult organs. Here we describe a system using genetic mosaics to screen for recessive mutations that impair compensatory growth. By generating clones of cells that carry a temperature-sensitive cell-lethal mutation, we conditionally ablate patches of tissue in the imaginal disc and assess the ability of the surviving sister clones to replace the lost tissue. We have used this system together with a modified whole-genome resequencing (WGS) strategy to identify several mutations that selectively compromise compensatory growth. We find specific alleles of bunched (bun) and Ribonucleoside diphosphate reductase large subunit (RnrL) reduce compensatory growth in the imaginal disc. Other genes identified in the screen, including two alleles of Topoisomerase 3-alpha (Top3α), while also required for developmental growth, appear to have an enhanced requirement during compensatory growth. Compensatory growth occurs at a higher rate than normal growth and may therefore have features in common with some types of overgrowth. Indeed, the RnrL allele identified compromises both these types of altered growth and mammalian ribonucleotide reductase and topoisomerases are targets of anticancer drugs. Finally, the approach we describe is applicable to the study of compensatory growth in diverse tissues in Drosophila.

  8. Hypoxia is a modifier of SMN2 splicing and disease severity in a severe SMA mouse model

    PubMed Central

    Bebee, Thomas W.; Dominguez, Catherine E.; Samadzadeh-Tarighat, Somayeh; Akehurst, Kristi L.; Chandler, Dawn S.

    2012-01-01

    Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease associated with low levels of the essential survival motor neuron (SMN) protein. Reduced levels of SMN is due to the loss of the SMN1 gene and inefficient splicing of the SMN2 gene caused by a C>T mutation in exon 7. Global analysis of the severe SMNΔ7 SMA mouse model revealed altered splicing and increased levels of the hypoxia-inducible transcript, Hif3alpha, at late stages of disease progression. Severe SMA patients also develop respiratory deficiency during disease progression. We sought to evaluate whether hypoxia was capable of altering SMN2 exon 7 splicing and whether increased oxygenation could modulate disease in a severe SMA mouse model. Hypoxia treatment in cell culture increased SMN2 exon 7 skipping and reduced SMN protein levels. Concordantly, the treatment of SMNΔ7 mice with hyperoxia treatment increased the inclusion of SMN2 exon 7 in skeletal muscles and resulted in improved motor function. Transfection splicing assays of SMN minigenes under hypoxia revealed that hypoxia-induced skipping is dependent on poor exon definition due to the SMN2 C>T mutation and suboptimal 5′ splice site. Hypoxia treatment in cell culture led to increased hnRNP A1 and Sam68 levels. Mutation of hnRNP A1-binding sites prevented hypoxia-induced skipping of SMN exon 7 and was found to bind both hnRNP A1 and Sam68. These results implicate hypoxic stress as a modulator of SMN2 exon 7 splicing in disease progression and a coordinated regulation by hnRNP A1 and Sam68 as modifiers of hypoxia-induced skipping of SMN exon 7. PMID:22763238

  9. The Conoid Associated Motor MyoH Is Indispensable for Toxoplasma gondii Entry and Exit from Host Cells.

    PubMed

    Graindorge, Arnault; Frénal, Karine; Jacot, Damien; Salamun, Julien; Marq, Jean Baptiste; Soldati-Favre, Dominique

    2016-01-01

    Many members of the phylum of Apicomplexa have adopted an obligate intracellular life style and critically depend on active invasion and egress from the infected cells to complete their lytic cycle. Toxoplasma gondii belongs to the coccidian subgroup of the Apicomplexa, and as such, the invasive tachyzoite contains an organelle termed the conoid at its extreme apex. This motile organelle consists of a unique polymer of tubulin fibres and protrudes in both gliding and invading parasites. The class XIV myosin A, which is conserved across the Apicomplexa phylum, is known to critically contribute to motility, invasion and egress from infected cells. The MyoA-glideosome is anchored to the inner membrane complex (IMC) and is assumed to translocate the components of the circular junction secreted by the micronemes and rhoptries, to the rear of the parasite. Here we comprehensively characterise the class XIV myosin H (MyoH) and its associated light chains. We show that the 3 alpha-tubulin suppressor domains, located in MyoH tail, are necessary to anchor this motor to the conoid. Despite the presence of an intact MyoA-glideosome, conditional disruption of TgMyoH severely compromises parasite motility, invasion and egress from infected cells. We demonstrate that MyoH is necessary for the translocation of the circular junction from the tip of the parasite, where secretory organelles exocytosis occurs, to the apical position where the IMC starts. This study attributes for the first time a direct function of the conoid in motility and invasion, and establishes the indispensable role of MyoH in initiating the first step of motility along this unique organelle, which is subsequently relayed by MyoA to enact effective gliding and invasion. PMID:26760042

  10. ENaC alpha-subunit variants are expressed in lung epithelial cells and are suppressed by oxidative stress.

    PubMed

    Xu, Haishan; Chu, Shijian

    2007-12-01

    Amiloride-sensitive epithelial sodium channel (ENaC) is a major sodium channel in the lung facilitating fluid absorption. ENaC is composed of alpha-, beta-, and gamma-subunits, and the alpha-subunit is indispensable for ENaC function in the lung. In human lungs, the alpha-subunit is expressed as various splice variants. Among them, alpha(1)- and alpha(2)-subunits are two major variants with different upstream regulatory sequences that possess similar channel characteristics when tested in Xenopus oocytes. Despite the importance of alpha-ENaC, little was known about the relative abundance of its variants in lung epithelial cells. Furthermore, lung infection and inflammation are often accompanied by reduced alpha-ENaC expression, oxidative stress, and pulmonary edema. However, it was not clear how oxidative stress affects expression of alpha-ENaC variants. In this study, we examined relative expression levels of alpha-subunit variants in four human lung epithelial cell lines. We also tested the hypothesis that oxidative stress inhibits alpha-ENaC expression. Our results show that both alpha(1)- and alpha(2)-ENaC variants are expressed in the cells we tested, but relative abundance varies. In the two monolayer-forming cell lines, H441 and Calu-3, alpha(2)-ENaC is the predominant variant. We also show that H(2)O(2) specifically suppresses alpha(1)- and alpha(2)-ENaC variant expression in H441 and Calu-3 cells in a dose-dependent fashion. This suppression is achieved by inhibition of their promoters and is attenuated by dexamethasone. These data demonstrate the importance of the alpha(2)-subunit variant and suggest that glucocorticoids and antioxidants may be useful in correcting infection/inflammation-induced lung fluid imbalance. PMID:17905853

  11. Regulation of human natural killer cell migration and proliferation by the exodus subfamily of CC chemokines.

    PubMed

    Robertson, M J; Williams, B T; Christopherson, K; Brahmi, Z; Hromas, R

    2000-01-10

    Natural killer (NK) cells play an important role in innate and adaptive immune responses to obligate intracellular pathogens. Nevertheless, the regulation of NK cell trafficking and migration to inflammatory sites is poorly understood. Exodus-1/MIP-3alpha/LARC, Exodus-2/6Ckine/SLC, and Exodus-3/MIP-3beta/ELC/CKbeta-11 are CC chemokines that share a unique aspartate-cysteine-cysteine-leucine motif near their amino terminus and preferentially stimulate the migration of T lymphocytes. The effects of Exodus chemokines on human NK cells were examined. Exodus-1, -2, and -3 did not induce detectable chemotaxis of resting peripheral blood NK cells. In contrast, Exodus-2 and -3 stimulated migration of polyclonal activated peripheral blood NK cells in a dose-dependent fashion. Exodus-2 and -3 also induced dose-dependent chemotaxis of NKL, an IL-2-dependent human NK cell line. Results of modified checkerboard assays indicate that migration of NKL cells in response to Exodus-2 and -3 represents true chemotaxis and not simply chemokinesis. Exodus-1, -2, and -3 did not induce NK cell proliferation in the absence of other stimuli. Nevertheless, Exodus-2 and -3 significantly augmented IL-2-induced proliferation of normal human CD56(dim) NK cells. In contrast, Exodus-1, -2, and -3 did not affect the cytolytic activity of resting or activated peripheral blood NK cells. Expression of message for CCR7, a shared receptor for Exodus-2 and -3, was detected in activated polyclonal NK cells and NKL cells but not resting NK cells. Taken together, these results indicate that Exodus-2 and -3 can participate in the recruitment and proliferation of activated NK cells. Exodus-2 and -3 may regulate interactions between T cells and NK cells that are crucial for the generation of optimal immune responses.

  12. A 28,000-dalton protein of normal mouse serum binds specifically to the inner core region of bacterial lipopolysaccharide.

