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Sample records for a1 a2 a3

  1. 47 CFR 80.1091 - Ship radio equipment-Sea areas A1, A2, and A3.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 5 2013-10-01 2013-10-01 false Ship radio equipment-Sea areas A1, A2, and A3... SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1091 Ship radio equipment—Sea areas A1, A2, and A3. This...

  2. 47 CFR 80.1091 - Ship radio equipment-Sea areas A1, A2, and A3.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 5 2011-10-01 2011-10-01 false Ship radio equipment-Sea areas A1, A2, and A3... SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1091 Ship radio equipment—Sea areas A1, A2, and A3. This...

  3. 47 CFR 80.1091 - Ship radio equipment-Sea areas A1, A2, and A3.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 5 2012-10-01 2012-10-01 false Ship radio equipment-Sea areas A1, A2, and A3... SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1091 Ship radio equipment—Sea areas A1, A2, and A3. This...

  4. 47 CFR 80.1091 - Ship radio equipment-Sea areas A1, A2, and A3.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 5 2014-10-01 2014-10-01 false Ship radio equipment-Sea areas A1, A2, and A3... SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1091 Ship radio equipment—Sea areas A1, A2, and A3. This...

  5. 47 CFR 80.1091 - Ship radio equipment-Sea areas A1, A2, and A3.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 5 2010-10-01 2010-10-01 false Ship radio equipment-Sea areas A1, A2, and A3... SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1091 Ship radio equipment—Sea areas A1, A2, and A3. This...

  6. Isolation and characterization of new onionins A2 and A3 from Allium cepa, and of onionins A1, A2, and A3 from Allium fistulosum.

    PubMed

    Nohara, Toshihiro; Fujiwara, Yukio; Kudo, Rino; Yamaguchi, Koki; Ikeda, Tsuyoshi; Murakami, Kotaro; Ono, Masateru; Kajimoto, Tetsuya; Takeya, Motohiro

    2014-01-01

    In this study, the new stable sulfur-containing compounds onionins A2 (1) and A3 (2) were isolated from the acetone extracts of the bulbs of Allium cepa L. and identified as the stereoisomers of onionin A1 discovered in our previous study. Their chemical structures, 3,4-dimethyl-5-(1E-propenyl)-tetrahydrothiophene-2-sulfenic acid-S-oxides, were characterized using various spectroscopic techniques. In addition, 1 and 2 together with onionin A1 were successfully isolated from the leaves of the Welsh onion, Allium fistulosum L. The onion-extracted fractions showed good potential to inhibit the polarization of M2 activated macrophages, indicating their possible ability to inhibit tumor cell proliferation.

  7. 47 CFR 80.1093 - Ship radio equipment-Sea areas A1, A2, A3, and A4.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 5 2013-10-01 2013-10-01 false Ship radio equipment-Sea areas A1, A2, A3, and... AND SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1093 Ship radio equipment—Sea areas A1, A2...

  8. 47 CFR 80.1093 - Ship radio equipment-Sea areas A1, A2, A3, and A4.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 5 2014-10-01 2014-10-01 false Ship radio equipment-Sea areas A1, A2, A3, and... AND SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1093 Ship radio equipment—Sea areas A1, A2...

  9. 47 CFR 80.1093 - Ship radio equipment-Sea areas A1, A2, A3, and A4.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 5 2012-10-01 2012-10-01 false Ship radio equipment-Sea areas A1, A2, A3, and... AND SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1093 Ship radio equipment—Sea areas A1, A2...

  10. 47 CFR 80.1093 - Ship radio equipment-Sea areas A1, A2, A3, and A4.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 5 2010-10-01 2010-10-01 false Ship radio equipment-Sea areas A1, A2, A3, and... AND SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1093 Ship radio equipment—Sea areas A1, A2...

  11. 47 CFR 80.1093 - Ship radio equipment-Sea areas A1, A2, A3, and A4.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 5 2011-10-01 2011-10-01 false Ship radio equipment-Sea areas A1, A2, A3, and... AND SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1093 Ship radio equipment—Sea areas A1, A2...

  12. The Functions of the A1A2A3 Domains in Von Willebrand Factor Include Multimerin 1 Binding

    PubMed Central

    Parker, D’Andra N.; Tasneem, Subia; Farndale, Richard W.; Bihan, Dominique; Sadler, J. Evan; Sebastian, Silvie; De Groot, Philip G.

    2016-01-01

    Summary Multimerin 1 (MMRN1) is a massive, homopolymeric protein that is stored in platelets and endothelial cells for activation-induced release. In vitro, MMRN1 binds to the outer surfaces of activated platelets and endothelial cells, the extracellular matrix (including collagen) and von Willebrand factor (VWF) to support platelet adhesive functions. VWF associates with MMRN1 at high shear, not static conditions, suggesting that shear exposes cryptic sites within VWF that support MMRN1 binding. Modified ELISA and surface plasmon resonance were used to study the structural features of VWF that support MMRN1 binding, and determine the affinities for VWF-MMRN1 binding. High shear microfluidic platelet adhesion assays determined the functional consequences for VWF-MMRN1 binding. VWF binding to MMRN1 was enhanced by shear exposure and ristocetin, and required VWF A1A2A3 region, specifically the A1 and A3 domains. VWF A1A2A3 bound to MMRN1 with a physiologically relevant binding affinity (KD: 2.0 ± 0.4 nM), whereas the individual VWF A1 (KD: 39.3 ± 7.7 nM) and A3 domains (KD: 229 ± 114 nM) bound to MMRN1 with lower affinities. VWF A1A2A3 was also sufficient to support the adhesion of resting platelets to MMRN1 at high shear, by a mechanism dependent on VWF-GPIbα binding. Our study provides new information on the molecular basis of MMRN1 binding to VWF, and its role in supporting platelet adhesion at high shear. We propose that at sites of vessel injury, MMRN1 that is released following activation of platelets and endothelial cells, binds to VWF A1A2A3 region to support platelet adhesion at arterial shear rates. PMID:27052467

  13. Activation of A1, A2A, or A3 adenosine receptors attenuates lung ischemia-reperfusion injury

    PubMed Central

    Gazoni, Leo M.; Walters, Dustin M.; Unger, Eric B.; Linden, Joel; Kron, Irving L.; Laubach, Victor E.

    2010-01-01

    Objective Adenosine and the activation of specific adenosine receptors are implicated in the attenuation of inflammation and organ ischemia-reperfusion (IR) injury. We hypothesized that activation of A1, A2A, or A3 adenosine receptors would provide protection against lung IR injury. Methods Using an isolated, ventilated, blood-perfused rabbit lung model, lungs underwent 18 hours cold ischemia followed by 2 hours reperfusion. Lungs were administered either vehicle, adenosine, or selective A1, A2A, or A3 receptor agonists (CCPA, ATL-313, or IB-MECA, respectively) alone or with their respective antagonists (DPCPX, ZM241385, or MRS1191) during reperfusion. Results Compared to the vehicle-treated control group, treatment with A1, A2A, or A3 agonists significantly improved function (increased lung compliance and oxygenation and decreased pulmonary artery pressure), decreased neutrophil infiltration by myeloperoxidase activity, decreased edema, and reduced TNF-α production. Adenosine treatment was also protective but not to the level of the agonists. When each agonist was paired with its respective antagonist, all protective effects were blocked. The A2A agonist reduced pulmonary artery pressure and myeloperoxidase activity and increased oxygenation to a greater degree than the A1 or A3 agonists. Conclusions Selective activation of A1, A2A, or A3 adenosine receptors provides significant protection against lung IR injury. The decreased elaboration of the potent proinflammatory cytokine, TNF-α, and decreased neutrophil sequestration likely contribute to the overall improvement in pulmonary function. These results provide evidence for the therapeutic potential of specific adenosine receptor agonists in lung transplant recipients. PMID:20398911

  14. Ligand-, structure- and pharmacophore-based molecular fingerprints: a case study on adenosine A1, A2A, A2B, and A3 receptor antagonists

    NASA Astrophysics Data System (ADS)

    Sirci, Francesco; Goracci, Laura; Rodríguez, David; van Muijlwijk-Koezen, Jacqueline; Gutiérrez-de-Terán, Hugo; Mannhold, Raimund

    2012-11-01

    FLAP fingerprints are applied in the ligand-, structure- and pharmacophore-based mode in a case study on antagonists of all four adenosine receptor (AR) subtypes. Structurally diverse antagonist collections with respect to the different ARs were constructed by including binding data to human species only. FLAP models well discriminate "active" (=highly potent) from "inactive" (=weakly potent) AR antagonists, as indicated by enrichment curves, numbers of false positives, and AUC values. For all FLAP modes, model predictivity slightly decreases as follows: A2BR > A2AR > A3R > A1R antagonists. General performance of FLAP modes in this study is: ligand- > structure- > pharmacophore- based mode. We also compared the FLAP performance with other common ligand- and structure-based fingerprints. Concerning the ligand-based mode, FLAP model performance is superior to ECFP4 and ROCS for all AR subtypes. Although focusing on the early first part of the A2A, A2B and A3 enrichment curves, ECFP4 and ROCS still retain a satisfactory retrieval of actives. FLAP is also superior when comparing the structure-based mode with PLANTS and GOLD. In this study we applied for the first time the novel FLAPPharm tool for pharmacophore generation. Pharmacophore hypotheses, generated with this tool, convincingly match with formerly published data. Finally, we could demonstrate the capability of FLAP models to uncover selectivity aspects although single AR subtype models were not trained for this purpose.

  15. Triple-Singlet Mixing in Si_3: the 1^3A_{1}^{''} - {a}{^3}A{^{'}_2} Transition

    NASA Astrophysics Data System (ADS)

    Zhang, Ruohan; Steimle, Timothy C.

    2013-06-01

    The electronic spectrum of the triplet states of the D_{3h} isomer of Si_3 recorded using both mass selected REMPI and LIF spectroscopy was recently reported. In that same study the dispersed laser induced fluorescence (DLIF) spectra resulting from excitation of various bands in the visible range were recorded. The DLIF spectra exhibited a progression with a 505 cm^{-1} spacing, which was assign to the breathing mode of the D_{3h}, equilateral triangle, Si_{3} molecule. In addition, and quite unexpectedly, the DLIF spectra exhibited a progression having a spacing of 173 cm^{-1}. This progression was tentatively assigned to transition involving the bending mode of the ^1A_1 state of the C_{2v} isomer. A possible explanation for the observation of transitions in the singlet manifold is that upon laser excitation in the D_{3h} triplet manifold there is rapid intersystem crossing to the singlet manifold followed by fluorescence to the ground state of C_{2v} isomer. Here we address the issue of possible intersystem crossing by recording the excitation on DLIF spectra in the present of a static magnetic field. Magnetic fields are known to enhance the singlet-triple mixing. Si_{3} was produced using a supersonic pulsed discharge source (900 V, 20 μs, 6kΩ) with a 1% SiH_{4} in argon mixture. Magnetic fields of approximately 500 and 950 Gauss were applied. We will report the interpretation of the magnetic field induced changes to the LIF and DLIF spectra and the implications for the singlet-triple mixing process. N. J. Reilly, X. Zhuang, V. Gupta, R. Nagarajan, R. C. Fortenberry, J. P. Maier, T. C. Steimle, J. F. Stanton, M. C. McCarthy; {J. Chem. Phys., {136(19)}, 194307, (2004). V. I. Makarov, I. V. Khmelinskii; {Advances in Chemical Phisics, {Volume 118}, 45-98, (2001). thanks

  16. Changes in expression of human serine protease HtrA1, HtrA2 and HtrA3 genes in benign and malignant thyroid tumors.

    PubMed

    Zurawa-Janicka, Dorota; Kobiela, Jarosław; Galczynska, Natalia; Stefaniak, Tomasz; Lipinska, Barbara; Lachinski, Andrzej; Skorko-Glonek, Joanna; Narkiewicz, Joanna; Proczko-Markuszewska, Monika; Sledzinski, Zbigniew

    2012-11-01

    Human HtrA proteins are serine proteases involved in essential physiological processes. HtrA1 and HtrA3 function as tumor suppressors and inhibitors of the TGF-β signaling pathway. HtrA2 regulates mitochondrial homeostasis and plays a pivotal role in the induction of apoptosis. The aim of the study was to determine whether the HtrA proteins are involved in thyroid carcinogenesis. We used the immunoblotting technique to estimate protein levels of HtrA1, HtrA2, long and short variants of HtrA3 (HtrA3-L and HtrA3-S) and TGF-β1 in tissues of benign and malignant thyroid lesions, and control groups. We found that the levels of HtrA2 and HtrA3-S were higher in thyroid malignant tumors compared to normal tissues and benign tumors. The HtrA3-L level was increased in malignant tumor tissues compared to benign tumor tissues and control tissues from patients with benign lesions, and elevated in normal tissues from patients with thyroid carcinoma compared to normal tissues from patients with benign lesions. We also compared levels of HtrA proteins in follicular thyroid carcinoma (FTC) and papillary thyroid carcinoma (PTC) and found that these types of carcinoma differed in the expression of HtrA3-S and HtrA1. These results indicate the implication of HtrA proteins in thyroid carcinogenesis suggest that HtrA3 variants may play different roles in cancer development, and that the increased HtrA3-L levels in thyroid tissue could be correlated with the development of malignant lesions. The TGF-β1 levels in tumor tissues were not significantly altered compared to control tissues.

  17. Basal adenosine modulates the functional properties of AMPA receptors in mouse hippocampal neurons through the activation of A1R A2AR and A3R

    PubMed Central

    Di Angelantonio, Silvia; Bertollini, Cristina; Piccinin, Sonia; Rosito, Maria; Trettel, Flavia; Pagani, Francesca; Limatola, Cristina; Ragozzino, Davide

    2015-01-01

    Adenosine is a widespread neuromodulator within the CNS and its extracellular level is increased during hypoxia or intense synaptic activity, modulating pre- and postsynaptic sites. We studied the neuromodulatory action of adenosine on glutamatergic currents in the hippocampus, showing that activation of multiple adenosine receptors (ARs) by basal adenosine impacts postsynaptic site. Specifically, the stimulation of both A1R and A3R reduces AMPA currents, while A2AR has an opposite potentiating effect. The effect of ARs stimulation on glutamatergic currents in hippocampal cultures was investigated using pharmacological and genetic approaches. A3R inhibition by MRS1523 increased GluR1-Ser845 phosphorylation and potentiated AMPA current amplitude, increasing the apparent affinity for the agonist. A similar effect was observed blocking A1R with DPCPX or by genetic deletion of either A3R or A1R. Conversely, impairment of A2AR reduced AMPA currents, and decreased agonist sensitivity. Consistently, in hippocampal slices, ARs activation by AR agonist NECA modulated glutamatergic current amplitude evoked by AMPA application or afferent fiber stimulation. Opposite effects of AR subtypes stimulation are likely associated to changes in GluR1 phosphorylation and represent a novel mechanism of physiological modulation of glutamatergic transmission by adenosine, likely acting in normal conditions in the brain, depending on the level of extracellular adenosine and the distribution of AR subtypes. PMID:26528137

  18. Pharmacological postconditioning of the rabbit heart with non-selective, A1 , A2A and A3 adenosine receptor agonists.

    PubMed

    Bibli, Sophia-Iris; Iliodromitis, Efstathios K; Lambertucci, Catia; Zoga, Anastasia; Lougiakis, Nikolaos; Dagres, Nikolaos; Volpini, Rosaria; Dal Ben, Diego; Kremastinos, Dimitrios Th; Tsantili Kakoulidou, Anna; Cristalli, Gloria; Andreadou, Ioanna

    2014-08-01

    We investigated the effects of novel selective and non-selective adenosine receptor agonists (ARs) on cardioprotection. Male rabbits divided into six groups were subjected to 30-min heart ischaemia and 3-h reperfusion: (1) control group, (2) postconditioning (PostC) group, (3) group A: treated with the non-selective agonist (S)-PHPNECA, (4) group B: treated with the A1 agonist CCPA, (5) group C: treated with the A2A agonist VT 7 and (6) group D: treated with the A3 agonist AR 170. The infarcted (I) and the areas at risk (R) were estimated as %I/R. In additional rabbits of all groups, heart samples were taken for determination of Akt, eNOS and STAT 3 at the 10th reperfusion minute. (S)-PHPNECA and CCPA reduced the infarct size (17.2 ± 2.9% and 17.9 ± 2.0% vs 46.8 ± 1.9% in control, P < 0.05), conferring a benefit similar to PostC (26.4 ± 0.3%). Selective A2A and A3 receptor agonists did not reduce the infarct size (39.5 ± 0.8% and 38.7 ± 3.5%, P = NS vs control). Akt, eNOS and STAT 3 were significantly activated after non-selective A1 ARs and PostC. Non-selective and A1 but not A2A and A3 ARs agonists are essential for triggering cardioprotection. The molecular mechanism involves both RISK and the JAK/STAT pathways. © 2014 Royal Pharmaceutical Society.

  19. Extraction and Inhibition of Enzymatic Activity of Botulinum Neurotoxins/A1, /A2, and /A3 by a Panel of Monoclonal Anti-BoNT/A Antibodies

    PubMed Central

    Kalb, Suzanne R.; Lou, Jianlong; Garcia-Rodriguez, Consuelo; Geren, Isin N.; Smith, Theresa J.; Moura, Hercules; Marks, James D.; Smith, Leonard A.; Pirkle, James L.; Barr, John R.

    2009-01-01

    Botulinum neurotoxins (BoNTs) are extremely potent toxins that are capable of causing death or respiratory failure leading to long-term intensive care. Treatment includes serotype-specific antitoxins, which must be administered early in the course of the intoxication. Rapidly determining human exposure to BoNT is an important public health goal. In previous work, our laboratory focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating BoNT/A–G serotypes in buffer and BoNT/A, /B, /E, and /F in clinical samples. We have previously reported the effectiveness of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. Because some antibodies inhibit or neutralize the activity of BoNT, the choice of antibody with which to extract the toxin is critical. In this work, we evaluated a panel of 16 anti-BoNT/A monoclonal antibodies (mAbs) for their ability to inhibit the in vitro activity of BoNT/A1, /A2, and /A3 complex as well as the recombinant LC of A1. We also evaluated the same antibody panel for the ability to extract BoNT/A1, /A2, and /A3. Among the mAbs, there were significant differences in extraction efficiency, ability to extract BoNT/A subtypes, and inhibitory effect on BoNT catalytic activity. The mAbs binding the C-terminal portion of the BoNT/A heavy chain had optimal properties for use in the Endopep-MS assay. PMID:19399171

  20. Human Organic Cation Transporters 1 (SLC22A1), 2 (SLC22A2), and 3 (SLC22A3) as Disposition Pathways for Fluoroquinolone Antimicrobials

    PubMed Central

    Mulgaonkar, Aditi; Venitz, Jürgen; Gründemann, Dirk

    2013-01-01

    Fluoroquinolones (FQs) are important antimicrobials that exhibit activity against a wide range of bacterial pathogens and excellent tissue permeation. They exist as charged molecules in biological fluids, and thus, their disposition depends heavily on active transport and facilitative diffusion. A recent review of the clinical literature indicated that tubular secretion and reabsorption are major determinants of their half-life in plasma, efficacy, and drug-drug interactions. In particular, reported in vivo interactions between FQs and cationic drugs affecting renal clearance implicated organic cation transporters (OCTs). In this study, 13 FQs, ciprofloxacin, enoxacin, fleroxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, pefloxacin, prulifloxacin, rufloxacin, and sparfloxacin, were screened for their ability to inhibit transport activity of human OCT1 (hOCT1) (SLC22A1), hOCT2 (SLC22A2), and hOCT3 (SLC22A3). All, with the exception of enoxacin, significantly inhibited hOCT1-mediated uptake under initial test conditions. None of the FQs inhibited hOCT2, and only moxifloxacin inhibited hOCT3 (∼30%), even at a 1,000-fold excess. Gatifloxacin, moxifloxacin, prulifloxacin, and sparfloxacin were determined to be competitive inhibitors of hOCT1. Inhibition constants (Ki) were estimated to be 250 ± 18 μM, 161 ± 19 μM, 136 ± 33 μM, and 94 ± 8 μM, respectively. Moxifloxacin competitively inhibited hOCT3-mediated uptake, with a Ki value of 1,598 ± 146 μM. Despite expression in enterocytes (luminal), hepatocytes (sinusoidal), and proximal tubule cells (basolateral), hOCT3 does not appear to contribute significantly to FQ disposition. However, hOCT1 in the sinusoidal membrane of hepatocytes, and potentially the basolateral membrane of proximal tubule cells, is likely to play a role in the disposition of these antimicrobial agents. PMID:23545524

  1. Identification and Quantification of Fumonisin A1, A2, and A3 in Corn by High-Resolution Liquid Chromatography-Orbitrap Mass Spectrometry

    PubMed Central

    Tamura, Masayoshi; Mochizuki, Naoki; Nagatomi, Yasushi; Harayama, Koichi; Toriba, Akira; Hayakawa, Kazuichi

    2015-01-01

    Three compounds, hypothesized as fumonisin A1 (FA1), fumonisin A2 (FA2), and fumonisin A3 (FA3), were detected in a corn sample contaminated with mycotoxins by high-resolution liquid chromatography-Orbitrap mass spectrometry (LC-Orbitrap MS). One of them has been identified as FA1 synthesized by the acetylation of fumonisin B1 (FB1), and established a method for its quantification. Herein, we identified the two remaining compounds as FA2 and FA3, which were acetylated fumonisin B2 (FB2) and fumonisin B3 (FB3), respectively. Moreover, we examined a method for the simultaneous analysis of FA1, FA2, FA3, FB1, FB2, and FB3. The corn samples were prepared by extraction using a QuEChERS kit and purification using a multifunctional cartridge. The linearity, recovery, repeatability, limit of detection, and limit of quantification of the method were >0.99, 82.9%–104.6%, 3.7%–9.5%, 0.02–0.60 μg/kg, and 0.05–1.98 μg/kg, respectively. The simultaneous analysis of the six fumonisins revealed that FA1, FA2, and FA3 were present in all corn samples contaminated with FB1, FB2, and FB3. The results suggested that corn marketed for consumption can be considered as being contaminated with both the fumonisin B-series and with fumonisin A-series. This report presents the first identification and quantification of FA1, FA2, and FA3 in corn samples. PMID:25690692

  2. Identification and quantification of fumonisin A1, A2, and A3 in corn by high-resolution liquid chromatography-orbitrap mass spectrometry.

    PubMed

    Tamura, Masayoshi; Mochizuki, Naoki; Nagatomi, Yasushi; Harayama, Koichi; Toriba, Akira; Hayakawa, Kazuichi

    2015-02-16

    Three compounds, hypothesized as fumonisin A1 (FA1), fumonisin A2 (FA2), and fumonisin A3 (FA3), were detected in a corn sample contaminated with mycotoxins by high-resolution liquid chromatography-Orbitrap mass spectrometry (LC-Orbitrap MS). One of them has been identified as FA1 synthesized by the acetylation of fumonisin B1 (FB1), and established a method for its quantification. Herein, we identified the two remaining compounds as FA2 and FA3, which were acetylated fumonisin B2 (FB2) and fumonisin B3 (FB3), respectively. Moreover, we examined a method for the simultaneous analysis of FA1, FA2, FA3, FB1, FB2, and FB3. The corn samples were prepared by extraction using a QuEChERS kit and purification using a multifunctional cartridge. The linearity, recovery, repeatability, limit of detection, and limit of quantification of the method were >0.99, 82.9%-104.6%, 3.7%-9.5%, 0.02-0.60 μg/kg, and 0.05-1.98 μg/kg, respectively. The simultaneous analysis of the six fumonisins revealed that FA1, FA2, and FA3 were present in all corn samples contaminated with FB1, FB2, and FB3. The results suggested that corn marketed for consumption can be considered as being contaminated with both the fumonisin B-series and with fumonisin A-series. This report presents the first identification and quantification of FA1, FA2, and FA3 in corn samples.

  3. Extraction and Inhibition of Enzymatic Activity of Botulinum Neurotoxins/A1, /A2, and /A3 by a Panel of Monoclonal Anti-BoNT/A Antibodies

    DTIC Science & Technology

    2009-04-01

    a disease that is contracted by ingestion of food containing the toxin [1,2], colonization of the bacteria in the gastrointestinal tract of infants...In addition, commer- cially purified BoNT/A1 (strain Hall) and BoNT/A2 (strain FRI- honey ) complex toxins from Metabiologics (Madison, WI) were used

  4. Production of novel antioxidative prenyl naphthalen-ols by combinational bioconversion with dioxygenase PhnA1A2A3A4 and prenyltransferase NphB or SCO7190.

    PubMed

    Shindo, Kazutoshi; Tachibana, Ayako; Tanaka, Ayumi; Toba, Shizuka; Yuki, Emi; Ozaki, Taro; Kumano, Takuto; Nishiyama, Makoto; Misawa, Norihiko; Kuzuyama, Tomohisa

    2011-01-01

    We performed combinational bioconversion of substituted naphthalenes with PhnA1A2A3A4 (an aromatic dihydroxylating dioxygenase from marine bacterium Cycloclasticus sp. strain A5) and prenyltransferase NphB (geranyltransferase from Streptomyces sp. strain CL190) or SCO7190 (dimethylallyltransferase from Streptomyces coelicolor A3(2)) to produce prenyl naphthalen-ols. Using 2-methylnaphthalene, 1-methoxynaphthalene, and 1-ethoxynaphthalene as the starting substrates, 10 novel prenyl naphthalen-ols were produced by combinational bioconversion. These novel prenyl naphthalen-ols each showed potent antioxidative activity against a rat brain homogenate model. 2-(2,3-Dihydroxyphenyl)-5,7-dihydroxy-chromen-4-one (2',3'-dihydroxychrysin) generated with another aromatic dihydroxylating dioxygenase and subsequent dehydrogenase was also geranylated at the C-5'-carbon by the action of NphB.

  5. Position of glycine substitutions in the triple helix of COL6A1, COL6A2, and COL6A3 is correlated with severity and mode of inheritance in collagen VI myopathies

    PubMed Central

    Butterfield, Russell J.; Foley, A. Reghan; Dastgir, Jahannaz; Asman, Stephanie; Dunn, Diane M.; Zou, Yaqun; Hu, Ying; Flanigan, Kevin M.; Swoboda, Kathryn J.; Winder, Thomas L.; Weiss, Robert B.; Bönnemann, Carsten G.

    2015-01-01

    Glycine substitutions in the conserved Gly-X-Y motif in the triple helical domain of collagen VI are the most commonly identified mutations in the collagen VI myopathies including Ullrich congenital muscular dystrophy, Bethlem myopathy, and intermediate phenotypes. We describe clinical and genetic characteristics of 97 individuals with glycine substitutions in the triple helical domain of COL6A1, COL6A2, or COL6A3 and add a review of 97 published cases, for a total of 194 cases. Clinical findings include severe, intermediate, and mild phenotypes even from patients with identical mutations. Intermediate phenotypes were most common, accounting for almost half of patients, emphasizing the importance of intermediate phenotypes to the overall phenotypic spectrum. Glycine substitutions in the triple helical domain are heavily clustered in a short segment N-terminal to the 17th Gly-X-Y triplet, where they are acting as dominants. The most severe cases are clustered in an even smaller region including Gly-X-Y triplets 10 to 15, accounting for only 5% of the triple helical domain. Our findings suggest that clustering of glycine substitutions in the N-terminal region of collagen VI is not based on features of the primary sequence. We hypothesize that this region may represent a functional domain within the triple helix. PMID:24038877

  6. Position of glycine substitutions in the triple helix of COL6A1, COL6A2, and COL6A3 is correlated with severity and mode of inheritance in collagen VI myopathies.

    PubMed

    Butterfield, Russell J; Foley, A Reghan; Dastgir, Jahannaz; Asman, Stephanie; Dunn, Diane M; Zou, Yaqun; Hu, Ying; Donkervoort, Sandra; Flanigan, Kevin M; Swoboda, Kathryn J; Winder, Thomas L; Weiss, Robert B; Bönnemann, Carsten G

    2013-11-01

    Glycine substitutions in the conserved Gly-X-Y motif in the triple helical (TH) domain of collagen VI are the most commonly identified mutations in the collagen VI myopathies including Ullrich congenital muscular dystrophy, Bethlem myopathy, and intermediate (INT) phenotypes. We describe clinical and genetic characteristics of 97 individuals with glycine substitutions in the TH domain of COL6A1, COL6A2, or COL6A3 and add a review of 97 published cases, for a total of 194 cases. Clinical findings include severe, INT, and mild phenotypes even from patients with identical mutations. INT phenotypes were most common, accounting for almost half of patients, emphasizing the importance of INT phenotypes to the overall phenotypic spectrum. Glycine substitutions in the TH domain are heavily clustered in a short segment N-terminal to the 17th Gly-X-Y triplet, where they are acting as dominants. The most severe cases are clustered in an even smaller region including Gly-X-Y triplets 10-15, accounting for only 5% of the TH domain. Our findings suggest that clustering of glycine substitutions in the N-terminal region of collagen VI is not based on features of the primary sequence. We hypothesize that this region may represent a functional domain within the triple helix.

  7. Renal tumours in a Tsc1+/- mouse model show epigenetic suppression of organic cation transporters Slc22a1, Slc22a2 and Slc22a3, and do not respond to metformin.

    PubMed

    Yang, Jian; Kalogerou, Maria; Gallacher, John; Sampson, Julian R; Shen, Ming Hong

    2013-04-01

    Metformin, a substrate of several poly-specific organic cation transporters, is a widely used biguanide for the treatment of type II diabetes. Recent studies suggest that metformin attenuates mTORC1 signalling by the activation of 5' adenosine monophosphate-activated protein kinase (AMPK) in the presence or absence of a functional hamartin/tuberin (TSC1/TSC2) complex. Metformin has also been reported to inhibit mTORC1 independent of AMPK through p53-dependent regulated in development and DNA damage responses 1 (REDD1) or by inhibiting Rag GTPases. These observations suggest that metformin could have therapeutic potential for tuberous sclerosis, an inherited disorder characterised by the aberrant activation of mTORC1 and the development of tumours in many organs, including the kidneys. In this study, we investigated the effect of metformin on renal lesions in a Tsc1(+/-) mouse model of tuberous sclerosis. Continuous treatment of metformin for 9 months at doses of up to 600 mg/kg/day had no significant effect on renal lesions in nine treated mice compared to 10 controls. Metformin treatment appeared to attenuate mTORC1 signalling in Tsc1(+/-) kidney tissues but not in renal tumours. Surprisingly, the expression of the organic cation transporters Slc22a1, Slc22a2 and Slc22a3 essential for the cellular uptake of metformin was highly suppressed in renal tumours. Treatment of cultured cells derived from a Tsc1-associated renal tumour with 5-aza-2-deoxycytidine or trichostatin A greatly increased the expression of these genes. These data suggest that the epigenetic suppression of the organic cation transporters in Tsc-associated mouse renal tumours may contribute to the lack of response to metformin treatment.

  8. ON THE DISTRIBUTION OF VITAMINS A1 AND A2

    PubMed Central

    Wald, George

    1939-01-01

    The distribution of vitamins A1 and A2 has been determined in the eye tissues and livers of a number of fishes. The vitamins were differentiated by means of the antimony chloride reaction, which yields with A1 a band at 615–620 mµ and with A2 a band at about 696 mµ. In the retina the presence of vitamin A1 is diagnostic of the operation of a rhodopsin, and vitamin A2 of a porphyropsin cycle. The eye tissues of all permanently marine fishes examined, except the tautog, contain vitamin A1 alone. Those of all permanently freshwater fishes possess only vitamin A2. Those of all euryhaline (potentially migratory) fishes, except possibly the alewife, contain mixtures of both vitamins A, and always predominantly that one which ordinarily is associated with the environment in which the fish is spawned. These correlations extend in part to the liver oils, but most livers contain mixtures of both vitamins A, and occasionally in proportions the reverse of those in the eye tissues. The vitamin A configuration does not depend upon environmental circumstances, but is determined genetically. The transfer from vitamin A1 to A2 metabolism appears associated phylogenetically with migration of marine teleosts into fresh water. PMID:19873110

  9. Milk A1 and A2 peptides and diabetes.

    PubMed

    Clemens, Roger A

    2011-01-01

    Food-derived peptides, specifically those derived from milk, may adversely affect health by increasing the risk of insulin-dependent diabetes. This position is based on the relationship of type 1 diabetes (T1D) and the consumption of variants A1 and B β-casein from cow's milk. It appears that β-casomorphin-7 (BCM-7) from β-casein may function as an immunosuppressant and impair tolerance to dietary antigens in the gut immune system, which, in turn, may contribute to the onset of T1D. There are thirteen genetic variants of β-casein in dairy cattle. Among those variants are A1, A2, and B, which are also found in human milk. The amino acid sequences of β-casomorphins among these bovine variants and those found in human milk are similar, often differing only by a single amino acid. In vitro studies indicate BCM-7 can be produced from A1 and B during typical digestive processes; however, BCM-7 is not a product of A2 digestion. Evidence from several epidemiological studies and animal models does not support the association of milk proteins, even proteins in breast milk, and the development of T1D. Ecological data, primarily based on A1/ A2 variations among livestock breeds, do not demonstrate causation, even among countries where there is considerable dairy consumption.

  10. Importance of UDP-glucuronosyltransferases 2A2 and 2A3 in tobacco carcinogen metabolism.

    PubMed

    Bushey, Ryan T; Dluzen, Douglas F; Lazarus, Philip

    2013-01-01

    UDP-glucuronosyltransferase A1 (UGT2A1) is expressed in the lung and exhibits activity against polycyclic aromatic hydrocarbons (PAHs), suggesting UGT2A1 involvement in the local metabolism of PAH tobacco carcinogens. The goal of the present study was to investigate the importance of two additional UGT2A enzymes, UGT2A2 and UGT2A3, in tobacco carcinogen metabolism. Real-time polymerase chain reaction suggested that wild-type UGT2A2 had the highest expression in the breast, followed by trachea > larynx > kidney. A novel splice variant of UGT2A2 lacking exon 3 (termed UGT2A2Δexon3) was investigated, with UGT2A2Δexon3 expression determined to be 25-50% that of wild-type UGT2A2 in all tissues examined. UGT2A3 was determined to be well expressed in the liver and colon, followed by pancreas > kidney > lung > tonsil > trachea > larynx. Cell homogenates prepared from human embryonic kidney (HEK)293 cells overexpressing wild-type UGT2A2 (termed UGT2A2_i1) exhibited glucuronidation activity, as observed by reverse-phase ultra-pressure liquid chromatography, against 1-hydroxy-(OH)-pyrene, 1-naphthol, and hydroxylated benzo(a)pyrene metabolites, whereas homogenates prepared from HEK293 cells overexpressing UGT2A3 only showed activity against simple PAHs like 1-OH-pyrene and 1-naphthol. Activity assays showed the UGT2A2Δexon3 protein (termed UGT2A2_i2) exhibited no detectable glucuronidation activity against all substrates examined; however, coexpression studies suggested that UGT2A2_i2 negatively modulates UGT2A2_i1 activity. Both UGT2A2 and UGT2A3 exhibited no detectable activity against complex PAH proximate carcinogens, tobacco-specific nitrosamines, or heterocyclic amines. These data suggest that, although UGT2A1 is the only UGT2A enzyme active against PAH proximate carcinogens (including PAH diols), both UGTs 2A1 and 2A2 play an important role in the local detoxification of procarcinogenic monohydroxylated PAH metabolites.

  11. Importance of UDP-Glucuronosyltransferases 2A2 and 2A3 in Tobacco Carcinogen Metabolism

    PubMed Central

    Bushey, Ryan T.; Dluzen, Douglas F.

    2013-01-01

    UDP-glucuronosyltransferase A1 (UGT2A1) is expressed in the lung and exhibits activity against polycyclic aromatic hydrocarbons (PAHs), suggesting UGT2A1 involvement in the local metabolism of PAH tobacco carcinogens. The goal of the present study was to investigate the importance of two additional UGT2A enzymes, UGT2A2 and UGT2A3, in tobacco carcinogen metabolism. Real-time polymerase chain reaction suggested that wild-type UGT2A2 had the highest expression in the breast, followed by trachea > larynx > kidney. A novel splice variant of UGT2A2 lacking exon 3 (termed UGT2A2Δexon3) was investigated, with UGT2A2Δexon3 expression determined to be 25–50% that of wild-type UGT2A2 in all tissues examined. UGT2A3 was determined to be well expressed in the liver and colon, followed by pancreas > kidney > lung > tonsil > trachea > larynx. Cell homogenates prepared from human embryonic kidney (HEK)293 cells overexpressing wild-type UGT2A2 (termed UGT2A2_i1) exhibited glucuronidation activity, as observed by reverse-phase ultra-pressure liquid chromatography, against 1-hydroxy-(OH)-pyrene, 1-naphthol, and hydroxylated benzo(a)pyrene metabolites, whereas homogenates prepared from HEK293 cells overexpressing UGT2A3 only showed activity against simple PAHs like 1-OH-pyrene and 1-naphthol. Activity assays showed the UGT2A2Δexon3 protein (termed UGT2A2_i2) exhibited no detectable glucuronidation activity against all substrates examined; however, coexpression studies suggested that UGT2A2_i2 negatively modulates UGT2A2_i1 activity. Both UGT2A2 and UGT2A3 exhibited no detectable activity against complex PAH proximate carcinogens, tobacco-specific nitrosamines, or heterocyclic amines. These data suggest that, although UGT2A1 is the only UGT2A enzyme active against PAH proximate carcinogens (including PAH diols), both UGTs 2A1 and 2A2 play an important role in the local detoxification of procarcinogenic monohydroxylated PAH metabolites. PMID:23086198

  12. Adenosine A1 and A3 receptors protect astrocytes from hypoxic damage.

    PubMed

    Björklund, Olga; Shang, Mingmei; Tonazzini, Ilaria; Daré, Elisabetta; Fredholm, Bertil B

    2008-10-31

    Brain levels of adenosine are elevated during hypoxia. Through effects on adenosine receptors (A(1), A(2A), A(2B) and A(3)) on astrocytes, adenosine can influence functions such as glutamate uptake, reactive gliosis, swelling, as well as release of neurotrophic and neurotoxic factors having an impact on the outcome of metabolic stress. We have studied the roles of these receptors in astrocytes by evaluating their susceptibility to damage induced by oxygen deprivation or exposure to the hypoxia mimic cobalt chloride (CoCl(2)). Hypoxia caused ATP breakdown and purine release, whereas CoCl(2) (0.8 mM) mainly reduced ATP by causing cell death in human D384 astrocytoma cells. Further experiments were conducted in primary astrocytes prepared from specific adenosine receptor knock-out (KO) and wild type (WT) mice. In WT cells purine release following CoCl(2) exposure was mainly due to nucleotide release, whereas hypoxia-induced intracellular ATP breakdown followed by nucleoside efflux. N-ethylcarboxamidoadenosine (NECA), an unselective adenosine receptor agonist, protected from cell death following hypoxia. Cytotoxicity was more pronounced in A(1)R KO astrocytes and tended to be higher in WT cells in the presence of the A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Genetic deletion of A(2A) receptor resulted in less prominent effects. A(3)R KO glial cells were more affected by hypoxia than WT cells. Accordingly, the A(3) receptor agonist 2-chloro-N(6)-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (CL-IB-MECA) reduced ATP depletion caused by hypoxic conditions. It also reduced apoptosis in human astroglioma D384 cells after oxygen deprivation. In conclusion, the data point to a cytoprotective role of adenosine mediated by both A(1) and A(3) receptors in primary mouse astrocytes.

  13. Identification of two metallothioneins as novel inhalative coffee allergens cof a 2 and cof a 3.

    PubMed

    Peters, Ulrike; Frenzel, Karsten; Brettschneider, Reinhold; Oldenburg, Marcus; Bittner, Cordula

    2015-01-01

    Dust of green coffee beans is known to be a relevant cause for occupational allergic disorders in coffee industry workers. Recently, we described the first coffee allergen (Cof a 1) establishing an allergenic potential of green coffee dust. Our aim was to identify allergenic components of green coffee in order to enhance inhalative coffee allergy diagnosis. A Coffea arabica pJuFo cDNA phage display library was created and screened for IgE binding with sera from allergic coffee workers. Two further coffee allergens were identified by sequence analysis, expressed in E. coli, and evaluated by Western blots. The prevalence of sensitization to recombinant Cof a 1, Cof a 2, and Cof a 3 and to commercially available extract was investigated by ELISA (enzyme-linked immunosorbent assay) respectively CAP (capacity test) screening in 18 sera of symptomatic coffee workers. In addition to the previously described chitinase Cof a 1, two Coffea arabica cysteine-rich metallothioneins of 9 and 7 kDa were identified and included in the IUIS Allergen Nomenclature as Cof a 2 and Cof a 3. Serum IgE antibodies to at least one of the recombinant allergens were found in 8 out of 18 symptomatic coffee workers (44%). Only 2 of the analysed sera (11%) had reacted previously to the commercial allergy test. In addition to the previously described Cof a 1 we have identified two further coffee proteins to be type I coffee allergens (Cof a 2 and Cof a 3) which may have a relevant potential for the specific diagnosis and/or therapy of coffee allergy.

  14. Identification of Two Metallothioneins as Novel Inhalative Coffee Allergens Cof a 2 and Cof a 3

    PubMed Central

    Peters, Ulrike; Frenzel, Karsten; Brettschneider, Reinhold; Oldenburg, Marcus; Bittner, Cordula

    2015-01-01

    Background Dust of green coffee beans is known to be a relevant cause for occupational allergic disorders in coffee industry workers. Recently, we described the first coffee allergen (Cof a 1) establishing an allergenic potential of green coffee dust. Objective Our aim was to identify allergenic components of green coffee in order to enhance inhalative coffee allergy diagnosis. Methods A Coffea arabica pJuFo cDNA phage display library was created and screened for IgE binding with sera from allergic coffee workers. Two further coffee allergens were identified by sequence analysis, expressed in E. coli, and evaluated by Western blots. The prevalence of sensitization to recombinant Cof a 1, Cof a 2, and Cof a 3 and to commercially available extract was investigated by ELISA (enzyme-linked immunosorbent assay) respectively CAP (capacity test) screening in 18 sera of symptomatic coffee workers. Results In addition to the previously described chitinase Cof a 1, two Coffea arabica cysteine-rich metallothioneins of 9 and 7 kDa were identified and included in the IUIS Allergen Nomenclature as Cof a 2 and Cof a 3. Serum IgE antibodies to at least one of the recombinant allergens were found in 8 out of 18 symptomatic coffee workers (44%). Only 2 of the analysed sera (11%) had reacted previously to the commercial allergy test. Conclusions In addition to the previously described Cof a 1 we have identified two further coffee proteins to be type I coffee allergens (Cof a 2 and Cof a 3) which may have a relevant potential for the specific diagnosis and/or therapy of coffee allergy. PMID:25962169

  15. Pla a 2 and Pla a 3 reactivities identify plane tree-allergic patients with respiratory symptoms or food allergy.

    PubMed

    Scala, E; Cecchi, L; Abeni, D; Guerra, E C; Pirrotta, L; Locanto, M; Giani, M; Asero, R

    2017-04-01

    Nine hundred and thirty-nine rPla a 1, nPla a 2, and rPla a 3 ImmunoCAP ISAC reactors were studied. nPla a 2(pos) MUXF3(pos) but Pla a 1/2(neg) subjects were excluded from the study because they were cross-reactive carbohydrate determinant reactors. Among the 764 remaining participants, 71.9% were Pla a 3(pos) , 54.1% Pla a 2(pos) , and 10.9% Pla a 1(pos) . Among Pla a 3 reactors, 89.6% were Pru p 3(pos) and 86.8% Jug 3(pos) , but the strongest IgE recognition relationship was observed between Pla a 3 and Jug r 3. Distinctive clinical subsets could be documented among plane tree-allergic patients. Pla a 3 reactors had both local and systemic food-induced reactions, but lower past respiratory symptoms occurrence. Pla a 2 reactivity was associated with respiratory symptoms but inversely related to systemic reactions to food. Cosensitization to Pla a 2 and Pla a 3 was associated with a lower past incidence of severe food-induced reactions. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Contextual view of building A3 showing building A1 at left ...

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

    Contextual view of building A3 showing building A1 at left background and building A3 to right (the last of three together); camera facing west. - Mare Island Naval Shipyard, Shell House No. 1, Railroad Avenue, west side near Maseda Road, Vallejo, Solano County, CA

  17. COL9A2 and COL9A3 mutations in canine autosomal recessive oculoskeletal dysplasia.

    PubMed

    Goldstein, Orly; Guyon, Richard; Kukekova, Anna; Kuznetsova, Tatyana N; Pearce-Kelling, Susan E; Johnson, Jennifer; Aguirre, Gustavo D; Acland, Gregory M

    2010-08-01

    Oculoskeletal dysplasia segregates as an autosomal recessive trait in the Labrador retriever and Samoyed canine breeds, in which the causative loci have been termed drd1 and drd2, respectively. Affected dogs exhibit short-limbed dwarfism and severe ocular defects. The disease phenotype resembles human hereditary arthro-ophthalmopathies such as Stickler and Marshall syndromes, although these disorders are usually dominant. Linkage studies mapped drd1 to canine chromosome 24 and drd2 to canine chromosome 15. Positional candidate gene analysis then led to the identification of a 1-base insertional mutation in exon 1 of COL9A3 that cosegregates with drd1 and a 1,267-bp deletion mutation in the 5' end of COL9A2 that cosegregates with drd2. Both mutations affect the COL3 domain of the respective gene. Northern analysis showed that RNA expression of the respective genes was reduced in affected retinas. These models offer potential for studies such as protein-protein interactions between different members of the collagen gene family, regulation and expression of these genes in retina and cartilage, and even opportunities for gene therapy.

  18. COL4A2 mutations impair COL4A1 and COL4A2 secretion and cause hemorrhagic stroke.

    PubMed

    Jeanne, Marion; Labelle-Dumais, Cassandre; Jorgensen, Jeff; Kauffman, W Berkeley; Mancini, Grazia M; Favor, Jack; Valant, Valerie; Greenberg, Steven M; Rosand, Jonathan; Gould, Douglas B

    2012-01-13

    Collagen, type IV, alpha 1 (COL4A1) and alpha 2 (COL4A2) form heterotrimers and are abundant components of basement membranes, including those of the cerebral vasculature. COL4A1 mutations are an increasingly recognized cause of multisystem disorders, including highly penetrant cerebrovascular disease and intracerebral hemorrhage (ICH). Because COL4A1 and COL4A2 are structurally and functionally associated, we hypothesized that variants in COL4A2 would also cause ICH. We sequence COL4A2 in 96 patients with ICH and identify three rare, nonsynonymous coding variants in four patients that are not present in a cohort of 144 ICH-free individuals. All three variants change evolutionarily conserved amino acids. Using a cellular assay, we show that these putative mutations cause intracellular accumulation of COL4A1 and COL4A2 at the expense of their secretion, which supports their pathogenecity. Furthermore, we show that Col4a2 mutant mice also have completely penetrant ICH and that mutations in mouse and human lead to retention of COL4A1 and COL4A2 within the endoplasmic reticulum (ER). Importantly, two of the three putative mutations found in patients trigger ER stress and activate the unfolded protein response. The identification of putative COL4A2 mutations that might contribute to ICH in human patients provides insight into the pathogenic mechanisms of this disease. Our data suggest that COL4A2 mutations impair COL4A1 and COL4A2 secretion and can also result in cytotoxicity. Finally, our findings suggest that, collectively, mutations in COL4A1 and COL4A2 contribute to sporadic cases of ICH.

  19. βA3/A1-crystallin and persistent fetal vasculature (PFV) disease of the eye☆

    PubMed Central

    Zigler, J. Samuel; Valapala, Mallika; Shang, Peng; Hose, Stacey; Goldberg, Morton F.; Sinha, Debasish

    2015-01-01

    Background Persistent fetal vasculature (PFV) is a human disease in which the fetal vasculature of the eye fails to regress normally. The fetal, or hyaloid, vasculature nourishes the lens and retina during ocular development, subsequently regressing after formation of the retinal vessels. PFV causes serious congenital pathologies and is responsible for as much as 5% of blindness in the United States. Scope of review The causes of PFV are poorly understood, however there are a number of animal models in which aspects of the disease are present. One such model results from mutation or elimination of the gene (Cryba1) encoding βA3/A1-crystallin. In this review we focus on the possible mechanisms whereby loss of functional βA3/A1-crystallin might lead to PFV. Major conclusions Cryba1 is abundantly expressed in the lens, but is also expressed in certain other ocular cells, including astrocytes. In animal models lacking βA3/A1-crystallin, astrocyte numbers are increased and they migrate abnormally from the retina to ensheath the persistent hyaloid artery. Evidence is presented that the absence of functional βA3/A1-crystallin causes failure of the normal acidification of endolysosomal compartments in the astrocytes, leading to impairment of certain critical signaling pathways, including mTOR and Notch/STAT3. General significance The findings suggest that impaired endolysosomal signaling in ocular astrocytes can cause PFV disease, by adversely affecting the vascular remodeling processes essential to ocular development, including regression of the fetal vasculature. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease. PMID:26022148

  20. 47 CFR 80.1089 - Ship radio equipment-Sea areas A1 and A2.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 5 2014-10-01 2014-10-01 false Ship radio equipment-Sea areas A1 and A2. 80... RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1089 Ship radio equipment—Sea areas A1 and A2. This section...

  1. 47 CFR 80.1089 - Ship radio equipment-Sea areas A1 and A2.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 5 2013-10-01 2013-10-01 false Ship radio equipment-Sea areas A1 and A2. 80... RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1089 Ship radio equipment—Sea areas A1 and A2. This section...

  2. 47 CFR 80.1089 - Ship radio equipment-Sea areas A1 and A2.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 5 2010-10-01 2010-10-01 false Ship radio equipment-Sea areas A1 and A2. 80... RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1089 Ship radio equipment—Sea areas A1 and A2. This section...

  3. 47 CFR 80.1089 - Ship radio equipment-Sea areas A1 and A2.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 5 2012-10-01 2012-10-01 false Ship radio equipment-Sea areas A1 and A2. 80... RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1089 Ship radio equipment—Sea areas A1 and A2. This section...

  4. 47 CFR 80.1089 - Ship radio equipment-Sea areas A1 and A2.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 5 2011-10-01 2011-10-01 false Ship radio equipment-Sea areas A1 and A2. 80... RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1089 Ship radio equipment—Sea areas A1 and A2. This section...

  5. [Determination of antigens A1 and A2 in erythrocytes by phytohemagglutinins].

    PubMed

    Potapov, M I

    2004-01-01

    Phytohemagglutinins antiH2 antiA1 and antiA2, when used jointly, ensure a reliable diagnosis of subantigens A1 and A2. Vegetable extracts are easier-to-find and safer for the purpose versus rare and hard-to-standardize corresponding isosera.

  6. Do mutations in COL4A1 or COL4A2 cause thin basement membrane nephropathy (TBMN)?

    PubMed

    Zhang, Ke Wei; Tonna, Stephen; Wang, Yan Yan; Rana, Kesha; Padavarat, Smitha; Savige, Judy

    2007-05-01

    Thin basement membrane nephropathy (TBMN) is the commonest cause of persistent glomerular haematuria and often presents in childhood. Only 40% of affected individuals have mutations identified in the COL4A3 and COL4A4 genes, but mutations in the genes for other COL4A isoforms also result in thinned membranes in humans (COL4A5) and mice (COL4A1). This study examined whether COL4A1/COL4A2 represented a further genetic locus for TBMN. Nine families with TBMN in whom haematuria did not segregate with COL4A3/COL4A4, were examined for linkage to COL4A1/COL4A2 using five micro-satellite markers. In addition, index cases from these families plus a further 14 unrelated individuals with TBMN that was not due to COL4A3 or COL4A4 mutations (n=23) were screened for mutations in each of the 52 exons of COL4A1 and the 47 exons of COL4A2 using single stranded conformational analysis (SSCA). DNA samples that demonstrated bandshifts were sequenced. Haplotype analysis demonstrated that haematuria segregated with the COL4A1/COL4A2 locus in only two small families (2/9, 22%). No definite COL4A1 or COL4A2 mutations were identified in the 23 unrelated individuals with TBMN although novel polymorphisms were demonstrated. This study indicates that COL4A1/COL4A2 does not represent a further major genetic locus for TBMN.

  7. Genetic diversity among Clostridium botulinum strains harboring bont/A2 and bont/A3 genes.

    PubMed

    Lúquez, Carolina; Raphael, Brian H; Joseph, Lavin A; Meno, Sarah R; Fernández, Rafael A; Maslanka, Susan E

    2012-12-01

    Clostridium botulinum type A strains are known to be genetically diverse and widespread throughout the world. Genetic diversity studies have focused mainly on strains harboring one type A botulinum toxin gene, bont/A1, although all reported bont/A gene variants have been associated with botulism cases. Our study provides insight into the genetic diversity of C. botulinum type A strains, which contain bont/A2 (n = 42) and bont/A3 (n = 4) genes, isolated from diverse samples and geographic origins. Genetic diversity was assessed by using bont nucleotide sequencing, content analysis of the bont gene clusters, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Sequences of bont genes obtained in this study showed 99.9 to 100% identity with other bont/A2 or bont/A3 gene sequences available in public databases. The neurotoxin gene clusters of the subtype A2 and A3 strains analyzed in this study were similar in gene content. C. botulinum strains harboring bont/A2 and bont/A3 genes were divided into six and two MLST profiles, respectively. Four groups of strains shared a similarity of at least 95% by PFGE; the largest group included 21 out of 46 strains. The strains analyzed in this study showed relatively limited genetic diversity using either MLST or PFGE.

  8. A(1) and A(3) adenosine receptors inhibit LPS-induced hypoxia-inducible factor-1 accumulation in murine astrocytes.

    PubMed

    Gessi, Stefania; Merighi, Stefania; Stefanelli, Angela; Fazzi, Debora; Varani, Katia; Borea, Pier Andrea

    2013-10-01

    Adenosine (Ado) exerts neuroprotective and anti-inflammatory functions by acting through four receptor subtypes A1, A2A, A2B and A3. Astrocytes are one of its targets in the central nervous system. Hypoxia-inducible factor-1 (HIF-1), a master regulator of oxygen homeostasis, is induced after hypoxia, ischemia and inflammation and plays an important role in brain injury. HIF-1 is expressed by astrocytes, however the regulatory role played by Ado on HIF-1α modulation induced by inflammatory and hypoxic conditions has not been investigated. Primary murine astrocytes were activated with lipopolysaccharide (LPS) with or without Ado, Ado receptor agonists, antagonists and receptor silencing, before exposure to normoxia or hypoxia. HIF-1α accumulation and downstream genes regulation were determined. Ado inhibited LPS-increased HIF-1α accumulation under both normoxic and hypoxic conditions, through activation of A1 and A3 receptors. In cells incubated with the blockers of p44/42 MAPK and Akt, LPS-induced HIF-1α accumulation was significantly decreased in normoxia and hypoxia, suggesting the involvement of p44/42 MAPK and Akt in this effect and Ado inhibited kinases phosphorylation. A series of angiogenesis and metabolism related genes were modulated by hypoxia in an HIF-1 dependent way, but not further increased by LPS, with the exception of GLUT-1 and hexochinase II that were elevated by LPS only in normoxia and inhibited by Ado receptors. Instead, genes involved in inflammation, like inducible nitric-oxide synthase (iNOS) and A2B receptors, were increased by LPS in normoxia, strongly stimulated by LPS in concert with hypoxia and inhibited by Ado, through A1 and A3 receptor subtypes. In conclusion A1 and A3 receptors reduce the LPS-mediated HIF-1α accumulation in murine astrocytes, resulting in a downregulation of genes involved in inflammation and hypoxic injury, like iNOS and A2B receptors, in both normoxic and hypoxic conditions.

  9. βA3/A1-crystallin in astroglial cells regulates retinal vascular remodeling during development

    PubMed Central

    Sinha, Debasish; Klise, Andrew; Sergeev, Yuri; Hose, Stacey; Bhutto, Imran A.; Hackler, Laszlo; Malpic-llanos, Tanya; Samtani, Sonia; Grebe, Rhonda; Goldberg, Morton F.; Hejtmancik, J. Fielding; Nath, Avindra; Zack, Donald J.; Fariss, Robert N.; McLeod, D. Scott; Sundin, Olof; Broman, Karl W.; Lutty, Gerard A.; Zigler, J. Samuel

    2016-01-01

    Vascular remodeling is a complex process critical to development of the mature vascular system. Astrocytes are known to be indispensable for initial formation of the retinal vasculature; our studies with the Nuc1 rat provide novel evidence that these cells are also essential in the retinal vascular remodeling process. Nuc1 is a spontaneous mutation in the Sprague–Dawley rat originally characterized by nuclear cataracts in the heterozygote and microphthalmia in the homozygote. We report here that the Nuc1 allele results from mutation of the βA3/A1-crystallin gene, which in the neural retina is expressed only in astrocytes. We demonstrate striking structural abnormalities in Nuc1 astrocytes with profound effects on the organization of intermediate filaments. While vessels form in the Nuc1 retina, the subsequent remodeling process required to provide a mature vascular network is deficient. Our data implicate βA3/A1-crystallin as an important regulatory factor mediating vascular patterning and remodeling in the retina. PMID:17931883

  10. Transgenic Restoration of Urea Transporter A1 Confers Maximal Urinary Concentration in the Absence of Urea Transporter A3.

    PubMed

    Klein, Janet D; Wang, Yanhua; Mistry, Abinash; LaRocque, Lauren M; Molina, Patrick A; Rogers, Richard T; Blount, Mitsi A; Sands, Jeff M

    2016-05-01

    Urea has a critical role in urinary concentration. Mice lacking the inner medullary collecting duct (IMCD) urea transporter A1 (UT-A1) and urea transporter A3 (UT-A3) have very low levels of urea permeability and are unable to concentrate urine. To investigate the role of UT-A1 in the concentration of urine, we transgenically expressed UT-A1 in knockout mice lacking UT-A1 and UT-A3 using a construct with a UT-A1 gene that cannot be spliced to produce UT-A3. This construct was inserted behind the original UT-A promoter to yield a mouse expressing only UT-A1 (UT-A1(+/+)/UT-A3(-/-)). Western blot analysis demonstrated UT-A1 in the inner medulla of UT-A1(+/+)/UT-A3(-/-) and wild-type mice, but not in UT-A1/UT-A3 knockout mice, and an absence of UT-A3 in UT-A1(+/+)/UT-A3(-/-) and UT-A1/UT-A3 knockout mice. Immunohistochemistry in UT-A1(+/+)/UT-A3(-/-) mice also showed negative UT-A3 staining in kidney and other tissues and positive UT-A1 staining only in the IMCD. Urea permeability in isolated perfused IMCDs showed basal permeability in the UT-A1(+/+)/UT-A3(-/-) mice was similar to levels in wild-type mice, but vasopressin stimulation of urea permeability in wild-type mice was significantly greater (100% increase) than in UT-A1(+/+)/UT-A3(-/-) mice (8% increase). Notably, basal urine osmolalities in both wild-type and UT-A1(+/+)/UT-A3(-/-) mice increased upon overnight water restriction. We conclude that transgenic expression of UT-A1 restores basal urea permeability to the level in wild-type mice but does not restore vasopressin-stimulated levels of urea permeability. This information suggests that transgenic expression of UT-A1 alone in mice lacking UT-A1 and UT-A3 is sufficient to restore urine-concentrating ability. Copyright © 2016 by the American Society of Nephrology.

  11. 49 CFR 173.435 - Table of A1 and A2 values for radionuclides.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ...-GENERAL REQUIREMENTS FOR SHIPMENTS AND PACKAGINGS Class 7 (Radioactive) Materials § 173.435 Table of A1... Radioactive Material, No. TS-R-1” (IBR, see § 171.7 of this subchapter). b The values of A1 and A2 in curies... rate of decay or a measurement of the radiation level at a prescribed distance from the source. d...