    PubMed Central

    Brade, L; Brade, H

    1985-01-01

    Normal mouse serum was found to contain a protein, referred to here as factor, which binds to the inner core region of lipopolysaccharides (LPSs) of various bacterial families. Since factor-LPS interactions resulted in activation of guinea pig complement, factor activity could be assayed by a passive hemolysis test with sheep erythrocytes coated with LPS or lipid A from Acinetobacter calcoaceticus (which was found earlier to bind particularly well to factor). Factor was purified by G-50 and hydroxyapatite chromatography whereby the specific hemolytic activity was enriched 1,675-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed the presence of a 28,000-dalton protein as the main band. The identity of this band was determined by absorption experiments with LPS-coated sheep erythrocytes or latex beads, whereby the 28,000-dalton band disappeared after specific absorption and could be recovered from the absorbent. The binding specificity of factor was determined in a passive hemolysis inhibition assay with defined oligosaccharides representative for the inner core region of LPS. Thus, the di- and trisaccharides alpha-D-mannoheptopyranosyl-(1----5)-2-keto-3-deoxy-D-mannoocto nic acid and alpha-D-mannoheptopyranosyl-(1----3)-alpha-D-mannoheptopyranosy l-(1----5)-2- keto-3-deoxy-D-mannooctonic acid, respectively, were able to inhibit binding of factor to LPS. The results are in accordance with our earlier observation that the heptose-2-keto-3-deoxy-D-mannooctonic acid region represents a common antigen of bacterial LPS. Rabbit hyperimmune serum directed against this common antigen and purified factor was found to exhibit the same specificity for LPS. Factor activity was followed in mice in vivo after injection of LPS; it disappeared completely 15 min after the injection of LPS and reappeared within 1 h. Images PMID:4066028

  13. Mechanisms of endothelial cell protection by blockade of the JAK2 pathway.

    PubMed

    Neria, Fernando; Caramelo, Carlos; Peinado, Héctor; González-Pacheco, Francisco R; Deudero, Juan J P; de Solis, Alain J; Fernández-Sánchez, Ruth; Peñate, Silvia; Cano, Amparo; Castilla, María Angeles

    2007-03-01

    Inhibition of the JAK2/STAT pathway has been implicated recently in cytoprotective mechanisms in both vascular smooth muscle cells and astrocytes. The advent of JAK2-specific inhibitors provides a practical tool for the study of this pathway in different cellular types. An interest in finding methods to improve endothelial cell (EC) resistance to injury led us to examine the effect of JAK2/STAT inhibition on EC protection. Furthermore, the signaling pathways involved in JAK2/STAT inhibition-related actions were examined. Our results reveal, for the first time, that blockade of JAK2 with the tyrosine kinase inhibitor AG490 strongly protects cultured EC against cell detachment-dependent death and serum deprivation and increases reseeding efficiency. Confirmation of the specificity of the effects of JAK2 inhibition was attained by finding protective effects on transfection with a dominant negative JAK2. Furthermore, AG490 blocked serum deprivation-induced phosphorylation of JAK2. In terms of mechanism, treatment with AG490 induces several relevant responses, both in monolayer and detached cells. These mechanisms include the following: 1) Increase and nuclear translocation of the active, dephosphorylated form of beta-catenin. In functional terms, this translocation is transcriptionally active, and its protective effect is further supported by the stimulation of EC cytoprotection by transfectionally induced excess of beta-catenin. 2) Increase of platelet endothelial cell adhesion molecule (PECAM)/CD31 levels. 3) Increase in total and phosphorylated AKT. 4) Increase in phosphorylated glycogen synthase kinase (GSK)3alpha/beta. The present findings imply potential practical applications of JAK2 inhibition on EC. These applications affect not only EC in the monolayer but also circulating detached cells and involve mechanistic interactions not previously described. PMID:17035297

  14. Lung macrophage uptake of unopsonized environmental particulates: Role of scavenger-type receptors

    SciTech Connect

    Kobzik, L.

    1995-07-01

    The receptors responsible for avid alveolar macrophage (AM) phagocytosis of unopsonized environmental particulates have not been well defined. This study used flow cytometry to quantitate the effects of a panel of soluble ligands for macrophage adhesion receptors on AM binding of unopsonized environmental dusts (titanium dioxide, TiO{sub 2}; iron oxide, Fe{sub 2}O{sub 3}; {alpha}-quartz, SiO{sub 2}; diesel engine exhaust dust) or fluorescent latex beads. Polyanionic ligands of the macrophage scavenger receptor (SR) for acetylated-LDL caused marked inhibition of AM binding of the oxide particles and latex beads (e.g., TiO{sub 2} binding; polyinosinic acid (polyl), 10 {mu}g/ml: 70.2 {+-} 1.5% inhibition, mean {+-} SE, n = 11). In contrast, no inhibition was seen with the polyanions heparin and chondroitin sulfate (chond-S), or dextran, consistent with the known inhibitor profile of macrophage SRs for acetylated-LDL. AM uptake of latex or SiO{sub 2} beads instilled into lungs of hamsters was inhibited by administration of polyl but not chondroitin sulfate (AM beads per cell: control, 6.1 {+-} 0.7; polyl, 3.5 {+-} 0.2; chond-S, 5.1 {+-} 0.7, n {ge} 4, p < 0.05 for control vs polyl) indicating macrophage SRs operate in vivo as well as in vitro. In contrast, AM binding of the carbonaceous diesel dust particles was not inhibited by any ligand tested. AM uptake of unopsonized TiO{sub 2}, SR ligands or acetylated LDL caused no significant activation of AM respiratory burst or TNF production, consistent with past observations that opsonin-independent phagocytosis of inert particles by normal AMs is not accompanied by pro-inflammatory activation. These data implicate macrophage-type SRs in AM binding of charged environmental particles and indicate that distinct mechanisms mediate binding of carbonaceous dusts. 54 refs., 7 figs., 4 tabs.

  15. The Conoid Associated Motor MyoH Is Indispensable for Toxoplasma gondii Entry and Exit from Host Cells

    PubMed Central

    Graindorge, Arnault; Salamun, Julien; Marq, Jean Baptiste; Soldati-Favre, Dominique

    2016-01-01

    Many members of the phylum of Apicomplexa have adopted an obligate intracellular life style and critically depend on active invasion and egress from the infected cells to complete their lytic cycle. Toxoplasma gondii belongs to the coccidian subgroup of the Apicomplexa, and as such, the invasive tachyzoite contains an organelle termed the conoid at its extreme apex. This motile organelle consists of a unique polymer of tubulin fibres and protrudes in both gliding and invading parasites. The class XIV myosin A, which is conserved across the Apicomplexa phylum, is known to critically contribute to motility, invasion and egress from infected cells. The MyoA-glideosome is anchored to the inner membrane complex (IMC) and is assumed to translocate the components of the circular junction secreted by the micronemes and rhoptries, to the rear of the parasite. Here we comprehensively characterise the class XIV myosin H (MyoH) and its associated light chains. We show that the 3 alpha-tubulin suppressor domains, located in MyoH tail, are necessary to anchor this motor to the conoid. Despite the presence of an intact MyoA-glideosome, conditional disruption of TgMyoH severely compromises parasite motility, invasion and egress from infected cells. We demonstrate that MyoH is necessary for the translocation of the circular junction from the tip of the parasite, where secretory organelles exocytosis occurs, to the apical position where the IMC starts. This study attributes for the first time a direct function of the conoid in motility and invasion, and establishes the indispensable role of MyoH in initiating the first step of motility along this unique organelle, which is subsequently relayed by MyoA to enact effective gliding and invasion. PMID:26760042

  16. Genetic epidemiology of pelvic organ prolapse: a systematic review.

    PubMed

    Ward, Renée M; Velez Edwards, Digna R; Edwards, Todd; Giri, Ayush; Jerome, Rebecca N; Wu, Jennifer M

    2014-10-01

    Given current evidence supporting a genetic predisposition for pelvic organ prolapse, we conducted a systematic review of published literature on the genetic epidemiology of pelvic organ prolapse. Inclusion criteria were linkage studies, candidate gene association and genome-wide association studies in adult women published in English and indexed in PubMed through Dec. 2012, with no limit on date of publication. Methodology adhered to the PRISMA guidelines. Data were systematically extracted by 2 reviewers and graded by the Venice criteria for studies of genetic associations. A metaanalysis was performed on all single nucleotide polymorphisms evaluated by 2 or more studies with similar methodology. The metaanalysis suggests that collagen type 3 alpha 1 (COL3A1) rs1800255 genotype AA is associated with pelvic organ prolapse (odds ratio, 4.79; 95% confidence interval, 1.91-11.98; P = .001) compared with the reference genotype GG in populations of Asian and Dutch women. There was little evidence of heterogeneity for rs1800255 (P value for heterogeneity = .94; proportion of variance because of heterogeneity, I(2) = 0.00%). There was insufficient evidence to determine whether other single nucleotide polymorphisms evaluated by 2 or more papers were associated with pelvic organ prolapse. An association with pelvic organ prolapse was seen in individual studies for estrogen receptor alpha (ER-α) rs2228480 GA, COL3A1 exon 31, chromosome 9q21 (heterogeneity logarithm of the odds score 3.41) as well as 6 single nucleotide polymorphisms identified by a genome-wide association study. Overall, individual studies were of small sample size and often of poor quality. Future studies would benefit from more rigorous study design as outlined in the Venice recommendations. PMID:24721264

  17. In vitro effects of glutamate and N-methyl-D-aspartate receptor (NMDAR) antagonism on human tendon derived cells.

    PubMed

    Dean, Benjamin John Floyd; Snelling, Sarah J B; Dakin, Stephanie Georgina; Javaid, Muhammad Kassim; Carr, Andrew Jonathan

    2015-10-01

    It is known that extracellular glutamate concentrations are increased in tendinopathy but the effects of glutamate upon human tendon derived cells are unknown. The primary purpose was to investigate the effect of glutamate exposure on human tendon-derived cells in terms of viability, protein, and gene expression. The second purpose was to assess whether NMDAR antagonism would affect the response of tendon-derived cells to glutamate exposure. Human tendon-derived cells were obtained from supraspinatus tendon tissue obtained during rotator cuff repair (tendon tear derived cells) and from healthy hamstring tendon tissue (control cells). The in vitro impact of glutamate exposure and NMDAR antagonism (MK-801) was measured using the Alamar blue cell viability assay, immunocytochemistry, and quantitative real-time PCR. Glutamate reduced cell viability at 24 h in tendon tear derived cells but not in control cells at concentrations of 7.5 mM and above. Cell viability was significantly reduced after 72 h of 1.875 mM glutamate in both cell groups; this deleterious effect was attenuated by NMDAR antagonism with 10 µM MK-801. Both 24 and 72 h of 1.875 mM glutamate exposure reduced Type 1 alpha 1 collagen (COL1A1) and Type 3 alpha 1 collagen (COL3A1) gene expression, but increased Aggrecan gene expression. We propose that these effects of glutamate on tendon derived cells including reduced cell viability and altered matrix gene expression contribute to the pathogenesis of tendinopathy. PMID:26041147

  18. Allopregnanolone inhibits learning in the Morris water maze.

    PubMed

    Johansson, Inga Maj; Birzniece, Vita; Lindblad, Charlotte; Olsson, Tommy; Bäckström, Torbjörn

    2002-05-01

    The progesterone metabolite allopregnanolone (3alpha-OH-5alpha-pregnane-20-one) inhibits neural functions, enhancing the GABA induced GABA(A) receptor activation. This effect is benzodiazepine like and benzodiazepines are known to impair memory. Acute effects of allopregnanolone on the hippocampus dependent spatial learning in the Morris water maze have not been studied. Adult male Wistar rats where injected (i.v.) with allopregnanolone (2 mg/kg), or vehicle, daily for 11 days. At 8 or 20 min after each injection, studies of place navigation were performed in the Morris water maze. Allopregnanolone concentrations in plasma and in nine different brain areas where analyzed by radioimmunoassay. The latency to find the platform was increased 8 min after the allopregnanolone injection, while normal learning was seen after 20 min. Swim speed did not differ between groups. A higher number of rats were swimming close to the pool wall (thigmotaxis) in the 8 min allopregnanolone group compared to the other groups. Allopregnanolone concentrations in the brain tissue at 8 min were 1.5 to 2.5 times higher then at 20 min after the allopregnanolone injections. After vehicle injections the brain concentrations of allopregnanolone were at control levels. Plasma concentrations of allopregnanolone followed the same pattern as in the brain, with the exception of an increase 8 min after vehicle injections. The natural progesterone metabolite allopregnanolone can inhibit learning in the Morris water maze, an effect not caused by motor impairment. The learning impairment might be due to a combination of changed swimming behavior and difficulties in navigation.

  19. Behavioural effects of fluoxetine and tianeptine, two antidepressants with opposite action mechanisms, in rats.

    PubMed

    Nowakowska, E; Kus, K; Chodera, A; Rybakowski, J

    2000-01-01

    The behavioural effects of two antidepressants with opposite molecular mechanisms, tianeptine 7-[(3-chloro-6,11-dihydro-6-methyldibenzo[c,f][1,2]thiazepin - 11-yl)amino]heptanoic acid S,S-dioxide, CAS 66981-73-5) 5 mg/kg p.o., a serotonin reuptake enhancer, and fluoxetine (+/-)-N-methyl-3-phenyl-3-[(alpha, alpha, alpha-trifluoro-p- tolyl)oxy]propylamine, CAS 54910-89-3) 5 mg/kg p.o., a serotonin reuptake inhibitor, were compared after single and prolonged administration (7 and 14 days) once daily). In all experiments the drug effects were noted at the peak activity time: 30 min after tianeptine and 60 min after fluoxetine administration. In the immobility time test both drugs had a shortening effect on immobility time only after prolonged administration or, in single treatment, after joint administration. A different pattern was observed in the two compartment test: both antidepressants showed anxiolytic effects after single and prolonged treatment. However, when the drugs were given in joint administration, the anxiolytic effects were entirely abolished after single as well as prolonged treatment. In reference spatial memory test (food finding time in the maze) tianeptine had no effect, whereas fluoxetine caused, after single and prolonged treatment, a very marked improvement of reference memory. Joint administration of both drugs resulted in worsening the effects on memory in comparison to fluoxetine alone, but the results were still significantly better vs. control. In the test for sedative action (in the Activity Meter AM-1, where the movements of the animals are counted electronically) only after prolonged treatment with tianeptine a diminished locomotor activity could be observed. It is concluded that in the action of the drugs (beside the effect on serotonin uptake) other mechanisms must play an important role. The diminished locomotor activity after tianeptine suggests an influence on the dopaminergic or GABA-Receptor system.

  20. Powerful beneficial effects of benfotiamine on cognitive impairment and beta-amyloid deposition in amyloid precursor protein/presenilin-1 transgenic mice.

    PubMed

    Pan, Xiaoli; Gong, Neng; Zhao, Jing; Yu, Zhe; Gu, Fenghua; Chen, Jia; Sun, Xiaojing; Zhao, Lei; Yu, Meijing; Xu, Zhiru; Dong, Wenxin; Qin, Yan; Fei, Guoqiang; Zhong, Chunjiu; Xu, Tian-Le

    2010-05-01

    Reduction of glucose metabolism in brain is one of the main features of Alzheimer's disease. Thiamine (vitamin B1)-dependent processes are critical in glucose metabolism and have been found to be impaired in brains from patients with Alzheimer's disease. However, thiamine treatment exerts little beneficial effect in these patients. Here, we tested the effect of benfotiamine, a thiamine derivative with better bioavailability than thiamine, on cognitive impairment and pathology alterations in a mouse model of Alzheimer's disease, the amyloid precursor protein/presenilin-1 transgenic mouse. We show that after a chronic 8 week treatment, benfotiamine dose-dependently enhanced the spatial memory of amyloid precursor protein/presenilin-1 mice in the Morris water maze test. Furthermore, benfotiamine effectively reduced both amyloid plaque numbers and phosphorylated tau levels in cortical areas of the transgenic mice brains. Unexpectedly, these effects were not mimicked by another lipophilic thiamine derivative, fursultiamine, although both benfotiamine and fursultiamine were effective in increasing the levels of free thiamine in the brain. Most notably, benfotiamine, but not fursultiamine, significantly elevated the phosphorylation level of glycogen synthase kinase-3alpha and -3beta, and reduced their enzymatic activities in the amyloid precursor protein/presenilin-1 transgenic brain. Therefore, in the animal Alzheimer's disease model, benfotiamine appears to improve the cognitive function and reduce amyloid deposition via thiamine-independent mechanisms, which are likely to include the suppression of glycogen synthase kinase-3 activities. These results suggest that, unlike many other thiamine-related drugs, benfotiamine may be beneficial for clinical Alzheimer's disease treatment.

  1. KDN-containing glycoprotein from loach skin mucus.

    PubMed

    Nakagawa, H; Hama, Y; Sumi, T; Li, S C; Li, Y T

    2001-01-01

    It has been widely recognized that the mucus coat of fish plays a variety of important physical, chemical, and physiological functions. One of the major constituents of the mucus coat is mucus glycoprotein. We found that sialic acids in the skin mucus of the loach, Misgurnus anguillicaudatus, consisted predominantly of KDN. Subsequently, we isolated KDN-containing glycoprotein from loach skin mucus and characterized its chemical nature and structure. Loach mucus glycoprotein was purified from the Tris-HCl buffer extract of loach skin mucus by DEAE-cellulose chromatography, Nuclease P1 treatment, and Sepharose CL-6B gel filtration. The purified mucus glycoprotein was found to contain 38.5 KDN, 0.5% NeuAc, 25.0% GalNAc, 3.5% Gal, 0.5% GlcNAc and 28% amino acids. Exhaustive Actinase digestion of the glycoprotein yielded a glycopeptide with a higher sugar content and higher Thr and Ser contents. The molecular size of this glycopeptide was approximately 1/12 of the intact glycoprotein. These results suggest that approximately 11 highly glycosylated polypeptide units are linked in tandem through nonglycosylated peptides to form the glycoporotein molecule. The oligosaccharide alditols liberated from the loach mucus glycoprotein by alkaline borohydride treatment were separated by Sephadex G-25 gel filtration and HPLC. The purified sugar chains were analyzed b --> 6GalNAc-ol, KDNalpha2 --> 3(GalNAcbeta1 --> 14)GalNAc-ol, KDNalpha2 --> 6(GalNAcalpha1 --> 3)GalNAc-ol, KDNalpha2 --> 6(Gal3alpha1--> 3)GalNAc-ol, and NeuAcalpha2 --> 6Gal NAc-ol. It is estimated that one loach mucus glycoprotein molecule contains more than 500 KDN-containing sugar chains that are linked to Thr and Ser residues of the protein core through GalNAc.