  12. Adenosine A1, but not A2, receptor blockade increases anxiety and arousal in Zebrafish.

    PubMed

    Maximino, Caio; Lima, Monica G; Olivera, Karen R M; Picanço-Diniz, Domingos L W; Herculano, Anderson M

    2011-09-01

    Adenosinergic systems have been implicated in anxiety-like states, as caffeine can induce a state of anxiety in human beings. Caffeine is an antagonist at A(1) and A(2) adenosine receptors but it remains unclear whether anxiety is mediated by one or both of these. As the adenosinergic system is rather conserved, we opted to pursue these questions using zebrafish, a widely used model organism in genetics and developmental biology. Zebrafish adenosine 1. 2A.1 and 2A.2 receptors conserve histidine residues in TM6 and TM7 that are responsible for affinity in bovine A1 receptor. We investigated the effects of caffeine, PACPX (an A(1) receptor antagonist) and 1,3-dimethyl-1-propargylxanthine (DMPX) (an A(2) receptor antagonist) on anxiety-like behaviour and locomotor activity of zebrafish in the scototaxis test as well as evaluated the effects of these drugs on pigment aggregation. Caffeine increased anxiety at the dose of 100 mg/kg, while locomotion at the dose of 10 mg/kg was increased. Both doses of 10 and 100 mg/kg induced pigment aggregation. PACPX, on the other hand, increased anxiety at a dose of 6 mg/kg and induced pigment aggregation at the doses of 0.6 and 6 mg/kg, but did not produce a locomotor effect. DMPX, in turn, increased locomotion at the dose of 6 mg/kg but did not produce any effect on pigment aggregation or anxiety-like behaviour. These results indicate that blockade of A(1)-R, but not A(2)-R, induces anxiety and autonomic arousal, while the blockade of A(2)-R induces hyperlocomotion. Thus, as in rodents, caffeine's anxiogenic and arousing effects are probably mediated by A(1) receptors in zebrafish and its locomotor activating effect is probably mediated by A(2) receptors. © 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society.

  13. No evidence for disturbed COL1A1 and A2 expression in otosclerosis.

    PubMed

    Csomor, Péter; Liktor, Balázs; Liktor, Bálint; Sziklai, István; Karosi, Tamás

    2012-09-01

    Otosclerosis is a complex bone remodeling disorder of the human otic capsule that might be associated with various mutations of A1 and A2 alleles of type-I collagen. The study herein presented, investigates the possibilty of the genetic involvement of type-I collagen in the pathogenesis of histologically confirmed otosclerosis. A total of 55 ankylotic stapes footplates were analyzed. Cortical bone fragments (n = 30), incus (n = 3) and malleus (n = 2) specimens were employed as negative controls. Specimens were divided into two groups. The first group was processed using conventional H.E. hematoxylin-eosin (H.E.) staining and type-I collagen-specific immunofluorescent assay (IFA), while the second group was examined by COL1A1 and A2-specific RT-PCR. Otosclerotic- (n = 31) and non-otosclerotic stapes footplates (n = 9) as well as cortical bones (n = 20), incus (n = 2) and malleus specimens (n = 1) showed normal and quite similar A1 and A2 allele expression confirmed by IFA. RT-PCR analysis revealed normal and consistent mRNA expression of both alleles in each specimen. Expression levels and patterns of COL1A1/A2 alleles did not show significant correlation with the histological diagnosis of otosclerosis. Type-I collagen is a highly conserved structure protein, which plays a fundamental role in the integritiy of various connective tissues. Mutations of A1 and A2 alleles result in serious systemic disorders of the skeleton, tendons and skin. Since otosclerosis is an organ-specific disease, it is difficult to explain its genetic association with type-I collagen. In conclusion, we found no evidence supporting the putative link of COL1A1 and COL1A2 alleles with otosclerosis.

  14. Boric acid increases the expression levels of human anion exchanger genes SLC4A2 and SLC4A3.

    PubMed

    Akbas, F; Aydin, Z

    2012-04-03

    Boron is an important micronutrient in plants and animals. The role of boron in living systems includes coordinated regulation of gene expression, growth and proliferation of higher plants and animals. There are several well-defined genes associated with boron transportation and tolerance in plants and these genes show close homology with human anion exchanger genes. Mutation of these genes also characterizes some genetic disorders. We investigated the toxic effects of boric acid on HEK293 cells and mRNA expression of anion exchanger (SLC4A1, SLC4A2 and SLC4A3) genes. Cytotoxicity of boric acid at different concentrations was tested by using the methylthiazolyldiphenyl-tetrazolium bromide assay. Gene expression profiles were examined using quantitative real-time PCR. In the HEK293 cells, the nontoxic upper concentration of boric acid was 250 μM; more than 500 μM caused cytotoxicity. The 250 μM boric acid concentration increased gene expression level of SLC4A2 up to 8.6-fold and SLC4A3 up to 2.6-fold, after 36-h incubation. There was no significant effect of boric acid on SLC4A1 mRNA expression levels.

  15. Optimization of arylindenopyrimidines as potent adenosine A(2A)/A(1) antagonists.

    PubMed

    Shook, Brian C; Rassnick, Stefanie; Chakravarty, Devraj; Wallace, Nathaniel; Ault, Mark; Crooke, Jeffrey; Barbay, J Kent; Wang, Aihua; Leonard, Kristi; Powell, Mark T; Alford, Vernon; Hall, Daniel; Rupert, Kenneth C; Heintzelman, Geoffrey R; Hansen, Kristen; Bullington, James L; Scannevin, Robert H; Carroll, Karen; Lampron, Lisa; Westover, Lori; Russell, Ronald; Branum, Shawn; Wells, Kenneth; Damon, Sandra; Youells, Scott; Beauchamp, Derek; Li, Xun; Rhodes, Kenneth; Jackson, Paul F

    2010-05-01

    Two reactive metabolites were identified in vivo for the dual A(2A)/A(1) receptor antagonist 1. Two strategies were implemented to successfully mitigate the metabolic liabilities associated with 1. Optimization of the arylindenopyrimidines led to a number of amide, ether, and amino analogs having comparable in vitro and in vivo activity. 2010 Elsevier Ltd. All rights reserved.

  16. [Simplified microdetermination of cerebral phospholipase A1, A2 and lysophopholipase].

    PubMed

    Hirashima, Y; Koshu, K; Kamiyama, K; Endo, S; Takaku, A; Honda, T; Takasaki, C

    1983-08-01

    The purpose of our study was to examine the ischemia induced enzymatic changes of decaylation-reacylation cycle of membrane phospholipids in dog brain. In this study, we developed new modified method for assay of phospholipase A1, A2 and lysophospholipase which is simpler and needs only a smaller amount of materials. For the first report, we introduced this new method and demonstrated some properties of phospholipase A1, A2 and lysophospholipase in dog brain. Crude enzyme solution for assays of phospholipase A1, A2 and lysophospholipase was gained from extraction of frozen brain with aceton, butanol and saline. The level of phosphorus in the enzyme extract was determined and only those extracts which had a level of phosphorus within a certain range were used. The substrates for assays were L-alpha-[beta-palmitoyl-1-14C] phosphatidylcholine, dipalmitoyl for phospholipase A1 and A2 and L-lysophosphatidylcholine-1-[1-14C] palmitoyl for lysophospolipase respectively. Each radioactive substrates was diluted with cold carrier lipid to give the proper specific activity. Reaction system including substrate, buffer [pH 7.0] and enzyme extract was incubated for 10 hours at 38 degrees C. But for the assay of phospholipase A1 and A2, enzyme solution was pre-incubated at 70 degrees C for 5 minutes. In our new method, reaction mixture was directly separated by TLC without extracting lipids. Enzyme activities were calculated from radio thin-layer chromatograms. Furthermore, we made a comparison between our method and the former one. The value of each enzyme activity was slightly higher in our method than in the former one. However, it was revealed that the results were reproducible in both methods.

  17. Mass spectrometry-based ligand binding assays on adenosine A1 and A2A receptors.

    PubMed

    Massink, A; Holzheimer, M; Hölscher, A; Louvel, J; Guo, D; Spijksma, G; Hankemeier, T; IJzerman, A P

    2015-12-01

    Conventional methods to measure ligand-receptor binding parameters typically require radiolabeled ligands as probes. Despite the robustness of radioligand binding assays, they carry inherent disadvantages in terms of safety precautions, expensive synthesis, special lab requirements, and waste disposal. Mass spectrometry (MS) is a method that can selectively detect ligands without the need of a label. The sensitivity of MS equipment increases progressively, and currently, it is possible to detect low ligand quantities that are usually found in ligand binding assays. We developed a label-free MS ligand binding (MS binding) assay on the adenosine A(1) and A(2A) receptors (A(1)AR and A(2A)AR), which are well-characterized members of the class A G protein-coupled receptor (GPCR) family. Radioligand binding assays for both receptors are well established, and ample data is available to compare and evaluate the performance of an MS binding assay. 1,3-Dipropyl-8-cyclopentyl-xanthine (DPCPX) and 4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol (ZM-241,385) are high-affinity ligands selective for the A(1)AR and A(2A)AR, respectively. To proof the feasibility of MS binding on the A(1)AR and A(2A)AR, we first developed an MS detection method for unlabeled DPCPX and ZM-241,385. To serve as internal standards, both compounds were also deuterium-labeled. Subsequently, we investigated whether the two unlabeled compounds could substitute for their radiolabeled counterparts as marker ligands in binding experiments, including saturation, displacement, dissociation, and competition association assays. Furthermore, we investigated the accuracy of these assays if the use of internal standards was excluded. The results demonstrate the feasibility of the MS binding assay, even in the absence of a deuterium-labeled internal standard, and provide great promise for the further development of label-free assays based on MS for other GPCRs.

  18. 40 CFR Table A-3 to Subpart A of... - Source Category List for § 98.2(a)(1)

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 22 2012-07-01 2012-07-01 false Source Category List for § 98.2(a)(1) A Table A-3 to Subpart A of Part 98 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) MANDATORY GREENHOUSE GAS REPORTING General Provision Pt. 98, Subpt....

  19. 40 CFR Table A-3 to Subpart A of... - Source Category List for § 98.2(a)(1)

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 21 2011-07-01 2011-07-01 false Source Category List for § 98.2(a)(1) A Table A-3 to Subpart A of Part 98 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) MANDATORY GREENHOUSE GAS REPORTING General Provision Pt. 98, Subpt....

  20. 40 CFR Table A-3 to Subpart A of... - Source Category List for § 98.2(a)(1)

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 22 2013-07-01 2013-07-01 false Source Category List for § 98.2(a)(1) A Table A-3 to Subpart A of Part 98 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) MANDATORY GREENHOUSE GAS REPORTING General Provision Pt. 98, Subpt....

  1. 40 CFR Table A-3 to Subpart A of... - Source Category List for § 98.2(a)(1)

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 21 2014-07-01 2014-07-01 false Source Category List for § 98.2(a)(1) A Table A-3 to Subpart A of Part 98 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) MANDATORY GREENHOUSE GAS REPORTING General Provision Pt. 98, Subpt....

  2. COL9A2 and COL9A3 mutations in canine autosomal recessive Oculo-skeletal Dysplasia

    PubMed Central

    Goldstein, Orly; Guyon, Richard; Kukekova, Anna; Pearce-Kelling, Sue; Johnson, Jennifer; Aguirre, Gustavo D.; Acland, Gregory M.

    2010-01-01

    Oculo-skeletal dysplasia segregates in two canine breeds, the Labrador retriever and samoyed, in which the causative loci have been termed drd1 and drd2, respectively. Affected dogs exhibit short-limbed dwarfism together with severe ocular defects, and this phenotype is inherited as an autosomal recessive trait in both breeds. The clinical and pathological appearance resembles human hereditary arthro-ophthalmopathies such as Stickler syndrome, or Marshall Syndrome, although these human disorders are usually dominant. Linkage studies in drd1-informative pedigrees mapped the locus to canine chromosome 24, and led to the identification of an insertional mutation in exon 1 of the gene COL9A3 that cosegregates with the disease. The drd2 locus was similarly mapped to canine chromosome 15 and shown to cosegregate with a 1,267 bp deletion mutation in the 5′ end of COL9A2. Both mutations affect the COL3 domain of the respective gene. Northern analysis showed reduced RNA expression in affected retina compared to normal. These models offer potential for studies such as protein-protein interactions between different members of the collagen gene family; regulation and expression of these genes in retina and cartilage, and even opportunities for gene therapy. PMID:20686772

  3. Temperature dependent quenching of CH(A 2Δ), NH(A 3Π), NH(c 1Π), and PH(A 3Π) by H 2

    NASA Astrophysics Data System (ADS)

    Heinrich, P.; Stuhl, F.

    1995-10-01

    The kinetics of the quenching of electronically excited NH(c 1Π), NH(A 3Π), PH(A 3Π), and CH(A 2Δ) radicals in collisions with H 2 was investigated in the temperature range 300 to about 1000 K. The reaction systems were heated by pulsed IR radiation from a CO 2 laser. The excited states were generated in the heated systems by ArF laser photolysis of appropriate parent molecules. Together with low temperature data, this study shows that the quenching of NH(c), NH(A), and PH(A) occurs without a barrier on the reaction paths while the removal of CH(A) exhibits an activation energy. These processes will be related to the corresponding dissociation/association systems AH3 ⇔ AH∗ + H2 and ab initio calculation thereof.

  4. A continuous spectrophotometric assay that distinguishes between phospholipase A1 and A2 activities[S

    PubMed Central

    El Alaoui, Meddy; Soulère, Laurent; Noiriel, Alexandre; Popowycz, Florence; Khatib, Abdallah; Queneau, Yves; Abousalham, Abdelkarim

    2016-01-01

    A new spectrophotometric assay was developed to measure, continuously and specifically, phospholipase A1 (PLA1) or phospholipase A2 (PLA2) activities using synthetic glycerophosphatidylcholines (PCs) containing α-eleostearic acid, either at the sn-1 position [1-α-eleostearoyl-2-octadecyl-rac-glycero-3-phosphocholine (EOPC)] or at the sn-2 position [1-octadecyl-2-α-eleostearoyl-rac-glycero-3-phosphocholine (OEPC)]. The substrates were coated onto the wells of microtiter plates. A nonhydrolyzable ether bond, with a non-UV-absorbing alkyl chain, was introduced at the other sn position to prevent acyl chain migration during lipolysis. Upon enzyme action, α-eleostearic acid is liberated and then solubilized into the micellar phase. The PLA1 or PLA2 activity was measured by the increase in absorbance at 272 nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. EOPC and OEPC differentiate, with excellent accuracy, between PLA1 and PLA2 activity. Lecitase®, guinea pig pancreatic lipase-related protein 2 (known to be a PLA1 enzyme), bee venom PLA2, and porcine pancreatic PLA2 were all used to validate the assay. Compared with current assays used for continuously measuring PLA1 or PLA2 activities and/or their inhibitors, the development of this sensitive enzymatic method, using coated PC substrate analogs to natural lipids and based on the UV spectroscopic properties of α-eleostearic acid, is a significant improvement. PMID:27194811

  5. ATPase activity of Mycobacterium tuberculosis SecA1 and SecA2 proteins and its importance for SecA2 function in macrophages.

    PubMed

    Hou, Jie M; D'Lima, Nadia G; Rigel, Nathan W; Gibbons, Henry S; McCann, Jessica R; Braunstein, Miriam; Teschke, Carolyn M

    2008-07-01

    The Sec-dependent translocation pathway that involves the essential SecA protein and the membrane-bound SecYEG translocon is used to export many proteins across the cytoplasmic membrane. Recently, several pathogenic bacteria, including Mycobacterium tuberculosis, were shown to possess two SecA homologs, SecA1 and SecA2. SecA1 is essential for general protein export. SecA2 is specific for a subset of exported proteins and is important for M. tuberculosis virulence. The enzymatic activities of two SecA proteins from the same microorganism have not been defined for any bacteria. Here, M. tuberculosis SecA1 and SecA2 are shown to bind ATP with high affinity, though the affinity of SecA1 for ATP is weaker than that of SecA2 or Escherichia coli SecA. Amino acid substitution of arginine or alanine for the conserved lysine in the Walker A motif of SecA2 eliminated ATP binding. We used the SecA2(K115R) variant to show that ATP binding was necessary for the SecA2 function of promoting intracellular growth of M. tuberculosis in macrophages. These results are the first to show the importance of ATPase activity in the function of accessory SecA2 proteins.

  6. Homologous Pairing Activities of Two Rice RAD51 Proteins, RAD51A1 and RAD51A2

    PubMed Central

    Ikawa, Shukuko; Mimida, Naozumi; Shimizu, Takeshi; Toki, Seiichi; Ichikawa, Hiroaki; Shibata, Takehiko; Kurumizaka, Hitoshi

    2013-01-01

    In higher eukaryotes, RAD51 functions as an essential protein in homologous recombination and recombinational repair of DNA double strand breaks. During these processes, RAD51 catalyzes homologous pairing between single-stranded DNA and double-stranded DNA. Japonica cultivars of rice (Oryza sativa) encode two RAD51 proteins, RAD51A1 and RAD51A2, whereas only one RAD51 exists in yeast and mammals. However, the functional differences between RAD51A1 and RAD51A2 have not been elucidated, because their biochemical properties have not been characterized. In the present study, we purified RAD51A1 and RAD51A2, and found that RAD51A2 robustly promotes homologous pairing in vitro. RAD51A1 also possesses homologous-pairing activity, but it is only about 10% of the RAD51A2 activity. Both RAD51A1 and RAD51A2 bind to ssDNA and dsDNA, and their DNA binding strictly requires ATP, which modulates the polymer formation activities of RAD51A1 and RAD51A2. These findings suggest that although both RAD51A1 and RAD51A2 have the potential to catalyze homologous pairing, RAD51A2 may be the major recombinase in rice. PMID:24124491

  7. Qualitative Differences in the N-Acetyl-D-galactosaminyltransferases Produced by Human A1 and A2 Genes

    PubMed Central

    Schachter, H.; Michaels, M. A.; Tilley, Christine A.; Crookston, Marie C.; Crookston, J. H.

    1973-01-01

    This study describes the kinetic properties of N-acetyl-D-galactosaminyltransferase in serum from subjects with blood groups A1 and A2. When the A1 and A2 enzymes were compared, with lacto-N-fucopentaose I and 2′-fucosyllactose as acceptors, the enzymes differed in their cation requirements, pH optima, and Km values. The two acceptors competed for the same transferase. Mixing experiments showed that the lower activity of the A2 enzyme could not be attributed to a modifier or inhibitor in serum. It was concluded that the A1 and A2 enzymes differ qualitatively. PMID:4509655

  8. βA3/A1-crystallin is required for proper astrocyte template formation and vascular remodeling in the retina

    PubMed Central

    Sinha, Debasish; Valapala, Mallika; Bhutto, Imran; Patek, Bonnie; Zhang, Cheng; Hose, Stacey; Yang, Fang; Cano, Marisol; Stark, Walter J.; Lutty, Gerard A.; Zigler, J. Samuel; Wawrousek, Eric F.

    2013-01-01

    Nuc1 is a spontaneous rat mutant resulting from a mutation in the Cryba1 gene, coding for βA3/A1-crystallin. Our earlier studies with Nuc1 provided novel evidence that astrocytes, which express βA3/A1-crystallin, have a pivotal role in retinal remodeling. The role of astrocytes in the retina is only beginning to be explored. One of the limitations in the field is the lack of appropriate animal models to better investigate the function of astrocytes in retinal health and disease. We have now established transgenic mice that overexpress the Nuc1 mutant form of Cryba1, specifically in astrocytes. Astrocytes in wild type mice show normal compact stellate structure, producing a honeycomb-like network. In contrast, in transgenics over-expressing the mutant (Nuc1) Cryba1 in astrocytes, bundle-like structures with abnormal patterns and morphology were observed. In the nerve fiber layer of the transgenic mice, an additional layer of astrocytes adjacent to the vitreous is evident. This abnormal organization of astrocytes affects both the superficial and deep retinal vascular density and remodeling. Fluorescein angiography showed increased venous dilation and tortuosity of branches in the transgenic retina, as compared to wild type. Moreover, there appear to be fewer interactions between astrocytes and endothelial cells in the transgenic retina than in normal mouse retina. Further, astrocytes overexpressing the mutant βA3/A1-crystallin migrate into the vitreous, and ensheath the hyaloid artery, in a manner similar to that seen in the Nuc1 rat. Together, these data demonstrate that developmental abnormalities of astrocytes can affect the normal remodeling process of both fetal and retinal vessels of the eye and that βA3/A1-crystallin is essential for normal astrocyte function in the retina. PMID:22427112

  9. Allosteric interactions at adenosine A1 and A3 receptors: new insights into the role of small molecules and receptor dimerization

    PubMed Central

    Hill, Stephen J; May, Lauren T; Kellam, Barrie; Woolard, Jeanette

    2014-01-01

    The purine nucleoside adenosine is present in all cells in tightly regulated concentrations. It is released under a variety of physiological and pathophysiological conditions to facilitate protection and regeneration of tissues. Adenosine acts via specific GPCRs to either stimulate cyclic AMP formation, as exemplified by Gs-protein-coupled adenosine receptors (A2A and A2B), or inhibit AC activity, in the case of Gi/o-coupled adenosine receptors (A1 and A3). Recent advances in our understanding of GPCR structure have provided insights into the conformational changes that occur during receptor activation following binding of agonists to orthosteric (i.e. at the same binding site as an endogenous modulator) and allosteric regulators to allosteric sites (i.e. at a site that is topographically distinct from the endogenous modulator). Binding of drugs to allosteric sites may lead to changes in affinity or efficacy, and affords considerable potential for increased selectivity in new drug development. Herein, we provide an overview of the properties of selective allosteric regulators of the adenosine A1 and A3 receptors, focusing on the impact of receptor dimerization, mechanistic approaches to single-cell ligand-binding kinetics and the effects of A1- and A3-receptor allosteric modulators on in vivo pharmacology. Linked ArticlesThis article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-5 PMID:24024783

  10. Site-specific Arylation of Rat Glutathione S-Transferase A1 and A2 by Bromobenzene Metabolites in vivo

    PubMed Central

    Koen, Yakov M.; Yue, Weimin; Galeva, Nadezhda A.; Williams, Todd D.; Hanzlik, Robert P.

    2006-01-01

    The hepatotoxicity of bromobenzene (BB) derives from its reactive metabolites (epoxides and quinones) which arylate cellular proteins. Application of proteomic methods to liver proteins from rats treated with an hepatotoxic dose of [14C]-BB has identified more than 40 target proteins, but no adducted peptides have yet been observed. Because such proteins are known to contain bromophenyl- and bromodihydroxyphenyl derivatives of cysteine, histidine and lysine, the failure to observe modified peptides has been attributed to the low level of total covalent binding and to the “dilution” effect of multiple metabolites reacting at multiple sites on multiple proteins. In this work glutathione transferase, a well known and abundant BB-target protein, was isolated from liver cytosol of rats treated with 14C-BB using a GSH-agarose affinity column and further resolved by reverse phase HPLC into subunits M1, M2, A1, A2 and A3. The subunits were identified by a combination of SDS-PAGE, whole-molecule mass spectrometry and peptide mass mapping and found to contain radioactivity corresponding to 0.01 - 0.05 adduct per molecule of protein. Examination of tryptic digests of these subunits by MALDI-TOF and ESI-MS again failed to reveal any apparent adducted peptides despite observed sequence coverages up to 87%. However, using HPLC-LTQ-FTMS to search for predicted modified tryptic peptides revealed peaks corresponding, with a high degree of mass accuracy, to a bromobenzoquinone adduct of peptide 89-119 in both GSTA1 and A2. The identity of these adducts and their location at Cys-111 was confirmed by MS-MS. No evidence for the presence of any putative BB-adducts in GST M1, M2 or A3 was obtained. This work highlights the challenges involved in the unambiguous identification of reactive metabolite adducts formed in vivo. PMID:17112229

  11. Genetic evidence that mutations in the COL1A1, COL1A2, COL3A1, or COL5A2 collagen genes are not responsible for mitral valve prolapse.

    PubMed Central

    Henney, A M; Tsipouras, P; Schwartz, R C; Child, A H; Devereux, R B; Leech, G J

    1989-01-01

    DNA markers were used to assess the segregation of genes encoding the collagen types that predominate in the mitral valve (types I, III, and V) in two family pedigrees that are phenotypically different but showed dominantly inherited mitral valve prolapse. The inheritance of these markers was compared with the segregation of the phenotype for mitral valve prolapse in both families. In one family it was shown that the COL1A1, COL1A2, COL3A1, and COL5A2 genes segregated independently of the phenotype; in the other family the results for COL1A1, COL1A2, and COL5A2 were similar but analysis at the COL3A1 locus was not possible. These data indicate that in these families mitral valve prolapse does not arise from a defect in one of these collagen genes. PMID:2930668

  12. Humanized SFTPA1 and SFTPA2 Transgenic Mice Reveal Functional Divergence of SP-A1 and SP-A2

    PubMed Central

    Wang, Guirong; Guo, Xiaoxuan; DiAngelo, Susan; Thomas, Neal J.; Floros, Joanna

    2010-01-01

    Surfactant protein A (SP-A) plays a role in lung innate immunity and surfactant-related functions. Two functional genes, SP-A1 (SFTPA1) and SP-A2 (SFTPA2), are present in humans and primates (rodents have one gene). Single gene SP-A1 or SP-A2 proteins expressed in vitro are functional. To study their role in vivo, we generated humanized transgenic (hTG) C57BL/6 mice, SP-A1(6A4) and SP-A2(1A3). The SP-A cDNA in experimental constructs was driven by the 3.7-kb SP-C promoter. Positive hTG mice were bred with SP-A knock-out mice to generate F8 offspring for study. Epithelial alveolar type II cells were SP-A-positive, and Clara cells were negative by immunohistochemistry in hTG mice. The levels of SP-A in lungs of two hTG lines used were comparable with those in human lungs. Southern blot analysis indicated that two cDNA copies of either SP-A1(6A4) or SP-A2(1A3) were integrated as a concatemer into the genome of each of the two hTG lines. Electron microscopy analysis revealed that hTG mice with a single SP-A1(6A4) or SP-A2(1A3) gene product lacked tubular myelin (TM), but hTG mice carrying both had TM. Furthermore, TM was observed in human bronchoalveolar lavage fluid only if both SP-A1 and SP-A2 gene products were present and not in those containing primarily (>99.7%) either SP-A1 or SP-A2 gene products. In vivo rescue study confirmed that TM can only be restored after administering exogenous SP-A containing both SP-A1 and SP-A2 into the lungs of SP-A knock-out mice. These observations indicate that SP-A1 and SP-A2 diverged functionally at least in terms of TM formation. PMID:20048345

  13. Outflow occlusion with A3A3 anastomosis for a doughnut-shaped partially thrombosed giant A2 aneurysm

    PubMed Central

    Ito, Hidemichi; Miyano, Ryotaro; Sase, Taigen; Wakui, Daisuke; Matsumori, Takashi; Takasuna, Hiroshi; Oshio, Kotaro; Tanaka, Yuichiro

    2016-01-01

    Background: A doughnut-shaped aneurysm, which is defined as a round-shaped aneurysm composed of an intraluminar thrombus and marginal parent artery, is an extremely uncommon subtype of partially thrombosed giant aneurysms. Surgical treatment of this characteristic aneurysm is technically challenging. Case Description: We report a rare case of a 79-year-old man with a symptomatic doughnut-shaped giant aneurysm at the A2 portion, which was successfully treated by outflow occlusion with an A3A3 side-to-side anastomosis. Postoperative angiograms demonstrated no filling of the doughnut-shaped aneurysm and perfusion in the distal right anterior cerebral artery territory via the anastomosis. Follow-up magnetic resonance imaging 1 year after the surgery demonstrated significant diminution of the aneurysm. Conclusions: Outflow occlusion with distal revascularization could be an effective surgical option for such a unique aneurysm. To the best of our knowledge, this is the first report of outflow occlusion as a therapy for doughnut-shaped aneurysms. PMID:28144486

  14. Prostaglandin A2 Enhances Cellular Insulin Sensitivity via a Mechanism that Involves the Orphan Nuclear Receptor NR4A3

    PubMed Central

    Zhu, X.; Walton, R. G.; Tian, L.; Luo, N.; Ho, S-R.; Fu, Y.; Garvey, W. T.

    2014-01-01

    We have previously reported that members of the NR4A family of orphan nuclear receptors can augment insulin’s ability to stimulate glucose transport in adipocytes. In the current study, we endeavored to test for an insulin-sensitizing effect in muscle cells and to identify a potential transactivator. Lentiviral constructs were used to engineer both hyperexpression and shRNA silencing of NR4A3 in C2C12 myocytes. The NR4A3 hyper-expression construct led to a significant increase in glucose transport rates in the presence of maximal insulin while the NR4A3 knock-down exhibited a significant reduction in insulin-stimulated glucose transport rates. Consistently, insulin-mediated AKT phosphorylation was increased by NR4A3 hyperexpression and decreased following shRNA NR4A3 suppression. Then, we examined effects of prostaglandin A2 (PGA2) on insulin action and NR4A3 transactivation. PGA2 augmented insulin-stimulated glucose uptake in C2C12 myocytes and AKT phosphorylation after 12-h treatment, without significant effects on basal transport or basal AKT phosphorylation. More importantly, we demonstrated that PGA2 led to a greater improvement in insulin-stimulated glucose rates in NR4A3 overexpressing C2C12 myocytes, when compared with Lac-Z controls stimulated with insulin and PGA2. Moreover, the sensitizing effect of PGA2 was significantly diminished in NR4A3 knockdown myocytes compared to scramble controls. These results show for the first time that: (i) PGA2 augments insulin action in myocytes as manifested by enhanced stimulation of glucose transport and AKT phosphorylation; and (ii) the insulin sensitizing effect is dependent upon the orphan nuclear receptor NR4A3. PMID:23104421

  15. Inhibition of phagocytosis by Haemophilus ducreyi requires expression of the LspA1 and LspA2 proteins.

    PubMed

    Vakevainen, Merja; Greenberg, Steven; Hansen, Eric J

    2003-10-01

    Haemophilus ducreyi previously has been shown to inhibit the phagocytosis of both secondary targets and itself by certain cells in vitro. Wild-type H. ducreyi strain 35000HP contains two genes, lspA1 and lspA2, whose encoded protein products are predicted to be 456 and 543 kDa, respectively. An isogenic mutant of H. ducreyi 35000HP with inactivated lspA1 and lspA2 genes has been shown to exhibit substantially decreased virulence in the temperature-dependent rabbit model for chancroid. This lspA1 lspA2 mutant was tested for its ability to inhibit phagocytosis of immunoglobulin G-opsonized particles by differentiated HL-60 and U-937 cells and by J774A.1 cells. The wild-type strain H. ducreyi 35000HP readily inhibited phagocytosis, whereas the lspA1 lspA2 mutant was unable to inhibit phagocytosis. Similarly, the wild-type strain was resistant to phagocytosis, whereas the lspA1 lspA2 mutant was readily engulfed by phagocytes. This inhibitory effect of wild-type H. ducreyi on phagocytic activity was primarily associated with live bacterial cells but could also be found, under certain conditions, in concentrated H. ducreyi culture supernatant fluids that lacked detectable outer membrane fragments. Both the wild-type strain and the lspA1 lspA2 mutant attached to phagocytes at similar levels. These results indicate that the LspA1 and LspA2 proteins of H. ducreyi are involved, directly or indirectly, in the antiphagocytic activity of this pathogen, and they provide a possible explanation for the greatly reduced virulence of the lspA1 lspA2 mutant.

  16. Comprehensive comparison of two protein family of P-ATPases (13A1 and 13A3) in insects.

    PubMed

    Seddigh, Samin

    2017-06-01

    The P-type ATPases (P-ATPases) are present in all living cells where they mediate ion transport across membranes on the expense of ATP hydrolysis. Different ions which are transported by these pumps are protons like calcium, sodium, potassium, and heavy metals such as manganese, iron, copper, and zinc. Maintenance of the proper gradients for essential ions across cellular membranes makes P-ATPases crucial for cell survival. In this study, characterization of two families of P-ATPases including P-ATPase 13A1 and P-ATPase 13A3 protein was compared in two different insect species from different orders. According to the conserved motifs found with MEME, nine motifs were shared by insects of 13A1 family but eight in 13A3 family. Seven different insect species from 13A1 and five samples from 13A3 family were selected as the representative samples for functional and structural analyses. The structural and functional analyses were performed with ProtParam, SOPMA, SignalP 4.1, TMHMM 2.0, ProtScale and ProDom tools in the ExPASy database. The tertiary structure of Bombus terrestris as a sample of each family of insects were predicted by the Phyre2 and TM-score servers and their similarities were verified by SuperPose server. The tertiary structures were predicted via the "c3b9bA" model (PDB Accession Code: 3B9B) in P-ATPase 13A1 family and "c2zxeA" model (PDB Accession Code: 2ZXE) in P-ATPase 13A3 family. A phylogenetic tree was constructed with MEGA 6.06 software using the Neighbor-joining method. According to the results, there was a high identity of P-ATPase families so that they should be derived from a common ancestor however they belonged to separate groups. In protein-protein interaction analysis by STRING 10.0, six common enriched pathways of KEGG were identified in B. terrestris in both families. The obtained data provide a background for bioinformatic studies of the function and evolution of other insects and organisms. Copyright © 2017 Elsevier Ltd. All rights

  17. 49 CFR 173.435 - Table of A1 and A2 values for radionuclides.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ...-GENERAL REQUIREMENTS FOR SHIPMENTS AND PACKAGINGS Class 7 (Radioactive) Materials § 173.435 Table of A1... Terabecquerels (TBq), (see § 171.10). c The quantity may be determined from a measurement of the rate of decay...

  18. Stabilizing interactions between D666-S1787 and T657-Y1792 at the A2-A3 interface support factor VIIIa stability in the blood clotting pathway.

    PubMed

    Monaghan, M; Wakabayashi, H; Griffiths, A E; Fay, P J

    2016-05-01

    Essentials Factor VIIIa (FVIIIa) is unstable due to loss of A2; D666 and Y1792 contribute to its stability. We conducted a study to identify the interactions made at these residues at the A2-A3 interface. We present evidence for stabilizing interactions between D666-S1787 and T657-Y1792 in FVIIIa. A D666C/S1788C variant with a disulfide A2-A3 linkage has a FVIIIa decay rate that is 1% of wild-type. Background Factor (F)VIIIa activity and stability depends on the non-covalent association of the A2 subunit with the A1/A3C1C2 dimer, but the interactions that contribute to A2 association are not well defined. Previous work had shown that D666A and Y1792F mutations at the A2-A3 interface resulted in increased FVIIIa decay, suggesting that the residues were involved in bonding interactions important for FVIIIa stability. Objectives Several potential hydrogen bonding partners of D666 and Y1792 across the A2-A3 interface were selected from the low-resolution FVIII crystal structure, and we used mutagenesis and biochemical analysis to examine the bonding interactions occurring at D666 and Y1792. Methods Using a series of stability and functional analyses, we analyzed FVIII variants in which D666 and Y1792 were each swapped with the residues of potential bonding partners. Results and conclusions We present evidence for hydrogen bonds between D666 and S1787 and between Y1792 and T657 that are important for FVIIIa stability. D666S/S1787D and T657Y/Y1792T variants each displayed wild-type (WT)-like FVIIIa stability and performed like WT FVIII in a series of functional analyses, whereas D666S, S1787D, and Y1792T single variants showed increased FVIIIa decay and a T657Y variant had little FVIIIa activity. These results suggest that WT hydrogen bonds are disrupted with the single mutations but maintained in the swap variants. Furthermore, mutation of D666 and S1788 to cysteine resulted in disulfide bond formation across the A2-A3 interface, confirming the close proximity of D666

  19. Involvement of cAMP-PKA pathway in adenosine A1 and A2A receptor-mediated regulation of acetaldehyde-induced activation of HSCs.

    PubMed

    Yang, Yaru; Wang, He; Lv, Xiongwen; Wang, Qi; Zhao, Han; Yang, Feng; Yang, Yan; Li, Jun

    2015-08-01

    The present study was undertaken to investigate the mechanism by which adenosine receptors (ARs)-mediated the cAMP/PKA/CREB signal pathway regulates the activation of acetaldehyde-induced hepatic stellate cells (HSCs). Primary HSCs were isolated from SD rats, cultured in vitro, and activated with different concentrations of acetaldehyde at different time points. Quantitative real-time PCR and Western blotting were used to quantify both protein and mRNA levels of the four AR (A1R, A2AR, A2BR, and A3R) in rat HSCs. Selective inhibitors of PDEs and the Gi/o protein pathway, general AR agonists, and AR subtype specific agents were used to study the AR signaling. The level of cAMP was measured by radio-immunoassay, and the expression of α-SMA, collagen type I and III, PKA and p-CREB were also detected by Western blotting. Acetaldehyde could significantly promote HSC proliferation, with a maximum stimulatory effect observed at 48 h after exposure to 200 μM acetaldehyde. All four AR subtypes could be present in rat HSCs, and the mRNA and protein expression levels for A2AR and A1R in much greater abundance than those for A2BR and A3R. The expression of A2AR and A1R was significantly increased in acetaldehyde-induced HSCs as compared with that of control group, whereas the expression of A2BR and A3R remained unaffected by the addition of acetaldehyde. Curiously, there is coupling of A2AR to the Gs-AC signaling, as well as coupling of A1R to the Gi/o-AC signaling pathway in acetaldehyde-induced HSCs. Both the A2AR and A1R antagonists could suppress the activation of HSC, although they have opposing effects on cAMP signal transduction. These results suggested that a combination of cAMP/PKA/CREB signals via A2AR and A1R likely mediate the activation of acetaldehyde-induced HSCs, and A1R coupled to the Gi/o-AC signaling pathway may be masked by the more predominant A2AR that coupled to the Gs-AC signaling pathway.

  20. StyA1 and StyA2B from Rhodococcus opacus 1CP: a Multifunctional Styrene Monooxygenase System▿

    PubMed Central

    Tischler, Dirk; Kermer, René; Gröning, Janosch A. D.; Kaschabek, Stefan R.; van Berkel, Willem J. H.; Schlömann, Michael

    2010-01-01

    Two-component flavoprotein monooxygenases are emerging biocatalysts that generally consist of a monooxygenase and a reductase component. Here we show that Rhodococcus opacus 1CP encodes a multifunctional enantioselective flavoprotein monooxygenase system composed of a single styrene monooxygenase (SMO) (StyA1) and another styrene monooxygenase fused to an NADH-flavin oxidoreductase (StyA2B). StyA1 and StyA2B convert styrene and chemical analogues to the corresponding epoxides at the expense of FADH2 provided from StyA2B. The StyA1/StyA2B system presents the highest monooxygenase activity in an equimolar ratio of StyA1 and StyA2B, indicating (transient) protein complex formation. StyA1 is also active when FADH2 is supplied by StyB from Pseudomonas sp. VLB120 or PheA2 from Rhodococcus opacus 1CP. However, in both cases the reductase produces an excess of FADH2, resulting in a high waste of NADH. The epoxidation rate of StyA1 heavily depends on the type of reductase. This supports that the FADH2-induced activation of StyA1 requires interprotein communication. We conclude that the StyA1/StyA2B system represents a novel type of multifunctional flavoprotein monooxygenase. Its unique mechanism of cofactor utilization provides new opportunities for biotechnological applications and is highly relevant from a structural and evolutionary point of view. PMID:20675468

  1. 17 CFR Appendix A to Part 3 - Interpretative Statement With Respect to Section 8a(2)(C) and (E) and Section 8a(3)(J) and (M) of...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Respect to Section 8a(2)(C) and (E) and Section 8a(3)(J) and (M) of the Commodity Exchange Act A Appendix... Section 8a(3)(J) and (M) of the Commodity Exchange Act Section 8a(2) (C) and (E) The provisions of... amend this interpretation if deemed appropriate. Section 8a(3) (J) and (M) Section 8a(3) authorizes...

  2. Successful ABO-Incompatible Renal Transplantation:  Blood Group A1B Donor Into A2B Recipient With Anti-A1 Isoagglutinins.

    PubMed

    Fadeyi, Emmanuel A; Stratta, Robert J; Farney, Alan C; Pomper, Gregory J

    2016-08-01

    Transplantation of the blood group A2B in a recipient was successfully performed in the setting of receiving a deceased donor kidney from an "incompatible" A1B donor. The donor and recipient were both typed for ABO blood group, including ABO genotyping. The donor and recipient were tested for ABO, non-ABO, and human leukocyte antigen (HLA) antibodies. The donor and recipient were typed for HLA antigens, including T- and B-flow cytometry crossmatch tests. The recipient's RBCs were negative with A1 lectin, and immunoglobulin G anti-A1 was demonstrated in the recipient's plasma. The donor-recipient pair was a four-antigen HLA mismatch, but final T- and B-flow cytometry crossmatch tests were compatible. The transplant procedure was uneventful; the patient experienced immediate graft function with no episodes of rejection or readmissions more than 2 years later. It may be safe to transplant across the A1/A2 blood group AB mismatch barrier in the setting of low titer anti-A1 isoagglutinins without the need for pretransplant desensitization even if the antibody produced reacts with anti-human globulin. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Interaction of model class A1, class A2, and class Y amphipathic helical peptides with membranes.

    PubMed

    Mishra, V K; Palgunachari, M N

    1996-08-27

    To test the hypothesis that differences in the lipid affinity of exchangeable apolipoproteins are due to the presence of different classes of amphipathic alpha-helical motifs which differ primarily in the distribution of charged amino acid residues, we designed and synthesized model peptides mimicking class A1, class A2, and class Y amphipathic helices present in these apolipoproteins. Both class A1 and class A2 helices have positive residues at the polar-nonpolar interface and negative residues at the center of the polar face. However, clustering of positive and negative residues is less exact in class A1 compared to class A2 helices. The class Y helices have two negative residue clusters on the polar face separating the two arms and the base of the Y motif formed by three positive residue clusters. The lipid affinities of three 18 residue model peptides representing these classes, Ac-18A1-NH2 (Ac-ELLEKWAEKLAALKEALK-NH2), Ac-18A2-NH2 (Ac-ELLEKWKEALAALAEKLK-NH2), and Ac-18Y-NH2 (Ac-ELLKAWKEALEALKEKLA-NH2), were determined by right-angle light scattering, circular dichroism spectroscopy, differential scanning calorimetry, and fluorescence spectroscopy. The observed rank order of lipid affinity of these three peptides is: Ac-18A2-NH2 > Ac-18Y-NH2 > Ac-18A1-NH2. This order is consistent with the known lipid affinity of exchangeable apolipoproteins containing class A1, class A2, and class Y helices (class A2 > class Y > class A1). Results of this study illustrate the important role of interfacial lysine residues in modulating the lipid affinity of amphipathic helices and suggest that the effect of interfacial lysine residues in increasing lipid affinity is additive. We propose that interfacial lysine residues, in addition to widening the hydrophobic face because of snorkeling, also help anchor the amphipathic helix in the lipid bilayer.

  4. Chimeric CYP21A1P/CYP21A2 genes identified in Czech patients with congenital adrenal hyperplasia.

    PubMed

    Vrzalová, Zuzana; Hrubá, Zuzana; Hrabincová, Eva Sťahlová; Vrábelová, Slávka; Votava, Felix; Koloušková, Stanislava; Fajkusová, Lenka

    2011-01-01

    Congenital adrenal hyperplasia (CAH) comprises a group of autosomal recessive disorders caused by an enzymatic deficiency which impairs the biosynthesis of cortisol and, in the majority of severe cases, also the biosynthesis of aldosterone. Approximately 95% of all CAH cases are caused by mutations in the steroid 21-hydroxylase gene (CYP21A2). The CYP21A2 gene and its inactive pseudogene (CYP21A1P) are located within the HLA class III region of the major histocompatibility complex (MHC) locus on chromosome 6p21.3. In this study, we describe chimeric CYP21A1P/CYP21A2 genes detected in our patients with 21-hydroxylase deficiency (21OHD). Chimeric CYP21A1P/CYP21A2 genes were present in 171 out of 508 mutated CYP21A2 alleles (33.8%). We detected four types of chimeric CYP21A1P/CYP21A2 genes: three of them have been described previously as CH-1, CH-3, CH-4, and one type is novel. The novel chimeric gene, termed CH-7, was detected in 21.4% of the mutant alleles. Possible causes of CYP21A1P/CYP21A2 formation are associated with 1) high recombination rate in the MHC locus, 2) high recombination rate between highly homologous genes and pseudogenes in the CYP21 gene area, and 3) the existence of chi-like sequences and repetitive minisatellite consensus sequences in CYP21A2 and CYP21A1P which play a role in promoting genetic recombination.

  5. Hall Effect Thruster Interactions Data From the Russian Express-A2 and Express-A3 Satellites. Part 11; Express/T-160E Project Express A2 and A3 Data Agreement Document

    NASA Technical Reports Server (NTRS)

    Sitnikova, N.; Volkov, D.; Maximov, I.; Petrusevich, V.; Allen, D.; Dunning, John (Technical Monitor)

    2003-01-01

    This 12-part report documents the data obtained from various sensor measurements taken aboard the Russian Express-A2 and Express-A3 spacecraft in Geosynchronous Earth Orbit (GEO). These GEO communications satellites, which were designed and built by NPO Prikladnoy Mekhaniki (NPO PM) of Zheleznogorsk, Russia, utilize Hall thruster propulsion systems for north-south and east-west stationkeeping and as of June 2002, were still operating at 80deg E. and 11deg W., respectively. Express-A2 was launched on March 12, 2000, while Express-A3 was launched on June 24, 2000. The diagnostic equipment from which these data were taken includes electric field strength sensors, ion current and energy sensors, and pressure sensors. The diagnostics and the Hall thruster propulsion systems are described in detail along with lists of tabular data from those diagnostics and propulsion system and other satellite systems. Space Power, Inc., now part of Pratt & Whitney's Chemical Systems Division, under contract NAS3-99151 to the NASA Glenn Research Center, obtained these data over several periods from March 12, 2000, through September 30, 2001. Each of the 12 individual reports describe, in detail, the propulsion systems as well as the diagnostic sensors utilized. Finally, parts 11 and 12 include the requirements to which NPO PM prepared and delivered these data.

  6. Hall Effect Thruster Interactions Data From the Russian Express-A2 and Express-A3 Satellites. Part 12; Express/T-160 Project Express A2 and A3 Sensors Operations Procedures Document

    NASA Technical Reports Server (NTRS)

    Dunning, John (Technical Monitor); Sitnikova, N.; Volkov, D.; Maximov, I.; Petrusevich, V.; Allen, D.

    2003-01-01

    This 12-part report documents the data obtained from various sensor measurements taken aboard the Russian Express-A2 and Express-A3 spacecraft in Geosynchronous Earth Orbit (GEO). These GEO communications satellites, which were designed and built by NPO Prikladnoy Mekhaniki (NPO PM) of Zheleznogorsk, Russia, utilize Hall thruster propulsion systems for north-south and east-west stationkeeping and as of June 2002, were still operating at 80 deg. E. and 11 deg. W respectively. Express-A2 was launched on March 12, 2000, while Express-A3 was launched on June 24, 2000. The diagnostic equipment from which these data were taken includes electric field strength sensors, ion current and energy sensors, and pressure sensors. The diagnostics and the Hall thruster propulsion systems are described in detail along with lists of tabular data from those diagnostics and propulsion system and other satellite systems. Space Power, Inc., now part of Pratt & Whitney's Chemical Systems Division, under contract NAS3 99151 to the NASA Glenn Research Center, obtained these data over several periods from March 12, 2000, through September 30, 2001. Each of the 12 individual reports describe, in detail, the propulsion systems as well as the diagnostic sensors utilized. Finally, parts 11 and 12 include the requirements to which NPO PM prepared and delivered these data.

  7. A1 and A2, two novel haloarchaeal isolates from bore cores of ancient Alpine rock salt deposits

    NASA Astrophysics Data System (ADS)

    Gruber, C.; Pfaffenhuemer, M.; Weidler, G.; Radax, C.; Stan-Lotter, H.

    2003-04-01

    Previously several novel halophilic archaea, for instance Haloccocus salifodinae BIp and Halococcus dombrowskii, were isolated from Permo-Triassic rock salt (age 200 - 250 million years) in our laboratory. By using molecular methods we found evidence for the presence of numerous additional haloarchaeal taxa. We investigated freshly drilled salt cores from a depth of about 600 m below surface in the salt mine of Altaussee, Austria, which were dissolved immediately in sterile water. After plating the dissolved salts on high salt nutrient agar, we were able to isolate, following incubation for 3 months, two red pigmented colonies, which were designated A1 and A2 and cultivated for further investigation. A1 and A2 showed the same antibiotic susceptibility as Halobacterium salinarum DSM 3754 and Halobacterium sp. NRC-1, which were cultivated from surface waters. Additionally, the cell morphology of the new isolates was highly similar to both reference strains. According to 16S rRNA gene sequences, whole cell protein patterns following SDS polyacrylamide gel electrophoresis, and restriction digestion patterns of their DNA following pulsed field gel electrophoresis, the isolates A1 and A2 could not be distinguished. 16S rRNA gene sequences indicated that the closest relative of strains A1 and A2 was Halobacterium salinarum DSM 3754 (sequence similarity 97,1%). Our results suggest that the isolates A1 and A2 might constitute a new haloarchaeal species, entrapped in ancient rock salt.

  8. Successful unintentional ABO-incompatible renal transplantation: Blood group A1B donor into an A2B recipient.

    PubMed

    Fadeyi, Emmanuel A; Stratta, Robert J; Farney, Alan C; Pomper, Gregory J

    2014-05-01

    To report a successful unintentional transplantation of a deceased donor kidney from an "incompatible" A1B donor into a recipient who was blood group A2B with unsuspected preformed anti-A1 antibodies. The donor and recipient were both typed for ABO antigens. The recipient was tested for ABO and non-ABO antibodies. The recipient was typed for HLA class I and class II antigens, including HLA antibody screen. The T-and B-flow cytometry crossmatch test was performed using standard protocol. The donor-recipient pair was a complete six-antigen human leukocyte antigen mismatch, but final T- and B-flow cytometry cross-match tests were compatible. The recipient was a 65-year-old woman with a medical history of end-stage renal disease secondary to diabetic nephropathy who underwent kidney transplantation from a 46-year-old brain-dead standard criteria donor. The recipient's RBCs were negative with A1 lectin, and the recipient was thus typed as an A2 subgroup. Anti-A1 could be demonstrated in the recipient's plasma. The donor's RBCs were positive with A1 lectin, thereby conferring an A1 blood type. It is safe to transplant across the A1/A2 blood group barrier provided that the preformed antibodies are not reactive at 37°C and with anti-human globulin.

  9. Chromomycins A2 and A3 from marine actinomycetes with TRAIL resistance-overcoming and Wnt signal inhibitory activities.

    PubMed

    Toume, Kazufumi; Tsukahara, Kentaro; Ito, Hanako; Arai, Midori A; Ishibashi, Masami

    2014-06-05

    A biological screening study of an actinomycetes strain assembly was conducted using a cell-based cytotoxicity assay. The CKK1019 strain was isolated from a sea sand sample. Cytotoxicity-guided fractionation of the CKK1019 strain culture broth, which exhibited cytotoxicity, led to the isolation of chromomycins A2 (1) and A3 (2). 1 and 2 showed potent cytotoxicity against the human gastric adenocarcinoma (AGS) cell line (IC50 1; 1.7 and 2; 22.1 nM), as well as strong inhibitory effects against TCF/β-catenin transcription (IC50 1; 1.8 and 2; 15.9 nM). 2 showed the ability to overcome tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) resistance. To the best of our knowledge, the effects of chromomycins A2 (1) and A3 (2) on TRAIL resistance-overcoming activity, and on the Wnt signaling pathway, have not been reported previously. Thus, 1 and 2 warrant potential drug lead studies in relation to TRAIL-resistant and Wnt signal-related diseases and offer potentially useful chemical probes for investigating TRAIL resistance and the Wnt signaling pathway.

  10. Hall Effect Thruster Interactions Data From the Russian Express-A2 and Express-A3 Satellites

    NASA Technical Reports Server (NTRS)

    Sitnikova, N.; Volkov, D.; Maximov, I.; Petrusevich, V.; Allen, D.

    2003-01-01

    This 12-part report documents the data obtained from various sensor measurements taken aboard the Russian Express-A2 and Express-A3 spacecraft in Geosynchronous Earth Orbit (GEO). These GEO communications satellites, which were designed and built by NPO Prikladnoy Mekhaniki (NPO PM) of Zheleznogorsk, Russia, utilize Hall thruster propulsion systems for north-south and east-west stationkeeping and as of June 2002, were still operating at 80 E. and 11 W., respectively. Express-A2 was launched on March 12, 2000, while Express-A3 was launched on June 24, 2000. The diagnostic equipment from which these data were taken includes electric field strength sensors, ion current and energy sensors, and pressure sensors. The diagnostics and the Hall thruster propulsion systems are described in detail along with lists of tabular data from those diagnostics and propulsion system and other satellite systems. Space Power, Inc., now part of Pratt & Whitney's Chemical Systems Division, under contract NAS3 99151 to the NASA Glenn Research Center, obtained these data over several periods from March 12, 2000, through September 30, 2001. Each of the 12 individual reports describe, in detail, the propulsion systems as well as the diagnostic sensors utilized. Finally, parts 11 and 12 include the requirements to which NPO PM prepared and delivered these data.

  11. Rational design of D-A1-D-A2 conjugated polymers with superior spectral coverage.

    PubMed

    Hedström, Svante; Tao, Qiang; Wang, Ergang; Persson, Petter

    2015-10-28

    The spectral coverage of a light-harvesting polymer largely determines the maximum achievable photocurrent in organic photovoltaics, and therefore constitutes a crucial parameter for improving their performance. The D-A1-D-A2 copolymer motif is a new and promising design strategy for extending the absorption range by incorporating two acceptor units with complementary photoresponses. The fundamental factors that promote an extended absorption are here determined for three prototype D-A1-D-A2 systems through a combination of experimental and computational methods. Systematic quantum chemical calculations are then used to reveal the intrinsic optical properties of ten further D-A1-D-A2 polymer candidates. These investigated polymers are all predicted to exhibit intense primary absorption peaks at 615-954 nm, corresponding to charge-transfer (CT) transitions to the stronger acceptor, as well as secondary absorption features at 444-647 nm that originate from CT transitions to the weaker acceptors. Realization of D-A1-D-A2 polymers with superior spectral coverage is thereby found to depend critically on the spatial and energetic separation between the two distinct acceptor LUMOs. Two promising D-A1-D-A2 copolymer candidates were finally selected for further theoretical and experimental study, and demonstrate superior light-harvesting properties in terms of significantly extended spectral coverage. This demonstrates great potential for enhanced light-harvesting in D-A1-D-A2 polymers via multiple absorption features compared to traditional D-A polymers.

  12. Local expression of CYP19A1 and CYP19A2 in developing and adult killifish (Fundulus heteroclitus)

    PubMed Central

    Dong, Wu; Willett, Kristine L.

    2008-01-01

    P450 aromatase (CYP19) is the terminal enzyme in the steroidogenic pathway and catalyzes the conversion of androgens to estrogens. Fundulus heteroclitus like other teleosts, express two CYP19 genes, CYP19A1 and CYP19A2. The expression of CYP19s in Fundulus was measured by in situ hybridization throughout development. In 90 dpf (day post-fertilization) fish and adult fish, CYP19A1 was expressed in the ooplasm of early stage I oocytes (primary growth stage). Expression of CYP19A1 was localized in the follicle cell layer of late stage I (previtellogenic stage) and stage II (vitellogenic stage) follicles, but by stage III (early maturational follicles) CYP19A1 expression was localized in the vitelline envelope. Overall, CYP19A1 oocyte membrane expression gradually declined from highest expression at late stage I to nondetectable levels by stage IV. Highest expression of CYP19A2 was detected in the brain including the hypothalamus from 4, 6, 8, 10, 14 dpf embryos, 90 dpf fry fish and adult fish brain. In females compared to males, there was higher CYP19A2 expression in olfactory bulb. In addition to the brain, there was strong CYP19A2 signal in adrenal/kidney cells in 6-14 dpf embryos. This work establishes the localization and constitutive expression of CYP19s in Fundulus which can then be compared with potential disruption of CYP19A1 and CYP19A2 expression and physiological consequences caused by environmental contaminants. PMID:17582409

  13. Genetic Complexity of the Human Innate Host Defense Molecules, Surfactant Protein A1 (SP-A1) and SP-A2—Impact on Function

    PubMed Central

    Floros, Joanna; Wang, Guirong; Mikerov, Anatoly N.

    2010-01-01

    Innate immunity mechanisms play a critical role in the primary response to invading pathogenic microorganisms and other insulting agents. The innate lung immune system includes lung surfactant, a lipoprotein complex that carries out a function essential for life, that is, reduction of the surface tension at the air–liquid interphase of the alveolar space. By means of this function, pulmonary surfactant prevents lung collapse, therefore ensuring normal lung function and lung health. Pulmonary surfactant contains a number of host-defense molecules that are involved in the elimination of pathogens, viruses, particles, allergens, and other insults, as well as in the control of inflammation. This review is concerned with one of the surfactant proteins, the human (h) surfactant protein A (hSP-A), which, in addition to its role in surfactant-related functions, plays an important role in the modulation of lung host defense. The hSP-A locus has been identified with extensive complexity that may have an impact on its function, structure, and regulation. In humans, two genes—SP-A1 (SFTPA1) and SP-A2 (SFTPA2)—encode SP-A, with SP-A2 gene products being more biologically active than SP-A1 in most of the in vitro assays investigated. Although the two hSP-A genes share a high level of sequence similarity, differences in the structure and function between SP-A1 and SP-A2 have been observed in recent studies. In this review, we discuss the human SP-A complexity and how this may affect SP-A function. PMID:19392648

  14. Genetic complexity of the human innate host defense molecules, surfactant protein A1 (SP-A1) and SP-A2--impact on function.

    PubMed

    Floros, Joanna; Wang, Guirong; Mikerov, Anatoly N

    2009-01-01

    Innate immunity mechanisms play a critical role in the primary response to invading pathogenic microorganisms and other insulting agents. The innate lung immune system includes lung surfactant, a lipoprotein complex that carries out a function essential for life, that is, reduction of the surface tension at the air-liquid interphase of the alveolar space. By means of this function, pulmonary surfactant prevents lung collapse, therefore ensuring normal lung function and lung health. Pulmonary surfactant contains a number of host-defense molecules that are involved in the elimination of pathogens, viruses, particles, allergens, and other insults, as well as in the control of inflammation. This review is concerned with one of the surfactant proteins, the human (h) surfactant protein A (hSP-A), which, in addition to its role in surfactant-related functions, plays an important role in the modulation of lung host defense. The hSP-A locus has been identified with extensive complexity that may have an impact on its function, structure, and regulation. In humans, two genes--SP-A1 (SFTPA1) and SP-A2 (SFTPA2)--encode SP-A, with SP-A2 gene products being more biologically active than SP-A1 in most of the in vitro assays investigated. Although the two hSP-A genes share a high level of sequence similarity, differences in the structure and function between SP-A1 and SP-A2 have been observed in recent studies. In this review, we discuss the human SP-A complexity and how this may affect SP-A function.

  15. Differences in adenosine A-1 and A-2 receptor density revealed by autoradiography in methylxanthine-sensitive and insensitive mice

    SciTech Connect

    Jarvis, M.F.; Williams, M.

    1988-07-01

    Two strains of inbred mice, CBA/J and SWR/J, have been identified which are, respectively, sensitive and insensitive to the behavioral and toxic effects of methylxanthines. Autoradiographic analyses of brain adenosine receptors were conducted with (/sup 3/H)CHA to label adenosine A-1 receptors and (/sup 3/H)NECA, in the presence of 50 nM CPA, to label adenosine A-2 receptors. For both mouse strains, adenosine A-1 receptors were most highly concentrated in the hippocampus and cerebellum whereas adenosine A-2 receptors were selectively localized in the striatum. CBA/J mice displayed a 30% greater density of adenosine A-1 receptors in the hippocampal CA-1 and CA-3 regions and in the cerebellum as compared to the SWR/J mice. The number of A-2 receptors (Bmax) was 40% greater in the striatum and olfactory tubercle of CBA/J as compared to SWR/J mice. No significant regional differences in A-1 or A-2 receptor affinities were observed between these inbred strains of mice. These results indicate that the differential sensitivity to methylxanthines between these mouse strains may reflect a genetically mediated difference in regional adenosine receptor densities.

  16. 75 FR 28188 - Airworthiness Directives; General Electric Company CF34-1A, -3A, -3A1, -3A2, -3B, and -3B1...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-20

    ...) numbers CF34-AL S/B 72 A0212, CF34-AL S/B 72 A0234, and CF34-AL S/B 72 A0235 in the regulatory section are... and eighth lines, ``CF34-AL'' is corrected to read ``CF34- BJ''. 2. On page 914, in the second column, in paragraph (k)(2)(iii), in the fifth line, ``CF34-AL'' is corrected to read ``CF34-BJ''. 3. On...

  17. 75 FR 910 - Airworthiness Directives; General Electric Company CF34-1A, -3A, -3A1, -3A2, -3B, and -3B1...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-07

    ... -3B1 turbofan engines. That AD currently requires a onetime visual and tactile inspection of certain..., removing from service fan disks with electrical arc-out indications, performing tactile and enhanced visual...-A0233, Revision 04, dated October 27, 2008, do the following: Tactile and Enhanced Visual...

  18. Three Novel Lantibiotics, Ticins A1, A3, and A4, Have Extremely Stable Properties and Are Promising Food Biopreservatives

    PubMed Central

    Xin, Bingyue; Zheng, Jinshui; Xu, Ziya; Li, Congzhi; Ruan, Lifang; Peng, Donghai

    2015-01-01

    Lantibiotics are antimicrobial peptides with potential applications as the next generation of antimicrobials in the food industry and/or the pharmaceutical industry. Nisin has successfully been used as a food preservative for over 40 years, but its major drawback is its limited stability under neutral and alkaline pH conditions. To identify alternatives with better biochemical properties, we screened more than 100 strains of the Bacillus cereus group. Three novel lantibiotics, ticins A1 (4,062.98 Da), A3 (4,048.96 Da), and A4 (4,063.02 Da), which were highly thermostable (121°C for 30 min) and extremely pH tolerant (pH 2.0 to 9.0), were identified in Bacillus thuringiensis BMB3201. They all showed potent antimicrobial activities against all tested Gram-positive bacteria and greater activities than those of nisin A against Bacillus cereus and Listeria monocytogenes, two important foodborne pathogens. These three novel lantibiotics, with their extremely stable properties and potent antimicrobial activities, have the potential for use as biopreservatives. PMID:26231642

  19. Three Novel Lantibiotics, Ticins A1, A3, and A4, Have Extremely Stable Properties and Are Promising Food Biopreservatives.

    PubMed

    Xin, Bingyue; Zheng, Jinshui; Xu, Ziya; Li, Congzhi; Ruan, Lifang; Peng, Donghai; Sun, Ming

    2015-10-01

    Lantibiotics are antimicrobial peptides with potential applications as the next generation of antimicrobials in the food industry and/or the pharmaceutical industry. Nisin has successfully been used as a food preservative for over 40 years, but its major drawback is its limited stability under neutral and alkaline pH conditions. To identify alternatives with better biochemical properties, we screened more than 100 strains of the Bacillus cereus group. Three novel lantibiotics, ticins A1 (4,062.98 Da), A3 (4,048.96 Da), and A4 (4,063.02 Da), which were highly thermostable (121°C for 30 min) and extremely pH tolerant (pH 2.0 to 9.0), were identified in Bacillus thuringiensis BMB3201. They all showed potent antimicrobial activities against all tested Gram-positive bacteria and greater activities than those of nisin A against Bacillus cereus and Listeria monocytogenes, two important foodborne pathogens. These three novel lantibiotics, with their extremely stable properties and potent antimicrobial activities, have the potential for use as biopreservatives. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  20. The occultation of beta Scorpii by Jupiter. VII - The angular diameters of beta Scorpii A1 and A2

    NASA Technical Reports Server (NTRS)

    Elliot, J. L.; Rages, K.; Veverka, J.

    1976-01-01

    A new technique for measuring angular diameters of stars, which makes use of stellar images formed by planetary atmospheres during occultations, is described. This method has been applied to light curves of the 1971 May 13 occultation of beta Sco by Jupiter, yielding the angular diameters of the two early B stars comprising the spectroscopic binary beta Sco A. An angular diameter of about .000422 arcsec for beta Sco A1 and about .000264 arcsec is found for beta Sco A2. These angular diameters are in good agreement with that obtained with the intensity interferometer for beta Sco, a star of nearly the same magnitude and spectral type as beta Sco A1. If the distance to beta Sco A were precisely known, beta Sco A1 and A2 would become fundamental standards of mass, luminosity, and radius for early B stars. Present constraints on the distance and methods by which it could be accurately determined are discussed.

  1. The Human UDP-glucuronosyltransferase UGT2A1 and UGT2A2 enzymes are highly active in bile acid glucuronidation.

    PubMed

    Perreault, Martin; Gauthier-Landry, Louis; Trottier, Jocelyn; Verreault, Mélanie; Caron, Patrick; Finel, Moshe; Barbier, Olivier

    2013-09-01

    Bile acids (BA) are essential modulators of lipid, glucose, and cholesterol homeostasis, but they exert cytotoxic effects in the cholestatic liver. Glucuronidation, catalyzed by the UDP-glucuronosyltransferase (UGT) enzymes is a pharmacologically relevant BA detoxification process. The present study characterized the BA-conjugating activity of the little-studied human UGTs of subfamily 2A: UGT2A1, 2A2, and 2A3. Recombinant UGT2As, expressed in baculovirus-infected insect cells, were assayed for the glucuronidation of six major bile acids: chenodeoxycholic acid (CDCA), cholic acid (CA), lithocholic acid (LCA), deoxycholic acid (DCA), hyocholic acid (HCA) and hyodeoxycholic acid (HDCA). UGT2A3 exhibited detectable but very low activity with all the tested BA substrates. UGT2A1 was highly efficient in forming LCA-3 and LCA-24G, CDCA-24, DCA-24, HCA-24, and HDCA-24G, whereas UGT2A2 was the most active enzyme for CA-24G and CDCA-24G formation and also was able to generate HDCA-6G, HDCA-24G, LCA-24G, and HCA-24G. The Km values of UGT2A1 varied between 102.2 ± 14.3 µM and 2.4 ± 1.2 mM. With the exception of CA-24G, a low affinity substrate for UGT2A2, all the Km values for UGT2A2 were in the 100 to 400 µM range. We demonstrate the high reactivity of the human UGT2A1 and UGT2A2 for bile acid glucuronidation. The physiologic importance of these reactions to BA disposition remains, however, to be clarified in vivo.

  2. The human UDP-glucuronosyltransferase UGT2A1 and UGT2A2 enzymes are highly active in bile acid glucuronidation

    PubMed Central

    Perreault, Martin; Gauthier-Landry, Louis; Trottier, Jocelyn; Verreault, Mélanie; Caron, Patrick; Finel, Moshe; Barbier, Olivier

    2013-01-01

    Bile acids (BA) are essential modulators of lipid, glucose and cholesterol homeostasis, but exert cytotoxic effects in the cholestatic liver. Glucuronidation, catalyzed by the UDP-glucuronosyltransferase (UGT) enzymes is a pharmacologically-relevant BA detoxification process. The present study aimed at characterizing the BA-conjugating activity of the little-studied human UGTs of subfamily 2A, UGT2A1, 2A2 and 2A3. Recombinant UGT2As, expressed in baculovirus-infected insect cells, were assayed for the glucuronidation of 6 major bile acids, chenodeoxycholic (CDCA), cholic (CA), lithocholic (LCA), deoxycholic (DCA), hyocholic (HCA) and hyodeoxycholic (HDCA) acids. UGT2A3 exhibited detectable, but very low, activity with all the tested BAs substrates. UGT2A1 was highly efficient in forming LCA-3 and -24G, CDCA-24, DCA-24, HCA-24 and HDCA-24G, while UGT2A2 was the most active enzyme for CA-24G and CDCA-24G formation, and was also able to generate HDCA-6G, HDCA-24G, LCA-24G and HCA-24G. The Km values of UGT2A1 varied between 102.2 ± 14.3 μM and 2.4 ± 1.2 mM. With the exception of CA-24G, a low affinity substrate for UGT2A2, all the Km values for UGT2A2 were in the 100 to 400 μM range. In conclusion, the present study demonstrates the high reactivity of the human UGT2A1 and UGT2A2 for bile acid glucuronidation. The physiological importance of these reactions to BA disposition remains, however, to be clarified in vivo. PMID:23756265

  3. A2B and A3 Adenosine Receptors Modulate Vascular Endothelial Growth Factor and Interleukin-8 Expression in Human Melanoma Cells Treated with Etoposide and Doxorubicin

    PubMed Central

    Merighi, Stefania; Simioni, Carolina; Gessi, Stefania; Varani, Katia; Mirandola, Prisco; Tabrizi, Mojgan Aghazadeh; Baraldi, Pier Giovanni; Borea, Pier Andrea

    2009-01-01

    Cancer patients undergoing treatment with systemic cancer chemotherapy drugs often have abnormal growth factor and cytokine profiles. Thus, serum levels of interleukin-8 (IL-8) are elevated in patients with malignant melanoma. In addition to IL-8, aggressive melanoma cells secrete, through its transcriptional regulator hypoxia-inducible factor 1 (HIF-1), vascular endothelial growth factor (VEGF), which promotes angiogenesis and metastasis of human cancerous cells. Whether these responses are related to adenosine, a ubiquitous mediator expressed at high concentrations in cancer and implicated in numerous inflammatory processes, is not known and is the focus of this study. We have examined whether the DNA-damaging agents etoposide (VP-16) and doxorubicin can affect IL-8, VEGF, and HIF-1 expressions in human melanoma cancer cells. In particular, we have investigated whether these responses are related to the modulation of the adenosine receptor subtypes, namely, A1, A2A, A2B, and A3. We have demonstrated that A2B receptor blockade can impair IL-8 production, whereas blocking A3 receptors, it is possible to further decrease VEGF secretion in melanoma cells treated with VP-16 and doxorubicin. This understanding may present the possibility of using adenosine antagonists to reduce chemotherapy-induced inflammatory cytokine production and to improve the ability of chemotherapeutic drugs to block angiogenesis. Consequently, we conclude that adenosine receptor modulation may be useful for refining the use of chemotherapeutic drugs to treat human cancer more effectively. PMID:19794965

  4. Homeostatic action of adenosine A3 and A1 receptor agonists on proliferation of hematopoietic precursor cells.

    PubMed

    Hofer, Michal; Pospísil, Milan; Znojil, Vladimír; Holá, Jirina; Streitová, Denisa; Vacek, Antonín

    2008-07-01

    Two adenosine receptor agonists, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) and N6-cyclopentyladenosine (CPA), which selectively activate adenosine A3 and A1 receptors, respectively, were tested for their ability to influence proliferation of granulocytic and erythroid cells in femoral bone marrow of mice using morphological criteria. Agonists were given intraperitoneally to mice in repeated isomolar doses of 200 nmol/kg. Three variants of experiments were performed to investigate the action of the agonists under normal resting state of mice and in phases of cell depletion and subsequent regeneration after treatment with the cytotoxic drug 5-fluorouracil. In the case of granulopoiesis, IB-MECA 1) increased by a moderate but significant level proliferation of cells under normal resting state; 2) strongly increased proliferation of cells in the cell depletion phase; but 3) did not influence cell proliferation in the regeneration phase. CPA did not influence cell proliferation under normal resting state and in the cell depletion phase, but strongly suppressed the overshooting cell proliferation in the regeneration phase. The stimulatory effect of IB-MECA on cell proliferation of erythroid cells was observed only when this agonist was administered during the cell depletion phase. CPA did not modulate erythroid proliferation in any of the functional states investigated, probably due to the lower demand for cell production as compared with granulopoiesis. The results indicate opposite effects of the two adenosine receptor agonists on proliferation of hematopoietic cells and suggest the plasticity and homeostatic role of the adenosine receptor expression.

  5. A1 and A2a receptors mediate inhibitory effects of adenosine on the motor activity of human colon.

    PubMed

    Fornai, M; Antonioli, L; Colucci, R; Ghisu, N; Buccianti, P; Marioni, A; Chiarugi, M; Tuccori, M; Blandizzi, C; Del Tacca, M

    2009-04-01

    Experimental evidence in animal models suggests that adenosine is involved in the regulation of digestive functions. This study examines the influence of adenosine on the contractile activity of human colon. Reverse transcription-polymerase chain reaction revealed A(1) and A(2a) receptor expression in colonic neuromuscular layers. Circular muscle preparations were connected to isotonic transducers to determine the effects of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; A(1) receptor antagonist), ZM 241385 (A(2a) receptor antagonist), CCPA (A(1) receptor agonist) and 2-[(p-2-carboxyethyl)-phenethylamino]-5'-N-ethyl-carboxamide-adenosine (CGS 21680; A(2a) receptor agonist) on motor responses evoked by electrical stimulation or carbachol. Electrically evoked contractions were enhanced by DPCPX and ZM 241385, and reduced by CCPA and CGS 21680. Similar effects were observed when colonic preparations were incubated with guanethidine (noradrenergic blocker), L-732,138, GR-159897 and SB-218795 (NK receptor antagonists). However, in the presence of guanethidine, NK receptor antagonists and N(omega)-propyl-L-arginine (NPA; neuronal nitric oxide synthase inhibitor), the effects of DPCPX and CCPA were still evident, while those of ZM 241385 and CGS 21680 no longer occurred. Carbachol-induced contractions were unaffected by A(2a) receptor ligands, but they were enhanced or reduced by DPCPX and CCPA, respectively. When colonic preparations were incubated with guanethidine, NK antagonists and atropine, electrically induced relaxations were partly reduced by ZM 241385 or NPA, but unaffected by DPCPX. Dipyridamole or application of exogenous adenosine reduced electrically and carbachol-evoked contractions, whereas adenosine deaminase enhanced such motor responses. In conclusion, adenosine exerts an inhibitory control on human colonic motility. A(1) receptors mediate direct modulating actions on smooth muscle, whereas A(2a) receptors operate through inhibitory nitrergic nerve pathways.

  6. Sequence variants in COL4A1 and COL4A2 genes in Ecuadorian families with keratoconus

    PubMed Central

    Karolak, Justyna A.; Kulinska, Karolina; Nowak, Dorota M.; Pitarque, Jose A.; Molinari, Andrea; Rydzanicz, Malgorzata; Bejjani, Bassem A.

    2011-01-01

    Purpose Keratoconus (KTCN) is a non-inflammatory, usually bilateral disorder of the eye which results in the conical shape and the progressive thinning of the cornea. Several studies have suggested that genetic factors play a role in the etiology of the disease. Several loci were previously described as possible candidate regions for familial KTCN; however, no causative mutations in any genes have been identified for any of these loci. The purpose of this study was to evaluate role of the collagen genes collagen type IV, alpha-1 (COL4A1) and collagen type IV, alpha-2 (COL4A2) in KTCN in Ecuadorian families. Methods COL4A1 and COL4A2 in 15 Ecuadorian KTCN families were examined with polymerase chain reaction amplification, and direct sequencing of all exons, promoter and intron-exon junctions was performed. Results Screening of COL4A1 and COL4A2 revealed numerous alterations in coding and non-coding regions of both genes. We detected three missense substitutions in COL4A1: c.19G>C (Val7Leu), c.1663A>C (Thr555Pro), and c.4002A>C (Gln1334His). Five non-synonymous variants were identified in COL4A2: c.574G>T (Val192Phe), c.1550G>A (Arg517Lys), c.2048G>C (Gly683Ala), c.2102A>G (Lys701Arg), and c.2152C>T (Pro718Ser). None of the identified sequence variants completely segregated with the affected phenotype. The Gln1334His variant was possibly damaging to protein function and structure. Conclusions This is the first mutation screening of COL4A1 and COL4A2 genes in families with KTCN and linkage to a locus close to these genes. Analysis of COL4A1 and COL4A2 revealed no mutations indicating that other genes are involved in KTCN causation in Ecuadorian families. PMID:21527998

  7. Behavioral Effects of A1- and A2-Selective Adenosine Agonists and Antagonists: Evidence for Synergism and Antagonism

    PubMed Central

    NIKODIJEVIĆ, OLGA; SARGES, REINHARD; DALY, JOHN W.; JACOBSON, KENNETH A.

    2012-01-01

    The locomotor effects in mice of selective A1 and A2 adenosine agonists, antagonists and combinations of agonists were investigated using a computerized activity monitor. The A2-selective agonist 2-[(2-aminoethylamino)carbonylethylphenylethylamino]-5'-N-ethylcarboxamidoadenosine (APEC), an amine derivative of 2-(carboxyethylphenylethylamino)adenosine-5'-carboxamide, was a more potent locomotor depressant than its amide conjugates. The rank order of potency after i.p. injection for adenosine agonists was 5'-N-ethylcarboxamidoadenosine (NECA) (ED50, 5.8 nmol/kg) > APEC (ED50, 25 nmol/kg) > N6-cyclohexyladenosine (CHA) (ED50, 270 nmol/kg). An A1-selective, centrally acting, adenosine antagonist, 8-cyclopentyltheophylline (10 mg/kg), completely reversed the locomotor depressant effects of CHA (A1-selective) and NECA (nonselective) at doses of agonists as high as twice the ED50, and shifted the dose-response curves to the right, suggesting a primary involvement of A1 receptors. 8-cyclopentyltheophylline did not affect the depressant effects of APEC at the ED50, consistent with the A2-selectivity of APEC. The locomotor effects of APEC and CHA were completely reversed by theophylline, but not by the peripherally active 8-p-sulfophenyltheophylline, indicating central action of the adenosine agonists. The depressant effects of APEC, but not of NECA or CHA, were reversed significantly by an A2-selective adenosine receptor antagonist, 4-amino-8-chloro-1-phenyl-[1,2,4]triazol[4,3-a]quinoxaline. Low or subthreshold doses of CHA potentiated the depressant effects of APEC. A subthreshold dose of CHA did not alter the depressant effect of NECA, whereas a subthreshold dose of APEC increased the depressant effects of low doses of NECA. Thus, it appears that A1- and A2-selective adenosine agonists have separate central depressant effects, which can be potentiative. The relatively high potency of NECA in vivo could be due to a synergism between central A1 and A2receptor activation by

  8. Theoretical Studies of Observable Transitions to Recoupled Pair Bonded States of Sulfur Halide Compounds: SF/SCl (X^2{Π}{→}A^2{Σ}^-), SF_2/SCl_2 (X^1A_1{→}1^1B_1, X^1A_1{→}1^1A_2), and SFCl (X^1A'{→}A^1A{'{'}})

    NASA Astrophysics Data System (ADS)

    Leiding, Jeff; Woon, David E.; Dunning, Thom H.; , Jr.

    2011-06-01

    In previous studies regarding the nature of hypervalent behavior, we identified low-lying excited states of SF(a^4{Σ}^-), SCl(a^4{Σ}^-), SF_2(a^3B_1,b^3A_2), SFCl(a^3A{'{'}}) and SCl_2(a^3B_1) that involve recoupled pair bonding (rpb), where the electrons of the S 3p^2 pair are made available to form bonds. While the transitions from the ground states to the quartet states of SF/SCl and the triplet states of SF_2/SFCl/SCl_2 are spin-forbidden, each of these excited states have analogs with formally spin- and dipole-allowed transitions (except ^1A_2). We performed high level MRCI+Q/aug-cc-pV(Q+d)Z calculations in order to characterize the electronic spectra, spectroscopic constants, and bonding of these species, and made comparisons to available experimental data. We found that excitation into the experimentally known and dipole-forbidden singlet rpb state, SCl_2(B^1A_2), can explain the well-known photodissociation behavior of SCl_2 used to produce SCl(X^2{Π}) radicals in the laboratory. Finally, we have also found a possible system of bond-stretch isomers on the SFCl(A^1A{'{'}}) potential energy surface that is analogous to the behavior on the triplet surface reported in our previous study. Howe, J. D.; Ashfold, M. N. R.; Morgan, R. A.;Western, C. M.; Buma, W. J.; Milan, J. B. and de Lang, C. A. J. Chem. Soc. Faraday Trans. 1995, 91, 773. Leiding, J.; Woon, D. E., and Dunning, T. H., Jr. J. Phys. Chem. A 2011, 115, 329.

  9. Immunoglobulin A1 and A2 subclass of salivary antibodies to Candida albicans in patients with oral candidosis.

    PubMed Central

    Jeganathan, S; Ufomata, D; Hobkirk, J A; Ivanyi, L

    1987-01-01

    Immunoglobulin A antibody titres to a cytoplasmic protein extract of Candida albicans were determined by ELISA in saliva from 20 patients with oral candidosis and 21 controls. Patients had significantly increased levels of salivary IgA anti-Candida antibodies when compared with controls (P less than 0.001). Antibody levels were associated with IgA1 subclass in 90% of the patients; in contrast, IgA2 subclass was predominant in 67% of the controls. Antifungal therapy resulted in significantly decreased IgA1 titres (P less than 0.05) whilst the mean IgA2 antibody titre remained unchanged. The results indicate that Candida infection may change the subclass pattern of salivary IgA antibodies. PMID:3322616

  10. Cloning and characterization of a novel CYP3A1 allelic variant: analysis of CYP3A1 and CYP3A2 sex-hormone-dependent expression reveals that the CYP3A2 gene is regulated by testosterone.

    PubMed

    Ribeiro, V; Lechner, M C

    1992-02-14

    A clone was isolated from a cDNA library constructed from phenobarbital-treated Wistar rat liver and proven to correspond to the full-length mRNA of a polymorphic variant of Sprague-Dawley CYP3A1. Eight nucleotide differences were detected in a single 76-nucleotide stretch and confirmed to be present in the genomic clone. They are seated in a region implicated in the definition of a substrate binding domain of the native P450. Three out of the eight nucleotide changes are nonconservative, implicating the replacement of Thr/Ala 207, Phe/Ile 213, and Ile/Val 232. This is the first report of an allelic variant of CYP3A1, a new example of interstrain P450 variability. The CYP3A subfamily is composed of several genes coding for active testosterone 6 beta-hydroxylases which are expressed in the liver. CYP3A genes are under strong and distinct developmental regulation. Conversely to CYP3A1, transiently expressed in immature animals, CYP3A2 is constitutively expressed in the liver early after birth and characterized by an extinction in the adult females. Castration of 90-day-old male rats causes a drastic reduction (80%) of CYP3A2 mRNA relative abundance. Administration of testosterone propionate restores the physiological levels of CYP3A2 mRNA characteristic of the male rat liver. Our results demonstrate the existence of a direct relationship between the male hormonal status and the constitutive expression of rat liver CYP3A2.

  11. Transplantation of ABO A2 kidneys into O recipients: do IgM anti-A1 titers matter?

    PubMed

    Tierney, Joshua; Shaffer, David

    2015-04-01

    The ABO blood subgroup A2 expresses lower levels of A antigen on the cell surface and is less immunogenic toward anti-A immunoglobulin present in blood type O or B recipients. Previous studies have shown successful kidney transplantation from A2 donors into O or B recipients with low pre-transplant anti-A titers. Previous studies suggest good results with recipient IgG titers <1:8. Few studies have specifically evaluated the importance of anti-A1 IgM titers on early outcomes following A2 to O or B kidney transplantation. We performed a single center, retrospective review of all A2 to O living donor kidney transplants. All recipients had pre-transplant anti-A IgG titers <1:8. IgM titers were measured in all recipients and were reported but not used to determine eligibility for transplant. From 2001 to 2013, we performed seven consecutive A2 to O living donor kidney transplants. Early allograft dysfunction, acute rejection or thrombotic microangiopathy, occurred in four patients and were associated with high IgM titers despite low IgG titers. Our data show a high incidence of early acute rejection or thrombotic microangiopathy in A2 to O kidney transplants with high recipient anti-A IgM titers despite low IgG titers. Steps to lower anti-IgM pre-transplant may reduce the risk of early allograft dysfunction in A2 to O or B kidney transplants. Attention should be paid to IgM titers in establishing individual center selection criteria for A2 to B kidney transplants under the new UNOS kidney allocation system. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Coronary-Heart-Disease-Associated Genetic Variant at the COL4A1/COL4A2 Locus Affects COL4A1/COL4A2 Expression, Vascular Cell Survival, Atherosclerotic Plaque Stability and Risk of Myocardial Infarction

    PubMed Central

    Pu, Xiangyuan; Ren, Meixia; An, Weiwei; Zhang, Ruoxin; Yan, Shunying; Situ, Haiteng; He, Xinjie; Chen, Yequn; Tan, Xuerui; Xiao, Qingzhong; Tucker, Arthur T.; Caulfield, Mark J.; Ye, Shu

    2016-01-01

    Genome-wide association studies have revealed an association between coronary heart disease (CHD) and genetic variation on chromosome 13q34, with the lead single nucleotide polymorphism rs4773144 residing in the COL4A2 gene in this genomic region. We investigated the functional effects of this genetic variant. Analyses of primary cultures of vascular smooth muscle cells (SMCs) and endothelial cells (ECs) from different individuals showed a difference between rs4773144 genotypes in COL4A2 and COL4A1 expression levels, being lowest in the G/G genotype, intermediate in A/G and highest in A/A. Chromatin immunoprecipitation followed by allelic imbalance assays of primary cultures of SMCs and ECs that were of the A/G genotype revealed that the G allele had lower transcriptional activity than the A allele. Electrophoretic mobility shift assays and luciferase reporter gene assays showed that a short DNA sequence encompassing the rs4773144 site interacted with a nuclear protein, with lower efficiency for the G allele, and that the G allele sequence had lower activity in driving reporter gene expression. Analyses of cultured SMCs from different individuals demonstrated that cells of the G/G genotype had higher apoptosis rates. Immunohistochemical and histological examinations of ex vivo atherosclerotic coronary arteries from different individuals disclosed that atherosclerotic plaques with the G/G genotype had lower collagen IV abundance and thinner fibrous cap, a hallmark of unstable, rupture-prone plaques. A study of a cohort of patients with angiographically documented coronary artery disease showed that patients of the G/G genotype had higher rates of myocardial infarction, a phenotype often caused by plaque rupture. These results indicate that the CHD-related genetic variant at the COL4A2 locus affects COL4A2/COL4A1 expression, SMC survival, and atherosclerotic plaque stability, providing a mechanistic explanation for the association between the genetic variant and CHD

  13. Conditional MHC class I ligands and peptide exchange technology for the human MHC gene products HLA-A1, -A3, -A11, and -B7.

    PubMed

    Bakker, Arnold H; Hoppes, Rieuwert; Linnemann, Carsten; Toebes, Mireille; Rodenko, Boris; Berkers, Celia R; Hadrup, Sine Reker; van Esch, Wim J E; Heemskerk, Mirjam H M; Ovaa, Huib; Schumacher, Ton N M

    2008-03-11

    Major histocompatibility complex (MHC) class I multimer technology has become an indispensable immunological assay system to dissect antigen-specific cytotoxic CD8(+) T cell responses by flow cytometry. However, the development of high-throughput assay systems, in which T cell responses against a multitude of epitopes are analyzed, has been precluded by the fact that for each T cell epitope, a separate in vitro MHC refolding reaction is required. We have recently demonstrated that conditional ligands that disintegrate upon exposure to long-wavelength UV light can be designed for the human MHC molecule HLA-A2. To determine whether this peptide-exchange technology can be developed into a generally applicable approach for high throughput MHC based applications we set out to design conditional ligands for the human MHC gene products HLA-A1, -A3, -A11, and -B7. Here, we describe the development and characterization of conditional ligands for this set of human MHC molecules and apply the peptide-exchange technology to identify melanoma-associated peptides that bind to HLA-A3 with high affinity. The conditional ligand technology developed here will allow high-throughput MHC-based analysis of cytotoxic T cell immunity in the vast majority of Western European individuals.

  14. Serum Levels of ApoA1 and ApoA2 Are Associated with Cognitive Status in Older Men

    PubMed Central

    Ma, Cheng; Li, Jin; Bao, Zhijun; Ruan, Qingwei; Yu, Zhuowei

    2015-01-01

    Background. Advancing age, chronic inflammation, oxidative damage, and disorders of lipid metabolism are positively linked to the late-life cognitive impairment. Serum biomarkers may be associated with the cognitive status in older men. Methods. 440 old male subjects with different cognitive functions were recruited to investigate probable serum markers. Pearson Chi-Squared test, univariate analysis, and multivariate logistic regression analysis were performed to evaluate biomarkers which may be associated with cognitive status. Results. Levels of fundus atherosclerosis (AS) (P < 0.001), age (P < 0.001), serum biomarkers peroxidase (POD) (P = 0.026) and interleukin-6 (IL-6) (P = 0.001), serum levels of high-density lipoprotein cholesterol (HDL-C) (P < 0.001), apolipoprotein A2 (ApoA2) (P = 0.001), and ApoC2 (P = 0.005) showed significant differences. Compared to group 3, ApoA1 in group 1 (OR = 1.30, 95% CI 1.01–1.67) and group 2 (OR = 1.47, 95% CI 1.11–1.94) were higher, while ApoA2 were lower (group 1: OR = 0.43, 95% CI 0.18–1.02; group 2: OR = 0.21, 95% CI 0.08–0.54) after adjusting for control variables. Conclusion. The results demonstrated that age, AS levels, POD, IL-6, HDL-C, ApoA2, and ApoC2 were significantly related to cognitive status. Moreover, ApoA1 and ApoA2 were independently associated with cognitive impairment and late-life dementia. PMID:26682220

  15. β-Cell deletion of Nr4a1 and Nr4a3 nuclear receptors impedes mitochondrial respiration and insulin secretion.

    PubMed

    Reynolds, Merrick S; Hancock, Chad R; Ray, Jason D; Kener, Kyle B; Draney, Carrie; Garland, Kevin; Hardman, Jeremy; Bikman, Benjamin T; Tessem, Jeffery S

    2016-07-01

    β-Cell insulin secretion is dependent on proper mitochondrial function. Various studies have clearly shown that the Nr4a family of orphan nuclear receptors is essential for fuel utilization and mitochondrial function in liver, muscle, and adipose. Previously, we have demonstrated that overexpression of Nr4a1 or Nr4a3 is sufficient to induce proliferation of pancreatic β-cells. In this study, we examined whether Nr4a expression impacts pancreatic β-cell mitochondrial function. Here, we show that β-cell mitochondrial respiration is dependent on the nuclear receptors Nr4a1 and Nr4a3. Mitochondrial respiration in permeabilized cells was significantly decreased in β-cells lacking Nr4a1 or Nr4a3. Furthermore, respiration rates of intact cells deficient for Nr4a1 or Nr4a3 in the presence of 16 mM glucose resulted in decreased glucose mediated oxygen consumption. Consistent with this reduction in respiration, a significant decrease in glucose-stimulated insulin secretion rates is observed with deletion of Nr4a1 or Nr4a3. Interestingly, the changes in respiration and insulin secretion occur without a reduction in mitochondrial content, suggesting decreased mitochondrial function. We establish that knockdown of Nr4a1 and Nr4a3 results in decreased expression of the mitochondrial dehydrogenase subunits Idh3g and Sdhb. We demonstrate that loss of Nr4a1 and Nr4a3 impedes production of ATP and ultimately inhibits glucose-stimulated insulin secretion. These data demonstrate for the first time that the orphan nuclear receptors Nr4a1 and Nr4a3 are critical for β-cell mitochondrial function and insulin secretion. Copyright © 2016 the American Physiological Society.

  16. Polymorphisms in the cytochrome P450 genes CYP1A2, CYP1B1, CYP3A4, CYP3A5, CYP11A1, CYP17A1, CYP19A1 and colorectal cancer risk

    PubMed Central

    Bethke, Lara; Webb, Emily; Sellick, Gabrielle; Rudd, Matthew; Penegar, Stephen; Withey, Laura; Qureshi, Mobshra; Houlston, Richard

    2007-01-01

    Background Cytochrome P450 (CYP) enzymes have the potential to affect colorectal cancer (CRC) risk by determining the genotoxic impact of exogenous carcinogens and levels of sex hormones. Methods To investigate if common variants of CYP1A2, CYP1B1, CYP3A4, CYP3A5, CYP11A1, CYP17A1 and CYP19A1 influence CRC risk we genotyped 2,575 CRC cases and 2,707 controls for 20 single nucleotide polymorphisms (SNPs) that have not previously been shown to have functional consequence within these genes. Results There was a suggestion of increased risk, albeit insignificant after correction for multiple testing, of CRC for individuals homozygous for CYP1B1 rs162558 and heterozygous for CYP1A2 rs2069522 (odds ratio [OR] = 1.36, 95% confidence interval [CI]: 1.03–1.80 and OR = 1.34, 95% CI: 1.00–1.79 respectively). Conclusion This study provides some support for polymorphic variation in CYP1A2 and CYP1B1 playing a role in CRC susceptibility. PMID:17615053

  17. 17 CFR 270.32a-3 - Exemption from provision of section 32(a)(1) regarding the time period during which a registered...

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... section 32(a)(1) regarding the time period during which a registered management investment company must....32a-3 Exemption from provision of section 32(a)(1) regarding the time period during which a registered management investment company must select an independent public accountant. (a) A registered management...

  18. 17 CFR 270.32a-3 - Exemption from provision of section 32(a)(1) regarding the time period during which a registered...

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... section 32(a)(1) regarding the time period during which a registered management investment company must....32a-3 Exemption from provision of section 32(a)(1) regarding the time period during which a registered management investment company must select an independent public accountant. (a) A registered management...

  19. 17 CFR 270.32a-3 - Exemption from provision of section 32(a)(1) regarding the time period during which a registered...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... section 32(a)(1) regarding the time period during which a registered management investment company must....32a-3 Exemption from provision of section 32(a)(1) regarding the time period during which a registered management investment company must select an independent public accountant. (a) A registered management...

  20. 17 CFR 270.32a-3 - Exemption from provision of section 32(a)(1) regarding the time period during which a registered...

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... section 32(a)(1) regarding the time period during which a registered management investment company must....32a-3 Exemption from provision of section 32(a)(1) regarding the time period during which a registered management investment company must select an independent public accountant. (a) A registered management...

  1. 17 CFR 270.32a-3 - Exemption from provision of section 32(a)(1) regarding the time period during which a registered...

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... section 32(a)(1) regarding the time period during which a registered management investment company must....32a-3 Exemption from provision of section 32(a)(1) regarding the time period during which a registered management investment company must select an independent public accountant. (a) A registered management...

  2. Membrane isoforms of human immunoglobulins of the A1 and A2 isotypes: structural and functional study.

    PubMed Central

    Leduc, I; Drouet, M; Bodinier, M C; Helal, A; Cogné, M

    1997-01-01

    As for IgM, human IgA occurs either as soluble molecules in plasma and various other body fluids, or as membrane-bound molecules on differentiated B cells, where they are part of the B-cell receptor for antigen (BCR). We studied the structure of transcripts encoding the membrane-anchored alpha-chain of the human BCR alpha, which may be present in two different forms resulting from alternate splicing of the alpha-chain mRNA (type I or type II). The ratio of type I versus type II did not vary upon stimulation of a B-cell line with various cytokines. Rather, it differed strikingly in cells expressing either the IgA1 or IgA2 isotype of the BCR alpha, with virtually no type II alpha-chain in the latter. Co-modulation experiments also yielded different results for both isotypes, since they demonstrated a physical association of both membrane (m)IgA1 and mIgA2 with CD79b, the beta component of the BCR Ig alpha/Ig beta heterodimer, but only of mIgA1 with CD19. Whatever the isotype, the BCR of the IgA class was able to carry out signal transduction upon cross-linking by specific monoclonal antibodies but, in contrast to mIgM, it relied mainly on the entry of extracellular Ca2+ rather than on the release of intracellular stocks. Images Figure 2 PMID:9155637

  3. Activation of J77A.1 macrophages by three phospholipases A2 isolated from Bothrops atrox snake venom.

    PubMed

    Furtado, Juliana L; Oliveira, George A; Pontes, Adriana S; Setúbal, Sulamita da S; Xavier, Caroline V; Lacouth-Silva, Fabianne; Lima, Beatriz F; Zaqueo, Kayena D; Kayano, Anderson M; Calderon, Leonardo A; Stábeli, Rodrigo G; Soares, Andreimar M; Zuliani, Juliana P

    2014-01-01

    In the present study, we investigated the in vitro effects of two basic myotoxic phospholipases A2 (PLA2), BaTX-I, a catalytically inactive Lys-49 variant, and BaTX-II, a catalytically active Asp-49, and of one acidic myotoxic PLA2, BaPLA2, a catalytically active Asp-49, isolated from Bothrops atrox snake venom, on the activation of J774A.1 macrophages. At noncytotoxic concentrations, the toxins did not affect the adhesion of the macrophages, nor their ability to detach. The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis. However, BaTX-I, BaTX-II, and BaPLA2 induced the release of the superoxide anion by J774A.1 macrophages. Additionally, only BaTX-I raised the lysosomal volume of macrophages after 15 min of incubation. After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min. Moreover, BaTX-I, BaTX-II, and BaPLA2 increased the number of lipid bodies on macrophages submitted to phagocytosis and not submitted to phagocytosis. However, BaTX-II and BaPLA2 induced the release of TNF-α by J774A.1 macrophages. Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects.

  4. Development of a 3He-hydraulic actuator for spin pump in superfluid 3He-A1

    NASA Astrophysics Data System (ADS)

    Yamaguchi, A.; Wada, M.; Tanaka, H.; Motoyama, G.; Sumiyama, A.; Aoki, Y.; Okuda, Y.; Murakawa, S.; Karaki, Y.; Kubota, M.; Kojima, H.

    2012-12-01

    The superfluid 3He A1 phase contains a spin-polarized condensate. This property allows novel superfluid spin current experiments. In the mechano-spin effect of the A1 phase a mechanically applied pressure gradient and a superleak-spin filter enable to directly boost spin polarization of 3He in a small chamber. Using a flexible membrane as an electrostatically actuated pump, we carried out such experiments and observed 50% enhancement of spin density. Here we report on a new 3He-hydraulic actuator for achieving greater enhancement of spin density. The actuator consists of two liquid 3He chambers located at a 4.2 K plate and in the interior of the cell. The pressure in the 4.2 K chamber is heater-controlled and it transmits a force onto a membrane in the cell. The motion of the membrane induces spin-polarized current into an accumulation chamber.

  5. Dipyridamole attenuates ischemia reperfusion induced acute kidney injury through adenosinergic A1 and A2A receptor agonism in rats.

    PubMed

    Puri, Nikkita; Mohey, Vinita; Singh, Manjinder; Kaur, Tajpreet; Pathak, Devendra; Buttar, Harpal Singh; Singh, Amrit Pal

    2016-04-01

    Dipyridamole (DYP) is an anti-platelet agent with marked vasodilator, anti-oxidant, and anti-inflammatory activity. The present study investigated the role of adenosine receptors in DYP-mediated protection against ischemia reperfusion-induced acute kidney injury (AKI) in rats. The rats were subjected to bilateral renal ischemia for 40 min followed by reperfusion for 24 h. The renal damage induced by ischemia reperfusion injury (IRI) was assessed by measuring creatinine clearance, blood urea nitrogen, uric acid, plasma potassium, fractional excretion of sodium, and microproteinuria in rats. The oxidative stress in renal tissues was assessed by quantification of thiobarbituric acid-reactive substances, superoxide anion generation, and reduced glutathione level. The hematoxylin-eosin staining was carried out to observe histopathological changes in renal tissues. DYP (10 and 30 mg/kg, intraperitoneal, i.p.) was administered 30 min before subjecting the rats to renal IRI. In separate groups, caffeine (50 mg/kg, i.p.), an adenosinergic A1 and A2A receptor antagonist was administered with and without DYP treatment before subjecting the rats to renal IRI. The ischemia reperfusion-induced AKI was demonstrated by significant changes in serum as well as urinary parameters, enhanced oxidative stress, and histopathological changes in renal tissues. The administration of DYP demonstrated protection against AKI. The prior treatment with caffeine abolished DYP-mediated reno-protection suggesting role of A1 and A2A adenosine receptors in DYP-mediated reno-protection in rats. It is concluded that adenosine receptors find their definite involvement in DYP-mediated anti-oxidative and reno-protective effect against ischemia reperfusion-induced AKI.

  6. Argon ion laser-induced BaS B1Σ +- A1Σ +, A' 1Π, a3Π, and X1Σ + fluorescence spectra: Analysis of A ˜ A', A ˜ a, and A' ˜ a perturbations

    NASA Astrophysics Data System (ADS)

    Cummins, P. G.; Field, Robert W.; Renhorn, Ingemar

    1981-12-01

    The 333.6-, 351.1-, and 363.8-nm lines of a cw argon ion laser are found to coincide with the BaS B1Σ +- X1Σ + (12, 0) R(17), (6, 0) P(35), and (3, 0) R(125) transitions, respectively. Fluorescence transitions from the laser-prepared upper levels terminating in X1Σ +v = 0-28, A1Σ +v = 1-3, A' 1Π v = 1-13, and a3Π 1v = 3-12 are assigned. These results are combined with a previous analysis of the extensively perturbed BaS A1Σ +- X1Σ + system [R. F. Barrow, W. G. Burton, and P. A. Jones, Trans. Farad. Soc.67, 902-906 (1971)]. Every observed perturbation of the BaS A1Σ + state is electronically and vibrationally assigned. The levels a3Π 0v = 10-13, a3Π 1v = 12-14, a3Π 2v = 15, and A' 1Π v = 10-13 are sampled via their perturbations of A1Σ +v = 0-2. Although the mutual interactions of the a3Π, A' 1Π, and A1Σ + states approach Hund's case ( c) limit, a complete deperturbation is performed from a case ( a) starting point. Of the five lowest energy electronic states of BaS, only b3Σ + remains uncharacterized. Principal deperturbed molecular constants are (in cm -1, 1σ uncertainties in parentheses):

  7. Antagonist Selective Modulation of Adenosine A1 and A3 Receptor Pharmacology by the Food Dye Brilliant Black BN: Evidence for Allosteric Interactions

    PubMed Central

    May, L. T.; Briddon, S. J.

    2010-01-01

    Allosteric binding sites on the adenosine receptor family represent potential therapeutic targets for a number of conditions involving metabolic stress. This study has identified Brilliant Black BN as a novel allosteric modulator of the adenosine A1 and A3 receptors. In addition to being a food dye and pharmaceutical excipient, Brilliant Black BN is commonly used within calcium mobilization assays to quench extracellular fluorescence. Brilliant Black BN (5–500 μM) had no significant effect on the calcium mobilization stimulated by the nonselective adenosine receptor agonist 5′-(N-ethylcarboxamido)adenosine in Chinese hamster ovary cells stably transfected with the human adenosine A1 or A3 receptor. Likewise, calcium mobilization and radioligand binding assays found that Brilliant Black BN (5–500 μM) did not significantly influence the antagonism mediated by 8-cyclopentyl-1,3-dipropylxanthine (100 nM) at the A1 receptor. In contrast, the affinity of N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-yl]benzene acetamide (MRS1220) at the A3 receptor and xanthine amine congener (XAC) and XAC-X-BY630 at the A1 and A3 receptors was significantly decreased in the presence of 500 μM Brilliant Black BN. A reduction in XAC potency at the A1 and A3 receptor was achieved within 1 min of Brilliant Black BN addition, despite receptors having been pre-equilibrated with antagonist. Dissociation kinetics of the fluorescent XAC derivative, XAC-X-BY630, revealed that the decrease in affinity is probably due to a significant increase in dissociation rate of the antagonist in the presence of Brilliant Black BN. Taken together, these results suggest that Brilliant Black BN can act allosterically to modify ligand affinity at A1 and A3 receptors. PMID:20086038

  8. Activation of J77A.1 Macrophages by Three Phospholipases A2 Isolated from Bothrops atrox Snake Venom

    PubMed Central

    Furtado, Juliana L.; Oliveira, George A.; Pontes, Adriana S.; Setúbal, Sulamita da S.; Xavier, Caroline V.; Lacouth-Silva, Fabianne; Lima, Beatriz F.; Zaqueo, Kayena D.; Kayano, Anderson M.; Calderon, Leonardo A.; Stábeli, Rodrigo G.; Soares, Andreimar M.; Zuliani, Juliana P.

    2014-01-01

    In the present study, we investigated the in vitro effects of two basic myotoxic phospholipases A2 (PLA2), BaTX-I, a catalytically inactive Lys-49 variant, and BaTX-II, a catalytically active Asp-49, and of one acidic myotoxic PLA2, BaPLA2, a catalytically active Asp-49, isolated from Bothrops atrox snake venom, on the activation of J774A.1 macrophages. At noncytotoxic concentrations, the toxins did not affect the adhesion of the macrophages, nor their ability to detach. The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis. However, BaTX-I, BaTX-II, and BaPLA2 induced the release of the superoxide anion by J774A.1 macrophages. Additionally, only BaTX-I raised the lysosomal volume of macrophages after 15 min of incubation. After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min. Moreover, BaTX-I, BaTX-II, and BaPLA2 increased the number of lipid bodies on macrophages submitted to phagocytosis and not submitted to phagocytosis. However, BaTX-II and BaPLA2 induced the release of TNF-α by J774A.1 macrophages. Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects. PMID:24592395

  9. Pulsed Electromagnetic Fields Increased the Anti-Inflammatory Effect of A2A and A3 Adenosine Receptors in Human T/C-28a2 Chondrocytes and hFOB 1.19 Osteoblasts

    PubMed Central

    Vincenzi, Fabrizio; Targa, Martina; Corciulo, Carmen; Gessi, Stefania; Merighi, Stefania; Setti, Stefania; Cadossi, Ruggero; Goldring, Mary B.; Borea, Pier Andrea; Varani, Katia

    2013-01-01

    Adenosine receptors (ARs) have an important role in the regulation of inflammation and their activation is involved in the inhibition of pro-inflammatory cytokine release. The effects of pulsed electromagnetic fields (PEMFs) on inflammation have been reported and we have demonstrated that PEMFs increased A2A and A3AR density and functionality in different cell lines. Chondrocytes and osteoblasts are two key cell types in the skeletal system that play important role in cartilage and bone metabolism representing an interesting target to study the effect of PEMFs. The primary aim of the present study was to evaluate if PEMF exposure potentiated the anti-inflammatory effect of A2A and/or A3ARs in T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts. Immunofluorescence, mRNA analysis and saturation binding assays revealed that PEMF exposure up-regulated A2A and A3AR expression. A2A and A3ARs were able to modulate cAMP production and cell proliferation. The activation of A2A and A3ARs resulted in the decrease of some of the most relevant pro-inflammatory cytokine release such as interleukin (IL)-6 and IL-8, following the treatment with IL-1β as an inflammatory stimuli. In human chondrocyte and osteoblast cell lines, the inhibitory effect of A2A and A3AR stimulation on the release of prostaglandin E2 (PGE2), an important lipid inflammatory mediator, was observed. In addition, in T/C-28a2 cells, the activation of A2A or A3ARs elicited an inhibition of vascular endothelial growth factor (VEGF) secretion. In hFOB 1.19 osteoblasts, PEMF exposure determined an increase of osteoprotegerin (OPG) production. The effect of the A2A or A3AR agonists in the examined cells was enhanced in the presence of PEMFs and completely blocked by using well-known selective antagonists. These results demonstrated that PEMF exposure significantly increase the anti-inflammatory effect of A2A or A3ARs suggesting their potential therapeutic use in the therapy of inflammatory bone and joint disorders

  10. Structures and absolute stereochemistry of dinapinones A1 and A2, inhibitors of triacylglycerol synthesis, produced by penicillium pinophilum FKI-3864.

    PubMed

    Uchida, Ryuji; Ohte, Satoshi; Kawamoto, Kyosuke; Yamazaki, Hiroyuki; Kawaguchi, Mio; Tomoda, Hiroshi

    2012-08-01

    During our screening program for microbial inhibitors of triacylglycerol synthesis in mammalian cells, four structurally related new compounds, dinapinones A1 (1) and A2 (2) and monapinones A (3) and B (4), were isolated from the culture broth of Penicillium pinophilum FKI-3864. Compounds 3 and 4 were produced by the fungus only when fermented in seawater-supplemented medium. The structures of 1 to 4 were elucidated by spectroscopic studies including various NMR experiments. Compounds 1 and 2 were atropisomers consisting of two monomers with the same planar structure of dihydronaphthopyranone as 3. The absolute stereochemistry of 3 was elucidated by NOE experiment and circular dichroism spectra. Furthermore, the stereochemistry of 1 and 2 was elucidated by in vitro conversion from the structure-defined 3 to its dimers 1 and 2.

  11. Purification and characterization of pepsins A1 and A2 from the Antarctic rock cod Trematomus bernacchii

    PubMed Central

    Brier, Sébastien; Maria, Giovanna; Carginale, Vincenzo; Capasso, Antonio; Wu, Yan; Taylor, Robert M.; Borotto, Nicholas B.; Capasso, Clemente; Engen, John R.

    2008-01-01

    SUMMARY The Antarctic notothenioid Trematomus bernacchii (rock cod) lives at a constant mean temperature of −1.9 °C. Gastric digestion under these conditions relies on the proteolytic activity of aspartic proteases such as pepsin. To understand the molecular mechanisms of Antarctic fish pepsins, T. bernacchii pepsins A1 and A2 were cloned, overexpressed in E. coli, purified and characterized with a number of biochemical and biophysical methods. The properties of these two Antarctic isoenzymes were compared to porcine pepsin and found to be unique in a number of ways. Fish pepsins were found to be more temperature sensitive, generally less active at lower pH and more sensitive to inhibition by pepstatin than the mesophilic counterpart. The specificity of Antarctic fish pepsins was similar but not identical to pig pepsin, likely owing to changes in the sequence of fish enzymes near the active site. Gene duplication of Antarctic rock cod pepsins is the likely mechanism for adaptation to the harsh temperature environment in which these enzymes must function. PMID:17976195

  12. Acute subdural hematoma without subarachnoid hemorrhage caused by ruptured A1-A2 junction aneurysm. Case report.

    PubMed

    Takada, Tomoya; Yamamoto, Tetsuya; Ishikawa, Eiichi; Zaboronok, Alexander; Kujiraoka, Yuji; Akutsu, Hiroyoshi; Ihara, Satoshi; Nakai, Kei; Matsumura, Akira

    2012-01-01

    A 54-year-old man was admitted to our hospital with complaint of sudden headache. The patient had suffered two episodes of transient headache before admission. Computed tomography (CT) revealed acute subdural hematoma (ASDH) on the right side of the cerebral convexity with bilateral extension along the tentorium cerebelli without signs of subarachnoid hemorrhage (SAH) or intracerebral hemorrhage (ICH). Three-dimensional CT angiography and conventional cerebral angiography revealed a left A1-A2 junction aneurysm. Neck clipping of the aneurysm was performed. The aneurysm extended inferiorly, with the dome embedded in the chiasmatic cistern and tightly adhered to the arachnoid membrane. There was no evidence of hematoma in the subarachnoid space. The patient was discharged without neurological deficit. Ruptured aneurysms resulting in ASDH without SAH or ICH are very rare. Radiological investigation such as three-dimensional CT angiography should be performed to find the causative aneurysm in a patient with ASDH with a history of repeated headaches and without traumatic signs or episodes, and the appropriate treatment should be planned with expediency.

  13. Study of proton conductivity of a 2D flexible MOF and a 1D coordination polymer at higher temperature.

    PubMed

    Sanda, Suresh; Biswas, Soumava; Konar, Sanjit

    2015-02-16

    We report the proton conduction properties of a 2D flexible MOF and a 1D coordination polymer having the molecular formulas {[Zn(C10H2O8)0.5(C10S2N2H8)]·5H2O]}n (1) and {[Zn(C10H2O8)0.5(C10S2N2H8)]·2H2O]}n (2), respectively. Compounds 1 and 2 show high conductivity values of 2.55 × 10(-7) and 4.39 × 10(-4) S cm(-1) at 80 °C and 95% RH. The conductivity value of compound 1 is in the range of those for previously reported flexible MOFs, and compound 2 shows the highest proton conductivity among the carboxylate-based 1D CPs. The dimensionality and the internal hydrogen bonding connectivity play a vital role in the resultant conductivity. Variable-temperature experiments of both compounds at high humidity reveal that the conductivity values increase with increasing temperature, whereas the variable humidity studies signify the influence of relative humidity on high-temperature proton conductivity. The time-dependent measurements for both compounds demonstrate their ability to retain conductivity up to 10 h.

  14. HsfA1d and HsfA1e involved in the transcriptional regulation of HsfA2 function as key regulators for the Hsf signaling network in response to environmental stress.

    PubMed

    Nishizawa-Yokoi, Ayako; Nosaka, Ryota; Hayashi, Hideki; Tainaka, Hitoshi; Maruta, Takanori; Tamoi, Masahiro; Ikeda, Miho; Ohme-Takagi, Masaru; Yoshimura, Kazuya; Yabuta, Yukinori; Shigeoka, Shigeru

    2011-05-01

    Heat shock transcription factor A2 (HsfA2) acts as a key component of the Hsf signaling network involved in cellular responses to various types of environmental stress. However, the mechanism governing the regulation of HsfA2 expression is still largely unknown. We demonstrated here that a heat shock element (HSE) cluster in the 5'-flanking region of the HsfA2 gene is involved in high light (HL)-inducible HsfA2 expression. Accordingly, to identify the Hsf regulating the expression of HsfA2, we analyzed the effect of loss-of-function mutations of class A Hsfs on the expression of HsfA2 in response to HL stress. Overexpression of an HsfA1d or HsfA1e chimeric repressor and double knockout of HsfA1d and HsfA1e Arabidopsis mutants (KO-HsfA1d/A1e) significantly suppressed the induction of HsfA2 expression in response to HL and heat shock (HS) stress. Transient reporter assays showed that HsfA1d and HsfA1e activate HsfA2 transcription through the HSEs in the 5'-flanking region of HsfA2. In the KO-HsfA1d/A1e mutants, 560 genes, including a number of stress-related genes and several Hsf genes, HsfA7a, HsfA7b, HsfB1 and HsfB2a, were down-regulated compared with those in the wild-type plants under HL stress. The PSII activity of KO-HsfA1d/A1e mutants decreased under HL stress, while the activity of wild-type plants remained high. Furthermore, double knockout of HsfA1d and HsfA1e impaired tolerance to HS stress. These findings indicated that HsfA1d and HsfA1e not only regulate HsfA2 expression but also function as key regulators of the Hsf signaling network in response to environmental stress.

  15. a 1+1' Resonance-Enhanced Multiphoton Ionization Scheme for Rotationally State-Selective Detection of Formaldehyde via the ˜{A}^1A_2←˜{X}^1A_1 Transition

    NASA Astrophysics Data System (ADS)

    Park, Barratt; Krueger, Bastian C.; Meyer, Sven; Wodtke, Alec; Schaefer, Tim

    2017-06-01

    The formaldehyde molecule is an important model system for understanding dynamical processes in small polyatomic molecules. However, prior to this work, there have been no reports of a resonance-enhanced multiphoton ionization (REMPI) detection scheme for formaldehyde suitable for rovibrationally state-selective detection in molecular beam scattering experiments. Previously reported tunable REMPI schemes are either non-rotationally resolved, involve multiple resonant steps, or involve many-photon ionization steps. In the current work, we present a new 1+1' REMPI scheme for formaldehyde. The first photon is tunable and provides rotational resolution via the vibronically allowed ˜{A}^1A_2←˜{X}^1A_1 transition. Molecules are then directly ionized from the ˜{A} state by one photon of 157 nm. The results indicate that the ionization cross section from the 4^1 vibrational level of the ˜{A} state is independent of the rotational level used as intermediate, to within experimental uncertainty. The 1+1' REMPI intensities are therefore directly proportional to the ˜{A}←˜{X} absorption intensities and can be used for quantitative measurement of ˜{X}-state population distributions.

  16. The infarct-sparing effect of IB-MECA against myocardial ischemia/reperfusion injury in mice is mediated by sequential activation of adenosine A3 and A 2A receptors.

    PubMed

    Tian, Yikui; Marshall, Melissa; French, Brent A; Linden, Joel; Yang, Zequan

    2015-03-01

    Conflicting results exist regarding the role of A3 adenosine receptors (A3ARs) in mediating cardioprotection during reperfusion following myocardial infarction. We hypothesized that the effects of the A3AR agonist IB-MECA to produce cardioprotection might involve activation of other adenosine receptor subtypes. C57Bl/6 (B6), A3AR KO, A2AAR KO, and A2AAR KO/WT bone marrow chimeric mice were assigned to 12 groups undergoing either hemodynamic studies or 45 min of LAD occlusion and 60 min of reperfusion. IB-MECA (100 μg/kg) or vehicle was administered by iv bolus 5 min before reperfusion. Radioligand binding assays showed that IB-MECA has high affinity for the mouse A3AR (K i = 0.17 ± 0.05 nM), but also can bind with lower affinity to the A1AR (9.0 ± 2.4 nM) or the A2AAR (56.5 ± 10.2 nM). IB-MECA caused bi-phasic hemodynamic changes, which were completely absent in A3AR KO mice and were modified by A2AAR blockade or deletion. IB-MECA stimulated histamine release, increased heart rate, and significantly reduced IF size in B6 mice from 61.5 ± 1.4 to 48.6 ± 2.4% of risk region (RR; 21% reduction, p < 0.05) but not in A3AR KO mice. Compared to B6, A3AR KO mice had significantly reduced IF size (p < 0.05). In B6/B6 bone marrow chimeras, IB-MECA caused a 47% reduction of IF size (from 47.3 ± 3.9 to 24.7 ± 4.5, p < 0.05). However, no significant cardioprotective effect of IB-MECA was observed in A2AARKO/B6 mice, which lacked A2AARs only on their bone marrow-derived cells. Activation of A3ARs induces a bi-phasic hemodynamic response, which is partially mediated by activation of A2AARs. The cardioprotective effect of IB-MECA is due to the initial activation of A3AR followed by activation of A2AARs in bone marrow-derived cells.

  17. Jet-cooled laser-induced dispersed fluorescence spectroscopy of TaN: Observation of a3Δ and A1Δ states

    NASA Astrophysics Data System (ADS)

    Mukund, Sheo; Bhattacharyya, Soumen; Nakhate, S. G.

    2016-07-01

    Laser-induced dispersed fluorescence spectra of TaN molecules, produced in a free-jet apparatus, have been studied. Two spin components of the lowest-lying a3Δ state along with their vibrational structure have been observed. The A1Δ state, which was predicted earlier by ab initio calculation has also been observed. The X1Σ+ ground state vibrational progression up to v = 9 has been recorded. The experimentally determined term energies and vibrational constants at equilibrium for the ground and a3Δ states are in fairly good agreement with the ab initio values reported earlier.

  18. Differential Roles for "Nr4a1" and "Nr4a2" in Object Location vs. Object Recognition Long-Term Memory

    ERIC Educational Resources Information Center

    McNulty, Susan E.; Barrett, Ruth M.; Vogel-Ciernia, Annie; Malvaez, Melissa; Hernandez, Nicole; Davatolhagh, M. Felicia; Matheos, Dina P.; Schiffman, Aaron; Wood, Marcelo A.