  2. Biochemical and immunological properties of cytokines conjugated to dendritic polymers.

    PubMed

    Lee, S C; Parthasarathy, R; Botwin, K; Kunneman, D; Rowold, E; Lange, G; Klover, J; Abegg, A; Zobel, J; Beck, T; Miller, T; Hood, W; Monahan, J; McKearn, J P; Jansson, R; Voliva, C F

    2004-09-01

    Here we describe a post-translational modification of SC-63032, a variant of the species restricted, multi-lineage hematopoeitic factor human interleukin-3 (hIL-3). We have made two new dendritic polymer (polyamidoamine or PAMAM dendrimers, generation 5)-SC-63032 bioconjugates. Using two distinct chemistries (one of which is novel to this work), we achieved site-specific conjugation with respect to the amino acid in the proteins ligated to the dendrimers. In both bioconjugates, conjugated cytokine maintains its ability to bind the hIL-3 alpha receptor subunit, but is significantly (about 10-fold) less potent in inducing hIL-3 dependent in vitro cell proliferation than is the free cytokine. In vivo data indicates that conjugation decreases the immunogenicity of the conjugated cytokine modestly. In the absence of pharmacokinetic or biodistribution effects associated with the bioconjugates that increase their potency in vivo (which can only be tested in a higher primate, due to the species restriction of hIL-3 and its derivatives), these immune mitigation effects may be too small to be therapeutically significant. Though unmodified PAMAM dendrimers fail to elicit an antibody response in mice, protein conjugation to dendrimers haptenizes them, and a dendrimer-specific antibody response is produced. In toto, the principal limitation of the dendrimer-cytokine bioconjugates herein is in their reduced receptor affinity and potency in vitro. Were the in vivo potency of the bioconjugates to parallel the in vitro potency of the conjugates reported here, it is likely that particular dendrimer bioconjugates could not justify their higher costs of goods relative to the parent SC-63032 molecule, though retention of SC-63032 biological activities in conjugates suggests that other cytokine-dendrimer bioconjugates may be bioactive. This is good news to the nanotechnology community, in as much as PAMAM dendrimers are among the monodisperse polymeric nanomaterials available, and these

  3. Cooperation of structural proteins during late events in the life cycle of polyomavirus.

    PubMed Central

    Forstová, J; Krauzewicz, N; Wallace, S; Street, A J; Dilworth, S M; Beard, S; Griffin, B E

    1993-01-01

    The polyomavirus minor late capsid antigen, VP2, is myristylated on its N-terminal glycine, this modification being required for efficient infection of mouse cells. To study further the functions of this antigen, as well as those of the other minor late antigen, VP3, recombinant baculoviruses carrying genes for VP1, VP2, and VP3 have been constructed and the corresponding proteins have been synthesized in insect cells. A monoclonal antibody recognizing VP1, alpha-PyVP1-A, and two monoclonal antibodies against the common region of VP2 and VP3, alpha-PyVP2/3-A and alpha-PyVP2/3-B, have been generated. Reactions of antibodies with antigens were characterized by indirect immunofluorescence, immunoprecipitation, and immunoblot analysis. Immunofluorescent staining of mouse cells infected with polyomavirus showed all antigens to be localized in nuclei. When the late polyomavirus proteins were expressed separately in insect cells, however, only VP1 was efficiently transported into the nucleus; VP2 was localized discretely around the outside of the nucleus, and VP3 exhibited a diffused staining pattern in the cytoplasm. Coexpression of VP2, or VP3, with VP1 restored nuclear localization. Immunoprecipitation of infected mouse cells with either anti-VP1 or anti-VP2/3 antibodies precipitated complexes containing all three species, consistent with the notion that VP1 is necessary for efficient transport of VP2 and VP3 into the nucleus. Purified empty capsid-like particles, formed in nuclei of insect cells coinfected with all three baculoviruses, contained VP2 and VP3 proteins in amounts comparable to those found in empty capsids purified from mouse cells infected with wild-type polyomavirus. Two-dimensional gel analysis of VP1 species revealed that coexpression with VP2 affects posttranslational modification of VP1. Images PMID:8382302

  4. Comparative analysis of the noncollagenous NC1 domain of type IV collagen: identification of structural features important for assembly, function, and pathogenesis.

    PubMed Central

    Netzer, K. O.; Suzuki, K.; Itoh, Y.; Hudson, B. G.; Khalifah, R. G.

    1998-01-01

    Type IV collagen alpha1-alpha6 chains have important roles in the assembly of basement membranes and are implicated in the pathogenesis of Goodpasture syndrome, an autoimmune disorder, and Alport syndrome, a hereditary renal disease. We report comparative sequence analyses and structural predictions of the noncollagenous C-terminal globular NC1 domain (28 sequences). The inferred tree verified that type IV collagen sequences fall into two groups, alpha1-like and alpha2-like, and suggested that vertebrate alpha3/alpha4 sequences evolved before alpha1/alpha2 and alpha5/alpha6. About one fifth of NC1 residues were identified to confer either the alpha1 or alpha2 group-specificity. These residues accumulate opposite charge in subdomain B of alpha1 (positive) and alpha2 (negative) sequences and may play a role in the stoichiometric chain selection upon type IV collagen assembly. Neural network secondary structure prediction on multiple aligned sequences revealed a subdomain core structure consisting of six hydrophobic beta-strands and one short alpha-helix with a significant hydrophobic moment. The existence of opposite charges in the alpha-helices may carry implications for intersubdomain interactions. The results provide a rationale for defining the epitope that binds Goodpasture autoantibodies and a framework for understanding how certain NC1 mutations may lead to Alport syndrome. A search algorithm, based entirely on amino acid properties, yielded a possible similarity of NC1 to tissue inhibitor of metalloproteinases (TIMP) and prompted an investigation of a possible functional relationship. The results indicate that NC1 preparations decrease the activity of matrix metalloproteinases 2 and 3 (MMP-2, MMP-3) toward a peptide substrate, though not to [14C]-gelatin. We suggest that an ancestral NC1 may have been incorporated into type IV collagen as an evolutionarily mobile domain carrying proteinase inhibitor function. PMID:9655338

  5. Atmospheric chemistry of CF{sub 3}C(O)OCH{sub 2}CF{sub 3}: UV spectra and kinetic data for CF{sub 3}C(O)OCH({sm_bullet})CF{sub 3} and CF{sub 3}C(O)OCH(OO{sm_bullet})CF{sub 3} radicals, and atmospheric fate of CF{sub 3}C(O)OCH(O{sm_bullet})CF{sub 3} radicals

    SciTech Connect

    Stein, T.N.N.; Christensen, L.K.; Platz, J.; Sehested, J.; Nielsen, O.J.; Wallington, T.J.

    1999-07-22

    Recognition of the adverse effect of chlorofluorocarbon (CFC) release into the atmosphere has led to an international effort to replace CFCs with environmentally acceptable alternatives. Hydrofluoroethers (HFEs) are fluids designed to replace CFCs in applications such as the cleaning of electronic equipment, heat transfer agents in refrigeration systems, and carrier fluids for lubricant deposition. HFEs are volatile compounds and will be released into the atmosphere during its use. In the atmosphere, photochemical oxidation of HFEs will lead to the formation of fluorinated esters and fluorinated formates. The atmospheric fate of these products is unknown at the present. To improve their understanding of the atmospheric chemistry of esters the authors have studied the atmospheric chemistry of 2,2,2-trifluoroethyltrifluoroacetate CF{sub 3}C(O)OCH{sub 2}CF{sub 3} (bp = 55.0 C). This compound provides insight into the behavior of alkyl, alkyl peroxy, and alkoxy radicals formed {alpha} to the ester functionality. The atmospheric fate of CF{sub 3}C(O)OCH(O{sup {sm_bullet}})CF{sub 3} radicals was investigated in a FTIR smog chamber. Three loss processes for the CF{sub 3}C(O)OCH(O{sup {sm_bullet}})CF{sub 3} radicals were identified at 296 K and 700 Torr total pressure, reaction with O{sub 2} to form CF{sub 3}C(O)OC(O)CF{sub 3}, {alpha}-rearrangement to form CF{sub 3}C(O){sm_bullet} radicals and CF{sub 3}C(O)OH, and decomposition via a mechanism which is unclear. In 760 Torr of air at 296 K, 65% of the CF{sub 3}C(O)OCH(O{sm_bullet})CF{sub 3} radicals react with oxygen, 18% undergo {alpha}-rearrangement, while the fate of the remaining 17% is unclear.

  6. Initial investigation of the potential of modified porcine erythrocytes for transfusion in primates.

    PubMed

    Eckermann, Jan M; Buhler, Leo H; Zhu, Alex; Dor, Frank J M F; Awwad, Michel; Cooper, David K C

    2004-01-01

    There is a shortage of human blood for transfusion. The possibility of using alpha-galactosidase-treated pig red blood cells (pRBCs) for transfusion into humans has been investigated. pRBCs were treated in vitro with alpha-galactosidase. In vitro binding of antibodies (Abs) in baboon or human sera to untreated/treated pRBCs was assessed by flow cytometry and serum cytotoxicity. In vivo clearance rates of (1) autologous baboon red blood cells (RBCs), (2) unmodified pRBCs, and (3) alpha-galactosidase-treated pRBCs were measured after transfusion into baboons receiving either no treatment or depletion of complement +/- depletion of anti-Gal alpha 1-3Gal (Gal) Ab or of macrophage phagocytes. In vitro binding of baboon or human Abs to treated pRBCs was absent or minimal compared with untreated pRBCs, and serum cytotoxicity was completely inhibited. In vivo autologous baboon RBCs survived for >16 days and unmodified pRBCs for <15 min in an untreated baboon. Treated pRBCs survived for 2 h in an untreated baboon, for 24 h in a complement-depleted baboon, and for 72 h when the baboon was depleted of both complement and anti-Gal Ab, or of complement and macrophage phagocytes. All baboons, however, became sensitized to Gal antigens. Failure to prolong the in vivo survival of treated pRBCs could be due to inadequate removal of Gal epitopes because sensitization to Gal developed, or could imply other, as yet unidentified, causes for RBC destruction. To fully assess the potential of pRBC transfusion in humans, more complete alpha-galactosidase treatment of pRBCs will be required.

  7. Cultured Ito cells of rat liver express the alpha 2-macroglobulin gene.

    PubMed

    Andus, T; Ramadori, G; Heinrich, P C; Knittel, T; Meyer zum Büschenfelde, K H

    1987-11-01

    Ito cells were isolated from rat liver and kept in culture for up to 13 days. The capability of the Ito cells to synthesize alpha 2-macroglobulin was analyzed at different times after isolation and by pulse-chase experiments. Newly synthesized alpha 2-macroglobulin was determined by immunoprecipitation followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. alpha 2-Macroglobulin synthesis was hardly detectable in Ito cells and their media 3 days after plating. However, 5-11 days after the isolation of the cells, increasing amounts of alpha 2-macroglobulin were synthesized. The results of pulse-chase experiments performed on day 7 showed that radioactively labeled alpha 2-macroglobulin decreased in the intracellular compartment and increased in the culture medium. alpha 2-Macroglobulin was identified by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions. Furthermore, when unlabeled alpha 2-macroglobulin was added during the immunoprecipitation, a competition was observed. Incubation of pancreatic elastase with culture medium of rat Ito cells or rat hepatocytes led to the same cleavage products as found with alpha 2-macroglobulin. alpha 2-Macroglobulin-specific mRNA could be demonstrated by Northern blot analysis of total RNA extracted from rat Ito cells. Under the conditions where alpha 2-macroglobulin was synthesized in Ito cells, no synthesis of alpha 1-macroglobulin, alpha 1-inhibitor 3, alpha 1-proteinase inhibitor, alpha 1-acid glycoprotein, alpha 1-acute-phase globulin (T-kininogen) and albumin could be demonstrated. It is concluded that alpha 2-macroglobulin is a true secretory protein of rat Ito cells in culture. This could be of importance for collagen metabolism in liver diseases. PMID:2444437

  8. Glucuronoxylomannan of Cryptococcus neoformans serotype B: structural analysis by gas-liquid chromatography-mass spectrometry and 13C-nuclear magnetic resonance spectroscopy.

    PubMed

    Turner, S H; Cherniak, R

    1991-04-01

    The major extracellular polysaccharide (glucuronoxylomannan, GXM) from six strains of Cryptococcus neoformans serotype B was characterized by gas-liquid chromatography (g.l.c.), g.l.c.-mass spectrometry (g.l.c.-m.s.), and nuclear magnetic resonance (n.m.r.) spectroscopy. Ultrasonic irradiation (u.i.) was used to reduce the mol.wt. of native GXM from 9.75 x 10(5) to 1.15 x 10(5) without apparent change in its composition (GXM-S). The Xylp:Manp:GlcpA molar ratio of the GXM and GXM-S from the six strains of C. neoformans serotype B is approximately 3.5:3.0:0.6. GXM-S was O-deacetylated (GXM-D) by treatment with NH4OH. The 13C-n.m.r. analysis of GXM-D gave spectra that served as characteristic fingerprints of the structure and also facilitated the assignment of the anomeric carbon resonances to specific structural moieties present in GXM-D. The GXM-D from each serotype B strain was found to be similar by 13C-n.m.r. spectroscopy. The structure contains a linear (1----3)-alpha-D-Manp backbone substituted with 2-O-beta-GlcpA and 2-O-beta-Xylp. beta-Xylp is also O-4 linked to the Manp substituted with GlcpA. In addition, a model for the disposition of the Xylp and GlcpA side chain substituents along the mannopyranan backbone is proposed, based upon results from the combination of g.l.c.-m.s. and 13C-n.m.r. spectroscopy. PMID:1773425

  9. Cross-talk between the two divergent insulin signaling pathways is revealed by the protein kinase B (Akt)-mediated phosphorylation of adapter protein APS on serine 588.

    PubMed

    Katsanakis, Kostas D; Pillay, Tahir S

    2005-11-11

    The APS adapter protein is recruited to the autophosphorylated kinase domain of the insulin receptor and initiates the phosphatidylinositol 3-kinase (PI3K)-independent pathway of insulin-stimulated glucose transport by recruiting CAP and c-Cbl. In this study, we have identified APS as a novel substrate for protein kinase B/Akt using an antibody that exhibits insulin-dependent immunoreactivity with a phosphospecific antibody raised against the protein kinase B substrate consensus sequence RXRXX(pS/pT) and a phosphospecific antibody that recognizes serine 21/9 of glycogen synthase kinase-3alpha/beta. This phosphorylation of APS is observed in both 3T3-L1 adipocytes and transfected cells. The insulin-stimulated serine phosphorylation of APS was inhibited by a PI3-kinase inhibitor, LY290004, a specific protein kinase B (PKB) inhibitor, deguelin, and knockdown of Akt. Serine 588 of APS is contained in a protein kinase B consensus sequence for phosphorylation conserved in APS across multiple species but not found in other members of this family, including SH2-B and Lnk. Mutation of serine 588 to alanine abolished the insulin-stimulated serine phosphorylation of APS and prevented the localization of APS to membrane ruffles. A glutathione S-transferase fusion protein containing amino acids 534-621 of APS was phosphorylated by purified PKB in vitro, and mutation of serine 588 abolished the PKB-mediated phosphorylation of APS in vitro. Taken together, this study identifies APS as a novel physiological substrate for PKB and the first serine phosphorylation site on APS. These data therefore reveal the molecular cross-talk between the insulin-activated PI3-kinase-dependent and -independent pathways previously thought to be distinct and divergent.

  10. Participation of mitochondrial diazepam binding inhibitor receptors in the anticonflict, antineophobic and anticonvulsant action of 2-aryl-3-indoleacetamide and imidazopyridine derivatives.