    2012-01-01

    "Nr4a1" and "Nr4a2" are transcription factors and immediate early genes belonging to the nuclear receptor Nr4a family. In this study, we examine their role in long-term memory formation for object location and object recognition. Using siRNA to block expression of either "Nr4a1" or "Nr4a2", we found that "Nr4a2" is necessary for both long-term…

  19. Differential Roles for "Nr4a1" and "Nr4a2" in Object Location vs. Object Recognition Long-Term Memory

    ERIC Educational Resources Information Center

    McNulty, Susan E.; Barrett, Ruth M.; Vogel-Ciernia, Annie; Malvaez, Melissa; Hernandez, Nicole; Davatolhagh, M. Felicia; Matheos, Dina P.; Schiffman, Aaron; Wood, Marcelo A.

    2012-01-01

    "Nr4a1" and "Nr4a2" are transcription factors and immediate early genes belonging to the nuclear receptor Nr4a family. In this study, we examine their role in long-term memory formation for object location and object recognition. Using siRNA to block expression of either "Nr4a1" or "Nr4a2", we found that "Nr4a2" is necessary for both long-term…

  20. Physiological roles of A1 and A2A adenosine receptors in regulating heart rate, body temperature, and locomotion as revealed using knockout mice and caffeine.

    PubMed

    Yang, Jiang-Ning; Chen, Jiang-Fan; Fredholm, Bertil B

    2009-04-01

    Heart rate (HR), body temperature (Temp), locomotor activity (LA), and oxygen consumption (O(2)C) were studied in awake mice lacking one or both of the adenosine A(1) or A(2A) receptors (A(1)R or A(2A)R, respectively) using telemetry and respirometry, before and after caffeine administration. All parameters were lower during day than night and higher in females than males. When compared with wild-type (WT) littermates, HR was higher in male A(1)R knockout (A(1)RKO) mice but lower in A(2A)RKO mice and intermediate in A(1)-A(2A)R double KO mice. A single dose of an unselective beta-blocker (timolol; 1 mg/kg) abolished the HR differences between these genotypes. Deletion of A(1)Rs had little effect on Temp, whereas deletion of A(2A)Rs increased it in females and decreased it in males. A(1)-A(2A)RKO mice had lower Temp than WT mice. LA was unaltered in A(1)RKO mice and lower in A(2A)RKO and A(1)-A(2A)RKO mice than in WT mice. Caffeine injection increased LA but only in mice expressing A(2A)R. Caffeine ingestion also increased LA in an A(2A)R-dependent manner in male mice. Caffeine ingestion significantly increased O(2)C in WT mice, but less in the different KO mice. Injection of 30 mg/kg caffeine decreased Temp, especially in KO mice, and hence in a manner unrelated to A(1)R or A(2A)R blockade. Selective A(2B) antagonism had little or no effect. Thus A(1)R and A(2A)R influence HR, Temp, LA, and O(2)C in mice in a sex-dependent manner, indicating effects of endogenous adenosine. The A(2A)R plays an important role in the modulation of O(2)C and LA by acute and chronic caffeine administration. There is also evidence for effects of higher doses of caffeine being independent of both A(1)R and A(2A)R.

  1. Physiological roles of A1 and A2A adenosine receptors in regulating heart rate, body temperature, and locomotion as revealed using knockout mice and caffeine

    PubMed Central

    Yang, Jiang-Ning; Chen, Jiang-Fan; Fredholm, Bertil B.

    2009-01-01

    Heart rate (HR), body temperature (Temp), locomotor activity (LA), and oxygen consumption (O2C) were studied in awake mice lacking one or both of the adenosine A1 or A2A receptors (A1R or A2AR, respectively) using telemetry and respirometry, before and after caffeine administration. All parameters were lower during day than night and higher in females than males. When compared with wild-type (WT) littermates, HR was higher in male A1R knockout (A1RKO) mice but lower in A2ARKO mice and intermediate in A1-A2AR double KO mice. A single dose of an unselective β-blocker (timolol; 1 mg/kg) abolished the HR differences between these genotypes. Deletion of A1Rs had little effect on Temp, whereas deletion of A2ARs increased it in females and decreased it in males. A1-A2ARKO mice had lower Temp than WT mice. LA was unaltered in A1RKO mice and lower in A2ARKO and A1-A2ARKO mice than in WT mice. Caffeine injection increased LA but only in mice expressing A2AR. Caffeine ingestion also increased LA in an A2AR-dependent manner in male mice. Caffeine ingestion significantly increased O2C in WT mice, but less in the different KO mice. Injection of 30 mg/kg caffeine decreased Temp, especially in KO mice, and hence in a manner unrelated to A1R or A2AR blockade. Selective A2B antagonism had little or no effect. Thus A1R and A2AR influence HR, Temp, LA, and O2C in mice in a sex-dependent manner, indicating effects of endogenous adenosine. The A2AR plays an important role in the modulation of O2C and LA by acute and chronic caffeine administration. There is also evidence for effects of higher doses of caffeine being independent of both A1R and A2AR. PMID:19218506

  2. Comparative evaluation of cow β-casein variants (A1/A2) consumption on Th2-mediated inflammatory response in mouse gut.

    PubMed

    Ul Haq, Mohammad Raies; Kapila, Rajeev; Sharma, Rohit; Saliganti, Vamshi; Kapila, Suman

    2014-06-01

    Recently, apprehension has been raised regarding "A1/A2 hypothesis" suggesting relationship between consumption of A1 "like" variants of cow β-casein and various physiological disorders. The information available is based on either the human epidemiological data of milk consumption or in vitro trials on cell lines with β-casomorphin peptides. The direct scientific evidence establishing the link between consumption of A1/A2 "like" milk and health is scanty. Thus, under present investigation, in vivo trials in mice were undertaken to study the effect of feeding three genetic variants (A1A1, A1A2 and A2A2) of cow β-casein milk on gastrointestinal immune system as it is the first and foremost site of immunological interactions. Animals were divided into four groups for feeding with basal diet (control) and β-casein isolated from milk of genotyped (A1A1, A1A2 and A2A2) dairy animals, respectively. Gut immune response was analyzed by spectrophotometric assessment of MPO activity, quantitative sandwich ELISA of inflammatory cytokines (MCP-1 and IL-4), antibodies (total IgE, IgG, sIgA, IgG1 and IgG2a) and qRT-PCR of mRNA expression for toll-like receptors (TLR-2 and TLR-4). Histological enumeration of goblet cells, total leukocytes and IgA(+) cells was also carried out. It was observed that consumption of A1 "like" variants (A1A1 and A1A2) significantly increased (p < 0.01) the levels of MPO, MCP-1, IL-4, total IgE, IgG, IgG1, IgG2a and leukocyte infiltration in intestine. TLR-2 and TLR-4 mRNA expression was also up-regulated (p < 0.01) on administration of A1 "like" variants. However, no changes in sIgA, IgA(+) and goblet cell numbers were recorded on consumption of any of the β-casein variants. It is reasonable to conclude that consumption of A1 "like" variants of β-casein induced inflammatory response in gut by activating Th2 pathway as compared to A2A2 variants. The present study thus supports the purported deleterious impacts of consumption of A1 "like

  3. Seasonal cycle in vitamin A1/A2-based visual pigment composition during the life history of coho salmon (Oncorhynchus kisutch).

    PubMed

    Temple, S E; Plate, E M; Ramsden, S; Haimberger, T J; Roth, W-M; Hawryshyn, C W

    2006-03-01

    Microspectrophotometry of rod photoreceptors was used to follow variations in visual pigment vitamin A1/A2 ratio at various life history stages in coho salmon. Coho parr shifted their A1/A2 ratio seasonally with A2 increasing during winter and decreasing in summer. The cyclical pattern was statistically examined by a least-squares cosine model, fit to the 12-month data sets collected from different populations. A1/A2 ratio varied with temperature and day length. In 1+ (>12 month old) parr the A2 to A1 shift in spring coincided with smoltification, a metamorphic transition preceding seaward migration in salmonids. The coincidence of the shift from A2 to A1 with both the spring increase in temperature and day length, and with the timing of seaward migration presented a challenge for interpretation. Our data show a shift in A1/A2 ratio correlated with season, in both 0+ (<12 months old) coho parr that remained in fresh water for another year and in oceanic juvenile coho. These findings support the hypothesis that the A1/A2 pigment pair system in coho is an adaptation to seasonal variations in environmental variables rather than to a change associated with migration or metamorphosis.

  4. Effects of centrally administered prostaglandin E(3) and thromboxane A(3) on plasma noradrenaline and adrenaline in rats: comparison with prostaglandin E(2) and thromboxane A(2).

    PubMed

    Shimizu, Takahiro; Yokotani, Kunihiko

    2009-06-02

    Previously, we reported the involvement of brain omega-6 prostanoids, especially prostaglandin E(2) and thromboxane A(2), in the activation of central sympatho-adrenomedullary outflow in rats. omega-3 Prostanoids, including prostaglandin E(3) and thromboxane A(3), are believed to be less bioactive than omega-6 prostanoids, although studies on the functions of omega-3 prostanoids in the central nervous system have not been reported. In the present study, therefore, we compared the effects of centrally administered omega-3 prostanoids, prostaglandin E(3) and thromboxane A(3), with those of omega-6 prostanoids, prostaglandin E(2) and thromboxane A(2), on the plasma catecholamines in anesthetized rats. Intracerebroventricularly (i.c.v.) administered prostaglandin E(2) (0.15, 0.3 and 1.5 nmol/animal) and prostaglandin E(3) (0.3 and 3 nmol/animal) predominantly elevated plasma noradrenaline but not adrenaline, but the latter was less efficient than the former. On the other hand, U-46619 (an analog of thromboxane A(2)) (30, 100 and 300 nmol/animal, i.c.v.) and Delta(17)-U-46619 (an analog of thromboxane A(3)) (100 and 300 nmol/animal, i.c.v.) both elevated plasma catecholamines (adrenaline>noradrenaline) to the same degree. These results suggest that centrally administered prostaglandin E(3) is less effective than prostaglandin E(2) to elevate plasma noradrenaline, and that thromboxane A(3) is almost as equipotent as thromboxane A(2) to elevate plasma catecholamines in rats.

  5. Comparative effects of A1 versus A2 beta-casein on gastrointestinal measures: a blinded randomised cross-over pilot study.

    PubMed

    Ho, S; Woodford, K; Kukuljan, S; Pal, S

    2014-09-01

    At present, there is debate about the gastrointestinal effects of A1-type beta-casein protein in cows' milk compared with the progenitor A2 type. In vitro and animal studies suggest that digestion of A1 but not A2 beta-casein affects gastrointestinal motility and inflammation through the release of beta-casomorphin-7. We aimed to evaluate differences in gastrointestinal effects in a human adult population between milk containing A1 versus A2 beta-casein. Forty-one females and males were recruited into this double-blinded, randomised 8-week cross-over study. Participants underwent a 2-week dairy washout (rice milk replaced dairy), followed by 2 weeks of milk (750 ml/day) that contained beta-casein of either A1 or A2 type before undergoing a second washout followed by a final 2 weeks of the alternative A1 or A2 type milk. The A1 beta-casein milk led to significantly higher stool consistency values (Bristol Stool Scale) compared with the A2 beta-casein milk. There was also a significant positive association between abdominal pain and stool consistency on the A1 diet (r=0.520, P=0.001), but not the A2 diet (r=-0.13, P=0.43). The difference between these two correlations (0.52 versus -0.13) was highly significant (P<0.001). Furthermore, some individuals may be susceptible to A1 beta-casein, as evidenced by higher faecal calprotectin values and associated intolerance measures. These preliminary results suggest differences in gastrointestinal responses in some adult humans consuming milk containing beta-casein of either the A1 or the A2 beta-casein type, but require confirmation in a larger study of participants with perceived intolerance to ordinary A1 beta-casein-containing milk.

  6. Transcriptomic Analysis Shows Decreased Cortical Expression of NR4A1, NR4A2 and RXRB in Schizophrenia and Provides Evidence for Nuclear Receptor Dysregulation

    PubMed Central

    Corley, Susan M.; Wilkins, Marc R.; Shannon Weickert, Cynthia

    2016-01-01

    Many genes are differentially expressed in the cortex of people with schizophrenia, implicating factors that control transcription more generally. Hormone nuclear receptors dimerize to coordinate context-dependent changes in gene expression. We hypothesized that members of two families of nuclear receptors (NR4As), and retinoid receptors (RARs and RXRs), are altered in the dorsal lateral prefrontal cortex (DLPFC) of people with schizophrenia. We used next generation sequencing and then qPCR analysis to test for changes in mRNA levels for transcripts encoding nuclear receptors: orphan nuclear receptors (3 in the NR4A, 3 in the RAR, 3 in the RXR families and KLF4) in total RNA extracted from the DLPFC from people with schizophrenia compared to controls (n = 74). We also correlated mRNA levels with demographic factors and with estimates of antipsychotic drug exposure (schizophrenia group only). We tested for correlations between levels of transcription factor family members and levels of genes putatively regulated by these transcription factors. We found significantly down regulated expression of NR4A1 (Nurr 77) and KLF4 mRNAs in people with schizophrenia compared to controls, by both NGS and qPCR (p = or <0.01). We also detected decreases in NR4A2 (Nurr1) and RXRB mRNAs by using qPCR in the larger cohort (p<0.05 and p<0.01, respectively). We detected decreased expression of RARG and NR4A2 mRNAs in females with schizophrenia (p<0.05). The mRNA levels of NR4A1, NR4A2 and NR4A3 were all negative correlated with lifetime estimates of antipsychotic exposure. These novel findings, which may be influenced by antipsychotic drug exposure, implicate the orphan and retinoid nuclear receptors in the cortical pathology found in schizophrenia. Genes down stream of these receptors can be dysregulated as well, but the direction of change is not immediately predictable based on the putative transcription factor changes. PMID:27992436

  7. Transcriptomic Analysis Shows Decreased Cortical Expression of NR4A1, NR4A2 and RXRB in Schizophrenia and Provides Evidence for Nuclear Receptor Dysregulation.

    PubMed

    Corley, Susan M; Tsai, Shan-Yuan; Wilkins, Marc R; Shannon Weickert, Cynthia

    2016-01-01

    Many genes are differentially expressed in the cortex of people with schizophrenia, implicating factors that control transcription more generally. Hormone nuclear receptors dimerize to coordinate context-dependent changes in gene expression. We hypothesized that members of two families of nuclear receptors (NR4As), and retinoid receptors (RARs and RXRs), are altered in the dorsal lateral prefrontal cortex (DLPFC) of people with schizophrenia. We used next generation sequencing and then qPCR analysis to test for changes in mRNA levels for transcripts encoding nuclear receptors: orphan nuclear receptors (3 in the NR4A, 3 in the RAR, 3 in the RXR families and KLF4) in total RNA extracted from the DLPFC from people with schizophrenia compared to controls (n = 74). We also correlated mRNA levels with demographic factors and with estimates of antipsychotic drug exposure (schizophrenia group only). We tested for correlations between levels of transcription factor family members and levels of genes putatively regulated by these transcription factors. We found significantly down regulated expression of NR4A1 (Nurr 77) and KLF4 mRNAs in people with schizophrenia compared to controls, by both NGS and qPCR (p = or <0.01). We also detected decreases in NR4A2 (Nurr1) and RXRB mRNAs by using qPCR in the larger cohort (p<0.05 and p<0.01, respectively). We detected decreased expression of RARG and NR4A2 mRNAs in females with schizophrenia (p<0.05). The mRNA levels of NR4A1, NR4A2 and NR4A3 were all negative correlated with lifetime estimates of antipsychotic exposure. These novel findings, which may be influenced by antipsychotic drug exposure, implicate the orphan and retinoid nuclear receptors in the cortical pathology found in schizophrenia. Genes down stream of these receptors can be dysregulated as well, but the direction of change is not immediately predictable based on the putative transcription factor changes.

  8. Alternating hemiplegia of childhood with a de novo mutation in ATP1A3 and changes in SLC2A1 responsive to a ketogenic diet.

    PubMed

    Ulate-Campos, Adriana; Fons, Carmen; Artuch, Rafael; Castejón, Esperanza; Martorell, Loreto; Ozelius, Laurie; Pascual, Juan; Campistol, Jaume

    2014-04-01

    Alternating hemiplegia of childhood (AHC) is a rare condition characterized by an early onset of hemiplegic episodes and other paroxysmal or permanent neurological dysfunctions. Recently, mutations in the ATP1A3 gene have been identified as the causal mechanism of AHC. Regarding the differential diagnosis of AHC, glucose transporter 1 deficiency syndrome may be considered because these two disorders share some paroxystic and nonparoxystic features. We report a typical case of AHC harboring a de novo mutation in the ATP1A3 gene, together with a duplication and insertion in the SLC2A1 gene who exhibited marked clinical improvement following ketogenic diet. Because the contribution of the SLC2A1 mutation to the clinical phenotype cannot be definitely demonstrated, the remarkable clinical response after ketogenic diet led us to the hypothesis that ketogenic diet might be effective in AHC as it provides an alternative energy source for the brain. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Omeprazole transactivates human CYP1A1 and CYP1A2 expression through the common regulatory region containing multiple xenobiotic-responsive elements.

    PubMed

    Yoshinari, Kouichi; Ueda, Rika; Kusano, Kazutomi; Yoshimura, Tsutomu; Nagata, Kiyoshi; Yamazoe, Yasushi

    2008-07-01

    Omeprazole induces human CYP1A1 and CYP1A2 in human hepatoma cells and human liver. Aryl hydrocarbon receptor (AHR) is shown to be involved in this induction. However, its precise molecular mechanism remains unknown because the chemical activates AHR without its direct binding in contrast to typical AHR ligands such as 3-methylcholanthrene (3MC) and beta-naphthoflavone (BNF). Human CYP1A1 and CYP1A2 genes are located in a head-to-head orientation sharing about 23 kb 5'-flanking region. Recently, we succeeded to measure CYP1A1 and CYP1A2 transcriptional activities simultaneously using dual reporter gene constructs containing the 23 kb sequence. In this study, transient transfection assays have been performed using numbers of single and dual reporter constructs to identify omeprazole-responsive region for CYP1A1 and CYP1A2 induction. Reporter assays with deletion constructs have demonstrated that the omeprazole-induced expression of both CYP1A1 and CYP1A2 is mediated via the common regulatory region containing multiple AHR-binding motifs (the nucleotides from -464 to -1829 of human CYP1A1), which is identical with the region for BNF and 3MC induction. Interestingly, omeprazole activated the transcription of CYP1A1 and CYP1A2 to similar extents while BNF and 3MC preferred CYP1A1 expression. We have also found that primaquine is an omeprazole-like CYP1A inducer, while lansoprazole and albendazole are 3MC/BNF-like in terms of the CYP1A1/CYP1A2 preference. The present results suggest that omeprazole as well as BNF and 3MC activates both human CYP1A1 and CYP1A2 expression through the common regulatory region despite that omeprazole may involve a different cellular signal(s) from BNF and 3MC.

  10. Identification of different promoters in the absA1-absA2 two-component system, a negative regulator of antibiotic production in Streptomyces coelicolor.

    PubMed

    Santos-Beneit, Fernando; Rodríguez-García, Antonio; Martín, Juan F

    2013-02-01

    The absA1-absA2 genes encode a two-component system that negatively regulates the transcription of multiple antibiotic gene clusters of Streptomyces coelicolor. The microarray dataset time series of a S. coelicolor M145 bioreactor culture indicated that the transcription values of absA2 were approximately four times higher than those of absA1 throughout the time course of the culture. The co-transcription of absA1 and absA2 genes has been previously shown, although an independent absA2 promoter was not detected. In this study, we show by different technical approaches that the absA1-absA2 operon is transcribed from at least two promoters, the first producing a read-through transcript that includes both absA1 and absA2 genes and the second including only the absA2 gene. An absA2 mRNA 5' end was mapped by primer extension and confirmed as TSS by deep sequencing in combination with TEX. Promoter-probe analyses detected promoter activity in both the absA1 and absA2 upstream regions. The absA2 upstream region showed a higher promoter activity, at least sevenfold higher than that of absA1. Furthermore, the absA2 gene may contain at least two additional promoters as shown by deep sequencing analyses. All together this work contributes to the understanding of the complex transcriptional regulation of these antibiotic regulators genes in S. coelicolor.

  11. Absolute stereostructures of hovenidulciosides A1 and A2, bioactive novel triterpene glycosides from hoveniae semen seu fructus, the seeds and fruit of Hovenia dulcis Thunb.

    PubMed

    Yoshikawa, M; Ueda, T; Muraoka, O; Aoyama, H; Matsuda, H; Shimoda, H; Yamahara, J; Murakami, N

    1995-03-01

    Two bioactive novel triterpene glycosides named hovenidulciosides A1 and A2 have been isolated from a Chinese natural medicine, Hoveniae Semen Seu Fructus, the seeds and fruit of Hovenia dulcis Thunb. (Rhamnaceae). The absolute stereostructures of hovenidulciosides A1 and A2 with a migrated 16,17-seco-dammarane skeleton have been determined on the basis of chemical and physicochemical evidence which included the X-ray crystallographic analysis of the p-bromobenzoate of their common aglycone, hovenidulcigenin A. Hovenidulciosides A1 and A2 exhibited inhibitory activity on the histamine release from rat mast cells induced by compound 48/80 or calcium ionophore A-23187.

  12. The Nonplanar Secretory IgA2 and Near Planar Secretory IgA1 Solution Structures Rationalize Their Different Mucosal Immune Responses*S⃞

    PubMed Central

    Bonner, Alexandra; Almogren, Adel; Furtado, Patricia B.; Kerr, Michael A.; Perkins, Stephen J.

    2009-01-01

    Secretory IgA (SIgA) is the most prevalent human antibody and is central to mucosal immunity. It exists as two subclasses, SIgA1 and SIgA2, where SIgA2 has a shorter hinge joining the Fab and Fc regions. Both forms of SIgA are predominantly dimeric and contain an additional protein called the secretory component (SC) that is attached during the secretory process and is believed to protect SIgA in harsh mucosal conditions. Here we locate the five SC domains relative to dimeric IgA2 within SIgA2 using constrained scattering modeling. The x-ray and sedimentation parameters showed that SIgA2 has an extended solution structure. The constrained modeling of SIgA2 was initiated using two IgA2 monomers that were positioned according to our best fit solution structure for dimeric IgA1. SC was best located along the convex edge of the Fc-Fc region. The best fit models showed that SIgA2 is significantly nonplanar in its structure, in distinction to our previous near planar SIgA1 structure. Both the shorter IgA2 hinges and the presence of SC appear to displace the four Fab regions out of the Fc plane in SIgA2. This may explain the noncovalent binding of SC in some SIgA2 molecules. This nonplanar structure is predicted to result in specific immune properties for SIgA2 and SIgA1. It may explain differences observed between the SIgA1 and SIgA2 subclasses in terms of their interactions with antigens, susceptibility to proteases, effects on receptors, and distribution in different tissues. The different structures account for the prevalence of both forms in mucosal secretions. PMID:19109255

  13. Deletion of adenosine A1 or A2A receptors reduces L-3,4-dihydroxyphenylalanine-induced dyskinesia in a model of Parkinson’s disease

    PubMed Central

    Xiao, Danqing; Cassin, Jared J.; Healy, Brian; Burdett, Thomas C.; Chen, Jiang-Fan; Fredholm, Bertil B.; Schwarzschild, Michael A.

    2010-01-01

    Adenosine A2A receptor antagonism provides a promising approach to developing nondopaminergic therapy for Parkinson’s disease (PD). Clinical trials of A2A antagonists have targeted PD patients with L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID) in an effort to improve parkinsonian symptoms. The role of adenosine in the development of LID is little known, especially regarding its actions via A1 receptors. We aimed to examine the effects of genetic deletion and pharmacological blockade of A1 and/or A2A receptors on the development of LID, on the induction of molecular markers of LID including striatal preprodynorphin and preproenkephalin (PPE), and on the integrity of dopaminergic nigrostriatal neurons in hemiparkinsonian mice. Following a unilateral 6-hydroxydopamine lesion A1, A2A and double A1-A2A knockout (KO) and wild-type littermate mice, and mice pretreated with caffeine (an antagonist of both A1 and A2A receptors) or saline were treated daily for 18–21 days with a low dose of L-DOPA. Total abnormal involuntary movements (AIMs, a measure of LID) were significantly attenuated (p<0.05) in A1 and A2A KOs, but not in A1-A2A KOs and caffeine-pretreated mice. An elevation of PPE mRNA ipsilateral to the lesion in WT mice was reduced in all KO mice. In addition, neuronal integrity assessed by striatal dopamine content was similar in all KOs and caffeine-pretreated mice following 6-hydroxydopamine lesioning. Our findings raise the possibility that A1 or A2A receptors blockade might also confer a disease-modifying benefit of reduced risk of disabling LID, whereas the effect of their combined inactivation is less clear. PMID:20828543

  14. Deactivation processes in PsbA1-Photosystem II and PsbA3-Photosystem II under photoinhibitory conditions in the cyanobacterium Thermosynechococcus elongatus.

    PubMed

    Ogami, Shogo; Boussac, Alain; Sugiura, Miwa

    2012-08-01

    The sensitivity to high light conditions of Photosystem II with either PsbA1 (WT*1) or PsbA3 (WT*3) as the D1 protein was studied in whole cells of the thermophilic cyanobacterium Thermosynechococcus elongatus. When the cells are cultivated under high light conditions the following results were found: (i) The O(2) evolution activity decreases faster in WT*1 cells than in WT*3 cells both in the absence and in the presence of lincomycin, a protein synthesis inhibitor; (ii) In WT*1 cells, the rate constant for the decrease of the O(2) evolution activity is comparable in the presence and in the absence of lincomycin; (iii) The D1 content revealed by western blot analysis decays similarly in both WT*1 and WT*3 cells and much slowly than O(2) evolution; (iv) The faster decrease in O(2) evolution in WT*1 than in WT*3 cells correlates with a much faster inhibition of the S(2)-state formation; (v) The shape of the WT*1 cells is altered. All these results are in agreement with a photo-inhibition process resulting in the loss of the O(2) activity much faster than the D1 turnover in PsbA1-PSII and likely to a greater production of reactive oxygen species under high light conditions in WT*1 than in WT*3. This latter result is discussed in view of the known effects of the PsbA1 to PsbA3 substitution on the redox properties of the Photosystem II cofactors. The observation that under low light conditions WT*3 cells are able to express the psbA(3) gene, whereas under similar conditions wild type cells are expressing mainly the psbA(1) gene is also discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. The Inhibitory Effect of α/β-Hydrolase Domain-Containing 6 (ABHD6) on the Surface Targeting of GluA2- and GluA3-Containing AMPA Receptors

    PubMed Central

    Wei, Mengping; Jia, Moye; Zhang, Jian; Yu, Lulu; Zhao, Yunzhi; Chen, Yingqi; Ma, Yimeng; Zhang, Wei; Shi, Yun S.; Zhang, Chen

    2017-01-01

    The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) are major excitatory receptors that mediate fast neurotransmission in the mammalian brain. The surface expression of functional AMPARs is crucial for synaptic transmission and plasticity. AMPAR auxiliary subunits control the biosynthesis, membrane trafficking, and synaptic targeting of AMPARs. Our previous report showed that α/β-hydrolase domain-containing 6 (ABHD6), an auxiliary subunit for AMPARs, suppresses the membrane delivery and function of GluA1-containing receptors in both heterologous cells and neurons. However, it remained unclear whether ABHD6 affects the membrane trafficking of glutamate receptor subunits, GluA2 and GluA3. Here, we examine the effects of ABHD6 overexpression in HEK293T cells expressing GluA1, GluA2, GluA3, and stargazin, either alone or in combination. The results show that ABHD6 suppresses the glutamate-induced currents and the membrane expression of AMPARs when expressing GluA2 or GluA3 in the HEK293T cells. We generated a series of GluA2 and GluA3 C-terminal deletion constructs and confirm that the C-terminus of GluAs is required for ABHD6’s inhibitory effects on glutamate-induced currents and surface expression of GluAs. Meanwhile, our pull-down experiments reveal that ABHD6 binds to GluA1–3, and deletion of the C-terminal domain of GluAs abolishes this binding. These findings demonstrate that ABHD6 inhibits the AMPAR-mediated currents and its surface expression, independent of the type of AMPAR subunits, and this inhibitor’s effects are mediated through the binding with the GluAs C-terminal regions. PMID:28303090

  16. A1R-A2AR heteromers coupled to Gs and G i/0 proteins modulate GABA transport into astrocytes.

    PubMed

    Cristóvão-Ferreira, Sofia; Navarro, Gemma; Brugarolas, Marc; Pérez-Capote, Kamil; Vaz, Sandra H; Fattorini, Giorgia; Conti, Fiorenzo; Lluis, Carmen; Ribeiro, Joaquim A; McCormick, Peter J; Casadó, Vicent; Franco, Rafael; Sebastião, Ana M

    2013-09-01

    Astrocytes play a key role in modulating synaptic transmission by controlling extracellular gamma-aminobutyric acid (GABA) levels via GAT-1 and GAT-3 GABA transporters (GATs). Using primary cultures of rat astrocytes, we show here that a further level of regulation of GABA uptake occurs via modulation of the GATs by the adenosine A1 (A1R) and A2A (A2AR) receptors. This regulation occurs through A1R-A2AR heteromers that signal via two different G proteins, Gs and Gi/0, and either enhances (A2AR) or inhibits (A1R) GABA uptake. These results provide novel mechanistic insight into how GPCR heteromers signal. Furthermore, we uncover a previously unknown mechanism where adenosine, in a concentration-dependent manner, acts via a heterocomplex of adenosine receptors in astrocytes to significantly contribute to neurotransmission at the tripartite (neuron-glia-neuron) synapse.

  17. Imidazo[1,2-a]pyrazin-8-amine core for the design of new adenosine receptor antagonists: Structural exploration to target the A3 and A2A subtypes.

    PubMed

    Poli, Daniela; Falsini, Matteo; Varano, Flavia; Betti, Marco; Varani, Katia; Vincenzi, Fabrizio; Pugliese, Anna Maria; Pedata, Felicita; Dal Ben, Diego; Thomas, Ajiroghene; Palchetti, Ilaria; Bettazzi, Francesca; Catarzi, Daniela; Colotta, Vittoria

    2017-01-05

    The imidazo[1,2-a]pyrazine ring system has been chosen as a new decorable core skeleton for the design of novel adenosine receptor (AR) antagonists targeting either the human (h) A3 or the hA2A receptor subtype. The N(8)-(hetero)arylcarboxyamido substituted compounds 4-14 and 21-30, bearing a 6-phenyl moiety or not, respectively, show good hA3 receptor affinity and selectivity versus the other ARs. In contrast, the 8-amino-6-(hetero)aryl substituted derivatives designed for targeting the hA2A receptor subtype (compounds 31-38) and also the 6-phenyl analogues 18-20 do not bind the hA2A AR, or show hA1 or balanced hA1/hA2A AR affinity in the micromolar range. Molecular docking of the new hA3 antagonists was carried out to depict their hypothetical binding mode to our refined model of the hA3 receptor. Some derivatives were evaluated for their fluorescent potentiality and showed some fluorescent emission properties. One of the most active hA3 antagonists herein reported, i.e. the 2,6-diphenyl-8-(3-pyridoylamino)imidazo[1,2-a]pyrazine 29, tested in a rat model of cerebral ischemia, delayed the occurrence of anoxic depolarization caused by oxygen and glucose deprivation in the hippocampus and allowed disrupted synaptic activity to recover. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  18. Temporo-spacial microanatomical distribution of the murine sodium-dependent ascorbic acid transporters Slc23a1 and Slc23a2 in the kidney throughout development.

    PubMed

    Eck, Peter K; Corpe, Christopher; Levine, Mark A

    2017-06-01

    The two membrane transporters Slc23a1 and Slc23a2 mediate ascorbic acid uptake into cells. We recently determined the key role of Slc23a1 in renal re-absorption of ascorbic acid in a knockout mouse model. However, the renal spatial and temporal expression patterns of murine Slc23a1 and Slc23a2 are not defined. This study utilizes database evidence combined with experimental confirmation via in-situ hybridization to define the spatial and temporal expression of Slc23a1 in the murine kidney. Slc23a1 is expressed in the early proximal tubule, but not in its precursors during embryonic development, and exclusive proximal tubular expression persists throughout the animal's lifetime. In contrast, Slc23a2 is uniformly expressed in metabolic cell types such as stromal cells. The expression patterns appear to be conserved from rodent lineages to humans.

  19. Effect of PlA1/A2 glycoprotein IIIa gene polymorphism on the long-term outcome after successful coronary stenting.

    PubMed

    Le Hello, Claire; Morello, Rémy; Lequerrec, Agnès; Duarte, Christine; Riddell, John; Hamon, Martial

    2007-11-16

    To prospectively determine the role of platelet glycoprotein IIIa (GP IIIa) gene PlA1/PlA2 polymorphism on the long-term clinical outcome in patients with coronary artery disease undergoing coronary stenting. Prospective observational study in the University Hospital of Caen (France). 1 111 symptomatic consecutive Caucasian patients treated with percutaneous coronary intervention including stent implantation underwent genotyping for GP IIIa PlA1/A2. Long-term clinical outcome in terms of the rate of major adverse cardiac events (MACE, ie death from any cause, non-fatal Q wave or non Q wave myocardial infarction, and need for coronary revascularisation) was obtained and subsequently stratified according to the GP IIIa PlA1/A2 polymorphism. Three groups of patients were determined according to the GP IIIa PlA1/A2 polymorphism (71.6% had the A1/A1, 25.8% had the A1/A2 and 2.6% had the A2/A2 genotype). These three groups were comparable for all clinical characteristics including sex ratio, mean age, vascular risk factors, previous coronary events, baseline angiographic exam, indication for the percutaneous coronary intervention and drug therapy). The incidence of MACE was similar in these 3 groups of patients during a mean follow-up period of 654+/-152 days. Independent risk factors for MACE were a left ventricular ejection fraction < 40%, absence of treatment with a beta-blocker and absence of treatment with an angiotensin converting enzyme inhibitor during follow-up. The GP IIIa PlA1/A2 polymorphism does not influence the clinical long-term outcome in patients with symptomatic coronary disease undergoing percutaneous coronary intervention with stent implantation.

  20. Effect of PlA1/A2 glycoprotein IIIa gene polymorphism on the long-term outcome after successful coronary stenting

    PubMed Central

    Le Hello, Claire; Morello, Rémy; Lequerrec, Agnès; Duarte, Christine; Riddell, John; Hamon, Martial

    2007-01-01

    Aim To prospectively determine the role of platelet glycoprotein IIIa (GP IIIa) gene PlA1/PlA2 polymorphism on the long-term clinical outcome in patients with coronary artery disease undergoing coronary stenting. Design and setting Prospective observational study in the University Hospital of Caen (France). Patients and methods 1 111 symptomatic consecutive Caucasian patients treated with percutaneous coronary intervention including stent implantation underwent genotyping for GP IIIa PlA1/A2. Main outcome measures Long-term clinical outcome in terms of the rate of major adverse cardiac events (MACE, ie death from any cause, non-fatal Q wave or non Q wave myocardial infarction, and need for coronary revascularisation) was obtained and subsequently stratified according to the GP IIIa PlA1/A2 polymorphism. Results Three groups of patients were determined according to the GP IIIa PlA1/A2 polymorphism (71.6% had the A1/A1, 25.8% had the A1/A2 and 2.6% had the A2/A2 genotype). These three groups were comparable for all clinical characteristics including sex ratio, mean age, vascular risk factors, previous coronary events, baseline angiographic exam, indication for the percutaneous coronary intervention and drug therapy). The incidence of MACE was similar in these 3 groups of patients during a mean follow-up period of 654+/-152 days. Independent risk factors for MACE were a left ventricular ejection fraction < 40%, absence of treatment with a beta-blocker and absence of treatment with an angiotensin converting enzyme inhibitor during follow-up. Conclusion The GP IIIa PlA1/A2 polymorphism does not influence the clinical long-term outcome in patients with symptomatic coronary disease undergoing percutaneous coronary intervention with stent implantation. PMID:18021403

  1. Rapid detection of HCV genotyping 1a, 1b, 2a, 3a, 3b and 6a in a single reaction using two-melting temperature codes by a real-time PCR-based assay.

    PubMed

    Athar, Muhammad Ammar; Xu, Ye; Xie, Xiaoting; Xu, Zhenxing; Ahmad, Vakil; Hayder, Zulfiqar; Hussain, Syed Sajid; Liao, Yiqun; Li, Qingge

    2015-09-15

    The genotype of the hepatitis C virus (HCV) is an important indicator for antiviral therapeutic response. We hereby described development of a rapid HCV genotyping approach that enabled the identification of the six most common HCV subtypes of Asia, i.e., 1a, 1b, 2a, 3a, 3b, and 6a, in a single reaction. Using two dual-labeled, self-quenched probes that target the core region of the HCV genome, the exact subtype could be accurately identified by two-melting temperature codes determined from the two respective probes in a real-time PCR assay. Analytical sensitivity studies using armored RNA samples representing each of the six HCV subtypes showed that 5 copies/reaction of HCV RNA could be detected. The assay was evaluated using 244 HCV-positive serum samples and the results were compared with sequencing analysis. Of the 224 samples, subtype 3a (127, 52.3%) was the dominant, followed by 1b (51, 20.9%), 3b (47, 19.3%), 2a (8, 3.3%), 6a (4, 1.6%) and the least was subtype 1a (1, 0.4%). Moreover, 6 (2.5%) mixed infection samples were also detected. These results were fully concordant with sequencing analysis. We concluded that this real-time PCR-based assay could provide a rapid and reliable tool for routine HCV genotyping in most Asian countries.

  2. An HLA-A3-binding prostate acid phosphatase-derived peptide can induce CTLs restricted to HLA-A2 and -A24 alleles.

    PubMed

    Terasaki, Yasunobu; Shichijo, Shigeki; Niu, Yamei; Komatsu, Nobukazu; Noguchi, Masanori; Todo, Satoru; Itoh, Kyogo

    2009-11-01

    We previously reported peptide vaccine candidates for HLA-A3 supertype (-A3, -A11, -A31, -A33)-positive cancer patients. In the present study, we examined whether those peptides can also induce cytotoxic T lymphocyte (CTL) activity restricted to HLA-A2, HLA-A24, and HLA-A26 alleles. Fourteen peptides were screened for their binding activity to HLA-A*0201, -A*0206, -A*0207, -A*2402, and -A*2601 molecules and then tested for their ability to induce CTL activity in peripheral blood mononuclear cells (PBMCs) from prostate cancer patients. Among these peptides, one from the prostate acid phosphatase protein exhibited binding activity to HLA-A*0201, -A*0206, and -A*2402 molecules. In addition, PBMCs stimulated with this peptide showed that HLA-A2 or HLA-A24 restricted CTL activity. Their cytotoxicity toward cancer cells was ascribed to peptide-specific and CD8+ T cells. These results suggest that this peptide could be widely applicable as a peptide vaccine for HLA-A3 supertype-, HLA-A2-, and -A24-positive cancer patients.

  3. Vasopressin increases expression of UT-A1, UT-A3, and ER chaperone GRP78 in the renal medulla of mice with a urinary concentrating defect.

    PubMed

    Cai, Qi; Nelson, Sarah K; McReynolds, Matthew R; Diamond-Stanic, Maggie Keck; Elliott, David; Brooks, Heddwen L

    2010-10-01

    Activation of V2 receptors (V2R) during antidiuresis increases the permeability of the inner medullary collecting duct to urea and water. Extracellular osmolality is elevated as the concentrating capacity of the kidney increases. Osmolality is known to contribute to the regulation of collecting duct water (aquaporin-2; AQP2) and urea transporter (UT-A1, UT-A3) regulation. AQP1KO mice are a concentrating mechanism knockout, a defect attributed to the loss of high interstitial osmolality. A V2R-specific agonist, deamino-8-D-arginine vasopressin (dDAVP), was infused into wild-type and AQP1KO mice for 7 days. UT-A1 mRNA and protein abundance were significantly increased in the medullas of wild-type and AQP1KO mice following dDAVP infusion. The mRNA and protein abundance of UT-A3, the basolateral urea transporter, was significantly increased by dDAVP in both wild-type and AQP1KO mice. Semiquantitative immunoblots revealed that dDAVP infusion induced a significant increase in the medullary expression of the endoplasmic reticulum (ER) chaperone GRP78. Immunofluorescence studies demonstrated that GRP78 expression colocalized with AQP2 in principal cells of the papillary tip of the renal medulla. Using immunohistochemistry and immunogold electron microscopy, we demonstrate that vasopressin induced a marked apical targeting of GRP78 in medullary principal cells. Urea-sensitive genes, GADD153 and ATF4 (components of the ER stress pathway), were significantly increased in AQP1KO mice by dDAVP infusion. These findings strongly support an important role of vasopressin in the activation of an ER stress response in renal collecting duct cells, in addition to its role in activating an increase in UT-A1 and UT-A3 abundance.

  4. Excitation of the \\tilde{a}\\,^3B_1 and \\tilde{A}\\,^1B_1 states of H2O by low-energy electron impact

    NASA Astrophysics Data System (ADS)

    Hargreaves, L.; Ralphs, K.; Serna, G.; Khakoo, M. A.; Winstead, C.; McKoy, V.

    2012-10-01

    We report measured and calculated differential cross-sections for inelastic scattering of low-energy electrons by water leading to excitation of the dissociative (1b1 → 4a1) 1, 3B1 states. The measurements were taken using conventional energy-loss spectroscopy at incident energies of 9, 10, 12, 15, and 20 eV for scattering angles from 10° to 130°. The calculations were carried out using the Schwinger multichannel method, with a Born-dipole correction applied in the singlet excitation channel. Integral excitation cross sections for the \\tilde{a}\\,^3B_1 and \\tilde{A}\\,^1B_1 states are also derived from the differential cross section results.

  5. Time and Sex-Dependent Effects of an Adenosine A2A/A1 Receptor Antagonist on Motivation to Self-Administer Cocaine in Rats

    PubMed Central

    Doyle, Susan E.; Breslin, Florence J.; Rieger, Jayson M.; Beauglehole, Anthony; Lynch, Wendy J.

    2012-01-01

    Adenosine is an important neuromodulator, known to interact with both dopaminergic and glutamatergic systems to influence psychostimulant action. In the present study, we examined the effects of ATL444, a novel adenosine receptor antagonist, on motivation for cocaine in male and female rats. Adult male and female Sprague-Dawley rats were trained to self-administer cocaine (1.5 mg/kg/infusion) on a fixed-ratio 1 schedule with a daily maximum of 20 infusions. Following 5 consecutive sessions during which all 20 available infusions were obtained, motivation for cocaine (0.5 mg/kg/infusion) was assessed under a progressive ratio (PR) schedule, and once responding stabilized, the effect of treatment with ATL444 (0, 15, and 30 mg/kg, i.p.) was examined. As a control, we also assessed its effects on PR responding for sucrose. Binding studies revealed that ATL 444 was 3-fold, 25-fold, and 400-fold more selective for the A2A receptor as compared to A1, A2B, and A3 receptors, respectively. ATL444 produced a significant increase in motivation for cocaine on the day of treatment in females with a trend for an increase in males. In addition, over the two PR sessions following ATL444 treatment a significant decrease in responding was observed in males but not females. Responding for sucrose was unaffected by ATL444 treatment. Our results reveal that adenosine receptor blockade may mediate both acute increases in the reinforcing effects of cocaine, and longer term inhibitory effects on cocaine reinforcement that differ according to sex. PMID:22579716

  6. Physiological Evaluation of A1 (Extreme-Cold-Weather) and A2 (Buoyant, Intermediate-Cold-Weather) Jackets.

    DTIC Science & Technology

    1983-08-01

    at -40*F than those with the Navy clothing . Exercise interspersed with the rest periods would significantly increase exposure time, the extent of...minutes. Following the exercise , the subjects again were told to sit quietly for another 60 minutes. The following clothing was worn during the 10...AD-A258 410 PHYSIOLOGICAL EVALUATION OF Al (EXTREME-COLD-WEATHER) AND A2 (BUOYANT, INTERMEDIATE-COLD-WEATHER) JACKETS NAVY CLOTHING AND TEXTILE

  7. Enzymatic characterization of in vitro-expressed Baikal seal cytochrome P450 (CYP) 1A1, 1A2, and 1B1: implication of low metabolic potential of CYP1A2 uniquely evolved in aquatic mammals.

    PubMed

    Iwata, Hisato; Yamaguchi, Keisuke; Takeshita, Yoko; Kubota, Akira; Hirakawa, Shusaku; Isobe, Tomohiko; Hirano, Masashi; Kim, Eun-Young

    2015-05-01

    This study aimed to elucidate the catalytic function of cytochrome P450 (CYP) 1 enzymes in aquatic mammals. Alkoxyresorufin O-dealkylation (AROD) activities including methoxy- (MROD), ethoxy- (EROD), pentoxy- (PROD), and benzyloxyresorufin O-dealkylation (BROD), and 2- and 4-hydroxylation activities of 17β-estradiol (E2) were measured by using yeast-expressed Baikal seal (Pusa sibirica) CYP1A1, 1A2, and 1B1 proteins. Heterologous protein expression of the Baikal seal CYP1s (bsCYP1s) in yeast microsomes was confirmed by reduced CO-difference spectra and immunoblotting. Heterologously expressed human CYP1 enzyme (hCYP1) activities were simultaneously measured and compared with those of bsCYP1 isozymes. Recombinant bsCYP1A1 protein showed the highest Vmax of EROD, followed by MROD, PROD, and BROD, similar to that of hCYP1A1. Vmax/Km ratios of all AROD activities catalyzed by bsCYP1A1 were lower than those catalyzed by hCYP1A1, suggesting less potential for AROD by bsCYP1A1. Enzymatic assays for bsCYP1A2 showed no or minimal AROD activities, while hCYP1A2 displayed MROD and EROD activities. bsCYP1B1 showed an AROD profile (EROD>BROD>MROD>PROD) similar to that of hCYP1B1; however, Vmax/Km ratios of all AROD activities by bsCYP1B1 were higher. Yeast microsomes containing bsCYP1A1 and 1B1 and hCYP1A1, 1A2, and 1B1 metabolized E2 to 2-OHE2 and 4-OHE2, whereas bsCYP1A2 showed no such activity. Comparison of 4- and 2-hydroxylations of E2 by CYP1As suggests that bsCYP1A1, hCYP1A1, and 1A2 preferentially catalyze 2- rather than 4-hydroxylation. As for CYP1B1, the Vmax/Km ratios suggest that both Baikal seal and human CYPs catalyze 4- rather than 2-hydroxylation. Interspecies comparison showed that bsCYP1B1 has higher metabolic potencies for both E2 hydroxylations than does hCYP1B1, whereas the activity of bsCYP1A1 was lower than that of hCYP1A1. Messenger RNA expression levels of bsCYP1s in the liver of Baikal seals indicated that bsCYP1A1 and 1A2 enzymes contributed to 16

  8. Hall Effect Thruster Interactions Data from the Russian Express-A2 and Express-A3 Satellites. Part 5; Acquire Express-A3 SPT?100 Based Propulsion Subsystem and Other Subsystem Flight Operation TM-Data, Task 31

    NASA Technical Reports Server (NTRS)

    Sitnikova, N.; Volkov, D.; Maximov, I.; Petrusevich, V.; Allen, D.

    2003-01-01

    This 12-part report documents the data obtained from various sensor measurements taken aboard the Russian Express-A2 and Express-A3 spacecraft in Geosynchronous Earth Orbit (GEO). These GEO communications satellites, which were designed and built by NPO Prikladnoy Mekhaniki (NPO PM) of Zheleznogorsk, Russia, utilize Hall thruster propulsion systems for north-south and east-west stationkeeping and as of June 2002, were still operating at 80deg E. and 11deg W., respectively. Express-A2 was launched on March 12, 2000, while Express-A3 was launched on June 24, 2000. The diagnostic equipment from which these data were taken includes electric field strength sensors, ion current and energy sensors, and pressure sensors. The diagnostics and the Hall thruster propulsion systems are described in detail along with lists of tabular data from those diagnostics and propulsion system and other satellite systems. Space Power, Inc., now part of Pratt & Whitney's Chemical Systems Division, under contract NAS3-99151 to the NASA Glenn Research Center, obtained these data over several periods from March 12, 2000, through September 30, 2001. Each of the 12 individual reports describe, in detail, the propulsion systems as well as the diagnostic sensors utilized. Finally, parts 11 and 12 include the requirements to which NPO PM prepared and delivered these data.

  9. Hall Effect Thruster Interactions Data From the Russian Express-A2 and Express-A3 Satellites. Acquire Express-A3 SPT 100 Based Propulsion Subsystem and Other Subsystem Flight Operation TM-Data, Task 33

    NASA Technical Reports Server (NTRS)

    Sitnikova, N.; Volkov, D.; Maximov, I.; Petrusevich, V.; Allen, D.

    2003-01-01

    This 12-part report documents the data obtained from various sensor measurements taken aboard the Russian Express-A2 and Express-A3 spacecraft in Geosynchronous Earth Orbit (GEO). These GEO communications satellites, which were designed and built by NPO Prikladnoy Mekhaniki (NPO PM) of Zheleznogorsk, Russia, utilize Hall thruster propulsion systems for north-south and east-west stationkeeping and as of June 2002, were still operating at 80 deg E and 11 deg W, respectively. Express-A2 was launched on March 12, 2000, while Express-A3 was launched on June 24, 2000. The diagnostic equipment from which these data were taken includes electric field strength sensors, ion current and energy sensors, and pressure sensors. The diagnostics and the Hall thruster propulsion systems are described in detail along with lists of tabular data from those diagnostics and propulsion system and other satellite systems. Space Power, Inc., now part of Pratt & Whitney's Chemical Systems Division, under contract NAS3-99151 to the NASA Glenn Research Center, obtained these data over several periods from March 12, 2000, through September 30, 2001. Each of the 12 individual reports describe, in detail, the propulsion systems as well as the diagnostic sensors utilized. Finally, parts 11 and 12 include the requirements to which NPO PM prepared and delivered these data.

  10. Hall Effect Thruster Interactions Data From the Russian Express-A2 and Express-A3 Satellites. Part 7; Acquire Express-A3 SPT-100 Based Propulsion Subsystem and Other Subsystem Flight Operation TM-Data, Task 32

    NASA Technical Reports Server (NTRS)

    Sitnikova, N.; Volkov, D.; Maximov, I.; Petrusevich, V.; Allen, D.

    2003-01-01

    This 12-part report documents the data obtained from various sensor measurements taken aboard the Russian Express-A2 and Express-A3 spacecraft in Geosynchronous Earth Orbit (GEO). These GEO communications satellites, which were designed and built by NPO Prikladnoy Mekhaniki (NPO PM) of Zheleznogorsk, Russia, utilize Hall thruster propulsion systems for north-south and east-west stationkeeping and as of June 2002, were still operating at 80 E. and 11 W., respectively. Express-A2 was launched on March 12, 2000, while Express-A3 was launched on June 24, 2000. The diagnostic equipment from which these data were taken includes electric field strength sensors, ion current and energy sensors, and pressure sensors. The diagnostics and the Hall thruster propulsion systems are described in detail along with lists of tabular data from those diagnostics and propulsion system and other satellite systems. Space Power, Inc., now part of Pratt & Whitney's Chemical Systems Division, to the NASA Glenn Research Center, obtained these data over several periods from March 12, 2000, through September 30, 2001. Each of the 12 individual reports describe, in detail, the propulsion systems as well as the diagnostic sensors utilized. Finally, parts 11 and 12 include the requirements to which NPO PM prepared and delivered these data.

  11. Retinoic acid homeostasis through aldh1a2 and cyp26a1 mediates meiotic entry in Nile tilapia (Oreochromis niloticus)

    PubMed Central

    Feng, Ruijuan; Fang, Lingling; Cheng, Yunying; He, Xue; Jiang, Wentao; Dong, Ranran; Shi, Hongjuan; Jiang, Dongneng; Sun, Lina; Wang, Deshou

    2015-01-01

    Meiosis is a process unique to the differentiation of germ cells. Retinoic acid (RA) is the key factor controlling the sex-specific timing of meiotic initiation in tetrapods; however, the role of RA in meiotic initiation in teleosts has remained unclear. In this study, the genes encoding RA synthase aldh1a2, and catabolic enzyme cyp26a1 were isolated from Nile tilapia (Oreochromis niloticus), a species without stra8. The expression of aldh1a2 was up-regulated and expression of cyp26a1 was down-regulated before the meiotic initiation in ovaries and in testes. Treatment with RA synthase inhibitor or disruption of Aldh1a2 by CRISPR/Cas9 resulted in delayed meiotic initiation, with simultaneous down-regulation of cyp26a1 and up-regulation of sycp3. By contrast, treatment with an inhibitor of RA catabolic enzyme and disruption of cyp26a1 resulted in earlier meiotic initiation, with increased expression of aldh1a2 and sycp3. Additionally, treatment of XY fish with estrogen (E2) and XX fish with fadrozole led to sex reversal and reversion of meiotic initiation. These results indicate that RA is indispensable for meiotic initiation in teleosts via a stra8 independent signaling pathway where both aldh1a2 and cyp26a1 are critical. In contrast to mammals, E2 is a major regulator of sex determination and meiotic initiation in teleosts. PMID:25976364

  12. Retinoic acid homeostasis through aldh1a2 and cyp26a1 mediates meiotic entry in Nile tilapia (Oreochromis niloticus).

    PubMed

    Feng, Ruijuan; Fang, Lingling; Cheng, Yunying; He, Xue; Jiang, Wentao; Dong, Ranran; Shi, Hongjuan; Jiang, Dongneng; Sun, Lina; Wang, Deshou

    2015-05-15

    Meiosis is a process unique to the differentiation of germ cells. Retinoic acid (RA) is the key factor controlling the sex-specific timing of meiotic initiation in tetrapods; however, the role of RA in meiotic initiation in teleosts has remained unclear. In this study, the genes encoding RA synthase aldh1a2, and catabolic enzyme cyp26a1 were isolated from Nile tilapia (Oreochromis niloticus), a species without stra8. The expression of aldh1a2 was up-regulated and expression of cyp26a1 was down-regulated before the meiotic initiation in ovaries and in testes. Treatment with RA synthase inhibitor or disruption of Aldh1a2 by CRISPR/Cas9 resulted in delayed meiotic initiation, with simultaneous down-regulation of cyp26a1 and up-regulation of sycp3. By contrast, treatment with an inhibitor of RA catabolic enzyme and disruption of cyp26a1 resulted in earlier meiotic initiation, with increased expression of aldh1a2 and sycp3. Additionally, treatment of XY fish with estrogen (E2) and XX fish with fadrozole led to sex reversal and reversion of meiotic initiation. These results indicate that RA is indispensable for meiotic initiation in teleosts via a stra8 independent signaling pathway where both aldh1a2 and cyp26a1 are critical. In contrast to mammals, E2 is a major regulator of sex determination and meiotic initiation in teleosts.

  13. Bacillus anthracis acetyltransferases PatA1 and PatA2 modify the secondary cell wall polysaccharide and affect the assembly of S-layer proteins.

    PubMed

    Lunderberg, J Mark; Nguyen-Mau, Sao-Mai; Richter, G Stefan; Wang, Ya-Ting; Dworkin, Jonathan; Missiakas, Dominique M; Schneewind, Olaf

    2013-03-01

    The envelope of Bacillus anthracis encompasses a proteinaceous S-layer with two S-layer proteins (Sap and EA1). Protein assembly in the envelope of B. anthracis requires S-layer homology domains (SLH) within S-layer proteins and S-layer-associated proteins (BSLs), which associate with the secondary cell wall polysaccharide (SCWP), an acetylated carbohydrate that is tethered to peptidoglycan. Here, we investigated the contributions of two putative acetyltransferases, PatA1 and PatA2, on SCWP acetylation and S-layer assembly. We show that mutations in patA1 and patA2 affect the chain lengths of B. anthracis vegetative forms and perturb the deposition of the BslO murein hydrolase at cell division septa. The patA1 and patA2 mutants are defective for the assembly of EA1 in the envelope but retain the ability of S-layer formation with Sap. SCWP isolated from the patA1 patA2 mutant lacked acetyl moieties identified in wild-type polysaccharide and failed to associate with the SLH domains of EA1. A model is discussed whereby patA1- and patA2-mediated acetylation of SCWP enables the deposition of EA1 as well as BslO near the septal region of the B. anthracis envelope.

  14. Differential roles for Nr4a1 and Nr4a2 in object location vs. object recognition long-term memory.

    PubMed

    McNulty, Susan E; Barrett, Ruth M; Vogel-Ciernia, Annie; Malvaez, Melissa; Hernandez, Nicole; Davatolhagh, M Felicia; Matheos, Dina P; Schiffman, Aaron; Wood, Marcelo A

    2012-11-16

    Nr4a1 and Nr4a2 are transcription factors and immediate early genes belonging to the nuclear receptor Nr4a family. In this study, we examine their role in long-term memory formation for object location and object recognition. Using siRNA to block expression of either Nr4a1 or Nr4a2, we found that Nr4a2 is necessary for both long-term memory for object location and object recognition. In contrast, Nr4a1 appears to be necessary only for object location. Indeed, their roles in these different types of long-term memory may be dependent on their expression in the brain, as NR4A2 was found to be expressed in hippocampal neurons (associated with object location memory) as well as in the insular and perirhinal cortex (associated with object recognition memory), whereas NR4A1 showed minimal neuronal expression in these cortical areas. These results begin to elucidate how NR4A1 and NR4A2 differentially contribute to object location versus object recognition memory.

  15. No association between polymorphisms and haplotypes of COL1A1 and COL1A2 genes and osteoporotic fracture in postmenopausal Chinese women

    PubMed Central

    Hu, Wei-wei; He, Jin-wei; Zhang, Hao; Wang, Chun; Gu, Jie-mei; Yue, Hua; Ke, Yao-hua; Hu, Yun-qiu; Fu, Wen-zhen; Li, Miao; Liu, Yu-juan; Zhang, Zhen-lin

    2011-01-01

    Aim: To study whether genetic polymorphisms of COL1A1 and COL1A2 genes affected the onset of fracture in postmenopausal Chinese women. Methods: SNPs in COL1A1 and COL1A2 genes were identified via direct sequencing in 32 unrelated postmenopausal Chinese women. Ten SNPs were genotyped in 1252 postmenopausal Chinese women. The associations were examined using both single-SNP and haplotype tests using logistic regression. Results: Twenty four (4 novel) and 28 (7 novel) SNPs were identified in COL1A1 and COL1A2 gene, respectively. The distribution frequencies of 2 SNPs in COL1A1 (rs2075554 and rs2586494) and 3 SNPs in COL1A2 (rs42517, rs1801182, and rs42524) were significantly different from those documented for the European Caucasian population. No significant difference was observed between fracture and control groups with respect to allele frequency or genotype distribution in 9 selected SNPs and haplotype. No significant association was found between fragility fracture and each SNP or haplotype. The results remained the same after additional corrections for other risk factors such as weight, height, and bone mineral density. Conclusion: Our results show no association between common genetic variations of COL1A1 and COL1A2 genes and fracture, suggesting the complex genetic background of osteoporotic fractures. PMID:21602843

  16. SpxA1 and SpxA2 Act Coordinately To Fine-Tune Stress Responses and Virulence in Streptococcus pyogenes

    PubMed Central

    Port, Gary C.; Cusumano, Zachary T.; Tumminello, Paul R.