    PubMed

    Auta, J; Romeo, E; Kozikowski, A; Ma, D; Costa, E; Guidotti, A

    1993-05-01

    The 2-hexyl-indoleacetamide derivative, FGIN-1-27 [N,N-di-n-hexyl-2- (4-fluorophenyl)indole-3-acetamide], and the imidazopyridine derivative, alpidem, both bind with high affinity to glial mitochondrial diazepam binding inhibitor receptors (MDR) and increase mitochondrial steroidogenesis. Although FGIN-1-27 is selective for the MDR, alpidem also binds to the allosteric modulatory site of the gamma-aminobutyric acidA receptor where the benzodiazepines bind. FGIN-1-27 and alpidem, like the neurosteroid 3 alpha,21-dehydroxy-5 alpha-pregnane-20-one (THDOC), clonazepam and zolpidem (the direct allosteric modulators of gamma-aminobutyric acidA receptors) delay the onset of isoniazid and metrazol-induced convulsions. The anti-isoniazid convulsant action of FGIN-1-27 and alpidem, but not that of THDOC, is blocked by PK 11195. In contrast, flumazenil blocked completely the anticonvulsant action of clonazepam and zolpidem and partially blocked that of alpidem, but it did not affect the anticonvulsant action of THDOC and FGIN-1-27. Alpidem, like clonazepam, zolpidem and diazepam, but not THDOC or FGIN-1-27, delay the onset of bicuculline-induced convulsions. In two animal models of anxiety, the neophobic behavior in the elevated plus maze test and the conflict-punishment behavior in the Vogel conflict test, THDOC and FGIN-1-27 elicited anxiolytic-like effects in a manner that is flumazenil insensitive, whereas alpidem elicited a similar anxiolytic effect, but is partially blocked by flumazenil. Whereas PK 11195 blocked the effect of FGIN-1-27 and partially blocked alpidem, it did not affect THDOC in both animal models of anxiety.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. 2-Aryl-3-indoleacetamides (FGIN-1): a new class of potent and specific ligands for the mitochondrial DBI receptor (MDR).

    PubMed

    Romeo, E; Auta, J; Kozikowski, A P; Ma, D; Papadopoulos, V; Puia, G; Costa, E; Guidotti, A

    1992-09-01

    The 2 aryl-3-indoleacetamides (FGIN-1) are a new class of compounds that potently (nM) and selectively bind to glial mitochondrial diazepam binding inhibitor (DBI) receptors (MDR), previously called peripheral benzodiazepine receptors, and increase mitochondrial steroidogenesis. The high-affinity binding of FGIN-1 to MDR derivatives depends on the following chemical characteristics: 1) the dialkylation of the amide; 2) the chain length of this alkyl substitution; and 3) the halogenation of aryl groups appended to the indole nucleus. FGIN-1 derivatives do not bind to gamma-aminobutyric acid (GABAA), GABAB, glycine, glutamate, dopamine, serotonin, opiate, cholecystokinin, beta adrenergic, cannabinoid or sigma receptors. FGIN-1-27 [N, N-di-n-hexyl 2-(4-fluorophenyl)indole-3-acetamide] enters the brain, and for this reason, this FGIN-1 compound is potent and efficacious behaviorally. Like the neurosteroid 3 alpha-5 alpha tetrahydrodeoxycorticosterone (THDOC), FGIN-1-27 delays the onset of isoniazid-induced convulsions, but fails to delay the onset of bicuculline-induced convulsions. However, differently from THDOC, the FGIN-1-27 anticonvulsant action is blocked by the isoquinoline carboxamide PK 11195. In the elevated plus maze test, FGIN-1-27 inhibits neophobia manner that is antagonized by PK 11195 but not by flumazenil. Because FGIN-1-27 binds to MDR and does not bind to the GABAA receptors, it is inferred that FGIN-1-27 may act on GABAA receptors indirectly, presumably via a stimulation of neurosteroid synthesis and release from glial cells.

  12. Apoptosis of human breast carcinoma cells in the presence of cis-platin and L-/D-PPMP: IV. Modulation of replication complexes and glycolipid: Glycosyltransferases.

    PubMed

    Boyle, Patrick J; Ma, Rui; Tuteja, Narendra; Banerjee, Sipra; Basu, Subhash

    2006-05-01

    Apoptosis of human breast carcinoma cells (SKBR-3, MCF-7, and MDA-468) has been observed after treatment of these cells with anti-cancer drug cis-platin and glycosphingolipid biosynthesis inhibitor L- and D-PPMP, respectively. These drugs initiated apoptosis in a dose-dependent manner as measured by phenotypic morphological changes, by binding of a fluorescent phophatidyl serine-specific dye (PSS-380) onto the outer leaflet of the cell membranes, and by activation of caspases, -3, -8, and -9. It was observed that in two hours very little apoptotic process had started but predominant biochemical changes occurred after 6 h. DNA degradation started after 24 hours of drug treatment. However, very little is known about the stability of the ';Replication Complexes'' during the apoptotic process. DNA helicases are motor proteins that catalyze the melting of genomic DNA during its replication, repair, and recombination processes. Previously, DNA helicase-III was characterized as a component of the replication complexes isolated from embryonic chicken brains as well as breast and colon carcinoma cells. Helicase activities were measured by a novel method (ROME assay), and DNA polymerase-alpha activities were determined by regular chain extension of the nicked ACT-DNA, by determining values obtained from +/- aphidicolin-treated incubation mixtures. In all three breast carcinoma cell lines, a common trend was observed: a decrease of activities of DNA polymerase-alpha and Helicase III. A sharp decrease of activities of the glycolipid sialyltransferases: SAT-2 (CMP-NeuAc; GD3 alpha2-8 sialyltransferase) and SAT-4 (CMP-NeuAc: GM1a alpha2-3 sialyltransferase) was observed in the apoptotic carcinoma cells treated with L-PPMP compared with cis-platin.

  13. Microarray-based gene expression profiling reveals genes and pathways involved in the oncogenic function of REG3A on pancreatic cancer cells.

    PubMed

    Xu, Qianqian; Fu, Rong; Yin, Guoxiao; Liu, Xiulan; Liu, Yang; Xiang, Ming

    2016-03-10

    We previously reported that regenerating islet-derived protein 3 alpha (REG3A) exacerbates pancreatic malignancies. The mechanism of this effect has not been clearly elucidated. Here we first identified key differentially expressed genes (DEGs) and signal pathways in the pancreatic cancer cell line SW1990, compared to two control cell lines, by microarray analysis. We then identified key genes and pathways regulated by REG3A or the cytokine IL6 in SW1990 cells. Afterwards, these DEGs induced by REG3A or IL6 were subjected to KEGG pathway enrichment analysis and GO function analysis by the DAVID online tool. Ultimately, we constructed protein-protein interaction networks among the DEGs by Cytoscape. Among the three pancreatic cell lines, SW1990 exhibited highly deterioration with the activation of genes and pathways related to proliferation, survival, angiogenesis, and invasion. As a result, 50 DEGs enriched in 11 pathways were identified in REG3A-treated SW1990 cells, and 28 DEGs enriched in 9 pathways were detected in IL6-treated cells. Overall, results of microarray analysis followed by qRT-PCR and Western blotting suggest that REG3A regulates pancreatic cell growth by increasing the expression of at least 8 genes: JAK1, STAT3, IL10, FOXM1, KRAS, MYC, CyclinD1, and c-fos; and activation of at least 4 signal pathways: TGFβ, PDGF, angiogenesis and RAS. Similar results were obtained with IL6 treatment. Regulation network analysis confirmed the cell growth related DEGs, and further uncovered three transcription factor families with immune functions regulated by REG3A.

  14. Melatonin decreases muscular oxidative stress and inflammation induced by strenuous exercise and stimulates growth factor synthesis.

    PubMed

    Borges, Leandro da Silva; Dermargos, Alexandre; da Silva Junior, Edenilson Pinto; Weimann, Eleine; Lambertucci, Rafael Herling; Hatanaka, Elaine

    2015-03-01

    Strenuous exercise is detrimental to athletes because of the overproduction of reactive oxygen species. Melatonin, a classic antioxidant, has been shown to exhibit beneficial effects regarding intense exercise and tissue repair. In this study, we evaluated the onset and resolution of inflammation in melatonin-treated and nontreated rats subjected to a strenuous exercise session. We also analyzed the formation of thiobarbituric acid reactive substances (TBARS) and the activities of catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD). Control and treated rats were subjected to exhaustive exercise after a period of 10 days of melatonin treatment (20 mg/dL). Plasma and muscle levels of tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), cytokine-induced neutrophil chemoattractant-2-alpha/beta (CINC-2α/β), l-selectin, macrophage inflammatory protein-3-alpha (MIP-3α), and vascular endothelial growth factor (VEGF) were measured prior to, immediately after, and 2 hr after exercise. Our data revealed decreases in the muscle concentrations of IL-1β (35%), TNF-α (13%), IL-6 (48%), and TBARS (40%) in the melatonin-treated group compared with the control group. We also observed decreases in the plasma concentrations of IL-1β (17%) in the melatonin-treated group. VEGF-α concentrations and SOD activity increased by 179% and 22%, respectively, in the melatonin-treated group compared with the control group. We concluded that muscle inflammation and oxidative stress resulting from exhaustive exercise were less severe in the muscles of melatonin-treated animals than in the muscles of control animals. Thus, melatonin treatment may reverse exercise-induced skeletal muscle inflammation and stimulate growth factor synthesis.

  15. The Conoid Associated Motor MyoH Is Indispensable for Toxoplasma gondii Entry and Exit from Host Cells.

    PubMed

    Graindorge, Arnault; Frénal, Karine; Jacot, Damien; Salamun, Julien; Marq, Jean Baptiste; Soldati-Favre, Dominique

    2016-01-01

    Many members of the phylum of Apicomplexa have adopted an obligate intracellular life style and critically depend on active invasion and egress from the infected cells to complete their lytic cycle. Toxoplasma gondii belongs to the coccidian subgroup of the Apicomplexa, and as such, the invasive tachyzoite contains an organelle termed the conoid at its extreme apex. This motile organelle consists of a unique polymer of tubulin fibres and protrudes in both gliding and invading parasites. The class XIV myosin A, which is conserved across the Apicomplexa phylum, is known to critically contribute to motility, invasion and egress from infected cells. The MyoA-glideosome is anchored to the inner membrane complex (IMC) and is assumed to translocate the components of the circular junction secreted by the micronemes and rhoptries, to the rear of the parasite. Here we comprehensively characterise the class XIV myosin H (MyoH) and its associated light chains. We show that the 3 alpha-tubulin suppressor domains, located in MyoH tail, are necessary to anchor this motor to the conoid. Despite the presence of an intact MyoA-glideosome, conditional disruption of TgMyoH severely compromises parasite motility, invasion and egress from infected cells. We demonstrate that MyoH is necessary for the translocation of the circular junction from the tip of the parasite, where secretory organelles exocytosis occurs, to the apical position where the IMC starts. This study attributes for the first time a direct function of the conoid in motility and invasion, and establishes the indispensable role of MyoH in initiating the first step of motility along this unique organelle, which is subsequently relayed by MyoA to enact effective gliding and invasion.

  16. Upregulation of apical sodium-chloride cotransporter and basolateral chloride channels is responsible for the maintenance of salt-sensitive hypertension.

    PubMed

    Capasso, Giovambattista; Rizzo, Maria; Garavaglia, Maria Lisa; Trepiccione, Francesco; Zacchia, Miriam; Mugione, Alessandra; Ferrari, Patrizia; Paulmichl, Markus; Lang, Florian; Loffing, Johannes; Carrel, Monique; Damiano, Sara; Wagner, Carsten A; Bianchi, Giuseppe; Meyer, Giuliano

    2008-08-01

    We investigated which of the NaCl transporters are involved in the maintenance of salt-sensitive hypertension. Milan hypertensive (MHS) rats were studied 3 mo after birth. In MHS, compared with normotensive strain (MNS), mRNA abundance, quantified by competitive PCR on isolated tubules, was unchanged, both for Na+/H+ isoform 3 (NHE3) and Na+-K+-2Cl- (NKCC2), but higher (119%, n = 5, P < 0.005) for Na+-Cl- (NCC) in distal convoluted tubules (DCT). These results were confirmed by Western blots, which revealed: 1) unchanged NHE3 in the cortex and NKCC2 in the outer medulla; 2) a significant increase (52%, n = 6, P < 0.001) of NCC in the cortex; 3) alpha- and beta-sodium channels [epithelial Na+ channel (ENaC)] unaffected in renal cortex and slightly reduced in the outer medulla, while gamma-ENaC remained unchanged. Pendrin protein expression was unaffected. The role of NCC was reinforced by immunocytochemical studies showing increased NCC on the apical membrane of DCT cells of MHS animals, and by clearance experiments demonstrating a larger sensitivity (P < 0.001) to bendroflumethiazide in MHS rats. Kidney-specific chloride channels (ClC-K) were studied by Western blot experiments on renal cortex and by patch-clamp studies on primary culture of DCT dissected from MNS and MHS animals. Electrophysiological characteristics of ClC-K channels were unchanged in MHS rats, but the number of active channels in a patch was 0.60 +/- 0.21 (n = 35) in MNS rats and 2.17 +/- 0.59 (n = 23) in MHS rats (P < 0.05). The data indicate that, in salt-sensitive hypertension, there is a strong upregulation, both of NCC and ClC-K along the DCT, which explains the persistence of hypertension.

  17. Effects of the apolipoprotein E polymorphism on levels of lipids, lipoproteins, and apolipoproteins among Mexican-Americans in Starr County, Texas.

    PubMed

    Hanis, C L; Hewett-Emmett, D; Douglas, T C; Bertin, T K; Schull, W J

    1991-01-01

    Genetic variability has been implicated as a significant contributor to the variation in levels of lipids, lipoproteins, and apolipoproteins (apos) through a variety of direct and indirect investigations. Among the direct investigations, apo E has been shown to be polymorphic and to explain a small but statistically significant proportion of the variability in cholesterol. The apo E polymorphism was typed in 964 randomly selected Mexican-Americans from Starr County, Tex., and its effects determined on levels of cholesterol, triglycerides, total high density lipoprotein (HDL) cholesterol, subfractions (HDL2 and HDL3), alpha- and beta-lipoprotein cholesterol, low density lipoprotein (LDL) cholesterol, and apos A-I, A-II, B, C-II, C-III, and E. Effects are reported for the entire sample and in each of three groups, namely, premenopausal females, postmenopausal women, and males. In the entire sample, significant effects were observed on cholesterol, beta-lipoprotein cholesterol, LDL, apo B, and apo E. There is evidence for significant physiological interaction of the apo E polymorphism effect in females by menopausal status. This is most evident for apo E levels, in which 5.9% of the variability in the entire sample is explained by the apo E polymorphism. In premenopausal females, however, the polymorphism accounts for 27.5% of the variability. In postmenopausal women and males, there is no significant effect. It is shown that the apo E polymorphism can be treated as a two-locus, two-allele system. Doing so identifies substitutions in amino acid position 158 as the mediators of most of the observed effects of this polymorphism. PMID:1998654

  18. Effects of chronic delta-9-tetrahydrocannabinol (THC) administration on neurotransmitter concentrations and receptor binding in the rat brain.

    PubMed

    Ali, S F; Newport, G D; Scallet, A C; Gee, K W; Paule, M G; Brown, R M; Slikker, W

    1989-01-01

    THC is the major psychoactive constituent of marijuana and is also known as an hallucinogenic compound. Numerous reports have shown that large doses of THC produce significant alterations in various neurotransmitter systems. The present study was designed to determine whether chronic exposure to THC produces significant alterations in selected neurotransmitter systems (dopamine, serotonin, acetylcholine, GABAergic, benzodiazepine, and opiate) in the rat brain. In Experiment 1, male Sprague-Dawley rats were gavaged with vehicle, 10 or 20 mg THC/kg body weight daily, 5 days/week for 90 days. Animals were killed either 24 hours or two months after the last dose. Brains were dissected into different regions for neurochemical analyses. Two months after the cessation of chronic administration, there was a significant decrease in GABA receptor binding in the hippocampus of animals in the high dose group. However, no other significant changes were found in neurotransmitter receptor binding characteristics in the hippocampus or in neurotransmitter concentrations in the caudate nucleus, hypothalamus or septum after chronic THC administration. In an attempt to replicate the GABA receptor binding changes and also to determine the [35S]TBPS binding in hippocampus, we designed Experiment 2. In this experiment, we dosed the animals by gavage with 0, 5, 10 or 20 mg THC/kg daily, 5 days/week or with 20 mg THC/kg Monday through Thursday and 60 mg/kg on Friday for 90 days. Results from this experiment failed to replicate the dose-dependent effect of THC on GABA receptor binding in hippocampus. Modulation of [35S]TBPS binding by GABA or 3 alpha-OH-DHP or inhibition by cold TBPS in frontal cortex did not show any significant dose-related effects. Results from these experiments suggest that chronic exposure to THC does not produce significant alterations in catecholamine or indoleamine neurotransmitter systems or in opiate or GABA receptor systems in the rat brain.

  19. The effects of pure density evolution on the brightness distribution of cosmological gamma-ray bursts

    NASA Technical Reports Server (NTRS)

    Horack, J. M.; Emslie, A. G.; Hartmann, D. H.

    1995-01-01

    In this work, we explore the effects of burst rate density evolution on the observed brightness distribution of cosmological gamma-ray bursts. Although the brightness distribution of gamma-ray bursts observed by the BATSE experiment has been shown to be consistent with a nonevolving source population observed to redshifts of order unity, evolution of some form is likely to be present in the gamma-ray bursts. Additionally, nonevolving models place significant constraints on the range of observed burst luminosities, which are relaxed if evolution of the burst population is present. In this paper, three analytic forms of density evolution are examined. In general, forms of evolution with densities that increase monotonically with redshift require that the BATSE data correspond to bursts at larger redshifts, or to incorporate a wider range of burst luminosities, or both. Independent estimates of the maximum observed redshift in the BATSE data and/or the range of luminosity from which a large fraction of the observed bursts are drawn therefore allow for constraints to be placed on the amount of evolution that may be present in the burst population. Specifically, if recent measurements obtained from analysis of the BATSE duration distribution of the actual limiting redshift in the BATSE data at z(sub lim) = 2 are correct, the BATSE N(P) distribution in a Lambda = 0 universe is inconsistent at a level of approximately 3 alpha with nonevolving gamma-ray bursts and some form of evolution in the population is required. The sense of this required source evolution is to provide a higher density, larger luminosities, or both with increasing redshift.