    2017-01-01

    ABSTRACT SpxA is a unique transcriptional regulator highly conserved among members of the phylum Firmicutes that binds RNA polymerase and can act as an antiactivator. Why some Firmicutes members have two highly similar SpxA paralogs is not understood. Here, we show that the SpxA paralogs of the pathogen Streptococcus pyogenes, SpxA1 and SpxA2, act coordinately to regulate virulence by fine-tuning toxin expression and stress resistance. Construction and analysis of mutants revealed that SpxA1− mutants were defective for growth under aerobic conditions, while SpxA2− mutants had severely attenuated responses to multiple stresses, including thermal and oxidative stresses. SpxA1− mutants had enhanced resistance to the cationic antimicrobial molecule polymyxin B, while SpxA2− mutants were more sensitive. In a murine model of soft tissue infection, a SpxA1− mutant was highly attenuated. In contrast, the highly stress-sensitive SpxA2− mutant was hypervirulent, exhibiting more extensive tissue damage and a greater bacterial burden than the wild-type strain. SpxA1− attenuation was associated with reduced expression of several toxins, including the SpeB cysteine protease. In contrast, SpxA2− hypervirulence correlated with toxin overexpression and could be suppressed to wild-type levels by deletion of speB. These data show that SpxA1 and SpxA2 have opposing roles in virulence and stress resistance, suggesting that they act coordinately to fine-tune toxin expression in response to stress. SpxA2− hypervirulence also shows that stress resistance is not always essential for S. pyogenes pathogenesis in soft tissue. PMID:28351920

  17. Molecular comparison of Slc11a1 and Slc11a2 genes of swamp- and riverine-type water buffaloes.

    PubMed

    Padiernos, R B C; Mingala, C N

    2016-06-01

    Solute-linked carrier 11a and 11a2 (Slc) have been associated with disease resistance and/or susceptibility across animal species. These genes have an important mechanism in the regulation against intracellular infection. This study analysed the genetic characteristic of Slc 11a and 11a2 in swamp-type and riverine-type water buffaloes to understand their immunological distinction. Characterization of Slc11a1 and Slc11a2 genes from swamp- and riverine-type water buffaloes was carried out by molecular cloning, sequencing and phylogenetic analysis. The cloned cDNA of Slc11a1 and Slc11a2 contained an open reading frame of 1647 and 1723 nucleotides, encoding 549 and 574 amino acids, respectively. Nucleotide sequence homology of both Slc11a1 and Slc11a2 had 99% in swamp and riverine type, which gives almost identical polypeptide. However, Slc11a1 and Slc11a2 have substitutions of 5 and 1 amino acid residues, correspondingly. These substitutions suggest as a potential gene markers for resistance and/or susceptibility to intracellular infection. Furthermore, phylogenetic analysis confirmed the degree of relationship between the bubaline species and justifies the distinctness of each breed by the bootstrap value generated. © 2016 John Wiley & Sons Ltd.

  18. Dietary A1 β-casein affects gastrointestinal transit time, dipeptidyl peptidase-4 activity, and inflammatory status relative to A2 β-casein in Wistar rats.

    PubMed

    Barnett, Matthew P G; McNabb, Warren C; Roy, Nicole C; Woodford, Keith B; Clarke, Andrew J

    2014-09-01

    We compared the gastrointestinal effects of milk-based diets in which the β-casein component was either the A1 or A2 type in male Wistar rats fed the experimental diets for 36 or 84 h. Gastrointestinal transit time was significantly greater in the A1 group, as measured by titanium dioxide recovery in the last 24 h of feeding. Co-administration of naloxone decreased gastrointestinal transit time in the A1 diet group but not in the A2 diet group. Colonic myeloperoxidase and jejunal dipeptidyl peptidase (DPP)-4 activities were greater in the A1 group than in the A2 group. Naloxone attenuated the increase in myeloperoxidase activity but not that in DPP-4 activity in the A1 group. Naloxone did not affect myeloperoxidase activity or DPP-4 activity in the A2 group. These results confirm that A1 β-casein consumption has direct effects on gastrointestinal function via opioid-dependent (gastrointestinal transit and myeloperoxidase activity) and opioid-independent (DPP-4 activity) pathways.

  19. A new CYP21A1P/CYP21A2 chimeric gene identified in an Italian woman suffering from classical congenital adrenal hyperplasia form.

    PubMed

    Concolino, Paola; Mello, Enrica; Minucci, Angelo; Giardina, Emiliano; Zuppi, Cecilia; Toscano, Vincenzo; Capoluongo, Ettore

    2009-07-22

    More than 90% of Congenital Adrenal Hyperplasia (CAH) cases are associated with mutations in the 21-hydroxylase gene (CYP21A2) in the HLA class III area on the short arm of chromosome 6p21.3. In this region, a 30 kb deletion produces a non functional chimeric gene with its 5' and 3' ends corresponding to CYP21A1P pseudogene and CYP21A2, respectively. To date, five different CYP21A1P/CYP21A2 chimeric genes have been found and characterized in recent studies. In this paper, we describe a new CYP21A1P/CYP21A2 chimera (CH-6) found in an Italian CAH patient. Southern blot analysis and CYP21A2 sequencing were performed on the patient. In addition, in order to isolate the new CH-6 chimeric gene, two different strategies were used. The CYP21A2 sequencing analysis showed that the patient was homozygote for the g.655C/A>G mutation and heterozygote for the p.P30L missense mutation. In addition, the promoter sequence revealed the presence, in heterozygosis, of 13 SNPs generally produced by microconversion events between gene and pseudogene. Southern blot analysis showed that the woman was heterozygote for the classic 30-kb deletion producing a new CYP21A1P/CYP21A2 chimeric gene (CH-6). The hybrid junction site was located between the end of intron 2 pseudogene, after the g.656C/A>G mutation, and the beginning of exon 3, before the 8 bp deletion. Consequently, CH-6 carries three mutations: the weak pseudogene promoter region, the p.P30L and the g.655C/A>G splice mutation. We describe a new CYP21A1P/CYP21A2 chimera (CH-6), associated with the HLA-B15, DR13 haplotype, in a young Italian CAH patient.

  20. Constitutive androstane receptor transcriptionally activates human CYP1A1 and CYP1A2 genes through a common regulatory element in the 5'-flanking region.

    PubMed

    Yoshinari, Kouichi; Yoda, Noriaki; Toriyabe, Takayoshi; Yamazoe, Yasushi

    2010-01-15

    Phenobarbital has long been known to increase cellular levels of CYP1A1 and CYP1A2 possibly through a pathway(s) independent of aryl hydrocarbon receptor. We have investigated the role of constitutive androstane receptor (CAR), a xenobiotic-responsive nuclear receptor, in the transactivation of human CYP1A1 and CYP1A2. These genes are located in a head-to-head orientation, sharing a 5'-flanking region. Reporter assays were thus performed with dual-reporter constructs, containing the whole or partially deleted human CYP1A promoter between two different reporter genes. In this system, human CAR (hCAR) enhanced the transcription of both genes through common promoter regions from -461 to -554 and from -18089 to -21975 of CYP1A1. With reporter assays using additional deleted and mutated constructs, electrophoresis mobility shift assays and chromatin immunoprecipitation assays, an ER8 motif (everted repeat separated by eight nucleotides), located at around -520 of CYP1A1, was identified as an hCAR-responsive element and a binding motif of hCAR/human retinoid X receptor alpha heterodimer. hCAR enhanced the transcription of both genes also in the presence of an aryl hydrocarbon receptor ligand. Finally, hCAR activation increased CYP1A1 and CYP1A2 mRNA levels in cultured human hepatocytes. Our results indicate that CAR transactivates human CYP1A1 and CYP1A2 in human hepatocytes through the common cis-element ER8. Interestingly, the ER8 motif is highly conserved in the CYP1A1 proximal promoter sequences of various species, suggesting a fundamental role of CAR in the xenobiotic-induced expression of CYP1A1 and CYP1A2 independent of aryl hydrocarbon receptor.

  1. Evaluation and comparison of a 1-month versus a 2-week community pediatrics and advocacy rotation for pediatric residents.

    PubMed

    Delago, Cynthia; Gracely, Edward

    2007-11-01

    This prospective study was conducted to assess the effects of a 4-week community pediatrics and advocacy rotation with a unique project (Curriculum A), a 2-week community pediatrics rotation with advocacy training and unique project throughout residency (Curriculum B), or no curriculum exposure on residents' attitudes, perceived competence, knowledge, and behaviors. A 27-item questionnaire was used to assess attitudes, competence, and knowledge. Examination of residents' patients' use of Early Intervention services during the 5-year period after curricula introduction assessed behaviors. Seventy percent of questionnaires distributed over several years were completed by 105 of 111 eligible residents. Residents exposed to Curriculum A or B demonstrated improved competence and knowledge but no significant increase in positive attitudes toward community pediatrics and advocacy. Residents' patients' use of Early Intervention services increased 65% during the 5-year period after curriculum introduction. No significant differences in outcome measures were observed between Curriculum A and Curriculum B.

  2. EphrinA1-EphA2 interaction-mediated apoptosis and Flt3L-induced immunotherapy inhibits tumor growth in a breast cancer mouse model

    PubMed Central

    Tandon, Manish; Vemula, Sai V.; Sharma, Anurag; Ahi, Yadvinder S.; Mittal, Shalini; Bangari, Dinesh S.; Mittal, Suresh K.

    2014-01-01

    Background The receptor tyrosine kinase EphA2 is overexpressed in several types of cancers and is currently being pursued as a target for breast cancer therapeutics. The EphA2 ligand EphrinA1 induces EphA2 phosphorylation and intracellular internalization and degradation, thus inhibiting tumor progression. The hematopoietic growth factor, FMS-like tyrosine kinase receptor ligand (Flt3L), promotes expansion and mobilization of functional dendritic cells. Methods We tested the EphrinA1-EphA2 interaction in MDA-MB-231 breast cancer cells focusing on the receptor-ligand-mediated apoptosis of breast cancer cells. In order to determine whether the EphrinA1-EphA2 interaction-associated apoptosis and Flt3L-mediated immunotherapy would have an additive effect in inhibiting tumor growth, we used an immunocompetent mouse model of breast cancer to evaluate intratumoral (i.t.) inoculation strategies with human adenovirus (HAd) vectors expressing either EphrinA1 (HAd-EphrinA1-Fc), Flt3L (HAd-Flt3L) or a combination of EphrinA1-Fc + Flt3L (HAd-EphrinA1-Fc + HAd-Flt3L). Results In vitro analysis demonstrated that an EphrinA1-EphA2 interaction led to apoptosis-related changes in breast cancer cells. In vivo, three i.t. inoculations of HAd-EphrinA1-Fc showed potent inhibition of tumor growth. Furthermore, increased inhibition in tumor growth was observed with the combination of HAd-EphrinA1-Fc and HAd-Flt3L accompanied by the generation of an anti-tumor adaptive immune response. Conclusions The results indicating induction of apoptosis and inhibition of mammary tumor growth show the potential therapeutic benefits of HAd-EphrinA1-Fc. In combination with HAd-Flt3L, this represents a promising strategy to effectively induce mammary tumor regression by HAd vector-based therapy. PMID:22228563

  3. Analysis of hepatic vitamins A1, A2, their fatty acyl esters, and vitamin E for biomonitoring mammals feeding on freshwater fish.

    PubMed

    Käkelä, Anne; Käkelä, Reijo; Hyvärinen, Heikki

    2002-02-01

    In tissues of freshwater fish-feeding mammals, 3,4-didehydroretinol (A2) is a major form of vitamin A. In mink liver, with organochlorine exposure, this analog has been found to decrease more than retinol (A1) and thus has potential as a sensitive freshwater biomarker. The presence of the analogs A1 and A2 as alcohol and different fatty acyl esters, which react to polychlorinated biphenyls differently, necessitates detailed analyses achieved by using direct extraction of tissue homogenate. In direct hexane extraction, compared to total levels of the vitamins obtained in the saponification procedure, a large proportion of the vitamins was released only after repeated and long-time vortex mixing with the extraction solvent. Thus, in tissue extraction, the use of internal standardization alone can lead to a rough underestimation of the levels of these fat-soluble vitamins. For analyses of vitamins A1 and A2 in liver, we applied the argentation high-performance liquid chromatography, which provided good separation of individual A1 and A2 fatty acyl esters. We report retention times for numerous esters of A1 and A2 and, to aid identification, the change in their retention properties after adding AgNO3 to the mobile phase. The argentation did not affect the recoveries of any forms of the retinoids studied but destroyed half the vitamin E. Despite selective acylation of fatty acids into the vitamin A esters, the fatty acids of the esters were the same as those found to be the major fatty acids in the gas-liquid chromatography of total lipids. The goal of this work was to create a methodology that is suitable for biomonitoring alcoholic and esterified vitamins A1 and A2 in tissues of freshwater fish-feeding mammals.

  4. Conversion of Bacillus subtilis OhrR from a 1-Cys to a 2-Cys Peroxide Sensor▿

    PubMed Central

    Soonsanga, Sumarin; Lee, Jin-Won; Helmann, John D.

    2008-01-01

    OhrR proteins can be divided into two groups based on their inactivation mechanism: 1-Cys (represented by Bacillus subtilis OhrR) and 2-Cys (represented by Xanthomonas campestris OhrR). A conserved cysteine residue near the amino terminus is present in both groups of proteins and is initially oxidized to the sulfenic acid. The B. subtilis 1-Cys OhrR protein is subsequently inactivated by formation of a mixed-disulfide bond with low-molecular-weight thiols or by cysteine overoxidation to sulfinic and sulfonic acids. In contrast, the X. campestris 2-Cys OhrR is inactivated when the initially oxidized cysteine sulfenate forms an intersubunit disulfide bond with a second Cys residue from the other subunit of the protein dimer. Here, we demonstrate that the 1-Cys B. subtilis OhrR can be converted into a 2-Cys OhrR by introducing another cysteine residue in either position 120 or position 124. Like the X. campestris OhrR protein, these mutants (G120C and Q124C) are inactivated by intermolecular disulfide bond formation. Analysis of oxidized 2-Cys variants both in vivo and in vitro indicates that intersubunit disulfide bond formation can occur simultaneously at both active sites in the protein dimer. Rapid formation of intersubunit disulfide bonds protects OhrR against irreversible overoxidation in the presence of strong oxidants much more efficiently than do the endogenous low-molecular-weight thiols. PMID:18586944

  5. Study on Microtexture and Martensite Formation of Friction Stir Lap-welded DP 590 Steel within A1 to A3 Temperature Range

    NASA Astrophysics Data System (ADS)

    Das, Hrishikesh; Lee, Kwang-Jin; Hong, Sung-Tae

    2017-07-01

    Friction stir lap welding of dual phase 590 steel has been successfully performed within the A1 to A3 temperature range upon adjusting the process parameters. The microstructures and the texture have been characterized using scanning electron microscopy and electron back scattered diffraction analysis. The martensite volume fraction increases with an increasing travel speed from 100 to 300 mm/min for a constant tool rotational speed of 300 rpm. Under severe deformation and high strain rate during friction stir welding, the grain orientation <101> shifts toward the <111> and <001> directions compared to the base metal. The base metal shows γ fiber components, whereas the friction stir welded specimen shows strong brass with weak cube fibers.

  6. A dispersed fluorescence and ab initio investigation of the X2B1 and A2A1 electronic states of the PH2 molecule.

    PubMed

    Jakubek, Z J; Bunker, P R; Zachwieja, M; Nakhate, S G; Simard, B; Yurchenko, S N; Thiel, W; Jensen, Per

    2006-03-07

    In this work, the X2B1 and A2A1 electronic states of the phosphino (PH2) free radical have been studied by dispersed fluorescence and ab initio methods. PH2 molecules were produced in a molecular free-jet apparatus by laser vaporizing a silicon rod in the presence of phosphine (PH3) gas diluted in helium. The laser-induced fluorescence, from the excited A2A1 electronic state down to the ground electronic state, was dispersed and analyzed. Ten (upsilon1upsilon2upsilon3) vibrationally excited levels of the ground electronic state, with upsilon1 < or = 2, upsilon2 < or = 6, and upsilon3 = 0, have been observed. Ab initio potential-energy surfaces for the X2B1 and A2A1 electronic states have been calculated at 210 points. These two states correlate with a 2Pi(u) state at linearity and they interact by the Renner-Teller coupling and spin-orbit coupling. Using the ab initio potential-energy surfaces with our RENNER computer program system, the vibronic structure and relative intensities of the A2A1 --> X2B1 emission band system have been calculated in order to corroborate the experimental assignments.

  7. Apparent affinity of 1,3-dipropyl-8-cyclopentylxanthine for adenosine A1 and A2 receptors in isolated tissues from guinea-pigs.

    PubMed Central

    Collis, M. G.; Stoggall, S. M.; Martin, F. M.

    1989-01-01

    1. The classification of adenosine receptor subtypes (A1 and A2) in intact tissues has been based on the order of agonist potency. In this study the apparent affinity of 1,3-dipropyl-8-cyclopentylxanthine (CPX), an antagonist which has been reported to be A1 selective, and the non-selective antagonist 1,3-dimethyl-8-phenylxanthine (8PT) has been evaluated on isolated tissues from the guinea-pig. 2. The isolated tissues used were atria (bradycardic response, proposed A1 sub-type), aorta and trachea (relaxant response, proposed A2 sub-type). 3. Both the xanthines antagonized responses to adenosine in the three tissues but had little or no effect on responses to carbachol (atria), sodium nitrite (aorta) or isoprenaline (trachea). 4. pA2 values for 8PT were similar on the three tissues (6.3-6.7), however, the pA2 value for CPX on the atria (7.9-8.4) was greater than that on the aorta (6.6) or trachea (6.6). 5. These results support the suggestion that the adenosine receptors which mediate bradycardia in the atrium are of the A1 sub-type and that those which mediate relaxation in the aorta and trachea are of the A2 type. PMID:2790383

  8. Selected C8 two-chain linkers enhance the adenosine A1/A2A receptor affinity and selectivity of caffeine.

    PubMed

    van der Walt, M M; Terre'Blanche, G

    2017-01-05

    Recent research exploring C8 substitution on the caffeine core identified 8-(2-phenylethyl)-1,3,7-trimethylxanthine as a non-selective adenosine receptor antagonist. To elaborate further, we included various C8 two-chain-length linkers to enhance adenosine receptor affinity. The results indicated that the unsubstituted benzyloxy linker (1e A1Ki = 1.52 μM) displayed the highest affinity for the A1 adenosine receptor and the para-chloro-substituted phenoxymethyl (1d A2AKi = 1.33 μM) linker the best A2A adenosine receptor affinity. The position of the oxygen revealed that the phenoxymethyl linker favoured A1 adenosine receptor selectivity over the benzyloxy linker and, by introducing a para-chloro substituent, A2A adenosine receptor selectivity was obtained. Selected compounds (1c, 1e) behaved as A1 adenosine receptor antagonists in GTP shift assays and therefore represent selective and non-selective A1 and A2A adenosine receptor antagonists that may have potential for treating neurological disorders.

  9. Hyperthermia-induced seizures alter adenosine A1 and A2A receptors and 5'-nucleotidase activity in rat cerebral cortex.

    PubMed

    León-Navarro, David Agustín; Albasanz, José L; Martín, Mairena

    2015-08-01

    Febrile seizure is one of the most common convulsive disorders in children. The neuromodulator adenosine exerts anticonvulsant actions through binding adenosine receptors. Here, the impact of hyperthermia-induced seizures on adenosine A1 and A2A receptors and 5'-nucleotidase activity has been studied at different periods in the cerebral cortical area by using radioligand binding, real-time PCR, and 5'-nucleotidase activity assays. Hyperthermic seizures were induced in 13-day-old rats using a warmed air stream from a hair dryer. Neonates exhibited rearing and falling over associated with hindlimb clonus seizures (stage 5 on Racine scale criteria) after hyperthermic induction. A significant increase in A1 receptor density was observed using [(3) H]DPCPX as radioligand, and mRNA coding A1 was observed 48 h after hyperthermia-induced seizures. In contrast, a significant decrease in A2A receptor density was detected, using [(3) H]ZM241385 as radioligand, 48 h after hyperthermia-evoked convulsions. These short-term changes in A1 and A2A receptors were also accompanied by a loss of 5'-nucleotidase activity. No significant variations either in A1 or A2A receptor density or 5'-nucleotidase were observed 5 and 20 days after hyperthermic seizures. Taken together, both regulation of A1 and A2A receptors and loss of 5'-nucleotidase in the cerebral cortex suggest the existence of a neuroprotective mechanism against seizures. Febrile seizure is one of the most common convulsive disorders in children. The consequences of hyperthermia-induced seizures (animal model of febrile seizures) on adenosine A1 and A2A receptors and 5'-nucleotidase activity have been studied at different periods in cerebral cortical area. A significant increase in A1 receptor density and mRNA coding A1 was observed 48 h after hyperthermia-induced seizures. In contrast, a significant decrease in A2A receptor density and 5'-nucleotidase activity was detected 48 h after convulsions evoked by hyperthermia

  10. Transcription of Oxidative Stress Genes Is Directly Activated by SpxA1 and, to a Lesser Extent, by SpxA2 in Streptococcus mutans

    PubMed Central

    Kajfasz, Jessica K.; Rivera-Ramos, Isamar; Scott-Anne, Kathleen; Gregoire, Stacy; Abranches, Jacqueline

    2015-01-01

    ABSTRACT The SpxA1 and SpxA2 (formerly SpxA and SpxB) transcriptional regulators of Streptococcus mutans are members of a highly conserved family of proteins found in Firmicutes, and they were previously shown to activate oxidative stress responses. In this study, we showed that SpxA1 exerts substantial positive regulatory influence over oxidative stress genes following exposure to H2O2, while SpxA2 appears to have a secondary regulatory role. In vitro transcription (IVT) assays using purified SpxA1 and/or SpxA2 showed that SpxA1 and, less often, SpxA2 directly activate transcription of some of the major oxidative stress genes. Addition of equimolar concentrations of SpxA1 and SpxA2 to the IVT reactions neither enhanced transcription of the tested genes nor disrupted the dominant role of SpxA1. Substitution of a conserved glycine residue (G52) present in both Spx proteins by arginine (SpxG52R) resulted in strains that phenocopied the Δspx strains. Moreover, addition of purified SpxA1G52R completely failed to activate transcription of ahpC, sodA, and tpx, further confirming that the G52 residue is critical for Spx functionality. IMPORTANCE Streptococcus mutans is a pathogen associated with the formation of dental caries in humans. Within the oral cavity, S. mutans routinely encounters oxidative stress. Our previous data revealed that two regulatory proteins, SpxA1 and SpxA2 (formerly SpxA and SpxB), bear high homology to the Spx regulator that has been characterized as a critical activator of oxidative stress genes in Bacillus subtilis. In this report, we prove that Spx proteins of S. mutans directly activate transcription of genes involved in the oxidative stress response, though SpxA1 appears to have a more dominant role than SpxA2. Therefore, the Spx regulators play a critical role in the ability of S. mutans to thrive within the oral cavity. PMID:25897032

  11. Identification of UDP-glucuronosyltransferases 1A1, 1A3 and 2B15 as the main contributors to glucuronidation of bakuchiol, a natural biologically active compound.

    PubMed

    Li, Feng; Wang, Shuai; Lu, Danyi; Wang, Yifei; Dong, Dong; Wu, Baojian

    2017-05-01

    1. Bakuchiol, one of the main active compounds of Psoralea corylifolia, possesses a variety of pharmacological activities such as anti-tumor and anti-aging effects. Here, we aimed to characterize the glucuronidation of bakuchiol using human liver microsomes (HLM) and expressed UDP-glucuronosyltransferase (UGT) enzymes. 2. The glucuronide of bakuchiol was confirmed by liquid chromatography-mass spectrometry (LC-MS) and β-glucuronidase hydrolysis assay. Glucuronidation rates and kinetic parameters were derived by enzymatic incubation and model fitting. Activity correlation analyses were performed to identify the main UGT isoforms contributing to hepatic metabolism of bakuchiol. 3. Among the three UGT enzymes (i.e., UGT1A1, UGT1A3 and UGT2B15) capable of catalyzing bakuchiol glucuronidation, UGT2B15 showed the highest activity with a CLint value of 100 μl/min/nmol. Bakuchiol glucuronidation was strongly correlated with glucuronidation of 5-hydroxyrofecoxib (r = 0.933; p < 0.001), 3-O-glucuronidation of β-estradiol (r = 0.719; p < 0.01) and significantly correlated with 24-O-glucuronidation of CDCA (r = 0.594; p < 0.05). In addition, a marked species difference existed in hepatic glucuronidation of bakuchiol. 4. In conclusion, UGT1A1, UGT1A3 and UGT2B15 were identified as the main contributors to glucuronidation of bakuchiol.

  12. Distribution of COL8A2 and COL8A1 gene variants in Caucasian primary open angle glaucoma patients with thin central corneal thickness

    PubMed Central

    Desronvil, T.; Logan-Wyatt, D.; Abdrabou, W.; Triana, M.; Jones, R.; Taheri, S.; Del Bono, E.; Pasquale, L.R.; Olivier, M.; Haines, J.L.; Fan, B.J.

    2010-01-01

    Purpose One approach to identify genes that contribute to common complex ocular disorders such as primary open angle glaucoma (POAG) is to study the genetic determinates of endophenotypes that are defined by underlying pre-disposing heritable quantitative traits such as central corneal thickness (CCT). Collagen VIII is a major component of Descemet’s membrane and studies in mice have indicated that targeted inactivation of the genes encoding the collagen type 8 alpha1 (Col8a1) and collagen type 8 alpha2 (Col8a2) subunits (COL8A1 and COL8A2) results in thinning of the corneal stroma and of Descemet’s membrane. The purpose of this study is to evaluate COL8A1 and COL8A2 as candidate genes for thin CCT in human POAG patients. Methods 100 Caucasian POAG patients were enrolled in this study. The entire COL8A1 and COL8A2 coding sequence was determined in 8 patients with CCT<513 µm (one standard deviation (36 microns) below the mean (550 microns) and 8 patients with CCT>586 µm (one standard deviation above the mean). Selected COL8A2 exons containing variants of interest were sequenced in the full POAG cohort. Association and quantitative trait analyses were performed. Results Three patients with CCT less than 513 µm and advanced POAG were found to have missense changes in COL8A2; two patients had a previously identified mutation, R155Q and one had a novel change, P678L (p=0.0035, Fisher’s exact test). Missense changes were not found in any of the patients with CCT>513 µm and missense changes in the COL8A1 gene were not found in any patient. One common COL8A2 SNP, rs274754 was also statistically associated with CCT (p=0.018). Conclusions In this study we have identified COL8A2 missense changes in a group of Caucasian patients with very thin CCT and advanced POAG. These results suggest that DNA sequence variants in the COL8A2 gene may be associated with thin corneas in some glaucoma patients. Further study of COL8A2 variants in other patient populations, especially

  13. Nkx6.1 regulates islet β-cell proliferation via Nr4a1 and Nr4a3 nuclear receptors.

    PubMed

    Tessem, Jeffery S; Moss, Larry G; Chao, Lily C; Arlotto, Michelle; Lu, Danhong; Jensen, Mette V; Stephens, Samuel B; Tontonoz, Peter; Hohmeier, Hans E; Newgard, Christopher B

    2014-04-08

    Loss of functional β-cell mass is a hallmark of type 1 and type 2 diabetes, and methods for restoring these cells are needed. We have previously reported that overexpression of the homeodomain transcription factor NK6 homeobox 1 (Nkx6.1) in rat pancreatic islets induces β-cell proliferation and enhances glucose-stimulated insulin secretion, but the pathway by which Nkx6.1 activates β-cell expansion has not been defined. Here, we demonstrate that Nkx6.1 induces expression of the nuclear receptor subfamily 4, group A, members 1 and 3 (Nr4a1 and Nr4a3) orphan nuclear receptors, and that these factors are both necessary and sufficient for Nkx6.1-mediated β-cell proliferation. Consistent with this finding, global knockout of Nr4a1 results in a decrease in β-cell area in neonatal and young mice. Overexpression of Nkx6.1 and the Nr4a receptors results in increased expression of key cell cycle inducers E2F transcription factor 1 and cyclin E1. Furthermore, Nkx6.1 and Nr4a receptors induce components of the anaphase-promoting complex, including ubiquitin-conjugating enzyme E2C, resulting in degradation of the cell cycle inhibitor p21. These studies identify a unique bipartite pathway for activation of β-cell proliferation, suggesting several unique targets for expansion of functional β-cell mass.

  14. Chromophore switch from 11-cis-dehydroretinal (A2) to 11-cis-retinal (A1) decreases dark noise in salamander red rods

    PubMed Central

    Ala-Laurila, Petri; Donner, Kristian; Crouch, Rosalie K; Cornwall, M Carter

    2007-01-01

    Dark noise, light-induced noise and responses to brief flashes of light were recorded in the membrane current of isolated rods from larval tiger salamander retina before and after bleaching most of the native visual pigment, which mainly has the 11-cis-3,4-dehydroretinal (A2) chromophore, and regenerating with the 11-cis-retinal (A1) chromophore in the same isolated rods. The purpose was to test the hypothesis that blue-shifting the pigment by switching from A2 to A1 will decrease the rate of spontaneous thermal activations and thus intrinsic light-like noise in the rod. Complete recordings were obtained in five cells (21°C). Based on the wavelength of maximum absorbance, λmax,A1 = 502 nm and λmax,A2 = 528 nm, the average A2 : A1 ratio determined from rod spectral sensitivities and absorbances was ∼0.74 : 0.26 in the native state and ∼0.09 : 0.91 in the final state. In the native (A2) state, the single-quantum response (SQR) had an amplitude of 0.41 ± 0.03 pA and an integration time of 3.16 ± 0.15 s (mean ± s.e.m.). The low-frequency branch of the dark noise power spectrum was consistent with discrete SQR-like events occurring at a rate of 0.238 ± 0.026 rod−1 s−1. The corresponding values in the final state were 0.57 ± 0.07 pA (SQR amplitude), 3.47 ± 0.26 s (SQR integration time), and 0.030 ± 0.006 rod−1 s−1 (rate of dark events). Thus the rate of dark events per rod and the fraction of A2 pigment both changed by ca 8-fold between the native and final states, indicating that the dark events originated mainly in A2 molecules even in the final state. By extrapolating the linear relation between event rates and A2 fraction to 0% A2 (100% A1) and 100% A2 (0% A1), we estimated that the A1 pigment is at least 36 times more stable than the A2 pigment. The noise component attributed to discrete dark events accounted for 73% of the total dark current variance in the native (A2) state and 46% in the final state. The power spectrum of the remaining

  15. Chromophore switch from 11-cis-dehydroretinal (A2) to 11-cis-retinal (A1) decreases dark noise in salamander red rods.

    PubMed

    Ala-Laurila, Petri; Donner, Kristian; Crouch, Rosalie K; Cornwall, M Carter

    2007-11-15

    Dark noise, light-induced noise and responses to brief flashes of light were recorded in the membrane current of isolated rods from larval tiger salamander retina before and after bleaching most of the native visual pigment, which mainly has the 11-cis-3,4-dehydroretinal (A2) chromophore, and regenerating with the 11-cis-retinal (A1) chromophore in the same isolated rods. The purpose was to test the hypothesis that blue-shifting the pigment by switching from A2 to A1 will decrease the rate of spontaneous thermal activations and thus intrinsic light-like noise in the rod. Complete recordings were obtained in five cells (21 degrees C). Based on the wavelength of maximum absorbance, lambda max,A1 = 502 nm and lambda max,A2 = 528 nm, the average A2 : A1 ratio determined from rod spectral sensitivities and absorbances was approximately 0.74 : 0.26 in the native state and approximately 0.09 : 0.91 in the final state. In the native (A2) state, the single-quantum response (SQR) had an amplitude of 0.41 +/- 0.03 pA and an integration time of 3.16 +/- 0.15 s (mean +/- s.e.m.). The low-frequency branch of the dark noise power spectrum was consistent with discrete SQR-like events occurring at a rate of 0.238 +/- 0.026 rod(-1) s(-1). The corresponding values in the final state were 0.57 +/- 0.07 pA (SQR amplitude), 3.47 +/- 0.26 s (SQR integration time), and 0.030 +/- 0.006 rod(-1) s(-1) (rate of dark events). Thus the rate of dark events per rod and the fraction of A2 pigment both changed by ca 8-fold between the native and final states, indicating that the dark events originated mainly in A2 molecules even in the final state. By extrapolating the linear relation between event rates and A2 fraction to 0% A2 (100% A1) and 100% A2 (0% A1), we estimated that the A1 pigment is at least 36 times more stable than the A2 pigment. The noise component attributed to discrete dark events accounted for 73% of the total dark current variance in the native (A2) state and 46% in the final

  16. Synthesis and characterization of monoisomeric 1,8,15,22-substituted (A3B and A2B2) phthalocyanines and phthalocyanine-fullerene dyads.

    PubMed

    Ranta, Jenni; Kumpulainen, Tatu; Lemmetyinen, Helge; Efimov, Alexander

    2010-08-06

    Synthesis and characterization of three phthalocyanine-fullerene (Pc-C(60)) dyads, corresponding monoisomeric phthalocyanines (Pc), and building blocks, phthalonitriles, are described. Six novel bisaryl phthalonitriles were prepared by the Suzuki-Miyaura coupling reaction from trifluoromethanesulfonic acid 2,3-dicyanophenyl ester and various oxaborolanes. Two phthalonitriles were selected for the synthesis of A(3)B- and A(2)B(2)-type phthalocyanines. Phthalonitrile 4 has a bulky 3,5-di-tert-butylphenyl substituent at the alpha-phthalo position, which forces only one regioisomer to form and greatly increases the solubility of phthalocyanine. Phthalonitrile 8 has a 3-phenylpropanol side chain at the alpha-position making further modifications of the side group possible. Synthesized monoisomeric A(3)B- and A(2)B(2)-type phthalocyanines are modified by attachment of malonic residues. Finally, fullerene is covalently linked to phthalocyanine with one or two malonic bridges to produce Pc-C(60) dyads. Due to the monoisomeric structure and increased solubility of phthalocyanines, the quality of NMR spectra of the compounds is enhanced significantly, making detailed NMR analysis of the structures possible. The synthesized dyads have different orientations of phthalocyanine and fullerene, which strongly influence the electron transfer (ET) from phthalocyanine to fullerene moiety. Fluorescence quenchings of the dyads were measured in both polar and nonpolar solvents, and in all cases, the quenching was more efficient in the polar environment. As expected, most efficient fluorescence quenching was observed for dyad 20b, with two linkers and phthalocyanine and fullerene in face-to-face orientation.

  17. Crystal structures of SULT1A2 and SULT1A1 *3: insights into the substrate inhibition and the role of Tyr149 in SULT1A2.

    PubMed

    Lu, Jinghua; Li, Haitao; Zhang, Jiping; Li, Mei; Liu, Ming-Yih; An, Xiaomin; Liu, Ming-Cheh; Chang, Wenrui

    2010-05-28

    The cytosolic sulfotransferases (SULTs) in vertebrates catalyze the sulfonation of endogenous thyroid/steroid hormones and catecholamine neurotransmitters, as well as a variety of xenobiotics, using 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfonate donor. In this study, we determined the structures of SULT1A2 and an allozyme of SULT1A1, SULT1A1 *3, bound with 3'-phosphoadenosine 5'-phosphate (PAP), at 2.4 and 2.3A resolution, respectively. The conformational differences between the two structures revealed a plastic substrate-binding pocket with two channels and a switch-like substrate selectivity residue Phe247, providing clearly a structural basis for the substrate inhibition. In SULT1A2, Tyr149 extends approximately 2.1A further to the inside of the substrate-binding pocket, compared with the corresponding His149 residue in SULT1A1 *3. Site-directed mutagenesis study showed that, compared with the wild-type SULT1A2, mutant Tyr149Phe SULT1A2 exhibited a 40 times higher K(m) and two times lower V(max) with p-nitrophenol as substrate. These latter data imply a significant role of Tyr149 in the catalytic mechanism of SULT1A2.

  18. Cambrian origin of the CYP27C1-mediated vitamin A1-to-A2 switch, a key mechanism of vertebrate sensory plasticity

    USGS Publications Warehouse

    Morshedian, Ala; Toomery, Matthew B.; Pollock, Gabriel E.; Frederiksen, Rikard; Enright, Jennifer; McCormick, Stephen; Cornwall, M. Carter; Fain, Gordon L.; Corbo, Joseph C.

    2017-01-01

    The spectral composition of ambient light varies across both space and time. Many species of jawed vertebrates adapt to this variation by tuning the sensitivity of their photoreceptors via the expression of CYP27C1, an enzyme that converts vitamin A1 into vitamin A2, thereby shifting the ratio of vitamin A1-based rhodopsin to red-shifted vitamin A2-based porphyropsin in the eye. Here, we show that the sea lamprey (Petromyzon marinus), a jawless vertebrate that diverged from jawed vertebrates during the Cambrian period (approx. 500 Ma), dynamically shifts its photoreceptor spectral sensitivity via vitamin A1-to-A2 chromophore exchange as it transitions between photically divergent aquatic habitats. We further show that this shift correlates with high-level expression of the lamprey orthologue of CYP27C1, specifically in the retinal pigment epithelium as in jawed vertebrates. Our results suggest that the CYP27C1-mediated vitamin A1-to-A2 switch is an evolutionarily ancient mechanism of sensory plasticity that appeared not long after the origin of vertebrates.

  19. Modulation of GABA transport by adenosine A1R-A2AR heteromers, which are coupled to both Gs- and G(i/o)-proteins.

    PubMed

    Cristóvão-Ferreira, Sofia; Navarro, Gemma; Brugarolas, Marc; Pérez-Capote, Kamil; Vaz, Sandra H; Fattorini, Giorgia; Conti, Fiorenzo; Lluis, Carmen; Ribeiro, Joaquim A; McCormick, Peter J; Casadó, Vicent; Franco, Rafael; Sebastião, Ana M

    2011-11-02

    Astrocytes play a key role in modulating synaptic transmission by controlling the available extracellular GABA via the GAT-1 and GAT-3 GABA transporters (GATs). Using primary cultures of rat astrocytes, we show here that an additional level of regulation of GABA uptake occurs via modulation of the GATs by the adenosine A(1) (A(1)R) and A(2A) (A(2A)R) receptors. This regulation occurs through a complex of heterotetramers (two interacting homodimers) of A(1)R-A(2A)R that signal via two different G-proteins, G(s) and G(i/o), and either enhances (A(2A)R) or inhibits (A(1)R) GABA uptake. These results provide novel mechanistic insight into how G-protein-coupled receptor heteromers signal. Furthermore, we uncover a previously unknown mechanism in which adenosine, in a concentration-dependent manner, acts via a heterocomplex of adenosine receptors in astrocytes to significantly contribute to neurotransmission at the tripartite (neuron-glia-neuron) synapse.

  20. Cambrian origin of the CYP27C1-mediated vitamin A1-to-A2 switch, a key mechanism of vertebrate sensory plasticity.

    PubMed

    Morshedian, Ala; Toomey, Matthew B; Pollock, Gabriel E; Frederiksen, Rikard; Enright, Jennifer M; McCormick, Stephen D; Cornwall, M Carter; Fain, Gordon L; Corbo, Joseph C

    2017-07-01

    The spectral composition of ambient light varies across both space and time. Many species of jawed vertebrates adapt to this variation by tuning the sensitivity of their photoreceptors via the expression of CYP27C1, an enzyme that converts vitamin A1 into vitamin A2, thereby shifting the ratio of vitamin A1-based rhodopsin to red-shifted vitamin A2-based porphyropsin in the eye. Here, we show that the sea lamprey (Petromyzon marinus), a jawless vertebrate that diverged from jawed vertebrates during the Cambrian period (approx. 500 Ma), dynamically shifts its photoreceptor spectral sensitivity via vitamin A1-to-A2 chromophore exchange as it transitions between photically divergent aquatic habitats. We further show that this shift correlates with high-level expression of the lamprey orthologue of CYP27C1, specifically in the retinal pigment epithelium as in jawed vertebrates. Our results suggest that the CYP27C1-mediated vitamin A1-to-A2 switch is an evolutionarily ancient mechanism of sensory plasticity that appeared not long after the origin of vertebrates.

  1. Measurement of HbA1c and HbA2 by Capillarys 2 Flex Piercing HbA1c programme for simultaneous management of diabetes and screening for thalassemia

    PubMed Central

    Ke, Peifeng; Liu, Jiawei; Chao, Yan; Wu, Xiaobin; Xiong, Yujuan; Lin, Li; Wan, Zemin; Wu, Xinzhong; Xu, Jianhua; Zhuang, Junhua; Huang, Xianzhang

    2017-01-01

    Introduction Thalassemia could interfere with some assays for haemoglobin A1c (HbA1c) measurement, therefore, it is useful to be able to screen for thalassemia while measuring HbA1c. We used Capillarys 2 Flex Piercing (Capillarys 2FP) HbA1c programme to simultaneously measure HbA1c and screen for thalassemia. Materials and methods Samples from 498 normal controls and 175 thalassemia patients were analysed by Capillarys 2FP HbA1c programme (Sebia, France). For method comparison, HbA1c was quantified by Premier Hb9210 (Trinity Biotech, Ireland) in 98 thalassaemia patients samples. For verification, HbA1c from eight thalassaemia patients was confirmed by IFCC reference method. Results Among 98 thalassaemia samples, Capillarys 2FP did not provide an HbA1c result in three samples with HbH due to the overlapping of HbBart’s with HbA1c fraction; for the remaining 95 thalassaemia samples, Bland-Altman plot showed 0.00 ± 0.35% absolute bias between two systems, and a significant positive bias above 7% was observed only in two HbH samples. The HbA1c values obtained by Capillarys 2FP were consistent with the IFCC targets (relative bias below ± 6%) in all of the eight samples tested by both methods. For screening samples with alpha (α-) thalassaemia silent/trait or beta (β-) thalassemia trait, the optimal HbA2 cut-off values were ≤ 2.2% and > 2.8%, respectively. Conclusions Our results demonstrated the Capillarys 2FP HbA1c system could report an accurate HbA1c value in thalassemia silent/trait, and HbA2 value (≤ 2.2% for α-thalassaemia silent/trait and > 2.8% for β-thalassemia trait) and abnormal bands (HbH and/or HbBart’s for HbH disease, HbF for β-thalassemia) may provide valuable information for screening. PMID:28900367

  2. Measurement of HbA1c and HbA2 by Capillarys 2 Flex Piercing HbA1c programme for simultaneous management of diabetes and screening for thalassemia.

    PubMed

    Ke, Peifeng; Liu, Jiawei; Chao, Yan; Wu, Xiaobin; Xiong, Yujuan; Lin, Li; Wan, Zemin; Wu, Xinzhong; Xu, Jianhua; Zhuang, Junhua; Huang, Xianzhang

    2017-10-01

    Thalassemia could interfere with some assays for haemoglobin A1c (HbA1c) measurement, therefore, it is useful to be able to screen for thalassemia while measuring HbA1c. We used Capillarys 2 Flex Piercing (Capillarys 2FP) HbA1c programme to simultaneously measure HbA1c and screen for thalassemia. Samples from 498 normal controls and 175 thalassemia patients were analysed by Capillarys 2FP HbA1c programme (Sebia, France). For method comparison, HbA1c was quantified by Premier Hb9210 (Trinity Biotech, Ireland) in 98 thalassaemia patients samples. For verification, HbA1c from eight thalassaemia patients was confirmed by IFCC reference method. Among 98 thalassaemia samples, Capillarys 2FP did not provide an HbA1c result in three samples with HbH due to the overlapping of HbBart's with HbA1c fraction; for the remaining 95 thalassaemia samples, Bland-Altman plot showed 0.00 ± 0.35% absolute bias between two systems, and a significant positive bias above 7% was observed only in two HbH samples. The HbA1c values obtained by Capillarys 2FP were consistent with the IFCC targets (relative bias below ± 6%) in all of the eight samples tested by both methods. For screening samples with alpha (α-) thalassaemia silent/trait or beta (β-) thalassemia trait, the optimal HbA2 cut-off values were ≤ 2.2% and > 2.8%, respectively. Our results demonstrated the Capillarys 2FP HbA1c system could report an accurate HbA1c value in thalassemia silent/trait, and HbA2 value (≤ 2.2% for α-thalassaemia silent/trait and > 2.8% for β-thalassemia trait) and abnormal bands (HbH and/or HbBart's for HbH disease, HbF for β-thalassemia) may provide valuable information for screening.

  3. Presymptomatic and symptomatic ALS SOD1(G93A) mice differ in adenosine A1 and A2A receptor-mediated tonic modulation of neuromuscular transmission.

    PubMed

    Nascimento, Filipe; Sebastião, Ana M; Ribeiro, Joaquim A

    2015-12-01

    Amyotrophic lateral sclerosis (ALS) is a disease leading to neuromuscular transmission impairment. A2A adenosine receptor (A2AR) function changes with disease stage, but the role of the A(1) receptors (A1Rs) is unknown and may have a functional cross-talk with A2AR. The role of A1R in the SOD1(G93A) mouse model of ALS in presymptomatic (4-6 weeks old) and symptomatic (12-14 weeks old) phases was investigated by recording endplate potentials (EPPs), miniature endplate potentials (MEPPs), and quantal content (q.c.) of EPPs, from Mg(2+) paralyzed hemidiaphragm preparations. In presymptomatic mice, the A1R agonist, N (6)-cyclopentyladenosine (CPA) (50 nM), decreased mean EPP amplitude, MEPP frequency, and q.c. of EPPs, an effect quantitatively similar to that in age-matched wild-type (WT) mice. However, coactivation of A2AR with CGS 21680 (5 nM) prevented the effects of CPA in WT mice but not in presymptomatic SOD1(G93A) mice, suggestive of A1R/A2AR cross-talk disruption in this phase of ALS. DPCPX (50 nM) impaired CGS 21680 facilitatory action on neuromuscular transmission in WT but not in presymptomatic mice. In symptomatic animals, CPA only inhibited transmission if added in the presence of adenosine deaminase (ADA, 1 U/mL). ADA and DPCPX enhanced more transmission in symptomatic mice than in age-matched WT mice, suggestive of increase in extracellular adenosine during the symptomatic phase of ALS. The data documents that at the neuromuscular junction of presymptomatic SOD1(G93A) mice, there is a loss of A1R-A2AR functional cross-talk, while in symptomatic mice there is increased A1R tonic activation, and that with disease progression, changes in A1R-mediated adenosine modulation may act as aggravating factors during the symptomatic phase of ALS.

  4. The role of 5-arylalkylamino- and 5-piperazino- moieties on the 7-aminopyrazolo[4,3-d]pyrimidine core in affecting adenosine A1 and A2A receptor affinity and selectivity profiles.

    PubMed

    Squarcialupi, Lucia; Betti, Marco; Catarzi, Daniela; Varano, Flavia; Falsini, Matteo; Ravani, Annalisa; Pasquini, Silvia; Vincenzi, Fabrizio; Salmaso, Veronica; Sturlese, Mattia; Varani, Katia; Moro, Stefano; Colotta, Vittoria

    2017-12-01

    New 7-amino-2-phenylpyrazolo[4,3-d]pyrimidine derivatives, substituted at the 5-position with aryl(alkyl)amino- and 4-substituted-piperazin-1-yl- moieties, were synthesized with the aim of targeting human (h) adenosine A1 and/or A2A receptor subtypes. On the whole, the novel derivatives 1-24 shared scarce or no affinities for the off-target hA2B and hA3 ARs. The 5-(4-hydroxyphenethylamino)- derivative 12 showed both good affinity (Ki = 150 nM) and the best selectivity for the hA2A AR while the 5-benzylamino-substituted 5 displayed the best combined hA2A (Ki = 123 nM) and A1 AR affinity (Ki = 25 nM). The 5-phenethylamino moiety (compound 6) achieved nanomolar affinity (Ki = 11 nM) and good selectivity for the hA1 AR. The 5-(N(4)-substituted-piperazin-1-yl) derivatives 15-24 bind the hA1 AR subtype with affinities falling in the high nanomolar range. A structure-based molecular modeling study was conducted to rationalize the experimental binding data from a molecular point of view using both molecular docking studies and Interaction Energy Fingerprints (IEFs) analysis.[Formula: see text].

  5. Effect of Caffeine Chronically Consumed During Pregnancy on Adenosine A1 and A2A Receptors Signaling in Both Maternal and Fetal Heart from Wistar Rats.

    PubMed

    Iglesias, Inmaculada; Albasanz, Jose Luis; Martín, Mairena

    2014-12-01

    Background: Caffeine is the most widely consumed psychoactive substance in the world, even during pregnancy. Its stimulatory effects are mainly due to antagonism of adenosine actions by blocking adenosine A1 and A2A receptors. Previous studies have shown that caffeine can cross the placenta and therefore modulate these receptors not only in the fetal brain but also in the heart. Methods: In the present work, the effect of caffeine chronically consumed during pregnancy on A1 and A2A receptors in Wistar rat heart, from both mothers and their fetuses, were studied using radioligand binding, Western-blotting, and adenylyl cyclase activity assays, as well as reverse transcription polymerase chain reaction. Results: Caffeine did not significantly alter A1R neither at protein nor at gene expression level in both the maternal and fetal heart. On the contrary, A2AR significantly decreased in the maternal heart, although mRNA was not affected. Gi and Gs proteins were also preserved. Finally, A1R-mediated inhibition of adenylyl cyclase activity did not change in the maternal heart, but A2AR mediated stimulation of this enzymatic activity significantly decreased according to the detected loss of this receptor. Conclusions: Opposite to the downregulation and desensitization of the A1R/AC pathway previously reported in the brain, these results show that this pathway is not affected in rat heart after caffeine exposure during pregnancy. In addition, A2AR is downregulated and desensitized in the maternal heart, suggesting a differential modulation of these receptor-mediated pathways by caffeine.

  6. Expression of CYP1A1 and CYP1A2 in the liver and kidney of rabbits after prolonged infusion of propofol.

    PubMed

    Campos, Sónia P; de Lurdes Pinto, Maria; Gomes, Gabriela; de Pinho, Paula Guedes; Monteiro, Joaquim A; Félix, Luis M; Branco, Paula S; Ferreira, Luísa M; Antunes, Luís M

    2016-10-01

    Propofol biotransformation occurs in the liver via hydroxylation by CYP450 enzymatic complex and by glucuronidation, however extra-hepatic metabolism has also been described. To better understand the metabolic pathways involved in propofol biotransformation, the expression of CYP1A1, CYP1A2, the enzymatic and non-enzymatic antioxidant activity and the amount of propofol and its non-conjugated metabolites were investigated. Twenty-one NewZealand rabbits were allocated into three groups continuously treated for 20h. Each group received: NaCl 0.9%, vehicle (SMOFlipid) and propofol 2% (Lipuro). At the end, liver and kidney samples were collected for histopathology and immunohistochemistry and plasma for quantification of propofol and its metabolites. CYP1A1 and CYP1A2 were observed in zone 1 and zone 3 regions of the liver acinus. The propofol and saline groups showed a higher expression of CYP1A1 when compared to vehicle group. Propofol significantly increased CYP1A2 expression, compared to saline. CYP1A1 and CYP1A2 immunoexpression were observed in the kidney but no differences were registered between groups. This suggests that propofol may act as selective inhibitor of CYP1A1 and an inducer of CYP1A2 expression in different regions of the liver. Propofol seems to have an antioxidative protective effect on liver parenchyma, comparatively to the emulsion alone. In the rabbit, extra-hepatic propofol biotransformation may also occur in the kidney. Copyright © 2016 Elsevier GmbH. All rights reserved.

  7. Differential Expression of Adenosine A1 and A2A Receptors After Upper Cervical (C2) Spinal Cord Hemisection in Adult Rats

    PubMed Central

    Petrov, Theodor; Kreipke, Christian; Alilain, Warren; Nantwi, Kwaku D

    2007-01-01

    Background: In an animal model of spinal cord injury, a latent respiratory motor pathway can be pharmacologically activated via adenosine receptors to restore respiratory function after cervical (C2) spinal cord hemisection that paralyzes the hemidiaphragm ipsilateral to injury. Although spinal phrenic motoneurons immunopositive for adenosine receptors have been demonstrated (C3–C5), it is unclear if adenosine receptor protein levels are altered after C2 hemisection and theophylline administration. Objective: To assess the effects of C2 spinal cord hemisection and theophylline administration on the expression of adenosine receptor proteins. Methods: Adenosine A1 and A2A receptor protein levels were assessed in adult rats classified as (a) noninjured and theophylline treated, (b) C2 hemisected, (c) C2 hemisected and administered theophylline orally (3× daily) for 3 days only, and (d) C2 hemisected and administered theophylline (3× daily for 3 days) and assessed 12 days after drug administration. Assessment of A1 protein levels was carried out via immunohistochemistry and A2A protein levels by densitometry. Results: Adenosine A1 protein levels decreased significantly (both ipsilateral and contralateral to injury) after C2 hemisection; however, the decrease was attenuated in hemisected and theophylline-treated animals. Attenuation in adenosine A1 receptor protein levels persisted when theophylline administration was stopped for 12 days prior to assessment. Adenosine A2A protein levels were unchanged by C2 hemisection; however, theophylline reduced the levels within the phrenic motoneurons. Furthermore, the decrease in A2A levels persisted 12 days after theophylline was withdrawn. Conclusion: Our findings suggest that theophylline mitigates the effects of C2 hemisection by attenuating the C2 hemisection–induced decrease in A1 protein levels. Furthermore, A2A protein levels are unaltered by C2 hemisection but decrease after continuous or interrupted theophylline

  8. Effect of Caffeine Chronically Consumed During Pregnancy on Adenosine A1 and A2A Receptors Signaling in Both Maternal and Fetal Heart from Wistar Rats

    PubMed Central

    Iglesias, Inmaculada; Albasanz, Jose Luis

    2014-01-01

    Background: Caffeine is the most widely consumed psychoactive substance in the world, even during pregnancy. Its stimulatory effects are mainly due to antagonism of adenosine actions by blocking adenosine A1 and A2A receptors. Previous studies have shown that caffeine can cross the placenta and therefore modulate these receptors not only in the fetal brain but also in the heart. Methods: In the present work, the effect of caffeine chronically consumed during pregnancy on A1 and A2A receptors in Wistar rat heart, from both mothers and their fetuses, were studied using radioligand binding, Western-blotting, and adenylyl cyclase activity assays, as well as reverse transcription polymerase chain reaction. Results: Caffeine did not significantly alter A1R neither at protein nor at gene expression level in both the maternal and fetal heart. On the contrary, A2AR significantly decreased in the maternal heart, although mRNA was not affected. Gi and Gs proteins were also preserved. Finally, A1R-mediated inhibition of adenylyl cyclase activity did not change in the maternal heart, but A2AR mediated stimulation of this enzymatic activity significantly decreased according to the detected loss of this receptor. Conclusions: Opposite to the downregulation and desensitization of the A1R/AC pathway previously reported in the brain, these results show that this pathway is not affected in rat heart after caffeine exposure during pregnancy. In addition, A2AR is downregulated and desensitized in the maternal heart, suggesting a differential modulation of these receptor-mediated pathways by caffeine. PMID:25538864

  9. Ah receptor, CYP1A1, CYP1A2 and CYP1B1 gene polymorphisms are not involved in the risk of recurrent pregnancy loss.

    PubMed

    Saijo, Y; Sata, F; Yamada, H; Suzuki, K; Sasaki, S; Kondo, T; Gong, Y Y; Kato, E H; Shimada, S; Morikawa, M; Minakami, H; Kishi, R

    2004-10-01

    The etiology of recurrent pregnancy loss (RPL) remains unclear, but it may be related to a possible genetic predisposition together with involvement of environmental factors. We examined the relation between RPL and polymorphisms in four genes, human aryl hydrocarbon (Ah) receptor, cytochrome P450 (CYP) 1A1, CYP1A2 and CYP1B1, which are involved in the metabolism of a wide range of environmental toxins and carcinogens. All cases and controls were women resident in Sapporo, Japan and the surrounding area. The Ah receptor, CYP1A1, CYP1A2 and CYP1B1 genotypes were assessed in 113 Japanese women with recurrent pregnancy loss (RPL) and 203 ethnically matched women experiencing at least one live birth and no spontaneous abortion (control). No significant differences in Ah receptor, CYP1A1, CYP1A2 and CYP1B1 genotype frequencies were found between the women with RPL and the controls [Ah receptor: Arg/Arg (reference); Arg/Lys and Lys/Lys, odds ratio (OR)=0.67; 95% confidence interval (CI)=0.40-1.11, CYP1A1: m1m1 (reference); m1m2 and m2m2, OR = 0.86; 95% CI = 0.53-1.40, CYP1A2: C/C and C/A (reference); A/A, OR = 1.16; 95% CI = 0.71-1.88, CYP1B1: Leu/Leu (reference); Leu/Val and Val/Val, OR = 1.18; 95% CI = 0.68-2.02]. The present study suggests that the Ah receptor, CYP1A1, CYP1A2 and CYP1B1 gene polymorphisms are not major genetic regulators in RPL.

  10. EphA2 receptor activation with ephrin-A1 ligand restores cetuximab efficacy in NRAS-mutant colorectal cancer cells.

    PubMed

    Cuyàs, Elisabet; Queralt, Bernardo; Martin-Castillo, Begoña; Bosch-Barrera, Joaquim; Menendez, Javier A

    2017-07-01

    Patients with wild-type KRAS metastatic colorectal cancer (mCRC) that harbors NRAS activating mutations do not benefit from anti-EGFR therapies. Very little is known about oncogenic NRAS signaling driving mCRC unresponsiveness to the EGFR-directed antibody cetuximab. Using a system of paired NRAS-mutant and wild-type isogenic mCRC cell lines to explore signaling pathways engaged by the common oncogenic NRAS Q61K variant upon challenge with cetuximab, we uncovered an unexpected mechanism of resistance to cetuximab involving dysregulation of the ephrin-A1/EphA2 signaling axis. Parental NRAS+/+ cells, but not NRASQ61K/+ cells, activated the ephrin receptor ephA1 in response to cetuximab treatment. Moreover, whereas cetuximab treatment significantly downregulated EPHA2 gene expression in NRAS+/+ cells, EPHA2 expression in NRASQ61K/+ cells was refractory to cetuximab. Remarkably, pharmacologically mimicked ephrin-A1 engagement to ephA2 converted NRAS-mutant into RAS wild-type mCRC cells in terms of cetuximab efficacy. Accordingly, activation of the ephA2 receptor by bioactive recombinant human ephrin-A1/Fc-fusion protein suppressed the cetuximab-unresponsive hyperactivation of MAPK and AKT and fully restored cetuximab activity in NRAS-mutant colorectal cells. Collectively, these findings reveal that the clinical benefit of cetuximab in mCRC might necessarily involve the suppression of the ligandless oncogenic signaling of the ephA2 receptor. Hence, ligand-dependent tumor suppressor signaling using therapeutic ephA2 agonists might offer new therapeutic opportunities to clinically widen the use of cetuximab in NRAS-mutated and/or ephA2-dependent mCRC tumors.

  11. N9-benzyl-substituted 1,3-dimethyl- and 1,3-dipropyl-pyrimido[2,1-f]purinediones: synthesis and structure-activity relationships at adenosine A1 and A2A receptors.