  20. The unusual estrogen-binding protein (UEBP) of male rat liver: structural determinants of ligands.

    PubMed

    Miroshnichenko, M L; Smirnova, O V; Smirnov, A N; Rozen, V B

    1983-04-01

    The unusual estrogen-binding protein (UEBP) found in a male rat liver is a sex dependent protein which differs from other known receptor and transport proteins by the high lability of its complexes with estradiol (E2) and also the unique specificity of affinity for hormones. In this work values of relative binding affinity (RBA) of the UEBP for 57 steroids and their analogs were determined. The affinity of steroids was characterised by the amount of the unlabeled compound needed for 50% inhibition of [3H]-E2 binding with the UEBP. A number of derivatives of estrane and androstane possess an ability to interact with this protein, in contrast to the derivatives of pregnane, stilbene and triphenylethane. Characterized by RBA values, natural steroids are found to have the following order: estriol larger than or equal to E2 greater than 16 alpha-hydroxyestrone = 2 alpha-hydroxytestosterone greater than 16-epiestriol greater than or equal to estetrol greater than or equal to 17-epiestriol greater than or equal to 2-methoxyestradiol greater than or equal to 5 alpha-androstane-3 alpha,17 beta-diol greater than or equal to estrone greater than testosterone greater than or equal to 2 beta-hydroxytestosterone greater than 5 alpha-dihydrotestosterone. Affinity of estrogens and androgens for the UEBP diminishes abruptly after removal of 3- and 17-hydroxy groups, masking of these by ether bonds or changing of 17 beta-hydroxyl to 17 alpha. All the investigated 17 oxo-C19-steroids, 5 beta-derivatives of testosterone, its 6 beta- and 16 alpha-hydroxy metabolites as well as 5 alpha-androstane-3 beta,17 beta-diol and 19-nortestosterone exhibit no essential affinity for the protein. On the basis of the results obtained it is suggested that the binding sites for estrogens and androgens in the UEBP molecule overlap but do not completely coincide. PMID:6834826

  1. Effects of NMDA receptor antagonists on morphine tolerance: a c-Fos study in the lumbar spinal cord of the rat.

    PubMed

    Le Guen, S; Catheline, G; Besson, J M

    1999-05-28

    This study investigated the contribution of NMDA receptors to the development of tolerance to the antinociceptive properties of morphine at the level of the spinal cord dorsal horn. The expression of c-Fos protein following intraplantar (i.pl.) injection of carrageenin (6 mg/150 microl of saline) was used. In naive rats, acute intravenous (i.v.) administration of morphine (3 mg/kg) decreased the total number per section of Fos-Like-Immunoreactive (Fos-LI) neurons by 51%, observed at 2 h after injection of carrageenin. In tolerant rats, acute morphine did not significantly modify the total number of Fos-like immunoreactive neurons/section. In rats receiving chronic morphine and chronic injections of the non-competitive ((+)-MK 801 maleate: (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,1 0-imine) or the competitive (LY 235959: [3S-(3alpha,4a alpha,6beta,8a alpha)]-Decahydro-6-(phosphonomethyl)-3-isoquinolinecarboxylic+ ++ acid) NMDA receptor antagonists, only partial tolerance to the acute effects of morphine were observed (decrease of 42% and 38%, respectively). Administration of an antagonist at the strychnine-insensitive glycine site of the NMDA receptor ((+)-HA-966: R(+)-3-Amino-1-hydroxypyrrolidin-2-one) did not affect the development of morphine tolerance. These findings suggest that compounds attenuating the actions of the NMDA receptor via blockade of the glycine modulatory site may be substantially different from those acting at the ion channel of the NMDA receptor complex. This in vivo experiment in freely moving animals demonstrates for the first time an attenuation of tolerance at the cellular level.

  2. Non-targeted Metabolite Profiling and Scavenging Activity Unveil the Nutraceutical Potential of Psyllium (Plantago ovata Forsk).

    PubMed

    Patel, Manish K; Mishra, Avinash; Jha, Bhavanath

    2016-01-01

    Non-targeted metabolomics implies that psyllium (Plantago ovata) is a rich source of natural antioxidants, PUFAs (ω-3 and ω-6 fatty acids) and essential and sulfur-rich amino acids, as recommended by the FAO for human health. Psyllium contains phenolics and flavonoids that possess reducing capacity and reactive oxygen species (ROS) scavenging activities. In leaves, seeds, and husks, about 76, 78, 58% polyunsaturated, 21, 15, 20% saturated, and 3, 7, 22% monounsaturated fatty acids were found, respectively. A range of FAs (C12 to C24) was detected in psyllium and among different plant parts, a high content of the nutritive indicators ω-3 alpha-linolenic acid CPS (57%) and ω-6 linoleic acid CPS (18%) was detected in leaves. Similarly, total content of phenolics and the essential amino acid valine were also detected utmost in leaves followed by sulfur-rich amino acids and flavonoids. In total, 36 different metabolites were identified in psyllium, out of which 26 (13 each) metabolites were detected in leaves and seeds, whereas the remaining 10 were found in the husk. Most of the metabolites are natural antioxidants, phenolics, flavonoids, or alkaloids and can be used as nutrient supplements. Moreover, these metabolites have been reported to have several pharmaceutical applications, including anti-cancer activity. Natural plant ROS scavengers, saponins, were also detected. Based on metabolomic data, the probable presence of a flavonoid biosynthesis pathway was inferred, which provides useful insight for metabolic engineering in the future. Non-targeted metabolomics, antioxidants and scavenging activities reveal the nutraceutical potential of the plant and also suggest that psyllium leaves can be used as a green salad as a dietary supplement to daily food. PMID:27092153

  3. Preparation and surface labeling of murine eosinophils.

    PubMed

    Burgess, A W; Cruise, K M; Mitchell, G F; Watt, S M

    1980-01-01

    Eosinophilic polymorphonuclear leukocytes were isolated from the peritoneal cavity of BALB/c mice infected with the parasite Mesocestoides corti. Approximately 4 X 10(7) eosinophils (purity, 50%) could be harvested from each mouse. A high yield and purity of eosinophils was obtained from the peritoneal cells of infected male BALB/c mice using density centrifugation on a gradient of slightly hypotonic colloidal silica sol (Percoll). After initial irradiation of the mice to lower the lymphocyte contamination, subsequent density gradient (and where necessary sedimentation velocity) centrifugation yielded 10(8) eosinophils (purity > 95%) from six to eight mice. It was also possible to isolate small numbers of eosinophils (2 X 10(4) cells/minute, purity > 99%) without irradiating the mice. This could be achieved by separating the density gradient purified peritoneal cells by light-scatter on a Becton-Dickinson cell sorter (FACS II). Analysis of proteins extracted from eosinophils using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed a group of high molecular weight proteins (bwtween 250K and 160K) which were not as distinctive in the neutrophil profile. Surface labeling was performed, before the cell separation by using 125I and 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril. Only five 125I-labeled proteins were detected initially (all with apparent molecular weights > 50,000). No 125I appeared to be associated with actin under the conditions used for surface labeling. Four of the eosinophil surface labeled proteins corresponded to surface labeled proteins on neutrophils, but the major surface component of the eosinophils (MW 79,000) appeared to be smaller than the major neutrophil protein (MW 90,000). PMID:7409032

  4. Gibbs-Thomson Law for Singular Step Segments: Thermodynamics Versus Kinetics

    NASA Technical Reports Server (NTRS)

    Chernov, A. A.

    2003-01-01

    Classical Burton-Cabrera-Frank theory presumes that thermal fluctuations are so fast that at any time density of kinks on a step is comparable with the reciprocal intermolecular distance, so that the step rate is about isotropic within the crystal plane. Such azimuthal isotropy is, however, often not the case: Kink density may be much lower. In particular, it was recently found on the (010) face of orthorhombic lysozyme that interkink distance may exceed 500-600 intermolecular distances. Under such conditions, Gibbs-Thomson law (GTL) may not be applicable: On a straight step segment between two corners, communication between the comers occurs exclusively by kink exchange. Annihilation between kinks of opposite sign generated at the comers results in the grain in step energy entering GTL. If the step segment length l much greater than D/v, where D and v are the kink diffusivity and propagation rate, respectively, the opposite kinks have practically no chance to annihilate and GTL is not applicable. The opposite condition of the GTL applicability, l much less than D/v, is equivalent to the requirement that relative supersaturation Delta(sub mu)/kT much less than alpha/l, where alpha is molecular size. Thus, GTL may be applied to a segment of 10(exp 3)alpha approx. 3 x 10(exp -5)cm approx 0.3 micron only if supersaturation is less than 0.1%, while practically used driving forces for crystallization are much larger. Relationships alternative to the GTL for different, but low, kink density have been discussed. They confirm experimental evidences that th