    PubMed

    Drabczyńska, Anna; Müller, Christa E; Karolak-Wojciechowska, Janina; Schumacher, Britta; Schiedel, Anke; Yuzlenko, Olga; Kieć-Kononowicz, Katarzyna

    2007-07-15

    Synthesis and physicochemical properties of N-benzyl pyrimido[2,1-f]purinediones are described. These derivatives were synthesized by the cyclization of 7-chloropropylo-8-bromo-1,3-dimethyl- or 1,3-dipropyl xanthine derivatives with corresponding (un)substituted benzylamines. Dipropyl derivatives were obtained under microwave irradiation conditions either. The obtained compounds (1-20) were evaluated for their affinity to adenosine A1 and A2A receptors, selected compounds were additionally investigated for affinity to the A3 receptor subtype. The results of the radioligand binding assays to A1 and A2A adenosine receptors showed that most of the 1,3-dimethyl-9-benzylpyrimidopurinediones exhibited selective affinity to A2A receptors at micromolar or submicromolar concentrations (for example, derivative 9 with o-methoxy substituent displayed a Ki value of 0.699 microM at rat A2A receptor with more than 36-fold selectivity). Contrary to previously described arylpyrimido[2,1-f]purinediones dipropyl derivatives (compounds 15-20) showed affinity to both kinds of receptors increased, however A1 affinity increased to a larger extent, with the result that A2A selectivity was abolished. The best adenosine A1 receptor ligand was m-chlorobenzyl derivative 18 (Ki=0.089 microM and 5-fold A1 selectivity). Structure-activity relationships were discussed with the analysis of lipophilic and spatial properties of the investigated compounds. Pharmacophore model of adenosine A1 receptor antagonist was adopted for this purpose.

  12. Induction of cytochromes P450 1A1 and 1A2 by tanshinones in human HepG2 hepatoma cell line

    SciTech Connect

    Zhang Rong; Sun Jianguo; Ma Liping; Wu Xiaolan; Pan Guoyu; Hao Haiping; Zhou Fang; Jiye, A; Liu Changhui; Ai Hua; Shang Lili; Gao Haiyan; Peng Ying; Wan Ping; Wu Hui; Wang Guangji

    2011-04-01

    Diterpenoid tanshinones including tanshinone IIA (TIIA), cryptotanshinone (CTS), tanshinone I (TI) and dihydrotanshinone I (DHTI) are the major bioactive components from Danshen. The major aim of our present study was to investigate the induction potential of these four main components of tanshinones (TIIA, CTS, TI, and DHTI) on the expression of CYP1A1 and CYP1A2 in HepG2 cells. Our results showed that all of these four tanshinones caused a significant time- and concentration-dependent increase in the amount of CYP1A1/2 expression in HepG2 cells. These induction effects were further characterized through transcriptional regulation: the induction of CYP1A1/2 mRNA level by tanshinones was completely blocked by the transcription inhibitor actinomycin D; the expression of CYP1A1/2 heterogeneous nuclear RNA was induced by tanshinone treatment; and CYP1A1 mRNA stability was not influenced by these tanshinones. Interestingly, tanshinones plus B[a]P produced additive/synergistic effect on CYP1A1/2 induction. In addition, the tanshinone-induced CYP1A1/2 expression was abolished by the aryl hydrocarbon receptor (AhR) antagonist resveratrol, suggesting an AhR dependent transcription mechanism. In the reporter gene assay, while TI and DHTI significantly induced AhR-dependent luciferase activity, TIIA and CTS failed to induce this activity. Collectively, the tanshinones could induce CYP1A1 and CYP1A2 expression through transcriptional activation mechanism and exert differential effects on activating AhR in HepG2 cells. Our findings suggest that rational administration of tanshinones should be considered with respect to their effect on AhR and CYP1A1/2 expression.

  13. Capable Reader Program: Lesson Plan Guide. Units A1; A2; A3; A4; [and] A5. Pilot Year 1979-1980, Final Edition 1980-1981.

    ERIC Educational Resources Information Center

    Casper, Donna; And Others

    Part of a curriculum series for academically gifted elementary students in the area of reading, the five lesson plan guides are intended to provide teachers with suggested activities stressing high levels of reading comprehension as well as encouraging teachers to use their own ideas. Each guide focuses on one of the following major objectives:…

  14. Operation JANGLE. Particle Studies. Projects 2.5a-1, 2.5a-2, 2.5a-3, 2. 8,

    DTIC Science & Technology

    1979-10-01

    stopping at half centimater intervals beginning 3 cm before, and 5 ca after departure of the center point of the standard from the central axis of the...of the station layout. The three papere from each of these tio sets were radioautor&phed for 17 days beginning February 4, 19520 in an attempt to...the disk and one-quarter its ridth. This opening is adjustable over a small range In aziuuthal position so that collection r-n start at the beginning

  15. Capable Reader Program: Lesson Plan Guide. Units A1; A2; A3; A4; [and] A5. Pilot Year 1979-1980, Final Edition 1980-1981.

    ERIC Educational Resources Information Center

    Casper, Donna; And Others

    Part of a curriculum series for academically gifted elementary students in the area of reading, the five lesson plan guides are intended to provide teachers with suggested activities stressing high levels of reading comprehension as well as encouraging teachers to use their own ideas. Each guide focuses on one of the following major objectives:…

  16. Modulation of CYP1A1 and CYP1A2 hepatic enzymes after oral administration of Chios mastic gum to male Wistar rats.

    PubMed

    Katsanou, Efrosini S; Kyriakopoulou, Katerina; Emmanouil, Christina; Fokialakis, Nikolas; Skaltsounis, Alexios-Leandros; Machera, Kyriaki

    2014-01-01

    Chios mastic gum (CMG), a resin derived from Pistacia lentiscus var. chia, is known since ancient times for its pharmacological activities. CYP1A1 and CYP1A2 enzymes are among the most involved in the biotransformation of chemicals and the metabolic activation of pro-carcinogens. Previous studies referring to the modulation of these enzymes by CMG have revealed findings of unclear biological and toxicological significance. For this purpose, the modulation of CYP1A1 and CYP1A2 enzymes in the liver of male Wistar rats following oral administration of CMG extract (CMGE), at the levels of mRNA and CYP1A1 enzyme activity, was compared to respective enzyme modulation following oral administration of a well-known bioactive natural product, caffeine, as control compound known to involve hepatic enzymes in its metabolism. mRNA levels of Cyp1a1 and Cyp1a2 were measured by reverse transcription real-time polymerase chain reaction and their relative quantification was calculated. CYP1A1 enzyme induction was measured through the activity of ethoxyresorufin-O-deethylase (EROD). The results indicated that administration of CMGE at the recommended pharmaceutical dose does not induce significant transcriptional modulation of Cyp1a1/2 and subsequent enzyme activity induction of CYP1A1 while effects of the same order of magnitude were observed in the same test system following the administration of caffeine at the mean daily consumed levels. The outcome of this study further confirms the lack of any toxicological or biological significance of the specific findings on liver following the administration of CMGE.

  17. Modulation of Ca2+-currents by sequential and simultaneous activation of adenosine A1 and A 2A receptors in striatal projection neurons.

    PubMed

    Hernández-González, O; Hernández-Flores, T; Prieto, G A; Pérez-Burgos, A; Arias-García, M A; Galarraga, E; Bargas, J

    2014-01-01

    D(1)- and D(2)-types of dopamine receptors are located separately in direct and indirect pathway striatal projection neurons (dSPNs and iSPNs). In comparison, adenosine A(1)-type receptors are located in both neuron classes, and adenosine A(2A)-type receptors show a preferential expression in iSPNs. Due to their importance for neuronal excitability, Ca(2+)-currents have been used as final effectors to see the function of signaling cascades associated with different G protein-coupled receptors. For example, among many other actions, D(1)-type receptors increase, while D(2)-type receptors decrease neuronal excitability by either enhancing or reducing, respectively, CaV1 Ca(2+)-currents. These actions occur separately in dSPNs and iSPNs. In the case of purinergic signaling, the actions of A(1)- and A(2A)-receptors have not been compared observing their actions on Ca(2+)-channels of SPNs as final effectors. Our hypotheses are that modulation of Ca(2+)-currents by A(1)-receptors occurs in both dSPNs and iSPNs. In contrast, iSPNs would exhibit modulation by both A(1)- and A2A-receptors. We demonstrate that A(1)-type receptors reduced Ca(2+)-currents in all SPNs tested. However, A(2A)-type receptors enhanced Ca(2+)-currents only in half tested neurons. Intriguingly, to observe the actions of A(2A)-type receptors, occupation of A(1)-type receptors had to occur first. However, A(1)-receptors decreased Ca(V)2 Ca(2+)-currents, while A(2A)-type receptors enhanced current through Ca(V)1 channels. Because these channels have opposing actions on cell discharge, these differences explain in part why iSPNs may be more excitable than dSPNs. It is demonstrated that intrinsic voltage-gated currents expressed in SPNs are effectors of purinergic signaling that therefore play a role in excitability.

  18. Regulation of intestinal hPepT1 (SLC15A1) activity by phosphodiesterase inhibitors is via inhibition of NHE3 (SLC9A3)

    PubMed Central

    Anderson, Catriona M.H.; Thwaites, David T.

    2007-01-01

    The H+-coupled transporter hPepT1 (SLC15A1) mediates the transport of di/tripeptides and many orally-active drugs across the brush-border membrane of the small intestinal epithelium. Incubation of Caco-2 cell monolayers (15 min) with the dietary phosphodiesterase inhibitors caffeine and theophylline inhibited Gly–Sar uptake across the apical membrane. Pentoxifylline, a phosphodiesterase inhibitor given orally to treat intermittent claudication, also decreased Gly–Sar uptake through a reduction in capacity (Vmax) without any effect on affinity (Km). The reduction in dipeptide transport was dependent upon both extracellular Na+ and apical pH but was not observed in the presence of the selective Na+/H+ exchanger NHE3 (SLC9A3) inhibitor S1611. Measurement of intracellular pH confirmed that caffeine was not directly inhibiting hPepT1 but rather having an indirect effect through inhibition of NHE3 activity. NHE3 maintains the H+-electrochemical gradient which, in turn, acts as the driving force for H+-coupled solute transport. Uptake of β-alanine, a substrate for the H+-coupled amino acid transporter hPAT1 (SLC36A1), was also inhibited by caffeine. The regulation of NHE3 by non-nutrient components of diet or orally-delivered drugs may alter the function of any solute carrier dependent upon the H+-electrochemical gradient and may, therefore, be a site for both nutrient–drug and drug–drug interactions in the small intestine. PMID:17498647

  19. A new CYP21A1P/CYP21A2 chimeric gene identified in an Italian woman suffering from classical congenital adrenal hyperplasia form

    PubMed Central

    Concolino, Paola; Mello, Enrica; Minucci, Angelo; Giardina, Emiliano; Zuppi, Cecilia; Toscano, Vincenzo; Capoluongo, Ettore

    2009-01-01

    Background More than 90% of Congenital Adrenal Hyperplasia (CAH) cases are associated with mutations in the 21-hydroxylase gene (CYP21A2) in the HLA class III area on the short arm of chromosome 6p21.3. In this region, a 30 kb deletion produces a non functional chimeric gene with its 5' and 3' ends corresponding to CYP21A1P pseudogene and CYP21A2, respectively. To date, five different CYP21A1P/CYP21A2 chimeric genes have been found and characterized in recent studies. In this paper, we describe a new CYP21A1P/CYP21A2 chimera (CH-6) found in an Italian CAH patient. Methods Southern blot analysis and CYP21A2 sequencing were performed on the patient. In addition, in order to isolate the new CH-6 chimeric gene, two different strategies were used. Results The CYP21A2 sequencing analysis showed that the patient was homozygote for the g.655C/A>G mutation and heterozygote for the p.P30L missense mutation. In addition, the promoter sequence revealed the presence, in heterozygosis, of 13 SNPs generally produced by microconversion events between gene and pseudogene. Southern blot analysis showed that the woman was heterozygote for the classic 30-kb deletion producing a new CYP21A1P/CYP21A2 chimeric gene (CH-6). The hybrid junction site was located between the end of intron 2 pseudogene, after the g.656C/A>G mutation, and the beginning of exon 3, before the 8 bp deletion. Consequently, CH-6 carries three mutations: the weak pseudogene promoter region, the p.P30L and the g.655C/A>G splice mutation. Conclusion We describe a new CYP21A1P/CYP21A2 chimera (CH-6), associated with the HLA-B15, DR13 haplotype, in a young Italian CAH patient. PMID:19624807

  20. On the dissociation of I2 by O2(a1Delta): Pathways involving the excited species I2(A'3Pi2u,A3Pi(1u)), I2(X1sigma,upsilon), and O2(a1Delta,upsilon).

    PubMed

    Azyazov, V N; Pichugin, S Yu; Heaven, Michael C

    2009-03-14

    Kinetic studies were carried out to explore the role of the excited species I(2)(A(') (3)Pi(2u),A (3)Pi(1u)), I(2)(X (1) summation operator,upsilon), and O(2)(a (1)Delta,upsilon) in the dissociation of I(2) by singlet oxygen. A flow tube apparatus that utilized a chemical singlet oxygen generator was used to measure the I(2) dissociation rate in O(2)(a (1)Delta)/I(2) mixtures. Vibrationally excited I(2)(X) is thought to be a significant intermediate in the dissociation process. Excitation probabilities (gamma(upsilon)) for population of the upsilonth I(2)(X) vibrational level in the reaction I(2)(X)+I((2)P(1/2))-->I(2)(X,upsilon>10)+I((2)P(3/2)) were estimated based on a comparison of calculated populations with experimentally determined values. Satisfactory agreement with the experimental data [Barnault et al., J. Phys. IV 1, C7/647 (1991)] was achieved for total excitation probabilities partitioned in two ranges, such that Gamma(25A(') (3)Pi(2u),A (3)Pi(1u)) and I(2)(X,upsilon). It was shown that the iodine dissociation process passes predominantly through the I(2)(A(') (3)Pi(2u),A (3)Pi(1u)) intermediate. These states are populated by collisions of I(2) with vibrationally excited O(2)(a (1)Delta,upsilon) at the initiation and the chain stages, when the mole fraction of I(2) is small (eta(I(2) )<1%). For higher I(2) concentrations (eta(I(2) )>/=1%) the excited states are populated in the chain stage by collisions of I(2)(X,15a (1)Delta).

  1. Inheritance and molecular mapping of Rf6 locus with pollen fertility restoration ability on A1 and A2 cytoplasms in sorghum.

    PubMed

    Praveen, M; Anurag Uttam, G; Suneetha, N; Umakanth, Av; Patil, J V; Madhusudhana, R

    2015-09-01

    Of the several male sterility cytoplasms available as an alternative to the widely exploited A1 (milo) cytoplasm in sorghum, A2 is more suitable for commercial exploitation. Diversification of genetic and cytoplasmic base of hybrids involving A2 cytoplasm necessitates mapping of fertility restorer (Rf) genes for use in marker-assisted restorer development. We mapped a major male fertility restoration locus on sorghum chromosome 4 tightly linked with SSR markers, SB2387 and SB2388. This new fertility locus, Rf6, was able to restore male fertility on both A1 and A2 cytoplasms. Analysis of the genomic region around the Rf6 locus identified six genes including a pentatricopeptide repeat (PPR) gene, Sobic.004G004100. With its similar restoration ability to Rf1, Rf2 and Rf5 loci in sorghum, it is most likely that the Rf6 is a member of the PPR gene family, and the PPR gene Sobic.004G004100 could be a candidate for fertility restoration on A1 and A2 cytoplasms.

  2. The a 1Δg(a2 g)→X 3Σ-g(X 21 g) transition of Se 2

    NASA Astrophysics Data System (ADS)

    Setzer, K. D.; Dorn, A.; Lorenz, M.; Fink, E. H.

    2003-09-01

    Emission spectra of the 0-0 and 1-1 bands of the a 1Δg(a2 g)→X 3Σ-g(X 21 g) magnetic dipole transition of Se 2 have been observed in the near-infrared spectral region near 3780 cm -1. The Se 2 molecules were generated in a fast-flow system by passing Se x vapor in Ar carrier gas through a microwave discharge and were excited by electronic-to-electronic energy transfer from metastable singlet oxygen O2(a 1Δg) . Medium-resolution spectra of the b 1Σ+g(b0 +g)→X 3Σ-g(X 21 g) and a 1Δg(a2 g)→X 3Σ-g(X 21 g) transitions were measured with a Fourier-transform spectrometer. By comparing the band shape of the 0-0 band of the a→ X2 system with computer simulations, the following spectroscopic constants of the a 1Δg(a2 g) state of 80Se 2 were derived (cm -1): Te=4303.7(1), B0=0.08888(5), and Δ G1/2=369.6(2), where the numbers in parentheses are estimated error limits of the parameters in units of the last digit.

  3. Butyric acid increases transepithelial transport of ferulic acid through upregulation of the monocarboxylate transporters SLC16A1 (MCT1) and SLC16A3 (MCT4).

    PubMed

    Ziegler, Kerstin; Kerimi, Asimina; Poquet, Laure; Williamson, Gary

    2016-06-01

    Ferulic acid is released by microbial hydrolysis in the colon, where butyric acid, a major by-product of fermentation, constitutes the main energy source for colonic enterocytes. We investigated how varying concentrations of this short chain fatty acid may influence the absorption of the phenolic acid. Chronic treatment of Caco-2 cells with butyric acid resulted in increased mRNA and protein abundance of the monocarboxylate transporters SLC16A1 (MCT1) and SLC16A3 (MCT4), previously proposed to facilitate ferulic acid absorption in addition to passive diffusion. Short term incubation with butyric acid only led to upregulation of MCT4 while both conditions increased transepithelial transport of ferulic acid in the apical to basolateral, but not basolateral to apical, direction. Chronic treatment also elevated intracellular concentrations of ferulic acid, which in turn gave rise to increased concentrations of ferulic acid metabolites. Immunofluorescence staining of cells revealed uniform distribution of MCT1 protein in the cell membrane, whereas MCT4 was only detected in the lateral plasma membrane sections of Caco-2 cells. We therefore propose that MCT1 may be acting as an uptake transporter and MCT4 as an efflux system across the basolateral membrane for ferulic acid, and that this process is stimulated by butyric acid.

  4. Downregulation of A(1) and A(2B) adenosine receptors in human trisomy 21 mesenchymal cells from first-trimester chorionic villi.

    PubMed

    Gessi, Stefania; Merighi, Stefania; Stefanelli, Angela; Mirandola, Prisco; Bonfatti, Alessandra; Fini, Sergio; Sensi, Alberto; Marci, Roberto; Varani, Katia; Borea, Pier Andrea; Vesce, Fortunato

    2012-11-01

    Human reproduction is complex and prone to failure. Though causes of miscarriage remain unclear, adenosine, a proangiogenic nucleoside, may help determine pregnancy outcome. Although adenosine receptor (AR) expression has been characterized in euploid pregnancies, no information is available for aneuploidies, which, as prone to spontaneous abortion (SA), are a potential model for shedding light on the mechanism regulating this event. AR expression was investigated in 71 first-trimester chorionic villi (CV) samples and cultured mesenchymal cells (MC) from euploid and TR21 pregnancies, one of the most frequent autosomal aneuploidy, with a view to elucidating their potential role in the modulation of vascular endothelial growth factor (VEGF) and nitric oxide (NO). Compared to euploid cells, reduced A(1) and A(2B) expression was revealed in TR21 CV and MCs. The non-selective adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased NO, by activating, predominantly, A(1)AR and A(2A)AR through a molecular pathway involving hypoxia-inducible-factor-1 (HIF-1α), and increased VEGF, mainly through A(2B). In conclusion the adenosine transduction cascade appears to be disturbed in TR21 through reduced expression of A(2B) and A(1)ARs. These anomalies may be implicated in complications such as fetal growth restriction, malformation and/or SA, well known features of aneuploid pregnancies. Therefore A(1) and A(2B)ARs could be potential biomarkers able to provide an early indication of SA risk and their stimulation may turn out to improve fetoplacental perfusion by increasing NO and VEGF.

  5. Involvement of adenosine A1 and A2A receptors in the adenosinergic modulation of the discriminative-stimulus effects of cocaine and methamphetamine in rats.

    PubMed

    Justinova, Zuzana; Ferre, Sergi; Segal, Pavan N; Antoniou, Katerina; Solinas, Marcello; Pappas, Lara A; Highkin, Jena L; Hockemeyer, Jorg; Munzar, Patrik; Goldberg, Steven R

    2003-12-01

    Adenosine, by acting on adenosine A1 and A2A receptors, is known to antagonistically modulate dopaminergic neurotransmission. We have recently reported that nonselective adenosine receptor antagonists (caffeine and 3,7-dimethyl-1-propargylxanthine) can partially substitute for the discriminative-stimulus effects of methamphetamine. In the present study, by using more selective compounds, we investigated the involvement of A1 and A2A receptors in the adenosinergic modulation of the discriminative-stimulus effects of both cocaine and methamphetamine. The effects of the A1 receptor agonist N6-cyclopentyladenosine (CPA; 0.01-0.1 mg/kg) and antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 1.3-23.7 mg/kg) and the A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680; 0.03-0.18 mg/kg) and antagonist 3-(3-hydroxypropyl)-8-(3-methoxystyryl)-7-methyl-1-propargylxanthin phosphate disodium salt (MSX-3; 1-56 mg/kg) were evaluated in rats trained to discriminate either 1 mg/kg methamphetamine or 10 mg/kg cocaine from saline under a fixed-ratio 10 schedule of food presentation. The A1 and A2A receptor antagonists (CPT and MSX-3) both produced high levels of drug-lever selection when substituted for either methamphetamine or cocaine and significantly shifted dose-response curves of both psychostimulants to the left. Unexpectedly, the A2A receptor agonist CGS 21680 also produced drug-appropriate responding (although at lower levels) when substituted for the cocaine-training stimulus, and both CGS 21680 and the A1 receptor agonist CPA significantly shifted the cocaine dose-response curve to the left. In contrast, both agonists did not produce significant levels of drug-lever selection when substituted for the methamphetamine-training stimulus and failed to shift the methamphetamine dose-response curve. Therefore, adenosine A1 and A2A receptors appear to play important but differential roles in the modulation of the

  6. Utilization of LAPAN Satellite (TUBSAT, A2, and A3) in supporting Indonesia’s potential as maritime center of the world

    NASA Astrophysics Data System (ADS)

    Julzarika, A.

    2017-01-01

    Indonesia has archipelago area of 2.8 million km2, territorial sea area of 0.4 km2. Indonesia have number of 13.466 islands. Coastline length of Indonesia reached 99.093 km2. Large areas can be monitored using remote sensing technology. Currently, Indonesia have research remote sensing satellites, namely LAPAN TUBSAT, LAPAN A2, LISAT (A3). All of these satellites could be used to monitor Indonesia. These satellites can be used to make the DSM using videogrammetry and depth cue perceptive methods. They also can be used for identification of geobiophysic parameter. Indonesian maritime territory which has sea highway planning can also be monitored using this satellites combination. AIS sensor on LAPAN A2 can be used to identify ships that pass in the territorial waters of Indonesia. It diagonally across the Indonesian region of west to east as much as 14 times a day. At this point it will have detection radius of over 100 km and has the ability to receive signals from maximum of 2000 vessels in the coverage area. Utilization of this satellites is expected to be helpful in supporting the ships cruise monitoring and their support sea highway also in making Indonesia as maritime center of the world.

  7. Integrated Advanced Microwave Sounding Unit-A (AMSU-A). Performance Verification Report: EOS AMSU-A1 and AMSU-A2 Receiver Assemblies

    NASA Technical Reports Server (NTRS)

    Ma, Y.

    1995-01-01

    The AMSU-A receiver subsystem comprises two separated receiver assemblies; AMSU-A1 and AMSU-A2 (P/N 1356441-1). The AMSU-A1 receiver contains 13 channels and the AMSU-A2 receiver 2 channels. The AMSU-A1 receiver assembly is further divided into two parts; AMSU-A1-1 (P/N 1356429-1) and AMSU-A1-2 (P/N 1356409-1), which contain 9 and 4 channels, respectively. The receiver assemblies are highlighted and illustrate the functional block diagrams of the AMSU-A1 and AMSU-A2 systems. The AMSU-A receiver subsystem stands in between the antenna and signal processing subsystems of the AMSU-A instrument and comprises the RF and IF components from isolators to attenuators. It receives the RF signals from the antenna subsystem, down-converts the RF signals to IF signals, amplifies and defines the IF signals to proper power level and frequency bandwidth as specified for each channel, and inputs the IF signals to the signal processing subsystem. This test report presents the test data of the EOS AMSU-A Flight Model No. 1 (FM-1) receiver subsystem. The tests are performed per the Acceptance Test Procedure for the AMSU-A Receiver Subsystem, AE-26002/6A. The functional performance tests are conducted either at the component or subsystem level. While the component-level tests are performed over the entire operating temperature range predicted by thermal analysis, the subsystem-level tests are conducted at ambient temperature only.

  8. Vitamin-D receptor agonist calcitriol reduces calcification in vitro through selective upregulation of SLC20A2 but not SLC20A1 or XPR1

    PubMed Central

    Keasey, M. P.; Lemos, R. R.; Hagg, T.; Oliveira, J. R. M.

    2016-01-01

    Vitamin D deficiency (hypovitaminosis D) causes osteomalacia and poor long bone mineralization. In apparent contrast, hypovitaminosis D has been reported in patients with primary brain calcifications (“Fahr’s disease”). We evaluated the expression of two phosphate transporters which we have found to be associated with primary brain calcification (SLC20A2, whose promoter has a predicted vitamin D receptor binding site, and XPR1), and one unassociated (SLC20A1), in an in vitro model of calcification. Expression of all three genes was significantly decreased in calcifying human bone osteosarcoma (SaOs-2) cells. Further, we confirmed that vitamin D (calcitriol) reduced calcification as measured by Alizarin Red staining. Cells incubated with calcitriol under calcifying conditions specifically maintained expression of the phosphate transporter SLC20A2 at higher levels relative to controls, by RT-qPCR. Neither SLC20A1 nor XPR1 were affected by calcitriol treatment and remained suppressed. Critically, knockdown of SLC20A2 gene and protein with CRISPR technology in SaOs2 cells significantly ablated vitamin D mediated inhibition of calcification. This study elucidates the mechanistic importance of SLC20A2 in suppressing the calcification process. It also suggests that vitamin D might be used to regulate SLC20A2 gene expression, as well as reduce brain calcification which occurs in Fahr’s disease and normal aging. PMID:27184385

  9. Hemizygous deletion of COL3A1, COL5A2, and MSTN causes a complex phenotype with aortic dissection: a lesson for and from true haploinsufficiency

    PubMed Central

    Meienberg, Janine; Rohrbach, Marianne; Neuenschwander, Stefan; Spanaus, Katharina; Giunta, Cecilia; Alonso, Sira; Arnold, Eliane; Henggeler, Caroline; Regenass, Stephan; Patrignani, Andrea; Azzarello-Burri, Silvia; Steiner, Bernhard; Nygren, Anders OH; Carrel, Thierry; Steinmann, Beat; Mátyás, Gábor

    2010-01-01

    Aortic dilatation/dissection (AD) can occur spontaneously or in association with genetic syndromes, such as Marfan syndrome (MFS; caused by FBN1 mutations), MFS type 2 and Loeys–Dietz syndrome (associated with TGFBR1/TGFBR2 mutations), and Ehlers–Danlos syndrome (EDS) vascular type (caused by COL3A1 mutations). Although mutations in FBN1 and TGFBR1/TGFBR2 account for the majority of AD cases referred to us for molecular genetic testing, we have obtained negative results for these genes in a large cohort of AD patients, suggesting the involvement of additional genes or acquired factors. In this study we assessed the effect of COL3A1 deletions/duplications in this cohort. Multiplex ligation-dependent probe amplification (MLPA) analysis of 100 unrelated patients identified one hemizygous deletion of the entire COL3A1 gene. Subsequent microarray analyses and sequencing of breakpoints revealed the deletion size of 3 408 306 bp at 2q32.1q32.3. This deletion affects not only COL3A1 but also 21 other known genes (GULP1, DIRC1, COL5A2, WDR75, SLC40A1, ASNSD1, ANKAR, OSGEPL1, ORMDL1, LOC100129592, PMS1, MSTN, C2orf88, HIBCH, INPP1, MFSD6, TMEM194B, NAB1, GLS, STAT1, and STAT4), mutations in three of which (COL5A2, SLC40A1, and MSTN) have also been associated with an autosomal dominant disorder (EDS classical type, hemochromatosis type 4, and muscle hypertrophy). Physical and laboratory examinations revealed that true haploinsufficiency of COL3A1, COL5A2, and MSTN, but not that of SLC40A1, leads to a clinical phenotype. Our data not only emphasize the impact/role of COL3A1 in AD patients but also extend the molecular etiology of several disorders by providing hitherto unreported evidence for true haploinsufficiency of the underlying gene. PMID:20648054

  10. Phellinins A1 and A2, new styrylpyrones from the culture broth of Phellinus sp. KACC93057P: I. Fermentation, taxonomy, isolation and biological properties.

    PubMed

    Lee, In-Kyoung; Seo, Geon-Sik; Jeon, Nak Beom; Kang, Hee-Wan; Yun, Bong-Sik

    2009-11-01

    Novel styrylpyrones, phellinins A1 and A2, were isolated together with known styrylpyrone compounds, hispidin and 1,1-distyrylpyrylethan, from the cultured broth of Phellinus sp. KACC93057P. These compounds were purified by solvent partition, Sephadex LH-20 column chromatography, C(18)-solid phase extraction and finally by reversed-phase (ODS) TLC. To identify the phellinin producer Phellinus sp. KACC93057P, the ribosomal DNA (rDNA) internal transcribed space regions containing 5.8 rDNA were sequenced and compared with those of the known Phellinus isolates. Phellinus sp. KACC93057P was 94.8% identical to P. baumii and P. linteus, all of which did not produce phellinins A1 and A2. These compounds significantly scavenged free radicals such as 1,1-diphenyl-2-picrylhydrazyl, 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) and superoxide.

  11. Highly Efficient Inverted D:A1:A2 Ternary Blend Organic Photovoltaics Combining a Ladder-type Non-Fullerene Acceptor and a Fullerene Acceptor.

    PubMed

    Chang, Shao-Ling; Cao, Fong-Yi; Huang, Wen-Chia; Huang, Po-Kai; Hsu, Chain-Shu; Cheng, Yen-Ju

    2017-07-26

    A formylated benzodi(cyclopentadithiophene) (BDCPDT) ladder-type structure with forced coplanarity is coupled with two 1,1-dicyanomethylene-3-indanone (IC) moieties via olefination to form a non-fullerene acceptor, BDCPDT-IC. The BDCPDT-IC, as an acceptor (A1) with broad light-absorbing ability and excellent solution processability, is combined with a second PC71BM acceptor (A2) and a medium band gap polymer, PBDB-T, as the donor (D) to form a ternary blend with gradient HOMO/LUMO energy alignments and panchromatic absorption. The device with the inverted architecture using the D:A1:A2 ternary blend has achieved a highest efficiency of 9.79% with a superior Jsc of 16.84 mA cm(-2).

  12. CYP1A1 and CYP1A2 expression: Comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

    SciTech Connect

    Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji; Tateno, Chise; Makishima, Makoto; Yoshizato, Katsutoshi; Nebert, Daniel W.

    2009-05-15

    Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how 'human-like' can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1{sub C}YP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+){sub s}evere-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.

  13. PacCYP707A2 negatively regulates cherry fruit ripening while PacCYP707A1 mediates drought tolerance.

    PubMed

    Li, Qian; Chen, Pei; Dai, Shengjie; Sun, Yufei; Yuan, Bing; Kai, Wenbin; Pei, Yuelin; He, Suihuan; Liang, Bin; Zhang, Yushu; Leng, Ping

    2015-07-01

    Sweet cherry is a non-climacteric fruit and its ripening is regulated by abscisic acid (ABA) during fruit development. In this study, four cDNAs (PacCYP707A1-4) encoding 8'-hydroxylase, a key enzyme in the oxidative catabolism of ABA, were identified in sweet cherry fruits using tobacco rattle virus-induced gene silencing (VIGS) and particle bombardment approaches. Quantitative real-time PCR confirmed significant down-regulation of target gene transcripts in VIGS-treated cherry fruits. In PacCYP707A2-RNAi-treated fruits, ripening and fruit colouring were promoted relative to control fruits, and both ABA accumulation and PacNCED1 transcript levels were up-regulated by 140%. Silencing of PacCYP707A2 by VIGS significantly altered the transcripts of both ABA-responsive and ripening-related genes, including the ABA metabolism-associated genes NCED and CYP707A, the anthocyanin synthesis genes PacCHS, PacCHI, PacF3H, PacDFR, PacANS, and PacUFGT, the ethylene biosynthesis gene PacACO1, and the transcription factor PacMYBA. The promoter of PacMYBA responded more strongly to PacCYP707A2-RNAi-treated fruits than to PacCYP707A1-RNAi-treated fruits. By contrast, silencing of PacCYP707A1 stimulated a slight increase in fruit colouring and enhanced resistance to dehydration stress compared with control fruits. These results suggest that PacCYP707A2 is a key regulator of ABA catabolism that functions as a negative regulator of fruit ripening, while PacCYP707A1 regulates ABA content in response to dehydration during fruit development. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  14. Functional analysis of Arabidopsis CYP714A1 and CYP714A2 reveals that they are distinct gibberellin modification enzymes.

    PubMed

    Nomura, Takahito; Magome, Hiroshi; Hanada, Atsushi; Takeda-Kamiya, Noriko; Mander, Lewis N; Kamiya, Yuji; Yamaguchi, Shinjiro

    2013-11-01

    Endogenous levels of bioactive gibberellins (GAs) are controlled by both biosynthetic and inactivation processes, and some cytochrome P450s are involved in this control mechanism. We have previously reported that CYP714B1 and CYP714B2 encode the enzyme GA 13-oxidase, which is required for GA1 biosynthesis, and that CYP714D1 encodes GA 16α,17-epoxidase, which inactivates the non-13-hydroxy GAs in rice. Arabidopsis has two CYP714 members, CYP714A1 and CYP714A2. To clarify the possible role of these genes in GA metabolism, enzymatic activities of their recombinant proteins were analyzed using a yeast expression system. We found that the recombinant CYP714A1 protein catalyzes the conversion of GA12 to 16-carboxylated GA12 (16-carboxy-16β,17-dihydro GA12), a previously unidentified GA metabolite. Bioassays of this GA product showed that CYP714A1 is an inactivation enzyme in Arabidopsis. This was confirmed by the extreme GA-deficient dwarf phenotype shown by CYP714A1-overexpressing plants. Intriguingly, the recombinant CYP714A2 protein catalyzed the conversion of ent-kaurenoic acid into steviol (ent-13-hydroxy kaurenoic acid). When GA12 was used as a substrate for CYP714A2, 12α-hydroxy GA12 (GA111) was produced as a major product and 13-hydroxy GA12 (GA53) as a minor product. Transgenic Arabidopsis plants overexpressing the CYP714A2 gene showed semi-dwarfism. GA analysis showed that the levels of non-13-hydroxy GAs, including GA4, were decreased, whereas those of 13-hydroxy GAs, including GA1 (which is less active than GA4), were increased in the transgenic plants. Our results suggest that the CYP714 family proteins contribute to the production of diverse GA compounds through various oxidations of C and D rings in both monocots and eudicots.

  15. Dual A1/A2B Receptor Blockade Improves Cardiac and Renal Outcomes in a Rat Model of Heart Failure with Preserved Ejection Fraction

    PubMed Central

    Tofovic, Stevan P.; Salah, Eman M.; Smits, Glenn J.; Whalley, Eric T.; Ticho, Barry; Deykin, Aaron

    2016-01-01

    Heart failure with preserved ejection fraction (HFpEF) is prevalent and often accompanied by metabolic syndrome. Current treatment options are limited. Here, we test the hypothesis that combined A1/A2B adenosine receptor blockade is beneficial in obese ZSF1 rats, an animal model of HFpEF with metabolic syndrome. The combined A1/A2B receptor antagonist 3-[4-(2,6-dioxo-1,3-dipropyl-7H-purin-8-yl)-1-bicyclo[2.2.2]octanyl]propanoic acid (BG9928) was administered orally (10 mg/kg/day) to obese ZSF1 rats (n = 10) for 24 weeks (from 20 to 44 weeks of age). Untreated ZSF1 rats (n = 9) served as controls. After 24 weeks of administration, BG9928 significantly lowered plasma triglycerides (in mg/dl: control group, 4351 ± 550; BG9928 group, 2900 ± 551) without adversely affecting plasma cholesterol or activating renin release. BG9928 significantly decreased 24-hour urinary glucose excretion (in mg/kg/day: control group, 823 ± 179; BG9928 group, 196 ± 80) and improved oral glucose tolerance, polydipsia, and polyuria. BG9928 significantly augmented left ventricular diastolic function in association with a reduction in cardiac vasculitis and cardiac necrosis. BG9928 significantly reduced 24-hour urinary protein excretion (in mg/kg/day: control group, 1702 ± 263; BG9928 group, 1076 ± 238), and this was associated with a reduction in focal segmental glomerulosclerosis, tubular atrophy, tubular dilation, and deposition of proteinaceous material in the tubules. These findings show that, in a model of HFpEF with metabolic syndrome, A1/A2B receptor inhibition improves hyperlipidemia, exerts antidiabetic actions, reduces HFpEF, improves cardiac histopathology, and affords renal protection. We conclude that chronic administration of combined A1/A2B receptor antagonists could be beneficial in patients with HFpEF, in particular those with comorbidities such as obesity, diabetes, and dyslipidemias. PMID:26585572

  16. Expression of the LspA1 and LspA2 proteins by Haemophilus ducreyi is required for virulence in human volunteers.

    PubMed

    Janowicz, Diane M; Fortney, Kate R; Katz, Barry P; Latimer, Jo L; Deng, Kaiping; Hansen, Eric J; Spinola, Stanley M

    2004-08-01

    Haemophilus ducreyi colocalizes with polymorphonuclear leukocytes and macrophages and evades phagocytosis during experimental infection of human volunteers. H. ducreyi contains two genes, lspA1 and lspA2, which encode predicted proteins of 456 and 543 kDa, respectively. Compared to its wild-type parent, an lspA1 lspA2 double mutant does not inhibit phagocytosis by macrophage and myelocytic cell lines in vitro and is attenuated in an experimental rabbit model of chancroid. To test whether expression of LspA1 and LspA2 was necessary for virulence in humans, six volunteers were experimentally infected. Each volunteer was inoculated with three doses (ranging from 85 to 112 CFU) of the parent (35000HP) in one arm and three doses (ranging from 60 to 822 CFU) of the mutant (35000HP Omega 12) in the other arm. The papule formation rates were 88% (95% confidence interval [95% CI], 76.8 to 99.9%) at 18 parent sites and 72% (95% CI, 44.4 to 99.9%) at 18 mutant sites (P = 0.19). However, papules were significantly smaller at mutant sites (mean size, 24.8 mm(2)) than at parent sites (mean size, 39.1 mm(2)) 24 h after inoculation (P = 0.0002). The pustule formation rates were 44% (95% CI, 5.8 to 77.6%) at parent sites and 0% (95% CI, 0 to 39.4%) at mutant sites (P = 0.009). With the caveat that biosafety regulations preclude testing of a complemented mutant in human subjects, these results indicate that expression of LspA1 and LspA2 facilitates the ability of H. ducreyi to initiate disease and to progress to pustule formation in humans.

  17. Phenotype of the Cyp1a1/1a2/1b1(−/−) Triple-Knockout Mouse*

    PubMed Central

    Dragin, Nadine; Shi, Zhanquan; Madan, Rajat; Karp, Christopher L.; Sartor, Maureen A.; Chen, Chi; Gonzalez, Frank J.; Nebert, Daniel W.

    2009-01-01

    Crossing the Cyp1a1/1a2(−/−) double-knockout mouse with the Cyp1b1(−/−) single-knockout mouse, we generated the Cyp1a1/1a2/1b1(−/−) triple-knockout mouse. In this triple-knockout mouse, statistically significant phenotypes (with incomplete penetrance) included slower weight gain and greater risk of embryolethality before gestational day 11, hydrocephalus, hermaphroditism, and cystic ovaries. Oral benzo[a]pyrene (BaP) daily for 18 days in the Cyp1a1/1a2(−/−) produced the same degree of marked immunosuppression as seen in the Cyp1a1(−/−) mouse; we believe this reflects the absence of intestinal CYP1A1. Oral BaP-treated Cyp1a1/1a2/1b1(−/−) mice showed the same “rescued” response as that seen in the Cyp1a1/1b1(−/−) mouse; we believe this reflects the absence of CYP1B1 in immune tissues. Urinary metabolite profiles were dramatically different between untreated triple-knockout and wild-type; principal components analysis showed that the shifts in urinary metabolite patterns in oral BaP-treated triple-knockout and wild-type mice were also strikingly different. Liver microarray cDNA differential expression (comparing triple-knockout with wild-type) revealed at least 89 genes up- and 62 genes down-regulated (P-value ≤0.00086). Gene Ontology “classes of genes” most perturbed in the untreated triple-knockout (compared with wild-type) include lipid, steroid, and cholesterol biosynthesis and metabolism; nucleosome and chromatin assembly; carboxylic and organic acid metabolism; metal-ion binding; and ion homeostasis. In the triple-knockout compared with the wild-type mice, response to zymosan-induced peritonitis was strikingly exaggerated, which may well reflect down-regulation of Socs2 expression. If a single common molecular pathway is responsible for all of these phenotypes, we suggest that functional effects of the loss of all three Cyp1 genes could be explained by perturbations in CYP1-mediated eicosanoid production, catabolism and

  18. Common and distinct functions of Arabidopsis class A1 and A2 heat shock factors in diverse abiotic stress responses and development.

    PubMed

    Liu, Hsiang-chin; Charng, Yee-yung

    2013-09-01

    There are 21 heat shock factor (HSF) homologs in Arabidopsis (Arabidopsis thaliana), of which members of class A1 (HSFA1a/HSFA1b/HSFA1d/HSFA1e) play the major role in activating the transcription of heat-induced genes, including HSFA2. Once induced, HSFA2 becomes the dominant HSF and is able to form heterooligomeric complexes with HSFA1. However, whether HSFA2 could function independently as a transcription regulator in the absence of the HSFA1s was undetermined. To address this question, we introduced a Cauliflower mosaic virus 35S promoter:HSFA2 construct into hsfa1a/hsfa1b/hsfa1d/hsfa1e quadruple knockout (QK) and wild-type (Wt) backgrounds to yield transgenic lines A2QK and A2Wt, respectively. Constitutive expression of HSFA2 rescued the developmental defects of the QK mutant and promoted callus formation in A2QK, but not in A2Wt, after heat treatment. Transcriptome analysis showed that heat stress response genes are differentially regulated by the HSFA1s and HSFA2; the genes involved in metabolism and redox homeostasis are preferentially regulated by HSFA2, while HSFA1-preferring genes are enriched in transcription function. Ectopic expression of HSFA2 complemented the defects of QK in tolerance to different heat stress regimes, and to hydrogen peroxide, but not to salt and osmotic stresses. Furthermore, we showed that HSFA1a/HSFA1b/HSFA1d are involved in thermotolerance to mild heat stress at temperatures as low as 27°C. We also noticed subfunctionalization of the four Arabidopsis A1-type HSFs in diverse abiotic stress responses. Overall, this study reveals the overlapping and distinct functions of class A1 and A2 HSFs and may enable more precise use of HSFs in engineering stress tolerance in the future.

  19. A correlated ab initio study of the X2A1 and A2E states of MgCH3

    NASA Technical Reports Server (NTRS)

    Woon, D. E.

    1996-01-01

    The X2A1 and A2E states of the MgCH3 radical have been studied with correlation consistent basis sets and the coupled cluster method RCCSD(T) in order to compare with two recent experimental efforts [M. A. Anderson and L. M. Ziurys, Astrophys. J. 452, L157 (1995); R. Rubino, J. M. Williamson, and T. A. Miller, J. Chem. Phys. 103, 5964 (1995)]. The best computed values [RCCSD(T)/cc-pCVTZ] for the X2A1 state are (experimental results in parentheses): Ae = 160.433 GHz, Be = 10.948 GHz (B0 = 11.008 GHz), and Mue = 1.011 D. The Mg-CH3 bond is weak, 26.3 kcal/mol. Values for the A2E state are Ae = 154.648 GHz (A0 = 149.666 GHz), Be = 10.87 GHz (B0 = 10.932 GHz), and Mue = 1.022 D. The excitation energy (Te) for the A2E <-- X2A1 transition is 19 999 cm-1 (T00 = 20 030 cm-1). A brief discussion of bonding trends in Mg-containing radials is included.

  20. Comparison of CYP106A1 and CYP106A2 from Bacillus megaterium - identification of a novel 11-oxidase activity.

    PubMed

    Kiss, Flora Marta; Schmitz, Daniela; Zapp, Josef; Dier, Tobias K F; Volmer, Dietrich A; Bernhardt, Rita

    2015-10-01

    The CYP106A subfamily hydroxylates steroids, diterpenes, and triterpenes in a regioselective and stereoselective manner, which is a challenging task for synthetic chemistry. The well-studied CYP106A2 enzyme, from the Bacillus megaterium strain ATCC 13368, is a highly promising candidate for the pharmaceutical industry. It shares 63 % amino acid sequence identity with CYP106A1 from B. megaterium DSM319, which was recently characterized. A focused steroid library was screened with both CYP106A1 and CYP106A2. Out of the 23 tested steroids, 19 were successfully converted by both enzymes during in vitro and in vivo reactions. Thirteen new substrates were identified for CYP106A1, while the substrate spectrum of CYP106A2 was extended by seven new members. Finally, six chosen steroids were further studied on a preparative scale employing a recombinant B. megaterium MS941 whole-cell system, yielding sufficient amounts of product for structure characterization by nuclear magnetic resonance. The hydroxylase activity was confirmed at positons 6β, 7β, 9α, and 15β. In addition, the CYP106A subfamily showed unprecedented 11-oxidase activity, converting 11β-hydroxysteroids to their 11-keto derivatives. This novel reaction and the diverse hydroxylation positions on pharmaceutically relevant compounds underline the role of the CYP106A subfamily in drug development and production.

  1. Refinement of the HIVAN1 Susceptibility Locus on Chr. 3A1-A3 via Generation of Sub-Congenic Strains.

    PubMed

    Papeta, Natalia; Patel, Ami; D'Agati, Vivette D; Gharavi, Ali G

    2016-01-01

    HIV-1 transgenic mice on the FVB/NJ background (TgFVB) represent a validated model of HIV-associated nephropathy (HIVAN). A major susceptibility locus, HIVAN1, was previously mapped to chromosome 3A1-A3 in a cross between TgFVB and CAST/EiJ (CAST) strains, and introgression of a 51.9 Mb segment encompassing HIVAN1 from CAST into TgFVB resulted in accelerated development of nephropathy. We generated three sub-congenic strains carrying CAST alleles in the proximal or distal regions of the HIVAN1 locus (Sub-II, 3.02-38.93 Mb; Sub-III, 38.45-55.1 Mb and Sub-IV, 47.7-55.1 Mb, build 38). At 5-10 weeks of age, histologic injury and proteinuria did not differ between HIV-1 transgenic Sub-II and TgFVB mice. In contrast, HIV-1 transgenic Sub-III and Sub-IV mice displayed up to 4.4 fold more histopathologic injury and 6-fold more albuminuria compared to TgFVB mice, similar in severity to the full-length congenic mice. The Sub-IV segment defines a maximal 7.4 Mb interval for HIVAN1, and encodes 31 protein coding genes: 15 genes have missense variants differentiating CAST from FVB, and 14 genes show differential renal expression. Of these, Frem1, Foxo1, and Setd7 have been implicated in the pathogenesis of nephropathy. HIVAN1 congenic kidneys are histologically normal without the HIV-1 transgene, yet their global transcriptome is enriched for molecular signatures of apoptosis, adenoviral infection, as well as genes repressed by histone H3 lysine 27 trimethylation, a histone modification associated with HIV-1 life cycle. These data refine HIVAN1to 7.4 Mb and identify latent molecular derangements that may predispose to nephropathy upon exposure to HIV-1.

  2. Refinement of the HIVAN1 Susceptibility Locus on Chr. 3A1-A3 via Generation of Sub-Congenic Strains

    PubMed Central

    Papeta, Natalia; Patel, Ami; D’Agati, Vivette D.; Gharavi, Ali G.

    2016-01-01

    HIV-1 transgenic mice on the FVB/NJ background (TgFVB) represent a validated model of HIV-associated nephropathy (HIVAN). A major susceptibility locus, HIVAN1, was previously mapped to chromosome 3A1-A3 in a cross between TgFVB and CAST/EiJ (CAST) strains, and introgression of a 51.9 Mb segment encompassing HIVAN1 from CAST into TgFVB resulted in accelerated development of nephropathy. We generated three sub-congenic strains carrying CAST alleles in the proximal or distal regions of the HIVAN1 locus (Sub-II, 3.02–38.93 Mb; Sub-III, 38.45–55.1 Mb and Sub-IV, 47.7–55.1 Mb, build 38). At 5–10 weeks of age, histologic injury and proteinuria did not differ between HIV-1 transgenic Sub-II and TgFVB mice. In contrast, HIV-1 transgenic Sub-III and Sub-IV mice displayed up to 4.4 fold more histopathologic injury and 6-fold more albuminuria compared to TgFVB mice, similar in severity to the full-length congenic mice. The Sub-IV segment defines a maximal 7.4 Mb interval for HIVAN1, and encodes 31 protein coding genes: 15 genes have missense variants differentiating CAST from FVB, and 14 genes show differential renal expression. Of these, Frem1, Foxo1, and Setd7 have been implicated in the pathogenesis of nephropathy. HIVAN1 congenic kidneys are histologically normal without the HIV-1 transgene, yet their global transcriptome is enriched for molecular signatures of apoptosis, adenoviral infection, as well as genes repressed by histone H3 lysine 27 trimethylation, a histone modification associated with HIV-1 life cycle. These data refine HIVAN1to 7.4 Mb and identify latent molecular derangements that may predispose to nephropathy upon exposure to HIV-1. PMID:27736906

  3. Roles of the AMPA receptor subunit GluA1 but not GluA2 in synaptic potentiation and activation of ERK in the anterior cingulate cortex.

    PubMed

    Toyoda, Hiroki; Zhao, Ming-Gao; Ulzhöfer, Bettina; Wu, Long-Jun; Xu, Hui; Seeburg, Peter H; Sprengel, Rolf; Kuner, Rohini; Zhuo, Min

    2009-08-10

    Cortical areas including the anterior cingulate cortex (ACC) are important for pain and pleasure. Recent studies using genetic and physiological approaches have demonstrated that the investigation of basic mechanism for long-term potentiation (LTP) in the ACC may reveal key cellular and molecular mechanisms for chronic pain in the cortex. Glutamate N-methyl D-aspartate (NMDA) receptors in the ACC are critical for the induction of LTP, including both NR2A and NR2B subunits. However, cellular and molecular mechanisms for the expression of ACC LTP have been less investigated. Here, we report that the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit, GluA1 but not GluA2 contributes to LTP in the ACC using genetic manipulated mice lacking GluA1 or GluA2 gene. Furthermore, GluA1 knockout mice showed decreased extracellular signal-regulated kinase (ERK) phosphorylation in the ACC in inflammatory pain models in vivo. Our results demonstrate that AMPA receptor subunit GluA1 is a key mechanism for the expression of ACC LTP and inflammation-induced long-term plastic changes in the ACC.

  4. Mapping and characterization of Rf 5: a new gene conditioning pollen fertility restoration in A1 and A2 cytoplasm in sorghum (Sorghum bicolor (L.) Moench).

    PubMed

    Jordan, D R; Klein, R R; Sakrewski, K G; Henzell, R G; Klein, P E; Mace, E S

    2011-08-01

    With an aim to further characterize the cytoplasmic male sterility-fertility restoration system in sorghum, a major fertility restoration gene was mapped along with a second locus capable of partial restoration of pollen fertility. The major fertility restoration gene, Rf(5), was located on sorghum chromosome SBI-05, and was capable of restoring pollen fertility in both A(1) and A(2) male sterile cytoplasms. Depending on the restorer parent, mapping populations exhibited fertility restoration phenotypes that ranged from nearly bimodal distribution due to the action of Rf(5), to a more normalized distribution reflecting the action of Rf(5) and additional modifier/partial restoration genes. A second fertility restoration locus capable of partially restoring pollen fertility in A(1) cytoplasm was localized to chromosome SBI-04. Unlike Rf(5), this modifier/partial restorer gene acting alone resulted in less than 10% seed set in both A(1) and A(2) cytoplasms, and modified the extent of restoration conditioned by the major restorer Rf(5) in A(1) cytoplasm. In examining the genomic regions spanning the Rf(5) locus, a cluster of pentatricopeptide gene family members with high homology to rice Rf (1) and sorghum Rf (2) were identified as potential candidates encoding Rf(5).

  5. The effect of knockout of sulfotransferases 1a1 and 1d1 and of transgenic human sulfotransferases 1A1/1A2 on the formation of DNA adducts from furfuryl alcohol in mouse models.

    PubMed

    Sachse, Benjamin; Meinl, Walter; Glatt, Hansruedi; Monien, Bernhard H

    2014-10-01

    Furfuryl alcohol is a rodent carcinogen present in numerous foodstuffs. Sulfotransferases (SULTs) convert furfuryl alcohol into the DNA reactive and mutagenic 2-sulfoxymethylfuran. Sensitive techniques for the isotope-dilution ultra performance liquid chromatography-tandem mass spectrometry quantification of resulting DNA adducts, e.g. N (2)-((furan-2-yl)methyl)-2'-deoxyguanosine (N (2)-MF-dG), were developed. To better understand the contribution of specific SULT forms to the genotoxicity of furfuryl alcohol in vivo, we studied the tissue distribution of N (2)-MF-dG in different mouse models. Earlier mutagenicity studies with Salmonella typhimurium strains expressing different human and murine SULT forms indicated that human SULT1A1 and murine Sult1a1 and 1d1 catalyze furfuryl alcohol sulfo conjugation most effectively. Here, we used three mouse lines to study the bioactivation of furfuryl alcohol by murine SULTs, FVB/N wild-type (wt) mice and two genetically modified models lacking either murine Sult1a1 or Sult1d1. The animals received a single dose of furfuryl alcohol, and the levels of the DNA adducts were determined in liver, kidney, lung, colon and small intestine. The effect of Sult1d1 gene disruption on the genotoxicity of furfuryl alcohol was moderate and limited to kidney and small intestine. In contrast, the absence of functional Sult1a1 had a massive influence on the adduct levels, which were lowered by 33-73% in all tissues of the female Sult1a1 null mice compared with the wt animals. The detection of high N (2)-MF-dG levels in a humanized mouse line expressing hSULT1A1/1A2 instead of endogeneous Sult1a1 and Sult1d1 supports the hypothesis that furfuryl alcohol is converted to the mutagenic 2-sulfoxymethylfuran also in humans.

  6. Isolation of nanomolar scFvs of non-human primate origin, cross-neutralizing botulinum neurotoxins A1 and A2 by targeting their heavy chain.

    PubMed

    Avril, Arnaud; Miethe, Sebastian; Popoff, Michel R; Mazuet, Christelle; Chahboun, Siham; Rasetti-Escargueil, Christine; Sesardic, Dorothea; Thullier, Philippe; Hust, Michael; Pelat, Thibaut

    2015-09-17

    Botulism is a naturally occurring disease, mainly caused by the ingestion of food contaminated by the botulinum neurotoxins (BoNTs). Botulinum neurotoxins are the most lethal. They are classified among the six major biological warfare agents by the Centers for Disease Control. BoNTs act on the cholinergic motoneurons, where they cleave proteins implicated in acetylcholine vesicle exocytosis. This exocytosis inhibition induces a flaccid paralysis progressively affecting all the muscles and generally engendering a respiratory distress. BoNTs are also utilized in medicine, mainly for the treatment of neuromuscular disorders, preventing large scale vaccination. Botulism specific treatment requires injections of antitoxins, usually of equine origin and thus poorly tolerated. Therefore, development of human or human-like neutralizing antibodies is of a major interest, and it is the subject of the European framework project called "AntiBotABE". In this study, starting from a macaque immunized with the recombinant heavy chain of BoNT/A1 (BoNT/A1-HC), an immune antibody phage-display library was generated and antibody fragments (single chain Fragment variable) with nanomolar affinity were isolated and further characterized. The neutralization capacities of these scFvs were analyzed in the mouse phrenic nerve-hemidiaphragm assay. After a three-round panning, 24 antibody fragments with affinity better than 10 nM were isolated. Three of them neutralized BoNT/A1 efficiently and two cross-neutralized BoNT/A1 and BoNT/A2 subtypes in the mouse phrenic nerve-hemidiaphragm assay. These are the first monoclonal human-like antibodies cross-neutralizing both BoNT/A1 and BoNT/A2. The antibody A1HC38 was selected for further development, and could be clinically developed for the prophylaxis and treatment of botulism.

  7. Single-cell analysis reveals differential regulation of the alveolar macrophage actin cytoskeleton by surfactant proteins A1 and A2: implications of sex and aging.

    PubMed

    Tsotakos, Nikolaos; Phelps, David S; Yengo, Christopher M; Chinchilli, Vernon M; Floros, Joanna

    2016-01-01

    Surfactant protein A (SP-A) contributes to lung immunity by regulating inflammation and responses to microorganisms invading the lung. The huge genetic variability of SP-A in humans implies that this protein is highly important in tightly regulating the lung immune response. Proteomic studies have demonstrated that there are differential responses of the macrophages to SP-A1 and SP-A2 and that there are sex differences implicated in these responses. Purified SP-A variants were used for administration to alveolar macrophages from SP-A knockout (KO) mice for in vitro studies, and alveolar macrophages from humanized SP-A transgenic mice were isolated for ex vivo studies. The actin cytoskeleton was examined by fluorescence and confocal microscopy, and the macrophages were categorized according to the distribution of polymerized actin. In accordance with previous data, we report that there are sex differences in the response of alveolar macrophages to SP-A1 and SP-A2. The cell size and F-actin content of the alveolar macrophages are sex- and age-dependent. Importantly, there are different subpopulations of cells with differential distribution of polymerized actin. In vitro, SP-A2 destabilizes actin in female, but not male, mice, and the same tendency is observed by SP-A1 in cells from male mice. Similarly, there are differences in the distribution of AM subpopulations isolated from SP-A transgenic mice depending on sex and age. There are marked sex- and age-related differences in the alveolar macrophage phenotype as illustrated by F-actin staining between SP-A1 and SP-A2. Importantly, the phenotypic switch caused by the different SP-A variants is subtle, and pertains to the frequency of the observed subpopulations, demonstrating the need for single-cell analysis approaches. The differential responses of alveolar macrophages to SP-A1 and SP-A2 highlight the importance of genotype in immune regulation and the susceptibility to lung disease and the need for development of

  8. Differential regulation of baboon SP-A1 and SP-A2 genes: structural and functional analysis of 5'-flanking DNA.

    PubMed

    Li, J; Gao, E; Seidner, S R; Mendelson, C R

    1998-12-01

    Surfactant protein (SP) A gene transcription is developmentally regulated and stimulated by hormones and factors that increase intracellular cAMP. The baboon (b) genome contains two highly similar SP-A genes, bSP-A1 and bSP-A2. With the use of a ribonuclease protection assay with gene-specific probes, the two bSP-A genes were found to be differentially regulated during baboon fetal lung development in that expression of the bSP-A2 gene appeared to be induced to a high level at a later time in gestation than that of the bSP-A1 gene. Both the bSP-A1 and bSP-A2 genes were found to be highly responsive to the inductive effects of cAMP in baboon fetal lung explants in culture. By DNase I footprinting and electrophoretic mobility shift assays with bacterially expressed thyroid transcription factor-1 (TTF-1) and type II cell nuclear extracts, three TTF-1 binding elements were identified within the 255-bp region flanking the 5'-end of each bSP-A gene; however, these differed in position and spacing for the two bSP-A genes. To functionally define the genomic regions that are required for cAMP regulation of bSP-A gene expression in type II cells, fusion genes composed of various amounts of 5'-flanking DNA from the bSP-A1 and bSP-A2 genes linked to the human growth hormone structural gene as a reporter were transfected into type II cells in primary culture. We found that 255 bp of 5'-flanking DNA, which contain three TTF-1 binding elements, from bSP-A1 and bSP-A2 genes were sufficient to mediate high basal and cAMP-inducible expression in type II cells. We also observed that there were no obvious differences in the magnitude of the responses of these fusion genes to cAMP treatment.

  9. The LspB protein is involved in the secretion of the LspA1 and LspA2 proteins by Haemophilus ducreyi.

    PubMed

    Ward, Christine K; Mock, Jason R; Hansen, Eric J

    2004-04-01

    The LspA1 and LspA2 proteins of Haemophilus ducreyi 35000 are two very large macromolecules that can be detected in concentrated culture supernatant fluid. Both of these proteins exhibit homology with the N-terminal region of the Bordetella pertussis filamentous hemagglutinin (FHA), which is involved in secretion of the latter macromolecule. The lspA2 open reading frame is flanked upstream by a gene, lspB, that encodes a predicted protein with homology to the B. pertussis FhaC outer membrane protein that is involved in secretion of FHA across the outer membrane. The H. ducreyi lspB gene encodes a protein with a predicted molecular mass of 66,573 Da. Reverse transcription-PCR analysis suggested that the lspB gene was transcribed together with the lspA2 gene on a single mRNA transcript. Polyclonal H. ducreyi LspB antiserum reacted with a 64-kDa antigen present in the Sarkosyl-insoluble cell envelope fraction of H. ducreyi 35000, which indicated that the LspB protein is likely an outer membrane protein. Concentrated culture supernatant fluids from H. ducreyi lspB and lspA1 lspB mutants did not contain detectable LspA1 and detectable LspA2, respectively. However, complementation of the lspB mutant with the wild-type lspB gene on a plasmid restored LspB protein expression and resulted in release of detectable amounts of the LspA1 protein into culture supernatant fluid. When evaluated in the temperature-dependent rabbit model of infection, the lspB mutant was attenuated in the ability to cause lesions and was never recovered in a viable form from lesions. These results indicated that the H. ducreyi LspB protein is involved in secretion of the LspA1 and LspA2 proteins across the outer membrane.

  10. Clinical and molecular characterization of 40 patients with classic Ehlers–Danlos syndrome: identification of 18 COL5A1 and 2 COL5A2 novel mutations

    PubMed Central

    2013-01-01

    Background Classic Ehlers–Danlos syndrome (cEDS) is a rare autosomal dominant connective tissue disorder that is primarily characterized by skin hyperextensibility, abnormal wound healing/atrophic scars, and joint hypermobility. A recent study demonstrated that more than 90% of patients who satisfy all of these major criteria harbor a type V collagen (COLLV) defect. Methods This cohort included 40 patients with cEDS who were clinically diagnosed according to the Villefranche nosology. The flowchart that was adopted for mutation detection consisted of sequencing the COL5A1 gene and, if no mutation was detected, COL5A2 analysis. In the negative patients the presence of large genomic rearrangements in COL5A1 was investigated using MLPA, and positive results were confirmed via SNP-array analysis. Results We report the clinical and molecular characterization of 40 patients from 28 families, consisting of 14 pediatric patients and 26 adults. A family history of cEDS was present in 9 patients. The majority of the patients fulfilled all the major diagnostic criteria for cEDS; atrophic scars were absent in 2 females, skin hyperextensibility was not detected in a male and joint hypermobility was negative in 8 patients (20% of the entire cohort). Wide inter- and intra-familial phenotypic heterogeneity was observed. We identified causal mutations with a detection rate of approximately 93%. In 25/28 probands, COL5A1 or COL5A2 mutations were detected. Twenty-one mutations were in the COL5A1 gene, 18 of which were novel (2 recurrent). Of these, 16 mutations led to nonsense-mediated mRNA decay (NMD) and to COLLV haploinsufficiency and 5 mutations were structural. Two novel COL5A2 splice mutations were detected in patients with the most severe phenotypes. The known p. (Arg312Cys) mutation in the COL1A1 gene was identified in one patient with vascular-like cEDS. Conclusions Our findings highlight that the three major criteria for cEDS are useful and sufficient for cEDS clinical

  11. Molecular Cloning, Tissue Distribution, and Functional Characterization of Marmoset Cytochrome P450 1A1, 1A2, and 1B1.

    PubMed

    Uehara, Shotaro; Uno, Yasuhiro; Inoue, Takashi; Sasaki, Erika; Yamazaki, Hiroshi

    2016-01-01

    The common marmoset (Callithrix jacchus), a New World monkey, has potential to be an animal model for drug metabolism studies. In this study, we identified and characterized cytochrome P450 (P450) 1A1 and 1B1 in addition to the known P450 1A2 in marmosets. Marmoset P450 1A1 and 1B1 cDNA contained open reading frames encoding 512 and 543 amino acids, respectively, with high sequence identities (90%-93%) to other primate P450 1A1s and 1B1s. A phylogenetic tree based on amino acid sequences showed close evolutionary relationships among marmoset, macaque, and human P450 1A and 1B enzymes. By mRNA quantification and immunoblot analyses in five marmoset tissues, P450 1A1 was mainly expressed in lungs and small intestines, and P450 1A2 was expressed predominantly in livers. In contrast, P450 1B1 was expressed in all tissues tested. Marmoset P450 1A1, 1A2, and 1B1 heterologously expressed in Escherichia coli catalyzed 7-ethoxyresorufin O-deethylation, 7-ethoxycoumarin O-deethylation, and phenacetin O-deethylation, similar to those of humans and cynomolgus monkeys. Notably, marmoset P450 1A1 and 1A2 more efficiently catalyzed 7-ethoxyresorufin O-deethylation than those of the human homologs, but were comparable to those of the cynomolgus monkey homologs. Additionally, marmoset P450 1B1 preferentially catalyzed estradiol 4-hydroxylation; however, rat P450 1B1 more favorably catalyzed estradiol 2-hydroxylation, indicating that the estradiol hydroxylation specificity of marmoset P450 1B1 was similar to those of human and cynomolgus monkey P450 1B1. These results indicated that marmoset P450 1A and 1B enzymes had functional characteristics similar to those of humans and cynomolgus monkeys, suggesting that P450 1A and 1B-dependent metabolism was similar among marmosets, cynomolgus monkeys, and humans. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  12. Persistent reduction of cocaine seeking by pharmacological manipulation of adenosine A1 and A2A receptors during extinction training in rats

    PubMed Central

    O’Neill, Casey E.; Hobson, Benjamin D.; Levis, Sophia C.; Bachtell, Ryan K.

    2014-01-01

    Rationale Adenosine receptor stimulation and blockade has been shown to modulate a variety of cocaine related behaviors. Objectives These studies identify the direct effects of adenosine receptor stimulation on cocaine seeking during extinction training and the persistent effects on subsequent reinstatement to cocaine seeking. Methods Rats self-administered cocaine on a fixed-ratio 1 schedule in daily sessions over 3 weeks. Following 1 week withdrawal, the direct effects of adenosine receptor modulation were tested by administering the adenosine A1 receptor agonist, CPA (0.03 mg/kg and 0.1 mg/kg), the adenosine A2A agonist, CGS 21680 (0.03 mg/kg and 0.1 mg/kg), the presynaptic adenosine A2A receptor antagonist, SCH 442416 (0.3 mg/kg, 1 mg/kg, and 3 mg/kg), or vehicle prior to each of 6 daily extinction sessions. The persistent effects of adenosine receptor modulation during extinction training were subsequently tested on reinstatement to cocaine seeking induced by cues, cocaine, and the dopamine D2 receptor agonist, quinpirole. Results All doses of CPA and CGS 21680 impaired initial extinction responding, however only CPA treatment during extinction produced persistent impairment in subsequent cocaine- and quinpirole-induced seeking. Dissociating CPA treatment from extinction did not alter extinction responding or subsequent reinstatement. Administration of SCH 442416 had no direct effects on extinction responding, but produced dose-dependent persistent impairment of cocaine- and quinpirole-induced seeking. Conclusions These findings demonstrate that adenosine A1 or A2A receptor stimulation directly impair extinction responding. Interestingly, adenosine A1 receptor stimulation or presynaptic adenosine A2A receptor blockade during extinction produces lasting changes in relapse susceptibility. PMID:24562064

  13. Transpedicular vertebral body augmentation reinforced with pedicle screw fixation in fresh traumatic A2 and A3 lumbar fractures: comparison between two devices and two bone cements.

    PubMed

    Korovessis, Panagiotis; Vardakastanis, Konstantinos; Repantis, Thomas; Vitsas, Vasilios

    2014-07-01

    This retrospective study compares efficacy and safety of balloon kyphoplasty (BK) with calcium phosphate (Group A) versus KIVA implant with PMMA (Group B) reinforced with three vertebrae pedicle screw constructs for A2 and A3 single fresh non-osteoporotic lumbar (L1-L4) fractures in 38 consecutive age- and diagnosis-matched patient populations. Extracanal leakage of both low-viscosity PMMA and calcium phosphate (CP) as well as the following roentgenographic parameters: segmental kyphosis (SKA), anterior (AVBHr) and posterior (PVBHr) vertebral body height ratio, spinal canal encroachment (SCE) clearance, and functional outcome measures: VAS and SF-36, were recorded and compared between the two groups. All patients in both groups were followed for a minimum 26 (Group A) and 25 (Group B) months. Extracanal CP and PMMA leakage was observed in four (18 %) and three (15 %) vertebrae/patients of group A and B, respectively. Hybrid fixation improved AVBHr, SKA, SCE, but PVBHr only in group B. VAS and SF-36 improved postoperatively in the patients of both groups. Short-segment construct with the novel KIVA implant restored better than BK-fractured lumbar vertebral body, but this had no impact in functional outcome. Since there was no leakage difference between PMMA and calcium phosphate and no short-term adverse related to PMMA use were observed, we advice the use of PMMA in fresh traumatic lumbar fractures.

  14. Pyrene-induced CYP1A2 and SULT1A1 may be regulated by CAR and not by AhR.

    PubMed

    Lee, Chul-Ho; Ito, Yuki; Yanagiba, Yukie; Yamanoshita, Osamu; Kim, Heon; Zhang, Shu-Yun; Kamijima, Michihiro; Gonzalez, Frank J; Nakajima, Tamie

    2007-09-05

    Aryl hydrocarbon receptor (AhR) plays important roles in the regulation and induction of xenobiotic-metabolizing enzymes including the cytochromes P450 1 family (CYP1) and UDP-glucuronosyltransferases 1A (UGT1As) by polycyclic aromatic hydrocarbons as well as chlorinated aromatic hydrocarbons. To determine whether pyrene-induced xenobiotic-metabolizing enzymes are regulated by AhR, male AhR (+/+) and (-/-) mice were used. Both genotyped mice were exposed to 0, 205, 300 or 410 mg/(kgday pyrene), once daily, for four consecutive days by gavage. Exposure to pyrene did not influence hepatic CYP1A1-mRNA in mice of both genotypes, whereas it induced hepatic CYP1A2 protein and mRNA expression and associated 7-ethoxyresorufin O-deethylase and pyrene 1-hydroxylation activities in both AhR (+/+) and (-/-) mice. Similar effects were also found with sulfotransferase 1A1 expression and the associated 1-hydroxypyrene sulfation activity. In contrast, pyrene exposure increased expression of the UGT1A1 and 1A6, and glucuronidation activities associated with 1-hydroxypyrene and 1-naphthol in the liver only in AhR (-/-) mice, although pyrene treatment dose-dependently decreased the latter activity. Pyrene exposure did not increase AhR-mRNA expression in AhR (+/+) mice. In contrast, pyrene-induced expression of the hepatic constitutive androstane receptor (CAR) and one of its target genes, CYP2B10, in both AhR (+/+) and (-/-) mice. These results strongly suggest that pyrene-induced CYP1A2 and SULT1A1 are regulated by CAR, not by AhR. However, the mechanisms of UGT1A1 and 1A6 induction by pyrene were not elucidated in this study.

  15. A2 Milk Enhances Dynamic Muscle Function Following Repeated Sprint Exercise, a Possible Ergogenic Aid for A1-Protein Intolerant Athletes?

    PubMed Central

    Kirk, Ben; Mitchell, Jade; Jackson, Matthew; Amirabdollahian, Farzad; Alizadehkhaiyat, Omid; Clifford, Tom

    2017-01-01

    Hyperaminoacidemia following ingestion of cows-milk may stimulate muscle anabolism and attenuate exercise-induced muscle damage (EIMD). However, as dairy-intolerant athletes do not obtain the reported benefits from milk-based products, A2 milk may offer a suitable alternative as it lacks the A1-protein. This study aimed to determine the effect of A2 milk on recovery from a sports-specific muscle damage model. Twenty-one male team sport players were allocated to three independent groups: A2 milk (n = 7), regular milk (n = 7), and placebo (PLA) (n = 7). Immediately following muscle-damaging exercise, participants consumed either A2 milk, regular milk or PLA (500 mL each). Visual analogue scale (muscle soreness), maximal voluntary isometric contraction (MVIC), countermovement jump (CMJ) and 20-m sprint were measured prior to and 24, 48, and 72 h post EIMD. At 48 h post-EIMD, CMJ and 20-m sprint recovered quicker in A2 (33.4 ± 6.6 and 3.3 ± 0.1, respectively) and regular milk (33.1 ± 7.1 and 3.3 ± 0.3, respectively) vs. PLA (29.2 ± 3.6 and 3.6 ± 0.3, respectively) (p < 0.05). Relative to baseline, decrements in 48 h CMJ and 20-m sprint were minimised in A2 (by 7.2 and 5.1%, respectively) and regular milk (by 6.3 and 5.2%, respectively) vs. PLA. There was a trend for milk treatments to attenuate decrements in MVIC, however statistical significance was not reached (p = 0.069). Milk treatments had no apparent effect on muscle soreness (p = 0.152). Following muscle-damaging exercise, ingestion of 500 mL of A2 or regular milk can limit decrements in dynamic muscle function in male athletes, thus hastening recovery and improving subsequent performance. The findings propose A2 milk as an ergogenic aid following EIMD, and may offer an alternative to athletes intolerant to the A1 protein. PMID:28134840

  16. A2 Milk Enhances Dynamic Muscle Function Following Repeated Sprint Exercise, a Possible Ergogenic Aid for A1-Protein Intolerant Athletes?

    PubMed

    Kirk, Ben; Mitchell, Jade; Jackson, Matthew; Amirabdollahian, Farzad; Alizadehkhaiyat, Omid; Clifford, Tom

    2017-01-28

    Hyperaminoacidemia following ingestion of cows-milk may stimulate muscle anabolism and attenuate exercise-induced muscle damage (EIMD). However, as dairy-intolerant athletes do not obtain the reported benefits from milk-based products, A2 milk may offer a suitable alternative as it lacks the A1-protein. This study aimed to determine the effect of A2 milk on recovery from a sports-specific muscle damage model. Twenty-one male team sport players were allocated to three independent groups: A2 milk (n = 7), regular milk (n = 7), and placebo (PLA) (n = 7). Immediately following muscle-damaging exercise, participants consumed either A2 milk, regular milk or PLA (500 mL each). Visual analogue scale (muscle soreness), maximal voluntary isometric contraction (MVIC), countermovement jump (CMJ) and 20-m sprint were measured prior to and 24, 48, and 72 h post EIMD. At 48 h post-EIMD, CMJ and 20-m sprint recovered quicker in A2 (33.4 ± 6.6 and 3.3 ± 0.1, respectively) and regular milk (33.1 ± 7.1 and 3.3 ± 0.3, respectively) vs. PLA (29.2 ± 3.6 and 3.6 ± 0.3, respectively) (p < 0.05). Relative to baseline, decrements in 48 h CMJ and 20-m sprint were minimised in A2 (by 7.2 and 5.1%, respectively) and regular milk (by 6.3 and 5.2%, respectively) vs. PLA. There was a trend for milk treatments to attenuate decrements in MVIC, however statistical significance was not reached (p = 0.069). Milk treatments had no apparent effect on muscle soreness (p = 0.152). Following muscle-damaging exercise, ingestion of 500 mL of A2 or regular milk can limit decrements in dynamic muscle function in male athletes, thus hastening recovery and improving subsequent performance. The findings propose A2 milk as an ergogenic aid following EIMD, and may offer an alternative to athletes intolerant to the A1 protein.

  17. Ethanol and Caffeine Effects on Social Interaction and Recognition in Mice: Involvement of Adenosine A2A and A1 Receptors.

    PubMed

    López-Cruz, Laura; San-Miguel, Noemí; Bayarri, Pilar; Baqi, Younis; Müller, Christa E; Salamone, John D; Correa, Mercé

    2016-01-01

    Ethanol and caffeine are frequently consumed in combination and have opposite effects on the adenosine system: ethanol metabolism leads to an increase in adenosine levels, while caffeine is a non-selective adenosine A1/A2A receptor antagonist. These receptors are highly expressed in striatum and olfactory tubercle, brain areas involved in exploration and social interaction in rodents. Ethanol modulates social interaction processes, but the role of adenosine in social behavior is still poorly understood. The present work was undertaken to study the impact of ethanol, caffeine and their combination on social behavior, and to explore the involvement of A1 and A2A receptors on those actions. Male CD1 mice were evaluated in a social interaction three-chamber paradigm, for preference of conspecific vs. object, and also for long-term recognition memory of familiar vs. novel conspecific. Ethanol showed a biphasic effect, with low doses (0.25 g/kg) increasing social contact and higher doses (1.0-1.5 g/kg) reducing social interaction. However, no dose changed social preference; mice always spent more time sniffing the conspecific than the object, independently of the ethanol dose. Ethanol, even at doses that did not change social exploration, produced amnestic effects on social recognition the following day. Caffeine reduced social contact (15.0-60.0 mg/kg), and even blocked social preference at higher doses (30.0-60.0 mg/kg). The A1 antagonist Cyclopentyltheophylline (CPT; 3-9 mg/kg) did not modify social contact or preference on its own, and the A2A antagonist MSX-3 (1.5-6 mg/kg) increased social interaction at all doses. Ethanol at intermediate doses (0.5-1.0 g/kg) was able to reverse the reduction in social exploration induced by caffeine (15.0-30.0 mg/kg). Although there was no interaction between ethanol and CPT or MSX-3 on social exploration in the first day, MSX-3 blocked the amnestic effects of ethanol observed on the following day. Thus, ethanol impairs the

  18. Ethanol and Caffeine Effects on Social Interaction and Recognition in Mice: Involvement of Adenosine A2A and A1 Receptors

    PubMed Central

    López-Cruz, Laura; San-Miguel, Noemí; Bayarri, Pilar; Baqi, Younis; Müller, Christa E.; Salamone, John D.; Correa, Mercé

    2016-01-01

    Ethanol and caffeine are frequently consumed in combination and have opposite effects on the adenosine system: ethanol metabolism leads to an increase in adenosine levels, while caffeine is a non-selective adenosine A1/A2A receptor antagonist. These receptors are highly expressed in striatum and olfactory tubercle, brain areas involved in exploration and social interaction in rodents. Ethanol modulates social interaction processes, but the role of adenosine in social behavior is still poorly understood. The present work was undertaken to study the impact of ethanol, caffeine and their combination on social behavior, and to explore the involvement of A1 and A2A receptors on those actions. Male CD1 mice were evaluated in a social interaction three-chamber paradigm, for preference of conspecific vs. object, and also for long-term recognition memory of familiar vs. novel conspecific. Ethanol showed a biphasic effect, with low doses (0.25 g/kg) increasing social contact and higher doses (1.0–1.5 g/kg) reducing social interaction. However, no dose changed social preference; mice always spent more time sniffing the conspecific than the object, independently of the ethanol dose. Ethanol, even at doses that did not change social exploration, produced amnestic effects on social recognition the following day. Caffeine reduced social contact (15.0–60.0 mg/kg), and even blocked social preference at higher doses (30.0–60.0 mg/kg). The A1 antagonist Cyclopentyltheophylline (CPT; 3–9 mg/kg) did not modify social contact or preference on its own, and the A2A antagonist MSX-3 (1.5–6 mg/kg) increased social interaction at all doses. Ethanol at intermediate doses (0.5–1.0 g/kg) was able to reverse the reduction in social exploration induced by caffeine (15.0–30.0 mg/kg). Although there was no interaction between ethanol and CPT or MSX-3 on social exploration in the first day, MSX-3 blocked the amnestic effects of ethanol observed on the following day. Thus, ethanol

  19. Non-operative vs. percutaneous stabilization in Magerl's A1 or A2 thoracolumbar spine fracture in adults: is it really advantageous for a good alignment of the spine? Preliminary data from a prospective study.

    PubMed

    Medici, Antonio; Meccariello, Luigi; Falzarano, Gabriele

    2014-10-01

    Percutaneous and non-operative stabilization are very controversial choices in the management of Magerl's A1 or A2 thoracolumbar spine fractures in adults. Our purpose is to figure out which of the two treatments is more suitable for the management and outcomes of these injuries. From 12/01/2011 to 06/30/2014 at the AO Orthopedics and Traumatology, Gaetano Rummo in Benevento, Italy, we treated 39 adult patients with thoracolumbar spinal fractures according to Magerl's A1 and A2. Twenty-four patients were treated with a 3-point orthopedic corset, and 15 patients were treated with percutaneous posterior stabilization without augmentation. The patients decided on treatment after extensive explanation of the pros and cons of the two treatments. The endpoint evaluation was set at the 6-month follow-up through the evaluation of the Visual Analogue Scale, Angle's Regional Kyphosis, Oswestry Low Back Pain Disability Questionnaire, and Denis work scale. The preliminary results of this prospective study show that there is a considerable advantage in functionality and pain in treating adults suffering from thoracolumbar fractures with Percutaneous technique at the expense of the bust with three points. Although the data are preliminary and based on data available in the literature, we can say that the Percutaneous posterior stabilization of thoracolumbar fractures in Magerl's A1 and A2 in adults is the ideal method for a good and functional alignment of the spine.

  20. Identification, characterization and genetic mapping of TLR7, TLR8a1 and TLR8a2 genes in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Palti, Yniv; Gahr, Scott A; Purcell, Maureen K; Hadidi, Sima; Rexroad, Caird E; Wiens, Gregory D

    2010-02-01

    Induction of the innate immune pathways is critical for early anti-viral defense but there is limited understanding of how teleost fish recognize viral molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 7 and 8 bind single-stranded RNA of viral origin and are activated by synthetic anti-viral imidazoquinoline compounds. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR7 and TLR8 gene orthologs and their mRNA expression. Two TLR7/8 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA fingerprinting and genetic linkage analyses. Direct sequencing of two representative BACs revealed intact omTLR7 and omTLR8a1 open reading frames (ORFs) located on chromosome 3 and a second locus on chromosome 22 that contains an omTLR8a2 ORF and a putative TLR7 pseudogene. We used the omTLR8a1/2 nomenclature for the two trout TLR8 genes as phylogenetic analysis revealed that they and all the other teleost TLR8 genes sequenced to date are similar to the zebrafish TLR8a, but are distinct from the zebrafish TLR8b. The duplicated trout loci exhibit conserved synteny with other fish genomes extending beyond the tandem of TLR7/8 genes. The trout TLR7 and 8a1/2 genes are composed of a single large exon similar to all other described TLR7/8 genes. The omTLR7 ORF is predicted to encode a 1049 amino acid (aa) protein with 84% similarity to the Fugu TLR7 and a conserved pattern of predicted leucine-rich repeats (LRR). The omTLR8a1 and omTLR8a2 are predicted to encode 1035- and 1034-aa proteins, respectively, and have 86% similarity to each other. omTLR8a1 is likely the ortholog of the only Atlantic salmon TLR8 gene described to date as they have 95% aa sequence similarity. The tissue expression profiles of omTLR7, omTLR8a1 and omTLR8a2 in healthy trout were highest in spleen tissue followed by anterior and then posterior kidney tissues. Rainbow trout anterior kidney leukocytes produced elevated

  1. Identification, characterization and genetic mapping of TLR7, TLR8a1 and TLR8a2 genes in rainbow trout (Oncorhynchus mykiss)

    USGS Publications Warehouse

    Palti, Yniv; Gahr, Scott A.; Purcell, Maureen K.; Hadidi, Sima; Rexroad, Caird E.; Wiens, Gregory A.

    2010-01-01

    Induction of the innate immune pathways is critical for early anti-viral defense but there is limited understanding of how teleost fish recognize viral molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 7 and 8 bind single-stranded RNA of viral origin and are activated by synthetic anti-viral imidazoquinoline compounds. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR7 and TLR8 gene orthologs and their mRNA expression. Two TLR7/8 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA fingerprinting and genetic linkage analyses. Direct sequencing of two representative BACs revealed intact omTLR7 and omTLR8a1 open reading frames (ORFs) located on chromosome 3 and a second locus on chromosome 22 that contains an omTLR8a2 ORF and a putative TLR7 pseudogene. We used the omTLR8a1/2 nomenclature for the two trout TLR8 genes as phylogenetic analysis revealed that they and all the other teleost TLR8 genes sequenced to date are similar to the zebrafish TLR8a, but are distinct from the zebrafish TLR8b. The duplicated trout loci exhibit conserved synteny with other fish genomes extending beyond the tandem of TLR7/8 genes. The trout TLR7 and 8a1/2 genes are composed of a single large exon similar to all other described TLR7/8 genes. The omTLR7 ORF is predicted to encode a 1049 amino acid (aa) protein with 84% similarity to the Fugu TLR7 and a conserved pattern of predicted leucine-rich repeats (LRR). The omTLR8a1 and omTLR8a2 are predicted to encode 1035- and 1034-aa proteins, respectively, and have 86% similarity to each other. omTLR8a1 is likely the ortholog of the only Atlantic salmon TLR8 gene described to date as they have 95% aa sequence similarity. The tissue expression profiles of omTLR7, omTLR8a1 and omTLR8a2 in healthy trout were highest in spleen tissue followed by anterior and then posterior kidney tissues. Rainbow trout anterior kidney leukocytes produced elevated

  2. Remifentanil-induced preconditioning has cross-talk with A1 and A2B adenosine receptors in ischemic-reperfused rat heart.

    PubMed

    Lee, Yong-Cheol; Jung, Jiyoon; Park, Sang-Jin

    2016-01-01

    The purpose of this study was to determine whether there is a cross-talk between opioid receptors (OPRs) and adenosine receptors (ADRs) in remifentanil preconditioning (R-Pre) and, if so, to investigate the types of ADRs involved in the cross-talk. Isolated rat hearts received 30 min of regional ischemia followed by 2 hr of reperfusion. OPR and ADR antagonists were perfused from 10 min before R-Pre until the end of R-Pre. The heart rate, left ventricular developed pressure (LVDP),velocity of contraction (+dP/dtmax), and coronary flow (CF) were recorded. The area at risk and area of necrosis were measured. After reperfusion, the LVDP, +dP/dtmax,and CF showed a significant increase in the R-Pre group compared with the control group (no intervention before or after regional ischemia). These increases in the R-Pre group were blocked by naloxone, a nonspecific ADR antagonist, an A1 ADR antagonist, and an A2B ADR antagonist. The infarct size was reduced significantly in the R-Pre group compared with the control group. The infarct-reducing effect in the R-Pre group was blocked by naloxone, the nonspecific ADR antagonist, the A1 ADR antagonist, and the A2B ADR antagonist. The results of this study demonstrate that there is cross-talk between ADRs and OPRs in R-Pre and that A1 ADR and A2B ADR appear to be involved in the cross-talk.

  3. Increased annexin A1 and A2 levels in bronchoalveolar lavage fluid are associated with resistance to respiratory disease in beef calves

    PubMed Central

    2013-01-01

    Strategies to control bovine respiratory disease depend on accurate classification of disease risk. An objective method to refine the risk classification of beef calves could be economically beneficial, improve welfare by preventing unexpected disease occurrences, refine and reduce the use of antibiotics in beef production, and facilitate alternative methods of disease control. The objective of this study was to identify proteins in bronchoalveolar lavage fluid (BALF) of stressed healthy calves that predict later disease outcome, serve as biomarkers of susceptibility to pneumonia, and play a role in pathogenesis. BALF was collected from 162 healthy beef calves 1–2 days after weaning and transportation. Difference in gel electrophoresis (DIGE) and mass spectrometry were used to compare proteins in samples from 7 calves that later developed respiratory disease compared to 7 calves that remained healthy. Calves that later developed pneumonia had significantly lower levels of annexin A1, annexin A2, peroxiredoxin I, calcyphosin, superoxide dismutase, macrophage capping protein and dihydrodiol dehydrogenase 3. Differences in annexin levels were partially confirmed by western blot analysis. Thus, lower levels of annexins A1 and A2 are potential biomarkers of increased susceptibility to pneumonia in recently weaned and transported feedlot cattle. Since annexins are regulated by glucocorticoids, this finding may reflect individual differences in the stress response that predispose to pneumonia. These findings also have implications in pathogenesis. Annexins A1 and A2 are known to prevent neutrophil influx and fibrin deposition respectively, and may thus act to minimize the harmful effects of the inflammatory response during development of pneumonia. PMID:23565988

  4. The role of adenosine A1 and A2A receptors in the caffeine effect on MDMA-induced DA and 5-HT release in the mouse striatum.

    PubMed

    Górska, A M; Gołembiowska, K

    2015-04-01

    3,4-Methylenedioxymethamphetamine (MDMA, "ecstasy") popular as a designer drug is often used with caffeine to gain a stronger stimulant effect. MDMA induces 5-HT and DA release by interaction with monoamine transporters. Co-administration of caffeine and MDMA may aggravate MDMA-induced toxic effects on DA and 5-HT terminals. In the present study, we determined whether caffeine influences DA and 5-HT release induced by MDMA. We also tried to find out if adenosine A1 and A2A receptors play a role in the effect of caffeine by investigating the effect of the selective adenosine A1 and A2A receptor antagonists, DPCPX and KW 6002 on DA and 5-HT release induced by MDMA. Mice were treated with caffeine (10 mg/kg) and MDMA (20 or 40 mg/kg) alone or in combination. DA and 5-HT release in the mouse striatum was measured using in vivo microdialysis. Caffeine exacerbated the effect of MDMA on DA and 5-HT release. DPCPX or KW 6002 co-administered with MDMA had similar influence as caffeine, but KW 6002 was more potent than caffeine or DPCPX. To exclude the contribution of MAO inhibition by caffeine in the caffeine effect on MDMA-induced increase in DA and 5-HT, we also tested the effect of the nonxanthine adenosine receptor antagonist CGS 15943A lacking properties of MAO activity modification. Our findings indicate that adenosine A1 and A2A receptor blockade may account for the caffeine-induced exacerbation of the MDMA effect on DA and 5-HT release and may aggravate MDMA toxicity.

  5. Modulation of dopamine-mediated facilitation at the neuromuscular junction of Wistar rats: A role for adenosine A1/A2A receptors and P2 purinoceptors.

    PubMed

    Elnozahi, Neveen A; AlQot, Hadir E; Mohy El-Din, Mahmoud M; Bistawroos, Azza E; Abou Zeit-Har, Mohamed S

    2016-06-21

    This study aims to understand how dopamine and the neuromodulators, adenosine and adenosine triphosphate (ATP) modulate neuromuscular transmission. Adenosine and ATP are well-recognized for their regulatory effects on dopamine in the central nervous system. However, if similar interactions occur at the neuromuscular junction is unknown. We hypothesize that the activation of adenosine A1/A2A and/or P2 purinoceptors may influence the action of dopamine on neuromuscular transmission. Using the rat phrenic nerve hemi-diaphragm, we assessed the influence of dopamine, adenosine and ATP on the height of nerve-evoked muscle twitches. We investigated how the selective blockade of adenosine A1 receptors (2.5nM DPCPX), adenosine A2A receptors (50nM CSC) and P2 purinoceptors (100μM suramin) modified the effects of dopamine. Dopamine alone increased indirect muscle contractions while adenosine and ATP either enhanced or depressed nerve-evoked muscle twitches in a concentration-dependent manner. The facilitatory effects of 256μM dopamine were significantly reduced to 29.62±2.79% or 53.69±5.45% in the presence of DPCPX or CSC, respectively, relative to 70.03±1.57% with dopamine alone. Alternatively, the action of 256μM dopamine was potentiated from 70.03±1.57, in the absence of suramin, to 86.83±4.36%, in the presence of suramin. It can be concluded that the activation of adenosine A1 and A2A receptors and P2 purinoceptors potentially play a central role in the regulation of dopamine effects at the neuromuscular junction. Clinically this study offers new insights for the indirect manipulation of neuromuscular transmission for the treatment of disorders characterized by motor dysfunction. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  6. Associations between polymorphisms in the AHR and CYP1A1-CYP1A2 gene regions and habitual caffeine consumption.

    PubMed

    Josse, Andrea R; Da Costa, Laura A; Campos, Hannia; El-Sohemy, Ahmed

    2012-09-01

    Recent genome-wide association studies (GWASs) from populations of European descent identified single nucleotide polymorphisms (SNPs) in aryl-hydrocarbon receptor (AHR) and cytochrome P450 1A1 and 1A2 (CYP1A1-CYP1A2) genes that are associated with habitual caffeine and coffee consumption. We examined whether these SNPs (AHR: rs6968865 and rs4410790; CYP1A1-CYP1A2: rs2472297 and rs2470893) and 6 additional tag SNPs in the AHR gene were associated with habitual caffeine consumption in a Costa Rican population. Subjects were from a case-control study of gene-diet interactions and myocardial infarction. Subjects with hypertension or missing information on smoking, caffeine intake, or genotype were excluded. Subjects were genotyped by using polymerase chain reaction with mass spectrometry-based detection, and caffeine intake was assessed by using a validated food-frequency questionnaire. Compared with subjects who consumed <100 mg caffeine/d, subjects who consumed >400 mg caffeine/d were more likely to be carriers of the T, C, or T allele for rs6968865, rs4410790, and rs2472297, respectively. The corresponding ORs and 95% CIs were 1.41 (1.03, 1.93), 1.41 (1.04, 1.92), and 1.55 (1.01, 2.36). Multivariate-adjusted ORs (95% CIs) for rs6968865 were 1.44 (1.03, 2.00) for all subjects, 1.75 (1.16, 2.65) for nonsmokers, 1.15 (0.58, 2.30) for current smokers, 2.42 (1.45, 4.04) for subjects >57 y old, and 1.00 (0.65, 1.56) for subjects ≤57 y old. A similar effect modification was observed for rs4410790 but not for rs2472297. Our findings show that previous associations between SNPs in AHR and CYP1A1-CYP1A2 and caffeine and coffee consumption from GWASs in European populations are also observed in an ethnically distinct Costa Rican population, but age and smoking are important effect modifiers.

  7. Hypervelocity Impact (HVI). Volume 2; WLE Small-Scale Fiberglass Panel Flat Multi-Layer Targets A-1, A-2, and B-1

    NASA Technical Reports Server (NTRS)

    Gorman, Michael R.; Ziola, Steven M.

    2007-01-01

    During 2003 and 2004, the Johnson Space Center's White Sands Testing Facility in Las Cruces, New Mexico conducted hypervelocity impact tests on the space shuttle wing leading edge. Hypervelocity impact tests were conducted to determine if Micro-Meteoroid/Orbital Debris impacts could be reliably detected and located using simple passive ultrasonic methods. The objective of Targets A-1, A-2, and B-2 was to study hypervelocity impacts through multi-layered panels simulating Whipple shields on spacecraft. Impact damage was detected using lightweight, low power instrumentation capable of being used in flight.

  8. Postsynaptic VAMP/Synaptobrevin Facilitates Differential Vesicle Trafficking of GluA1 and GluA2 AMPA Receptor Subunits

    PubMed Central

    Hussain, Suleman; Davanger, Svend

    2015-01-01

    Vertebrate organisms adapt to a continuously changing environment by regulating the strength of synaptic connections between brain cells. Excitatory synapses are believed to increase their strength by vesicular insertion of transmitter glutamate receptors into the postsynaptic plasma membrane. These vesicles, however, have never been demonstrated or characterized. For the first time, we show the presence of small vesicles in postsynaptic spines, often closely adjacent to the plasma membrane and PSD (postsynaptic density). We demonstrate that they harbor vesicle-associated membrane protein 2 (VAMP2/synaptobrevin-2) and glutamate receptor subunit 1 (GluA1). Disrupting VAMP2 by tetanus toxin treatment reduces the concentration of GluA1 in the postsynaptic plasma membrane. GluA1/VAMP2-containing vesicles, but not GluA2/VAMP2-vesicles, are concentrated in postsynaptic spines relative to dendrites. Our results indicate that small postsynaptic vesicles containing GluA1 are inserted directly into the spine plasma membrane through a VAMP2-dependent mechanism. PMID:26488171

  9. UDP-galactose (SLC35A2) and UDP-N-acetylglucosamine (SLC35A3) Transporters Form Glycosylation-related Complexes with Mannoside Acetylglucosaminyltransferases (Mgats)*

    PubMed Central

    Maszczak-Seneczko, Dorota; Sosicka, Paulina; Kaczmarek, Beata; Majkowski, Michał; Luzarowski, Marcin; Olczak, Teresa; Olczak, Mariusz

    2015-01-01

    UDP-galactose transporter (UGT; SLC35A2) and UDP-N-acetylglucosamine transporter (NGT; SLC35A3) form heterologous complexes in the Golgi membrane. NGT occurs in close proximity to mannosyl (α-1,6-)-glycoprotein β-1,6-N-acetylglucosaminyltransferase (Mgat5). In this study we analyzed whether NGT and both splice variants of UGT (UGT1 and UGT2) are able to interact with four different mannoside acetylglucosaminyltransferases (Mgat1, Mgat2, Mgat4B, and Mgat5). Using an in situ proximity ligation assay, we found that all examined glycosyltransferases are in the vicinity of these UDP-sugar transporters both at the endogenous level and upon overexpression. This observation was confirmed via the FLIM-FRET approach for both NGT and UGT1 complexes with Mgats. This study reports for the first time close proximity between endogenous nucleotide sugar transporters and glycosyltransferases. We also observed that among all analyzed Mgats, only Mgat4B occurs in close proximity to UGT2, whereas the other three Mgats are more distant from UGT2, and it was only possible to visualize their vicinity using proximity ligation assay. This strongly suggests that the distance between these protein pairs is longer than 10 nm but at the same time shorter than 40 nm. This study adds to the understanding of glycosylation, one of the most important post-translational modifications, which affects the majority of macromolecules. Our research shows that complex formation between nucleotide sugar transporters and glycosyltransferases might be a more common phenomenon than previously thought. PMID:25944901

  10. Detection of cfxA2, cfxA3, and cfxA6 genes in beta-lactamase producing oral anaerobes

    PubMed Central

    BINTA, Buhle; PATEL, Mrudula

    2016-01-01

    ABSTRACT Purpose The aim of this study was to identify β-lactamase-producing oral anaerobic bacteria and screen them for the presence of cfxA and BlaTEM genes that are responsible for β-lactamase production and resistance to β-lactam antibiotics. Material and Methods Periodontal pocket debris samples were collected from 48 patients with chronic periodontitis and anaerobically cultured on blood agar plates with and without β-lactam antibiotics. Presumptive β-lactamase-producing isolates were evaluated for definite β-lactamase production using the nitrocefin slide method and identified using the API Rapid 32A system. Antimicrobial susceptibility was performed using disc diffusion and microbroth dilution tests as described by CLSI Methods. Isolates were screened for the presence of the β-lactamase-TEM (BlaTEM) and β-lactamase-cfxA genes using Polymerase Chain Reaction (PCR). Amplified PCR products were sequenced and the cfxA gene was characterized using Genbank databases. Results Seventy five percent of patients carried two species of β-lactamase-producing anaerobic bacteria that comprised 9.4% of the total number of cultivable bacteria. Fifty one percent of β-lactamase-producing strains mainly Prevotella, Porphyromonas, and Bacteroides carried the cfxA gene, whereas none of them carried blaTEM. Further characterization of the cfxA gene showed that 76.7% of these strains carried the cfxA2 gene, 14% carried cfxA3, and 9.3% carried cfxA6. The cfxA6 gene was present in three Prevotella spp. and in one Porphyromonas spp. Strains containing cfxA genes (56%) were resistant to the β-lactam antibiotics. Conclusion This study indicates that there is a high prevalence of the cfxA gene in β-lactamase-producing anaerobic oral bacteria, which may lead to drug resistance and treatment failure. PMID:27119762

  11. Increased Slc12a1 expression in β-cells and improved glucose disposal in Slc12a2 heterozygous mice

    PubMed Central

    Alshahrani, Saeed; Almutairi, Mohammed Mashari; Kursan, Shams; Dias-Junior, Eduardo; Almiahuob, Mohamed Mahmoud; Aguilar-Bryan, Lydia; Di Fulvio, Mauricio

    2015-01-01

    The products of the Slc12a1 and Slc12a2 genes, commonly known as Na+-dependent K+2Cl− co-transporters NKCC2 and NKCC1, respectively, are the targets for the diuretic bumetanide. NKCCs are implicated in the regulation of intracellular chloride concentration ([Cl−]i) in pancreatic β-cells, and as such, they may play a role in glucose-stimulated plasma membrane depolarization and insulin secretion. Unexpectedly, permanent elimination of NKCC1 does not preclude insulin secretion, an event potentially linked to the homeostatic regulation of additional Cl− transporters expressed in β-cells. In this report we provide evidence for such a mechanism. Mice lacking a single allele of Slc12a2 exhibit lower fasting glycemia, increased acute insulin response (AIR) and lower blood glucose levels 15–30 min after a glucose load when compared to mice harboring both alleles of the gene. Furthermore, heterozygous expression or complete absence of Slc12a2 associates with increased NKCC2 protein expression in rodent pancreatic β-cells. This has been confirmed by using chronic pharmacological down-regulation of NKCC1 with bumetanide in the mouse MIN6 β-cell line or permanent molecular silencing of NKCC1 in COS7 cells, which results in increased NKCC2 expression. Furthermore, MIN6 cells chronically pretreated with bumetanide exhibit increased initial rates of Cl− uptake while preserving glucose-stimulated insulin secretion. Together, our results suggest that NKCCs are involved in insulin secretion and that a single Slc12a2 allele may protect β-cells from failure due to increased homeostatic expression of Slc12a1. PMID:26400961

  12. Inosine reduces pain-related behavior in mice: involvement of adenosine A1 and A2A receptor subtypes and protein kinase C pathways.

    PubMed

    Nascimento, Francisney P; Figueredo, Sonia M; Marcon, Rodrigo; Martins, Daniel F; Macedo, Sérgio J; Lima, Denise A N; Almeida, Rúbia C; Ostroski, Rosana M; Rodrigues, Ana Lúcia S; Santos, Adair Roberto Soares

    2010-08-01

    Inosine, an endogenous purine, is the first metabolite of adenosine in a reaction catalyzed by adenosine deaminase. This study aimed to investigate the antinociceptive effects of inosine against several models of pain in mice and rats. In mice, inosine given by systemic or central routes inhibited acetic acid-induced nociception. Furthermore, inosine also decreased the late phase of formalin-induced licking and the nociception induced by glutamate. Inosine produced inhibition (for up to 4 h) of mechanical allodynia induced by complete Freund's adjuvant (CFA) injected into the mouse's paw. Given chronically for 21 days, inosine reversed the mechanical allodynia caused by CFA. Moreover, inosine also reduced the thermal (cold stimuli) and mechanical allodynia caused by partial sciatic nerve ligation (PSNL) for 4 h; when inosine was chronically administered, it decreased the mechanical allodynia induced by PSNL for 22 days. Antinociception caused by inosine in the acetic acid test was attenuated by treatment of mice with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; a selective adenosine A(1) receptor antagonist), 8-phenyltheophylline (8-PT; a nonselective adenosine A(1) receptor antagonist), and 4-{2- [7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-yl- amino]ethyl}phenol (ZM241385; a selective adenosine A(2A) receptor antagonist). In rats, inosine inhibited the mechanical and heat hyperalgesia induced by bradykinin and phorbol 12-myristate 13-acetate, without affecting similar responses caused by prostaglandin E(2) or forskolin. These results indicate that inosine induces antinociceptive, antiallodynic, and antihyperalgesic effects in rodents. The precise mechanisms through which inosine produces antinociception are currently under investigation, but involvement of adenosine A(1) and A(2A) receptors and blockade of the protein kinase C pathway seem to largely account for inosine's antinociceptive effect.

  13. Genetic link with cholelithiasis among pediatric SCA Tunisian patients: Examples of UGT1A1, SLCO1A2 and SLCO1B1.

    PubMed

    Chaouch, Leila; Kalai1, Miniar; Darragi, Imen; Boudrigua, Imen; Chaouachi, Dorra; Ammar, Slim Ben; Mellouli, F; Bjaoui, M; Abbes, Salem

    2016-03-01

    Hyperbilirubinemia is often observed in chronic hemolysis and results in the formation of pigment cholelithiasis that could be increased by the presence of defected enzymes involved in the bilirubin metabolism. Indeed, this is the first report that interested in the study of polymorphisms in genes encoded for enzymes involved in the bilirubin metabolism: rs 4149056 of SLCO1B1 and rs4149000 of SLCO1A2 in combination with rs8175347 and rs887829 of UGT1A1 in order to find a correlation between the polymorphisms studied and the presence of gallstones in a population of sickle cell anemia (SCA) pediatric Tunisians. Our study involved 102 unrelated Tunisian subjects. All SCA patients are children (less than 16 years old) and were characterized by hyperbilirubinemia and 52 of them have cholelithiasis. The polymorphisms of the candidate genes were analyzed for all subjects by PCR/sequencing. Genotype and allele frequencies between cases and controls were compared using Pearson's chi-square test with a significance threshold of P < 0.05 (compare 2, version 1.02). The novelty of this report is that children carrying the combined genotype of the rs studied: (TA7TA7)/TT/TC/GA have a higher risk to develop gallstones (P = 0.0027, RR = 18.27 (20.0061-915.28)). Altogether our data provide the implication of UGT1A1 and SLCO1A2 in sickle cell anemia-related cholelithiasis.

  14. A D-π-A1-π-A2 push-pull small molecule donor for solution processed bulk heterojunction organic solar cells.

    PubMed

    Gautam, Prabhat; Misra, Rajneesh; Biswas, Subhayan; Sharma, Ganesh D

    2016-05-18

    Herein, benzothiadiazole (BTD), as an acceptor A1, has been used as a backbone to link triphenylamine (TPA) as donor and naphthalimide (NPI) as acceptor (A2) moieties through ethylene linkers to design a small molecule. The donor-π-acceptor-π-acceptor (D-π-A1-π-A2) type small molecule denoted as was synthesized. In order to use it as an electron donor for solution processed bulk heterojunction small molecule solar cells its photonic and electronic properties were explored. The small molecule organic solar cells based on the optimized blend of with PC71BM processed in chloroform showed a power conversion efficiency (PCE) of 2.21%, which was significantly improved up to 6.67%, when a two-step annealing (TSA) treated blend was used as an active layer. The increase in the PCE was due to the enhancement in both Jsc and FF. The improvement in Jsc was related to the enhancement in the light harvesting efficiency of a TSA treated active layer relative to the as-cast layer, which is reflected in a better IPCE and better charge collection. The TSA treatment also leads to better nanoscale morphology for exciton dissociation into free charge carriers and improved crystallinity for balanced charge transport.

  15. The antidepressant-like effect of inosine in the FST is associated with both adenosine A1 and A 2A receptors.

    PubMed

    Kaster, Manuella P; Budni, Josiane; Gazal, Marta; Cunha, Mauricio P; Santos, Adair R S; Rodrigues, Ana Lúcia S

    2013-09-01

    Inosine is an endogenous purine nucleoside, which is formed during the breakdown of adenosine. The adenosinergic system was already described as capable of modulating mood in preclinical models; we now explored the effects of inosine in two predictive models of depression: the forced swim test (FST) and tail suspension test (TST). Mice treated with inosine displayed higher anti-immobility in the FST (5 and 50 mg/kg, intraperitoneal route (i.p.)) and in the TST (1 and 10 mg/kg, i.p.) when compared to vehicle-treated groups. These antidepressant-like effects started 30 min and lasted for 2 h after intraperitoneal administration of inosine and were not accompanied by any changes in the ambulatory activity in the open-field test. Both adenosine A1 and A2A receptor antagonists prevented the antidepressant-like effect of inosine in the FST. In addition, the administration of an adenosine deaminase inhibitor (1 and 10 mg/kg, i.p.) also caused an antidepressant-like effect in the FST. These results indicate that inosine possesses an antidepressant-like effect in the FST and TST probably through the activation of adenosine A1 and A2A receptors, further reinforcing the potential of targeting the purinergic system to the management of mood disorders.

  16. Gene sequences for cytochromes p450 1A1 and 1A2: the need for biomarker development in sea otters (Enhydra lutris).

    PubMed

    Hook, Sharon E; Cobb, Michael E; Oris, James T; Anderson, Jack W

    2008-11-01

    There has been recent public concern regarding the impacts of environmental pollution on populations of otters. Population level impacts have been seen with otter (Lutra lutra) populations in Europe due to polychlorinated biphenyls, and with some segments of the Prince William Sound, AK, sea otter (Enhydra lutris) population following the Exxon Valdez oil spill. Despite public interest in these animals and their ecological significance, there are few tools that allow for the study of otter's response to contaminant exposure. Cytochrome p450 1A (CYP1A) performs the first step in metabolizing many xenobiotics, including many polychlorinated biphenyls and polycyclic aromatic hydrocarbons. CYP1A induction is a frequently used biomarker of exposure to these compounds. Despite the potential importance of this gene in ecological risk assessment, the complete coding sequence has not been published for any otter species. This study's objective was to isolate the gene for CYP1A1 and CYP1A2 in sea otters using a series of PCR-based approaches. The coding sequences from CYP1A1 and CYP1A2 from sea otters were identified and published in GenBank. Both CYP1A sequences are homologous to those obtained from marine mammals and other carnivores. These sequences will be useful as tools for researchers assessing contaminant exposure in mustelid populations.

  17. A widespread sequence-specific mRNA decay pathway mediated by hnRNPs A1 and A2/B1

    PubMed Central

    Geissler, Rene; Simkin, Alfred; Floss, Doreen; Patel, Ravi; Fogarty, Elizabeth A.; Scheller, Jürgen; Grimson, Andrew

    2016-01-01

    3′-untranslated regions (UTRs) specify post-transcriptional fates of mammalian messenger RNAs (mRNAs), yet knowledge of the underlying sequences and mechanisms is largely incomplete. Here, we identify two related novel 3′ UTR motifs in mammals that specify transcript degradation. These motifs are interchangeable and active only within 3′ UTRs, where they are often preferentially conserved; furthermore, they are found in hundreds of transcripts, many encoding regulatory proteins. We found that degradation occurs via mRNA deadenylation, mediated by the CCR4–NOT complex. We purified trans factors that recognize the motifs and identified heterogeneous nuclear ribonucleoproteins (hnRNPs) A1 and A2/B1, which are required for transcript degradation, acting in a previously unknown manner. We used RNA sequencing (RNA-seq) to confirm hnRNP A1 and A2/B1 motif-dependent roles genome-wide, profiling cells depleted of these factors singly and in combination. Interestingly, the motifs are most active within the distal portion of 3′ UTRs, suggesting that their role in gene regulation can be modulated by alternative processing, resulting in shorter 3′ UTRs. PMID:27151978

  18. Two families with Leber's hereditary optic neuropathy carrying G11778A and T14502C mutations with haplogroup H2a2a1 in mitochondrial DNA.

    PubMed

    Qiao, Chen; Wei, Tanwei; Hu, Bo; Peng, Chunyan; Qiu, Xueping; Wei, Li; Yan, Ming

    2015-08-01

    The mitochondrial haplogroup has been reported to affect the clinical expression of Leber's hereditary optic neuropathy (LHON). The present study aimed to investigate the interaction between mutations and the haplogroup of mitochondrial DNA (mtDNA) in families. Two unrelated families with LHON were enrolled in the study, and clinical, genetic and molecular characterizations were determined in the affected and unaffected family members. Polymerase chain reaction direct sequencing was performed using 24 pairs of overlapping primers for whole mtDNA to screen for mutations and haplogroup. Bioinformatics analysis was performed to evaluate the pathogenic effect of these mtDNA mutations and the haplogroup. The G11778A mutation was identified in the two families. In addition, the members of family 2 exhibited the T14502C mutation and those in family 1 exhibited the T3394C and T14502C mutations, which were regarded as secondary mutations. The penetrance of visual loss in families 1 and 2 were 30.8 and 33.3%, respectively. In addition, the two families were found to be in the H2a2a1 haplogroup. In this limited sample size, it was demonstrated that the H2a2a1 haplogroup had a possible protective effect against LHON. Additional modifying factors, including environmental factors, lifestyle, estrogen levels and nuclear genes may also be important in LHON.

  19. Dual blockade of the A1 and A2A adenosine receptor prevents amyloid beta toxicity in neuroblastoma cells exposed to aluminum chloride.

    PubMed

    Giunta, Salvatore; Andriolo, Violetta; Castorina, Alessandro

    2014-09-01

    In a previous work we have shown that exposure to aluminum (Al) chloride (AlCl3) enhanced the neurotoxicity of the amyloid beta(25-35) fragment (Abeta(25-35)) in neuroblastoma cells and affected the expression of Alzheimer's disease (AD)-related genes. Caffein, a compound endowed with beneficial effects against AD, exerts neuroprotection primarily through its antagonist activity on A2A adenosine receptors (A2AR), although it also inhibits A1Rs with similar potency. Still, studies on the specific involvement of these receptors in neuroprotection in a model of combined neurotoxicity (Abeta(25-35)+AlCl3) are missing. To address this issue, cultured SH-SY5Y cells exposed to Abeta(25-35)+AlCl3 were assessed for cell viability, morphology, intracellular ROS activity and expression of apoptosis-, stress- and AD-related proteins. To define the role of A1R and A2ARs, pretreatment with caffein, specific receptor antagonists (DPCPX or SCH58261) or siRNA-mediated gene knockdown were delivered. Results indicate that AlCl3 treatment exacerbated Abeta(25-35) toxicity, increased ROS production, lipid peroxidation, β-secretase-1 (BACE1) and amyloid precursor protein (APP). Interestingly, SCH58261 successfully prevented toxicity associated to Abeta(25-35) only, whereas pretreatment with both DPCPX and SCH58261 was required to fully avert Abeta(25-35)+AlCl3-induced damage, suggesting that A1Rs might also be critically involved in protection during combined toxicity. The effects of caffein were mimicked by both N-acetyl cysteine, an antioxidant, and desferrioxamine, likely acting through distinct mechanisms. Altogether, our data establish a novel protective function associated with A1R inhibition in the setting of combined Abeta(25-35)+AlCl3 neurotoxicity, and expand our current knowledge on the potential beneficial role of caffein to prevent AD progression in subjects environmentally exposed to aluminum. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Actions of adenosine A1 and A2 receptor antagonists on CFTR antibody-inhibited β-adrenergic mucin secretion response

    PubMed Central

    Pereira, M M C; Lloyd Mills, C; Dormer, R L; McPherson, M A

    1998-01-01

    The cystic fibrosis gene protein, the cystic fibrosis transmembrane conductance regulator (CFTR) acts as a chloride channel and is a key regulator of mucin secretion. The mechanism by which 3-isobutyl-1-methylxanthine (IBMX) corrects the defect in CFTR mediated β-adrenergic stimulation of mucin secretion has not been determined. The present study has investigated the actions of adenosine A1 and A2 receptor antagonists to determine whether ability to stimulate mucin secretion correlates with correction of CFTR antibody inhibited β-adrenergic response and whether excessive cyclic AMP rise is required.CFTR antibodies were introduced into living rat submandibular acini by hypotonic swelling. Following recovery, mucin secretion in response to isoproterenol was measured.The adenosine A1 receptor antagonist, 8 cyclopentyltheophylline (CPT) was a less potent stimulator of mucin secretion than was the A2 receptor antagonist dimethylpropargylxanthine (DMPX). A concentration of CPT close to the Ki for A1 receptor antagonism (10 nM) did not stimulate mucin secretion.DMPX, although a potent stimulator of mucin secretion, did not correct CFTR antibody inhibited mucin secretion.CPT corrected defective CFTR antibody inhibited mucin secretion at a high (1 mM) concentration, suggesting a mechanism other than adenosine receptor antagonism.DMPX potentiated the isoproterenol induced cyclic AMP rise, whereas CPT did not.Correction of the defective CFTR mucin secretion response did not correlate with ability to stimulate mucin secretion and did not require potentiation of β-adrenergic induced increases in cyclic AMP. This affords real promise for the development of a selective drug treatment for cystic fibrosis. PMID:9831904

  1. Genome-wide association analysis of coffee drinking suggests association with CYP1A1/CYP1A2 and NRCAM

    PubMed Central

    Amin, N; Byrne, E; Johnson, J; Chenevix-Trench, G; Walter, S; Nolte, I M; Vink, J M; Rawal, R; Mangino, M; Teumer, A; Keers, J C; Verwoert, G; Baumeister, S; Biffar, R; Petersmann, A; Dahmen, N; Doering, A; Isaacs, A; Broer, L; Wray, N R; Montgomery, G W; Levy, D; Psaty, B M; Gudnason, V; Chakravarti, A; Sulem, P; Gudbjartsson, D F; Kiemeney, L A; Thorsteinsdottir, U; Stefansson, K; van Rooij, F J A; Aulchenko, Y S; Hottenga, J J; Rivadeneira, F R; Hofman, A; Uitterlinden, A G; Hammond, C J; Shin, S-Y; Ikram, A; Witteman, J C M; Janssens, A C J W; Snieder, H; Tiemeier, H; Wolfenbuttel, B H R; Oostra, B A; Heath, A C; Wichmann, E; Spector, T D; Grabe, H J; Boomsma, D I; Martin, N G; van Duijn, C M

    2012-01-01

    Coffee consumption is a model for addictive behavior. We performed a meta-analysis of genome-wide association studies (GWASs) on coffee intake from 8 Caucasian cohorts (N=18 176) and sought replication of our top findings in a further 7929 individuals. We also performed a gene expression analysis treating different cell lines with caffeine. Genome-wide significant association was observed for two single-nucleotide polymorphisms (SNPs) in the 15q24 region. The two SNPs rs2470893 and rs2472297 (P-values=1.6 × 10−11 and 2.7 × 10−11), which were also in strong linkage disequilibrium (r2=0.7) with each other, lie in the 23-kb long commonly shared 5′ flanking region between CYP1A1 and CYP1A2 genes. CYP1A1 was found to be downregulated in lymphoblastoid cell lines treated with caffeine. CYP1A1 is known to metabolize polycyclic aromatic hydrocarbons, which are important constituents of coffee, whereas CYP1A2 is involved in the primary metabolism of caffeine. Significant evidence of association was also detected at rs382140 (P-value=3.9 × 10−09) near NRCAM—a gene implicated in vulnerability to addiction, and at another independent hit rs6495122 (P-value=7.1 × 10−09)—an SNP associated with blood pressure—in the 15q24 region near the gene ULK3, in the meta-analysis of discovery and replication cohorts. Our results from GWASs and expression analysis also strongly implicate CAB39L in coffee drinking. Pathway analysis of differentially expressed genes revealed significantly enriched ubiquitin proteasome (P-value=2.2 × 10−05) and Parkinson's disease pathways (P-value=3.6 × 10−05). PMID:21876539

  2. Down-regulation of adenosine A1 and A2A receptors in peripheral cells from idiopathic normal-pressure hydrocephalus patients.

    PubMed

    Casati, Martina; Arosio, Beatrice; Gussago, Cristina; Ferri, Evelyn; Magni, Lorenzo; Assolari, Lara; Scortichini, Valeria; Nani, Carolina; Rossi, Paolo Dionigi; Mari, Daniela

    2016-02-15

    Idiopathic normal-pressure hydrocephalus (iNPH) is a neurological disease that usually develops in the elderly. Natural history of iNPH is still unknown. It has been hypothesized that cerebrovascular diseases could have a role in etiology of chronic hydrocephalus and studies show an increased prevalence of cardiovascular diseases in iNPH patients. Moreover, evidences show a possible alteration of immune system in iNPH patients. Adenosine (Ado) is a metabolite produced in response to metabolic stress and injury. Adenosine and its receptors play an important role in vascular protection and in the modulation of inflammatory reactions and neuroinflammation. Our aim is to evaluate gene and protein expression of A1R and A2AR in the peripheral blood mononuclear cells (PBMCs) from iNPH patients compared to control subjects. We investigate if Ado system, that plays an important role in central nervous system, in vascular system, and also in inflammation, is involved in pathophysiology of iNPH disease. Our analysis showed that A1R mRNA levels and A1R density in PBMCs from iNPH patients were significantly lower than CT subjects (0.84 ± 0.12 and 2.42 ± 0.42, p<0.001 and 0.31 ± 0.02 and 0.42 ± 0.04, p=0.043; respectively). About A2AR, the gene expression in PBMCs was significantly lower in iNPH than CT (0.65 ± 0.09 and 1.5 ± 0.14, p<0.001) as well as there was a trend in protein expression: iNPH and CT (0.51 ± 0.05 and 0.62 ± 0.03; p=0.172). This preliminary study underlines the involvement of Ado system in iNPH disease whose pathophysiology is still unclear.

  3. Scoliosis in osteogenesis imperfecta caused by COL1A1/COL1A2 mutations - genotype-phenotype correlations and effect of bisphosphonate treatment.

    PubMed

    Sato, Atsuko; Ouellet, Jean; Muneta, Takeshi; Glorieux, Francis H; Rauch, Frank

    2016-05-01

    Bisphosphonates are widely used to treat children with osteogenesis imperfecta (OI), a bone fragility disorder that is most often caused by mutations in COL1A1 or COL1A2. However, it is unclear whether this treatment decreases the risk of developing scoliosis. We retrospectively evaluated spine radiographs and charts of 437 patients (227 female) with OI caused by mutations in COL1A1 or COL1A2 and compared the relationship between scoliosis, genotype and bisphosphonate treatment history. At the last follow-up (mean age 11.9 [SD: 5.9] years), 242 (55%) patients had scoliosis. The prevalence of scoliosis was highest in OI type III (89%), followed by OI type IV (61%) and OI type I (36%). Moderate to severe scoliosis (Cobb angle ≥25°) was rare in individuals with COL1A1 haploinsufficiency mutations but was present in about two fifth of patients with triple helical glycine substitutions or C-propeptide mutations. During the first 2 to 4years of bisphosphonate therapy, patients with OI type III had lower Cobb angle progression rates than before bisphosphonate treatment, whereas in OI types I and IV bisphosphonate treatment was not associated with a change in Cobb angle progression rates. At skeletal maturity, the prevalence of scoliosis (Cobb angle >10°) was similar in patients who had started bisphosphonate treatment early in life (before 5.0years of age) and in patients who had started therapy later (after the age of 10.0years) or had never received bisphosphonate therapy. Bisphosphonate treatment decreased progression rate of scoliosis in OI type III but there was no evidence of a positive effect on scoliosis in OI types I and IV. The prevalence of scoliosis at maturity was not influenced by the bisphosphonate treatment history in any OI type. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Mutations in COL1A1 and COL1A2 and dental aberrations in children and adolescents with osteogenesis imperfecta – A retrospective cohort study

    PubMed Central

    Dahllöf, Göran; Lindahl, Katarina; Kindmark, Andreas; Grigelioniene, Giedre; Åström, Eva; Malmgren, Barbro

    2017-01-01

    Osteogenesis imperfecta (OI) is a heterogeneous group of disorders of connective tissue, caused mainly by mutations in the collagen I genes (COL1A1 and COL1A2). Dentinogenesis imperfecta (DGI) and other dental aberrations are common features of OI. We investigated the association between collagen I mutations and DGI, taurodontism, and retention of permanent second molars in a retrospective cohort of 152 unrelated children and adolescents with OI. The clinical examination included radiographic evaluations. Teeth from 81 individuals were available for histopathological evaluation. COL1A1/2 mutations were found in 104 individuals by nucleotide sequencing. DGI was diagnosed clinically and radiographically in 29% of the individuals (44/152) and through isolated histological findings in another 19% (29/152). In the individuals with a COL1A1 mutation, 70% (7/10) of those with a glycine substitution located C-terminal of p.Gly305 exhibited DGI in both dentitions while no individual (0/7) with a mutation N-terminal of this point exhibited DGI in either dentition (p = 0.01). In the individuals with a COL1A2 mutation, 80% (8/10) of those with a glycine substitution located C terminal of p.Gly211 exhibited DGI in both dentitions while no individual (0/5) with a mutation N-terminal of this point (p = 0.007) exhibited DGI in either dentition. DGI was restricted to the deciduous dentition in 20 individuals. Seventeen had missense mutations where glycine to serine was the most prevalent substitution (53%). Taurodontism occurred in 18% and retention of permanent second molars in 31% of the adolescents. Dental aberrations are strongly associated with qualitatively changed collagen I. The varying expressivity of DGI is related to the location of the collagen I mutation. Genotype information may be helpful in identifying individuals with OI who have an increased risk of dental aberrations. PMID:28498836

  5. Mutations in COL1A1 and COL1A2 and dental aberrations in children and adolescents with osteogenesis imperfecta - A retrospective cohort study.

    PubMed

    Andersson, Kristofer; Dahllöf, Göran; Lindahl, Katarina; Kindmark, Andreas; Grigelioniene, Giedre; Åström, Eva; Malmgren, Barbro

    2017-01-01

    Osteogenesis imperfecta (OI) is a heterogeneous group of disorders of connective tissue, caused mainly by mutations in the collagen I genes (COL1A1 and COL1A2). Dentinogenesis imperfecta (DGI) and other dental aberrations are common features of OI. We investigated the association between collagen I mutations and DGI, taurodontism, and retention of permanent second molars in a retrospective cohort of 152 unrelated children and adolescents with OI. The clinical examination included radiographic evaluations. Teeth from 81 individuals were available for histopathological evaluation. COL1A1/2 mutations were found in 104 individuals by nucleotide sequencing. DGI was diagnosed clinically and radiographically in 29% of the individuals (44/152) and through isolated histological findings in another 19% (29/152). In the individuals with a COL1A1 mutation, 70% (7/10) of those with a glycine substitution located C-terminal of p.Gly305 exhibited DGI in both dentitions while no individual (0/7) with a mutation N-terminal of this point exhibited DGI in either dentition (p = 0.01). In the individuals with a COL1A2 mutation, 80% (8/10) of those with a glycine substitution located C terminal of p.Gly211 exhibited DGI in both dentitions while no individual (0/5) with a mutation N-terminal of this point (p = 0.007) exhibited DGI in either dentition. DGI was restricted to the deciduous dentition in 20 individuals. Seventeen had missense mutations where glycine to serine was the most prevalent substitution (53%). Taurodontism occurred in 18% and retention of permanent second molars in 31% of the adolescents. Dental aberrations are strongly associated with qualitatively changed collagen I. The varying expressivity of DGI is related to the location of the collagen I mutation. Genotype information may be helpful in identifying individuals with OI who have an increased risk of dental aberrations.

  6. A Dynamical Systems Approach to Analysis of Earthquakes on a 2D Discrete Fault Zone Embedded in a 3D Elastic Half-Space

    NASA Astrophysics Data System (ADS)

    Anghel, M.; Ben-Zion, Y.

    2001-05-01

    Numerical simulations of slip along a 2D cellular fault zone in a 3D elastic half-space are performed for several models [Ben-Zion and Rice, 1993; Ben-Zion, 1996] that belong to a generalized phase space of models. Each model consists of a planar computational grid with uniform cells where slip is governed by brittle and creep processes, surrounded by regions with constant slip rate representing the tectonic loading. Brittle failures are governed by a static/kinetic friction law with spatially varying coefficients chosen to represent different cases of quenched heterogeneities. The creep process is given by a power law dependency of creep velocity on stress with spatially varying coefficients chosen to produce, when activated, gradual (brittle-ductile type) stress transitions at the edges of the computational grid. Quasi-static stress transfer due to slip anywhere on the fault is calculated with 3D elastic dislocation theory. Inertial effects during brittle failures are accounted for approximately by a dynamic overshoot coefficient. The phase space of models is described by generalized coordinates that measure: a) the form of slip transitions at the edges of the computational grid (we employ two different limits, one in which the creep process in the computational grid is deactivated leading to abrupt transitions, and one with gradual transitions), b) the dynamic weakening, c) the distribution of brittle fault properties, d) the fault aspect ratio, e) the depth dependence of the static friction (we simulate two different limits, one in which the static friction does not have a trend with depth and one in which it grows linearly with depth). We analyze the interplay between temporal chaos and spatial scales in the simulated patterns by measuring various quantifiers of the chaotic dynamics (such as Lyapunov exponents, entropies and dimensions of the attractor describing the motion) as we explore the phase space of models. A key tool in the analysis of the results is

  7. Adenosine A1 and A2A receptors are not upstream of caffeine's dopamine D2 receptor-dependent aversive effects and dopamine-independent rewarding effects

    PubMed Central

    Sturgess, Jessica E; Ting-A-Kee, Ryan A; Podbielski, Dominik; Sellings, Laurie HL; Chen, Jiang-Fan; van der Kooy, Derek

    2010-01-01

    Caffeine is widely consumed throughout the world, yet little is known about the mechanisms underlying its rewarding and aversive properties. We show that pharmacological antagonism of dopamine not only blocks conditioned place aversions to caffeine, but reveals dopamine blockade-induced conditioned place preferences. These aversions are mediated by the dopamine D2 receptor since knockout mice showed conditioned place preferences to doses of caffeine that C57Bl/6 mice found aversive. Further, these aversions appear to be centrally-mediated since a quaternary analogue to caffeine failed to produce conditioned place aversions. While the adenosine A2A receptor is important for caffeine's physiological effects, this receptor seems only to modulate the appetitive and aversive effects of caffeine. A2A receptor knockout mice showed stronger dopamine-dependent aversions to caffeine than C57Bl/6 animals, which partially obscured the dopamine- and A2A receptor-independent preferences. Additionally, the A1 receptor, alone or in combination with the A2A receptor, does not seem to be important for caffeine's rewarding or aversive effects. Finally, excitotoxic lesions of the tegmental pedunculopontine nucleus revealed that this brain region is not involved in dopamine blockade-induced caffeine reward. This data provides surprising new information on the mechanism of action of caffeine, indicating that adenosine receptors do not mediate caffeine's appetitive and aversive effects. We show that caffeine has an atypical reward mechanism, independent of the dopaminergic system and the tegmental pedunculopontine nucleus and provide additional evidence in support of a role for the dopaminergic system in aversive learning. PMID:20576036

  8. Oxidation of the flavonoids galangin and kaempferide by human liver microsomes and CYP1A1, CYP1A2, and CYP2C9.

    PubMed

    Otake, Yoko; Walle, Thomas

    2002-02-01

    There is very limited information on cytochrome P450 (P450)-mediated oxidative metabolism of dietary flavonoids in humans. In this study, we used human liver microsomes and recombinant P450 isoforms to examine the metabolism of two flavonols, galangin and kaempferide, and one flavone, chrysin. Both galangin and kaempferide, but not chrysin, were oxidized by human liver microsomes to kaempferol, with K(m) values of 9.5 and 17.8 microM, respectively. These oxidations were catalyzed mainly by CYP1A2 but also by CYP2C9. Consistent with these observations, the human liver microsomal metabolism of galangin and kaempferide were inhibited by the P450 inhibitors furafylline and sulfaphenazole. In addition, CYP1A1, although less efficient, was also able to oxidize the two flavonols. Thus, dietary flavonols are likely to undergo oxidative metabolism mainly in the liver but also extrahepatically.

  9. Dynamic hip screw versus proximal femur locking compression plate in intertrochanteric femur fractures (AO 31A1 and 31A2): A prospective randomized study

    PubMed Central

    Agrawal, Prabhat; Gaba, Sahil; Das, Saubhik; Singh, Ranjit; Kumar, Arvind; Yadav, Gajanand

    2017-01-01

    Introduction: Intertrochanteric fractures are common in elderly population and pose a significant financial burden to the society. Anatomically contoured proximal femur locking compression plate (PFLCP) is the latest addition in the surgeons’ armamentarium to deal with these fractures. It creates an angular stable construct, which will theoretically lessen the risk of failure by screw cut-out and varus collapse, the common mode of DHS failure. We compared DHS with PFLCP in AO type 31A1 and 31A2 intertrochanteric fractures. Materials and Methods: A randomized prospective study was carried out between June 2011 and June 2013. 26 cases each of DHS and PFLCP were included. Results: Functional and radiological outcome was similar in both groups. Conclusion: Both DHS and PFLCP are good choices for stable intertrochanteric fractures, and both lead to excellent functional outcomes, but non-union might be more common with PFLCP.

  10. Pharmacokinetic and pharmacodynamic consequences of inhibition of terazosin metabolism via CYP3A1 and/or 3A2 by DA-8159, an erectogenic, in rats

    PubMed Central

    Oh, E Y; Bae, S K; Kwon, J W; You, M; Lee, D C; Lee, M G

    2007-01-01

    Background and purpose: Recently, orthostatic hypotension was observed in patients with benign prostatic hyperplasia who are taking vardenafil (a PDE 5 inhibitor) and terazosin (a long acting alpha blocker). Therefore, this study was performed with DA-8159 (a long acting PDE 5 inhibitor) and terazosin in rats to find whether or not pharmacokinetic and pharmacodynamic interactions between the two drugs were observed. Experimental approach: Pharmacokinetic and pharmacodynamic (changes in blood pressure) interactions between DA-8159 and terazosin were evaluated after simultaneous i.v. and p.o. administration of DA-8159 (30 mg kg−1) and terazosin (5 mg kg−1) to male Sprague–Dawley rats. Key results: After simultaneous i.v. and p.o. administration of terazosin and DA-8159, the total area under the plasma concentration–time curve from time zero to time infinity (AUC) of terazosin became significantly greater (57.4 and 75.4% increase for i.v. and p.o. administration, respectively) than those of without DA-8159. The blood pressure dropping effect was considerable after simultaneous p.o. administration of DA-8159 and terazosin compared with each drug alone. Conclusions and implications: The significantly greater AUC of terazosin after both simultaneous i.v. and p.o. administration of both drugs could be due to the hepatic (both i.v. and p.o.) and intestinal (p.o.) inhibition of the metabolism of terazosin via CYP3A1 and/or 3A2 by DA-8159, since both DA-8159 and terazosin are metabolized via CYP3A1 and/or 3A2 in rats. The blood pressure lowering effect after simultaneous p.o. administration of both drugs could be due to significant increase in plasma concentrations of terazosin. PMID:17351661

  11. [Thulium vapoenucleation of prostates larger than 80 ml using a 1.9-µm and a 2-µm thulium laser. Early perioperative results from two centres].

    PubMed

    Netsch, C; Knoll, T; Gross, A J; Wendt-Nordahl, G

    2015-10-01

    Numerous studies have shown that thulium vapoenucleation of the prostate (ThuVEP) is a size-independent minimally invasive procedure for the treatment of benign prostatic enlargement. All ThuVEP series have been performed with a 2-µm thulium laser device so far. The aim of this study was to evaluate the complications and early postoperative results of two thulium-devices with different wavelengths for ThuVEP in prostates larger than 80 ml. A retrospective bi-centric matched-paired analysis with 296 patients was performed. Based on prostate size, 148 were matched at each centre and laser device, respectively. A 2-µm (RevoLix, LISA Laser products, Katlenburg, Germany n=148) and a 1.9-µm (vela XL, starmedtec, Starnberg, Germany, n=148) thulium laser with a power output of 90 and 80 W was used. Patients' data were assessed and compared. The median prostate volume (interquartile) was 100 ml (range 86.25-120 ml). At discharge, Qmax (preoperative 7.9 and 9 ml/s vs. postoperative 19.35 and 16.2 ml/s) and postvoiding-residual urine (preoperative 130 and 45 ml vs. postoperative 20 and 25 ml) were significantly improved after 2-µm and 1.9-µm ThuVEP (p<0.001). The median catheterization time and hospitalization times were 2 and 4 days in both groups. Perioperative complications occurred in 89 patients (30.1%): Clavien 1 (12.2%), Clavien 2 (9.1%), Clavien 3a (0.7%), Clavien 3b (7.1%), and Clavien 4a (1%). Regarding the occurrence of complications, there were no differences between the two thulium devices. ThuVEP represents a safe and effective treatment for prostates larger than 80 ml. Both thulium laser devices give satisfactory immediate micturition improvement with low perioperative morbidity.

  12. Pulmonary surfactant synthesis in miRNA-26a-1/miRNA-26a-2 double knockout mice generated using the CRISPR/Cas9 system

    PubMed Central

    Zhang, Ying-Hui; Wu, Li-Zhi; Liang, Hong-Lu; Yang, Yang; Qiu, Jie; Kan, Qing; Zhu, Wen; Ma, Cheng-Ling; Zhou, Xiao-Yu

    2017-01-01

    Pulmonary surfactant (PS), which is synthesized by type II alveolar epithelial cells (AECIIs), maintains alveolar integrity by reducing surface tension. Many premature neonates who lack adequate PS are predisposed to developing respiratory distress syndrome (RDS), one of the leading causes of neonatal morbidity and mortality. PS synthesis is influenced and regulated by various factors, including microRNAs. Previous in vitro studies have shown that PS synthesis is regulated by miR-26a in fetal rat AECIIs. This study aimed to investigate the role of miR-26a in PS synthesis in vivo. To obtain a miR-26a-1/miR-26a-2 double knockout mouse model, we used the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) system, an important genome editing technology. Real-time PCR was performed to determine the miR-26a levels in various organs, as well as the mRNA levels of surfactant-associated proteins. Moreover, AECIIs and surfactant-associated proteins in lung tissues were analyzed by hematoxylin-eosin staining and immunohistochemistry. Homozygous offspring of miR-26a-1/miR-26a-2 double knockout mice generated using the CRISPR/Cas9 system were successfully obtained, and PS synthesis and the number of AECIIs were significantly increased in the miR-26a knockout mice. These results indicate that miR-26a plays an important role in PS synthesis in AECIIs. PMID:28337265

  13. What difference would a 1.5°C vs a 2°C warming target make to precipitation?

    NASA Astrophysics Data System (ADS)

    Rogers, Megan; Hegerl, Gabi; Iles, Carley

    2017-04-01

    In December 2015, the member states of the United Nations signed the Paris agreement, committing to limit the rise in global temperature above pre-industrial levels to between 1.5 and 2°C. Precipitation changes are a major impact of climate change, and so understanding how these are likely to differ between a 1.5°C and a 2°C warming scenario is very important. In this study, we examine the precipitation changes at these temperature thresholds in CMIP5 simulations. We compare results for three climate models for the RCP 4.5 scenario: HadGEM2-ES, CCSM4 and CanESM2. There are expected to be opposite precipitation changes in some regions under global warming in keeping with the 'wet gets wetter, dry gets drier' paradigm. These patterns may also shift seasonally, following the seasonal cycle of precipitation, particularly in the tropics. Therefore, both global and regional, and annual and seasonal data are analysed to provide a comprehensive insight into the differences in precipitation changes. Finally, the time of emergence of precipitation trends at a regional and gridpoint scale is determined, and analysed within the context of a 1.5°C vs a 2°C scenario. Time of emergence is a valuable metric as it describes when a particular change becomes significant with respect to natural variability, and therefore when said change will begin to affect local ecosystems and human societies. The smaller the scale over which time of emergence is calculated the more relevant it is for local impacts. However, for precipitation, natural variability at gridpoint level is very high and signals may not emerge before the end of the century. Therefore, emergence of regional average precipitation is also analysed.

  14. Annexin A1 reduces inflammatory reaction and tissue damage through inhibition of phospholipase A2 activation in adult rats following spinal cord injury.

    PubMed

    Liu, Nai-Kui; Zhang, Yi Ping; Han, Shu; Pei, Jiong; Xu, Lisa Y; Lu, Pei-Hua; Shields, Christopher B; Xu, Xiao-Ming

    2007-10-01

    Annexin A1 (ANXA1) has been suggested to be a mediator of the anti-inflammatory actions of glucocorticoids and more recently an endogenous neuroprotective agent. In the present study, we investigated the anti-inflammatory and neuroprotective effects of ANXA1 in a model of contusive spinal cord injury (SCI). Here we report that injections of ANXA1 (Ac 2-26) into the acutely injured spinal cord at 2 concentrations (5 and 20 microg) inhibited SCI-induced increases in phospholipase A2 and myeloperoxidase activities. In addition, ANXA1 administration reduced the expression of interleukin-1beta and activated caspase-3 at 24 hours, and glial fibrillary acidic protein at 4 weeks postinjury. Furthermore, ANXA1 administration significantly reversed phospholipase A2-induced spinal cord neuronal death in vitro and reduced tissue damage and increased white matter sparing in vivo, compared to the vehicle-treated controls. Fluorogold retrograde tracing showed that ANXA1 administration protected axons of long descending pathways at 6 weeks post-SCI. ANXA1 administration also significantly increased the number of animals that responded to transcranial magnetic motor-evoked potentials. However, no measurable behavioral improvement was found after these treatments. These results, particularly the improvements obtained in tissue sparing and electrophysiologic measures, suggest a neuroprotective effect of ANXA1.

  15. Cloning and structural analysis of two highly divergent IgA isotypes, IgA1 and IgA2 from the duck billed platypus, Ornithorhynchus anatinus.

    PubMed

    Vernersson, M; Belov, K; Aveskogh, M; Hellman, L

    2010-01-01

    To trace the emergence of modern IgA isotypes during vertebrate evolution we have studied the immunoglobulin repertoire of a model monotreme, the platypus. Two highly divergent IgA-like isotypes (IgA1 and IgA2) were identified and their primary structures were determined from full-length cDNAs. A comparative analysis of the amino acid sequences for IgA from various animal species showed that the two platypus IgA isotypes form a branch clearly separated from their eutherian (placental) counterparts. However, they still conform to the general structure of eutherian IgA, with a hinge region and three constant domains. This indicates that the deletion of the second domain and the formation of a hinge region in IgA did occur very early during mammalian evolution, more than 166 million years ago. The two IgA isotypes in platypus differ in primary structure and appear to have arisen from a very early gene duplication, possibly preceding the metatherian eutherian split. Interestingly, one of these isotypes, IgA1, appears to be expressed in only the platypus, but is present in the echidna based on Southern blot analysis. The platypus may require a more effective mucosal immunity, with two highly divergent IgA forms, than the terrestrial echidna, due to its lifestyle, where it is exposed to pathogens both on land and in the water.

  16. Differential effects of cytokines on the inducible expression of CYP1A1, CYP1A2, and CYP3A4 in human hepatocytes in primary culture.

    PubMed

    Muntané-Relat, J; Ourlin, J C; Domergue, J; Maurel, P

    1995-10-01

    We have investigated the effect of cytokines, including interleukin-6 (Il-6), interleukin-1 alpha (Il-1 alpha), and tumor necrosis factor-alpha (TNF-alpha), on the inducible expression of cytochrome P450s (CYP) CYP1A1, CYP1A2, and CYP3A4 in human hepatocytes in primary culture. The ability of these cultures to mimic the acute phase response when stimulated with cytokines was evaluated using immunoblotting to measure the production of albumin, ferritin, fibrinogen, and ceruloplasmin. The cytokines exhibited specific patterns of action on the production of these proteins. Albumin was depressed by all the cytokines. In contrast to Il-6 and Il-1 alpha, TNF-alpha reduced the production of fibrinogen and ceruloplasmin but stimulated the production of ferritin. When cells were treated with the CYP inducer alone, large increases in the expression of CYP1A1 and CYP1A2 by beta-naphthoflavone and of CYP3A4 by rifampicin were observed at messenger RNA (mRNA) and protein levels, by ribonuclease protection and immunoblotting, respectively. When the cells were treated with the inducer plus cytokines, the induction of mRNA was greatly reduced. Again, specific patterns of action were revealed: Il-6 had the most potent effect on CYP3A4, whereas TNF-alpha was the most potent with CYP1A genes. In all cases, changes at the protein levels paralleled changes at the mRNA levels. In cells preinduced with beta-naphthoflavone or rifampicin, the decay with time of the levels of the CYP1A2 or CYP3A4 proteins, after the removal of the inducer, was not affected by cytokines. We conclude that cytokines strongly repress the inducibility of CYP1As and CYP3A4 genes at a transcriptional or a posttranscriptional level, but affect neither the rate of translation of CYP mRNAs nor the rate of degradation of the CYP proteins in these cultures.

  17. Analysis of the Rotationally Resolved, Non-Degenerate (a''_1) and Degenerate (e') Vibronic Bands in the tilde{A}^2E'' ← tilde{X}^2A'_2 Transition of NO_3.

    NASA Astrophysics Data System (ADS)

    Tran, Henry; Miller, Terry A.

    2016-06-01

    The magnitude of the Jahn-Teller (JT) effect in NO_3 has been the subject of considerable research in our group and other groups around the world. The rotational contour of the 4^1_0 vibronic band was first described by Hirota and coworkers using an oblate symmetric top. Near-infrared band of the nitrate radical NO_3 observed by diode laser spectroscopy. J. Chem. Phys., 107:2829, 1997.} Deev et al. argued that an asymmetric top was required to describe the 2^1_0 band, although their spectrum was not completely rotationally resolved. These discrepancies suggest that a rotational analysis will provide considerable experimental information on the geometry of NO_3. Our group has collected high-resolution, rotationally resolved spectra of the vibronic tilde{A}^2E'' ← tilde{X}^2A'_2 transitions. We have completed analysis of the 3^1_0 and 3^1_04^1_0 parallel bands with a_1'' symmetry by using an oblate symmetric top with spin-rotation and centrifugal distortions. Several other parallel bands are now also reasonably understood. This analysis is consistent with a D3h geometry for NO_3. In order to analyze the perpendicular bands with e' symmetry, we have adapted the oblate symmetric top Hamiltonian from the previous analysis to include spin-orbit coupling, coriolis coupling, and Watson Terms (JT distortions) that allow the oblate symmetric top Hamiltonian to transition continuously to the distorted limit of C2v symmetry. Preliminary analysis of the 2^1_0 and 2^1_04^2_0 bands has shown generally good agreement between model and experimental spectra. Our results indicate only modest JT distortions, although we do find evidence of multiple perturbations between these bands and high vibrational levels of the tilde{X} state. We will present our adapted Hamiltonian and the analysis of the 3^1_0, 3^1_04^1_0, 2^1_0, and 2^1_04^2_0 bands. E. Hirota, T. Ishiwata, K. Kawaguchi, M. Fujitake, N. Ohashi, and I. Tanaka. Near-infrared band of the nitrate radical NO_3 observed by diode

  18. 17 CFR Appendix A to Part 3 - Interpretative Statement With Respect to Section 8a(2)(C) and (E) and Section 8a(3)(J) and (M) of...

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... cause” under Section 8a(3)(M) of the Act in issuing guidance letters on assessing the fitness of floor... registration review process during the past three years. This guidance will help ensure that NFA exercises its... action in the past and endeavor to seek similar registration restrictions, conditions,...

  19. Formation of DNA adducts in wild-type and transgenic mice expressing human sulfotransferases 1A1 and 1A2 after oral exposure to furfuryl alcohol

    PubMed Central

    Høie, Anja Hortemo; Monien, Bernhard Hans; Sakhi, Amrit Kaur; Glatt, Hansruedi; Hjertholm, Hege; Husøy, Trine

    2015-01-01

    Furfuryl alcohol (FFA) is present in many heat-treated foods as a result of its formation via dehydration of pentoses. It is also used legally as a flavouring agent. In an inhalation study conducted in the National Toxicology Program, FFA showed some evidence of carcinogenic activity in rats and mice. FFA was generally negative in conventional genotoxicity assays, which suggests that it may be a non-genotoxic carcinogen. However, it was recently found that FFA is mutagenic in Salmonella strains expressing appropriate sulfotransferases (SULTs), such as human or mouse SULT1A1. The same DNA adducts that were formed by FFA in these strains, mainly N 2-((furan-2-yl)methyl)-2′-deoxyguanosine (N 2-MF-dG), were also detected in tissues of FFA-exposed mice and even in human lung specimens. In the present study, a single oral dose of FFA (250mg/kg body weight) or saline was administered to FVB/N mice and transgenic mice expressing human SULT1A1/1A2 on the FVB/N background. The transgenic mice were used, since human and mouse SULT1A1 substantially differ in substrate specificity and tissue distribution. DNA adducts were studied in liver, kidney, proximal and distal small intestine as well as colon, using isotope-dilution ultra performance liquid chromatography (UPLC–MS/MS). Surprisingly, low levels of adducts that may represent N 2-MF-dG were detected even in tissues of untreated mice. FFA exposure enhanced the adduct levels in colon and liver, but not in the remaining investigated tissues of wild-type (wt) mice. The situation was similar in transgenic mice, except that N 2-MF-dG levels were also strongly enhanced in the proximal small intestine. These different results between wt and transgenic mice may be attributed to the fact that human SULT1A1, but not the orthologous mouse enzyme, is strongly expressed in the small intestine. PMID:25904584

  20. Formation of DNA adducts in wild-type and transgenic mice expressing human sulfotransferases 1A1 and 1A2 after oral exposure to furfuryl alcohol.

    PubMed

    Høie, Anja Hortemo; Monien, Bernhard Hans; Sakhi, Amrit Kaur; Glatt, Hansruedi; Hjertholm, Hege; Husøy, Trine

    2015-09-01

    Furfuryl alcohol (FFA) is present in many heat-treated foods as a result of its formation via dehydration of pentoses. It is also used legally as a flavouring agent. In an inhalation study conducted in the National Toxicology Program, FFA showed some evidence of carcinogenic activity in rats and mice. FFA was generally negative in conventional genotoxicity assays, which suggests that it may be a non-genotoxic carcinogen. However, it was recently found that FFA is mutagenic in Salmonella strains expressing appropriate sulfotransferases (SULTs), such as human or mouse SULT1A1. The same DNA adducts that were formed by FFA in these strains, mainly N (2)-((furan-2-yl)methyl)-2'-deoxyguanosine (N (2)-MF-dG), were also detected in tissues of FFA-exposed mice and even in human lung specimens. In the present study, a single oral dose of FFA (250 mg/kg body weight) or saline was administered to FVB/N mice and transgenic mice expressing human SULT1A1/1A2 on the FVB/N background. The transgenic mice were used, since human and mouse SULT1A1 substantially differ in substrate specificity and tissue distribution. DNA adducts were studied in liver, kidney, proximal and distal small intestine as well as colon, using isotope-dilution ultra performance liquid chromatography (UPLC-MS/MS). Surprisingly, low levels of adducts that may represent N (2)-MF-dG were detected even in tissues of untreated mice. FFA exposure enhanced the adduct levels in colon and liver, but not in the remaining investigated tissues of wild-type (wt) mice. The situation was similar in transgenic mice, except that N (2)-MF-dG levels were also strongly enhanced in the proximal small intestine. These different results between wt and transgenic mice may be attributed to the fact that human SULT1A1, but not the orthologous mouse enzyme, is strongly expressed in the small intestine.

  1. The Pseudomonas aeruginosa PA14 ABC Transporter NppA1A2BCD Is Required for Uptake of Peptidyl Nucleoside Antibiotics.

    PubMed

    Pletzer, Daniel; Braun, Yvonne; Dubiley, Svetlana; Lafon, Corinne; Köhler, Thilo; Page, Malcolm G P; Mourez, Michael; Severinov, Konstantin; Weingart, Helge

    2015-07-01

    Analysis of the genome sequence of Pseudomonas aeruginosa PA14 revealed the presence of an operon encoding an ABC-type transporter (NppA1A2BCD) showing homology to the Yej transporter of Escherichia coli. The Yej transporter is involved in the uptake of the peptide-nucleotide antibiotic microcin C, a translation inhibitor that targets the enzyme aspartyl-tRNA synthetase. Furthermore, it was recently shown that the Opp transporter from P. aeruginosa PAO1, which is identical to Npp, is required for uptake of the uridyl peptide antibiotic pacidamycin, which targets the enzyme translocase I (MraY), which is involved in peptidoglycan synthesis. We used several approaches to further explore the substrate specificity of the Npp transporter. Assays of growth in defined minimal medium containing peptides of various lengths and amino acid compositions as sole nitrogen sources, as well as Biolog Phenotype MicroArrays, showed that the Npp transporter is not required for di-, tri-, and oligopeptide uptake. Overexpression of the npp operon increased susceptibility not just to pacidamycin but also to nickel chloride and the peptidyl nucleoside antibiotic blasticidin S. Furthermore, heterologous expression of the npp operon in a yej-deficient mutant of E. coli resulted in increased susceptibility to albomycin, a naturally occurring sideromycin with a peptidyl nucleoside antibiotic. Additionally, heterologous expression showed that microcin C is recognized by the P. aeruginosa Npp system. Overall, these results suggest that the NppA1A2BCD transporter is involved in the uptake of peptidyl nucleoside antibiotics by P. aeruginosa PA14. One of the world's most serious health problems is the rise of antibiotic-resistant bacteria. There is a desperate need to find novel antibiotic therapeutics that either act on new biological targets or are able to bypass known resistance mechanisms. Bacterial ABC transporters play an important role in nutrient uptake from the environment. These uptake

  2. Energetics and kinetics of phase transition between a 2H and a 1T MoS2 monolayer-a theoretical study.

    PubMed

    Zhao, Wen; Ding, Feng

    2017-02-09

    Phase transitions between semiconducting 2H and metallic 1T (or 1T') molybdenum disulphides (MoS2) are explored comprehensively by first-principles calculations. The nucleation of a 1T (or 1T') nucleus in a 2H MoS2 lattice, the formation of the 2H-1T (1T') interfaces and the kinetics of interface propagation during phase transition were thoroughly investigated in this study. It was found that, among various potential interface structures between the two phases, Mo-rich and two S-rich zigzag (ZZ) boundaries are energetically more preferable than others. Therefore, triangular or hexagonal 1T MoS2 domains with the three types of ZZ boundaries are expected to be the equilibrium structures of the 1T nucleus in a 2H lattice. Further exploration reveals a very high nucleation energy of the 1T phase which suggests that the nucleation may start from a defect site or the edge of the 2H phase. Besides, the kinetics of 2H-1T (1T') boundary propagation show that the growth rate of the ZZ boundaries is significantly slower than the armchair (AC) ones'. Therefore, the ZZ-boundary dominated domains are expected to be observed during the growth of the 1T phase while the rounded domains with various high-index edges are expected to be seen throughout shrinkage. This study reveals both the energetics and the kinetics of the phase transition of transition metal dichalcogenide materials and also sheds light on other 2D materials.

  3. Evaluation of toxic equivalency factors for induction of cytochromes P450 CYP1A1 and CYP1A2 enzyme activity by dioxin-like compounds.

    PubMed

    Toyoshiba, Hiroyoshi; Walker, Nigel J; Bailer, A John; Portier, Christopher J

    2004-01-15

    The toxic equivalency factor (TEF) method has been used to characterize the toxicity of human mixtures of dioxin-like compounds and is being considered for use with other classes of potentially toxic agents. TEFs are estimated by examining the relative potencies of the various congeners for a series of biological and toxicological effects. In this paper, we consider changes in activity for two enzymes, cytochrome P450 1A1 (CYP1A1)-associated 7-ethoxyresorufin-O-deethylase (EROD) and CYP1A2-associated acetanilide-4-hydroxylase (A4H) activity, resulting from exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4',5-pentachlorobiphenyl (PCB), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) or a mixture of these agents. The ratio of median effective dose (ED50) is one way to estimate the relative potencies, especially for gene expression and protein endpoints. ED50's were estimated with a nonlinear regression model in which dose-related changes in mean responses are described by a Hill function. ED50's along with other model parameters were estimated by fitting this model to a given data set. Significant differences in estimated model parameters were tested by likelihood ratio methods. The estimated parameters indicated that congener-specific dose-response shapes were significantly different, that additivity failed for these congeners, and that the ratios of ED50's did not predict the response seen for the mixture. These results indicate that for some biological responses, the use of a single relative potency factor (RPF) is not appropriate for the comparison of the dose response behavior of different dioxin-like congeners.

  4. Genetic studies on Vysyas of Andhra Pradesh, S. India: A1A2BO, Rh (O) D, transferrin, group specific component, haptoglobin and pseudocholinesterase types.

    PubMed

    Gopalam, K B; Rao, P R

    1981-01-01

    Vysya population, an endogamous Hindu caste group, was sampled from five distant localities of Andhra Pradesh, S. India and examined for A1A2BO, Rh blood groups, Tf, Hp, Gc, cholinesterase E1 and E2 loci, albumin and ceruloplasmin types. The blood group A has shown an exceptionally low value in these groups and consequently there is a rise in O group frequencies (0.7429 to 0.8144). The incidence of Rh negative individuals is very low (0.1115-0.1571), being absent from one of the groups. Tf DChi is found with a frequency ranging from 0.0043 to 0.0333 with a single fast moving Tf B variant in one of the sub-populations. Hp1 gene frequencies ranged from 0.1271 to 0.2130 and Gc2 from 0.1504 to 0.2773. Silent variants at E1 locus of pseudocholinesterase were present in very high frequencies (0.0115 to 0.1925), the overall frequency being 0.1040. Only a single C+5 was found and dibucaine as well as fluoride resistant variants were rare. No variants were found at the loci of albumin and ceruloplasmin. Differentiation in the distribution of these variants in the five sub-populations of Vysyas reported is evident from these studies.

  5. Targeted disruption of Col8a1 and Col8a2 genes in mice leads to anterior segment abnormalities in the eye.

    PubMed

    Hopfer, Ulrike; Fukai, Naomi; Hopfer, Helmut; Wolf, Gunter; Joyce, Nancy; Li, En; Olsen, Bjorn R

    2005-08-01

    Collagen VIII is localized in subendothelial and subepithelial extracellular matrices. It is a major component of Descemet's membrane, a thick basement membrane under the corneal endothelium, where it forms a hexagonal lattice structure; a similar structure, albeit less extensive, may be formed in other basement membranes. We have examined the function of collagen VIII in mice by targeted inactivation of the genes encoding the two polypeptide subunits, Col8a1 and Col8a2. Analysis of these mice reveals no major structural defects in most organs, but demonstrates that type VIII collagen is required for normal anterior eye development, particularly the formation of a corneal stroma with the appropriate number of fibroblastic cell layers and Descemet's membrane of appropriate thickness. Complete lack of type VIII collagen leads to dysgenesis of the anterior segment of the eye: a globoid, keratoglobus-like protrusion of the anterior chamber with a thin corneal stroma. Descemet's membrane is markedly thinned. The corneal endothelial cells are enlarged and reduced in number, and show a decreased ability to proliferate in response to different growth factors in vitro. An important function of collagen VIII may therefore be to generate a peri- or subcellular matrix environment that permits or stimulates cell proliferation.

  6. Bilateral Leg Replantation in a 3-Month-Old Baby After a Knee Level Crush Amputation-A 2-Year Follow-up.

    PubMed

    Bulic, Kresimir; Antabak, Anko; Dujmovic, Anto; Kisic, Hrvoje; Lorencin, Mia

    2017-03-01

    We present a case of a successful bilateral leg replantation in a 3-month-old baby after a knee-level crush amputation with the loss of both knee joints. The legs were replanted after 4 hours of warm and an additional 2.5 and 3.5 hours of cold ischemia time. Both legs show motor and sensory reinnervation, without additional procedures performed on the right leg, and after a nerve reconstruction with cadaveric allografts on the left leg. Both replanted legs exhibit excellent bony and soft tissue growth. Two years after the injury, the patient is progressing well with rehabilitation, with favourable odds of having knee reconstructions performed at a later age. This is the youngest patient reported to have had successful replantation of both legs.

  7. Cloning and expression analysis of two ROR-γ homologues (ROR-γa1 and ROR-γa2) in rainbow trout Oncorhynchus mykiss.

    PubMed

    Monte, Milena M; Wang, Tiehui; Costa, Maria M; Harun, Nor Omaima; Secombes, Chris J

    2012-08-01

    This paper describes the cloning and characterisation of two retinoid-related orphan receptor (ROR)-γ homologues (ROR-γa1 and -γa2) in rainbow trout (Oncorhynchus mykiss). The coding region predicted for both homologues consists of 1410 base pairs (bp), which translate into two 469 amino acid (aa) proteins. The trout ROR-γs revealed a high conservation of both DNA- and ligand-binding domains (functional regions of the nuclear receptor family), and shared a high homology to mammalian ROR-γt. A phylogenetic tree containing ROR family members confirmed that both trout homologues clustered within the ROR-γ group. Both results suggested that these molecules are likely to be ROR-γ homologues, more similar to the mammalian splice variant ROR-γt than the full length ROR-γ. Expression analysis of tissues obtained from healthy fish revealed highest constitutive expression of trout ROR-γ in muscle, followed by the brain, heart and skin. This suggests that these genes may play an important role in such tissues. In vitro studies, using trout cell lines, demonstrated that ROR-γ is induced significantly by LPS and down-regulated by the presence of PolyI:C and recombinant interferon (IFN)-γ. Moreover, analysis of this gene in head kidney macrophages and mixed primary leucocyte cultures indicated that differences were apparent between the different cell types/sources used, indicating that its expression may be cell-type dependent. Additional studies to investigate the regulation of this gene in vivo demonstrated that its expression was significantly higher in vaccinated vs unvaccinated fish following bacterial (Yersinia ruckeri) challenge but it was down-regulated after a viral (VHSV) infection. This suggests a potential role of trout ROR-γ, a putative T(H)17 transcription factor, in protection against extracellular bacteria.

  8. In vitro digestion of purified β-casein variants A(1), A(2), B, and I: effects on antioxidant and angiotensin-converting enzyme inhibitory capacity.

    PubMed

    Petrat-Melin, B; Andersen, P; Rasmussen, J T; Poulsen, N A; Larsen, L B; Young, J F

    2015-01-01

    Genetic polymorphisms of bovine milk proteins affect the protein profile of the milk and, hence, certain technological properties, such as casein (CN) number and cheese yield. However, reports show that such polymorphisms may also affect the health-related properties of milk. Therefore, to gain insight into their digestion pattern and bioactive potential, β-CN was purified from bovine milk originating from cows homozygous for the variants A(1), A(2), B, and I by a combination of cold storage, ultracentrifugation, and acid precipitation. The purity of the isolated β-CN was determined by HPLC, variants were verified by mass spectrometry, and molar extinction coefficients at λ=280nm were determined. β-Casein from each of the variants was subjected to in vitro digestion using pepsin and pancreatic enzymes. Antioxidant and angiotensin-converting enzyme (ACE) inhibitory capacities of the hydrolysates were assessed at 3 stages of digestion and related to that of the undigested samples. Neither molar extinction coefficients nor overall digestibility varied significantly between these 4 variants; however, clear differences in digestion pattern were indicated by gel electrophoresis. In particular, after 60min of pepsin followed by 5min of pancreatic enzyme digestion, one ≈4kDa peptide with the N-terminal sequence (106)H-K-E-M-P-F-P-K- was absent from β-CN variant B. This is likely a result of the (122)Ser to (122)Arg substitution in variant B introducing a novel trypsin cleavage site, leading to the changed digestion pattern. All investigated β-CN variants exhibited a significant increase in antioxidant capacity upon digestion, as measured by the Trolox-equivalent antioxidant capacity assay. After 60min of pepsin + 120min of pancreatic enzyme digestion, the accumulated increase in antioxidant capacity was ≈1.7-fold for the 4 β-CN variants. The ACE inhibitory capacity was also significantly increased by digestion, with the B variant reaching the highest inhibitory

  9. Breadth of neutralization and synergy of clinically relevant human monoclonal antibodies against HCV genotypes 1a, 1b, 2a, 2b, 2c, and 3a.

    PubMed

    Carlsen, Thomas H R; Pedersen, Jannie; Prentoe, Jannick C; Giang, Erick; Keck, Zhen-Yong; Mikkelsen, Lotte S; Law, Mansun; Foung, Steven K H; Bukh, Jens

    2014-11-01

    Human monoclonal antibodies (HMAbs) with neutralizing capabilities constitute potential immune-based treatments or prophylaxis against hepatitis C virus (HCV). However, lack of cell culture-derived HCV (HCVcc) harboring authentic envelope proteins (E1/E2) has hindered neutralization investigations across genotypes, subtypes, and isolates. We investigated the breadth of neutralization of 10 HMAbs with therapeutic potential against a panel of 16 JFH1-based HCVcc-expressing patient-derived Core-NS2 from genotypes 1a (strains H77, TN, and DH6), 1b (J4, DH1, and DH5), 2a (J6, JFH1, and T9), 2b (J8, DH8, and DH10), 2c (S83), and 3a (S52, DBN, and DH11). Virus stocks used for in vitro neutralization analysis contained authentic E1/E2, with the exception of full-length JFH1 that acquired the N417S substitution in E2. The 50% inhibition concentration (IC50) for each HMAb against the HCVcc panel was determined by dose-response neutralization assays in Huh7.5 cells with antibody concentrations ranging from 0.0012 to 100 μg/mL. Interestingly, IC50 values against the different HCVcc's exhibited large variations among the HMAbs, and only three HMAbs (HC-1AM, HC84.24, and AR4A) neutralized all 16 HCVcc recombinants. Furthermore, the IC50 values for a given HMAb varied greatly with the HCVcc strain, which supports the use of a diverse virus panel. In cooperation analyses, HMAbs HC84.24, AR3A, and, especially HC84.26, demonstrated synergistic effects towards the majority of the HCVcc's when combined individually with AR4A. Through a neutralization analysis of 10 clinically relevant HMAbs against 16 JFH1-based Core-NS2 recombinants from genotypes 1a, 1b, 2a, 2b, 2c, and 3a, we identified at least three HMAbs with potent and broad neutralization potential. The neutralization synergism obtained when pooling the most potent HMAbs could have significant implications for developing novel strategies to treat and control HCV. © 2014 by the American Association for the Study of Liver

  10. Heterologous expression of fungal cytochromes P450 (CYP5136A1 and CYP5136A3) from the white-rot basidiomycete Phanerochaete chrysosporium: Functionalization with cytochrome b5 in Escherichia coli.

    PubMed

    Hatakeyama, Mayumi; Kitaoka, Takuya; Ichinose, Hirofumi

    2016-07-01

    Cytochromes P450 from the white-rot basidiomycete Phanerochaete chrysosporium, CYP5136A1 and CYP5136A3, are capable of catalyzing oxygenation reactions of a wide variety of exogenous compounds, implying their significant roles in the metabolism of xenobiotics by the fungus. It is therefore interesting to explore their biochemistry to better understand fungal biology and to enable the use of fungal enzymes in the biotechnology sector. In the present study, we developed heterologous expression systems for CYP5136A1 and CYP5136A3 using the T7 RNA polymerase/promoter system in Escherichia coli. Expression levels of recombinant P450s were dramatically improved by modifications and optimization of their N-terminal amino acid sequences. A CYP5136A1 reaction system was reconstructed in E. coli whole cells by coexpression of CYP5136A1 and a redox partner, NADPH-dependent P450 reductase (CPR). The catalytic activity of CYP5136A1 was significantly increased when cytochrome b5 (Cyt-b5) was further coexpressed with CPR, indicating that Cyt-b5 supports electron transfer reactions from NAD(P)H to CYP5136A1. Notably, P450 reaction occurred in E. coli cells that harbored CYP5136A1 and Cyt-b5 but not CPR, implying that the reducing equivalents required for the P450 catalytic cycle were transferred via a CPR-independent pathway. Such an "alternative" electron transfer system in CYP5136A1 reaction was also demonstrated using purified enzymes in vitro. The fungal P450 reaction system may be associated with sophisticated electron transfer pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Transient up-regulation of retinal EphA3 and EphA5, but not ephrin-A2, coincides with re-establishment of a topographic map during optic nerve regeneration in goldfish.

    PubMed

    King, Carolyn E; Wallace, Amy; Rodger, Jennifer; Bartlett, Carole; Beazley, Lyn D; Dunlop, Sarah A

    2003-10-01

    Eph tyrosine kinase receptors and their ligands, the ephrins, play a key role in the establishment of retinotectal topography during development. Tectal up-regulation of ephrin-A2 in goldfish, coincident with the reestablishment of a retinotectal map, suggests a similar role during optic nerve regeneration. Here we report a complementary study of EphA3, EphA5 and ephrin-A2 expression in the retina. EphA3 and EphA5 are transiently up-regulated as ascending naso-temporal gradients, whereas ephrin-A2 remains uniform. The expression profiles differ from those in developing chick and mouse, suggesting that different combinations of retinal Eph receptors and ligands can generate topographic guidance information.

  12. Tomato SlERF.A1, SlERF.B4, SlERF.C3 and SlERF.A3, Members of B3 Group of ERF Family, Are Required for Resistance to Botrytis cinerea.

    PubMed

    Ouyang, Zhigang; Liu, Shixia; Huang, Lihong; Hong, Yongbo; Li, Xiaohui; Huang, Lei; Zhang, Yafen; Zhang, Huijuan; Li, Dayong; Song, Fengming

    2016-01-01

    The Ethylene-Responsive Factors (ERFs) comprise a large family of transcriptional factors that play critical roles in plant immunity. Gray mold disease caused by Botrytis cinerea, a typical necrotrophic fungal pathogen, is the serious disease that threatens tomato production worldwide. However, littler is known about the molecular mechanism regulating the immunity to B. cinerea in tomato. In the present study, virus-induced gene silencing (VIGS)-based functional analyses of 18 members of B3 group (also called Group IX) in tomato ERF family were performed to identify putative ERFs that are involved in disease resistance against B. cinerea. VIGS-based silencing of either SlERF.B1 or SlERF.C2 had lethal effect while silencing of SlERF.A3 (Pit4) significantly suppressed vegetative growth of tomato plants. Importantly, silencing of SlERF.A1, SlERF.A3, SlERF.B4, or SlERF.C3 resulted in increased susceptibility to B. cinerea, attenuated the B. cinerea-induced expression of jasmonic acid/ethylene-mediated signaling responsive defense genes and promoted the B. cinerea-induced H2O2 accumulation. However, silencing of SlERF.A3 also decreased the resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 but silencing of SlERF.A1, SlERF.B4 or SlERF.C3 did not affect the resistance to this bacterial pathogen. Expression of SlERF.A1, SlERF.A3, SlERF.B4, or SlERF.C3 was induced by B. cinerea and by defense signaling hormones such as salicylic acid, methyl jasmonate, and 1-aminocyclopropane-1-carboxylic acid (an ethylene precursor). SlERF.A1, SlERF.B4, SlERF.C3, and SlERF.A3 proteins were found to localize in nucleus of cells and possess transactivation activity in yeasts. These data suggest that SlERF.A1, SlERF.B4, and SlERF.C3, three previously uncharacterized ERFs in B3 group, and SlERF.A3, a previously identified ERF with function in immunity to Pst DC3000, play important roles in resistance against B. cinerea in tomato.

  13. Tomato SlERF.A1, SlERF.B4, SlERF.C3 and SlERF.A3, Members of B3 Group of ERF Family, Are Required for Resistance to Botrytis cinerea

    PubMed Central

    Ouyang, Zhigang; Liu, Shixia; Huang, Lihong; Hong, Yongbo; Li, Xiaohui; Huang, Lei; Zhang, Yafen; Zhang, Huijuan; Li, Dayong; Song, Fengming

    2016-01-01

    The Ethylene-Responsive Factors (ERFs) comprise a large family of transcriptional factors that play critical roles in plant immunity. Gray mold disease caused by Botrytis cinerea, a typical necrotrophic fungal pathogen, is the serious disease that threatens tomato production worldwide. However, littler is known about the molecular mechanism regulating the immunity to B. cinerea in tomato. In the present study, virus-induced gene silencing (VIGS)-based functional analyses of 18 members of B3 group (also called Group IX) in tomato ERF family were performed to identify putative ERFs that are involved in disease resistance against B. cinerea. VIGS-based silencing of either SlERF.B1 or SlERF.C2 had lethal effect while silencing of SlERF.A3 (Pit4) significantly suppressed vegetative growth of tomato plants. Importantly, silencing of SlERF.A1, SlERF.A3, SlERF.B4, or SlERF.C3 resulted in increased susceptibility to B. cinerea, attenuated the B. cinerea-induced expression of jasmonic acid/ethylene-mediated signaling responsive defense genes and promoted the B. cinerea-induced H2O2 accumulation. However, silencing of SlERF.A3 also decreased the resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 but silencing of SlERF.A1, SlERF.B4 or SlERF.C3 did not affect the resistance to this bacterial pathogen. Expression of SlERF.A1, SlERF.A3, SlERF.B4, or SlERF.C3 was induced by B. cinerea and by defense signaling hormones such as salicylic acid, methyl jasmonate, and 1-aminocyclopropane-1-carboxylic acid (an ethylene precursor). SlERF.A1, SlERF.B4, SlERF.C3, and SlERF.A3 proteins were found to localize in nucleus of cells and possess transactivation activity in yeasts. These data suggest that SlERF.A1, SlERF.B4, and SlERF.C3, three previously uncharacterized ERFs in B3 group, and SlERF.A3, a previously identified ERF with function in immunity to Pst DC3000, play important roles in resistance against B. cinerea in tomato. PMID:28083004

  14. Spectroscopic investigations on NO+(X1Σ+, a3Σ+, A1Π) ion using multi-reference configuration interaction method and correlation-consistent sextuple basis set augmented with diffuse functions

    NASA Astrophysics Data System (ADS)

    Zhang, Jin-Ping; Cheng, Xin-Lu; Zhang, Hong; Yang, Xiang-Dong

    2011-06-01

    Three low-lying electronic states (X1Σ+, a3Σ+, and A1Π) of NO+ ion are studied using the complete active space self-consistent-field (CASSCF) method followed by highly accurate valence internally contracted multi-reference configuration interaction (MRCI) approach in combination of the correlation-consistent sextuple basis set augmented with diffuse functions, aug-cc-pV6Z. The potential energy curves (PECs) of the NO+(X1Σ+, a3Σ+, A1Π) are calculated. Based on the PECs, the spectroscopic parameters Re, De, ωe, ωeχe, αe, Be, and D0 are reproduced, which are in excellent agreement with the available measurements. By numerically solving the radial Schrödinger equation of nuclear motion using the Numerov method, the first 20 vibrational levels, inertial rotation and centrifugal distortion constants of NO+(X1Σ+, a3Σ+, A1Π) ion are derived when the rotational quantum number J is equal to zero (J = 0) for the first time, which accord well with the available measurements. Finally, the analytical potential energy functions of these states are fitted, which are used to accurately derive the first 20 classical turning points when J = 0. These results are compared in detail with those of previous investigations reported in the literature.

  15. The Localization of Cytochrome P450s CYP1A1 and CYP1A2 into Different Lipid Microdomains Is Governed by Their N-terminal and Internal Protein Regions.

    PubMed

    Park, Ji Won; Reed, James R; Backes, Wayne L

    2015-12-04

    In cellular membranes, different lipid species are heterogeneously distributed forming domains with different characteristics. Ordered domains are tightly packed with cholesterol, sphingomyelin, and saturated fatty acids, whereas disordered domains contain high levels of unsaturated fatty acids. Our laboratory has shown that membrane heterogeneity affects the organization of cytochrome P450s and their cognate redox partner, the cytochrome P450 reductase (CPR). Despite the high degree of sequence similarity, CYP1A1 was found to localize to disordered regions, whereas CYP1A2 resided in ordered domains. We hypothesized that regions of amino acid sequence variability may contain signal motifs that direct CYP1A proteins into ordered or disordered domains. Thus, chimeric constructs of CYP1A1 and CYP1A2 were created, and their localization was tested in HEK293T cells. CYP1A2, containing the N-terminal regions from CYP1A1, no longer localized in ordered domains, whereas the N terminus of CYP1A2 partially directed CYP1A1 into ordered regions. In addition, intact CYP1A2 containing a 206-302-residue peptide segment of CYP1A1 had less affinity to bind to ordered microdomains. After expression, the catalytic activity of CYP1A2 was higher than that of the CYP1A1-CYP1A2 chimera containing the N-terminal end of CYP1A1 with subsaturating CPR concentrations, but it was approximately equal with excess CPR suggesting that the localization of the CYP1A enzyme in ordered domains favored its interaction with CPR. These data demonstrate that both the N-terminal end and an internal region of CYP1A2 play roles in targeting CYP1A2 to ordered domains, and domain localization may influence P450 function under conditions that resemble those found in vivo. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. The Ruminococcus albus pilA1-pilA2 locus: expression and putative role of two adjacent pil genes in pilus formation and bacterial adhesion to cellulose.

    PubMed

    Rakotoarivonina, Harivony; Larson, Marilynn A; Morrison, Mark; Girardeau, Jean-Pierre; Gaillard-Martinie, Brigitte; Forano, Evelyne; Mosoni, Pascale

    2005-04-01

    Ruminococcus albus produces fimbria-like structures that are involved with the bacterium's adhesion to cellulose. The subunit protein has been identified in strain 8 (CbpC) and strain 20 (GP25) and both are type IV fimbrial (Pil) proteins. The presence of a pil locus that is organized similarly in both strains is reported here together with the results of an initial examination of a second Pil protein. Downstream of the cbpC/gp25 gene (hereafter referred to as pilA1) is a second pilin gene (pilA2). Northern blot analysis of pilA1 and pilA2 transcripts showed that the pilA1 transcript is much more abundant in R. albus 8, and real-time PCR was used to measure pilA1 and pilA2 transcript abundance in R. albus 20 and its adhesion-defective mutant D5. Similar to the findings with R. albus 8, the relative expression of pilA1 in the wild-type strain was 73-fold higher than that of pilA2 following growth with cellobiose, and there were only slight differences between the wild-type and mutant strain in pilA1 and pilA2 transcript abundances, indicating that neither pilA1 nor pilA2 transcription is adversely affected in the mutant strain. Western immunoblots showed that the PilA2 protein is localized primarily to the membrane fraction, and the anti-PilA2 antiserum does not inhibit bacterial adhesion to cellulose. These results suggest that the PilA2 protein plays a role in the synthesis and assembly of type IV fimbriae-like structures by R. albus, but its role is restricted to cell-associated functions, rather than as part of the externalized fimbrial structure.

  17. Design synthesis and evaluation of the inhibitory selectivity of novel trans-resveratrol analogues on human recombinant CYP1A1 CYP1A2 and CYP1B1

    USDA-ARS?s Scientific Manuscript database

    A series of trans-stilbene derivatives containing 4’-thiomethyl substituent were synthesized and evaluated for inhibitory activities on human recombinant cytochrome P450(s): CYP1A1, CYP1A2, and CYP1B1. CYP1A2-related metabolism of stilbene derivatives was estimated by using NADPH oxidation assay. A...

  18. 76 FR 15820 - Airworthiness Directives; B-N Group Ltd. Model BN-2, BN-2A, BN-2A-2, BN-2A-3, BN-2A-6, BN-2A-8...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-22

    .... Model BN-2, BN-2A, BN- 2A-2, BN-2A-3, BN-2A-6, BN-2A-8, BN-2A-9, BN-2A-20, BN-2A-21, BN-2A-26, BN-2A-27, BN-2B-20, BN-2B-21, BN-2B-26, BN-2B-27, BN-2T, and BN-2T-4R Airplanes AGENCY: Federal Aviation.... Affected ADs (b) None. Applicability (c) This AD applies to B-N Group Ltd. Models BN-2, BN-2A, BN-2A- 2, BN...

  19. Active Site Mutations as a Suitable Tool Contributing to Explain a Mechanism of Aristolochic Acid I Nitroreduction by Cytochromes P450 1A1, 1A2 and 1B1

    PubMed Central

    Milichovský, Jan; Bárta, František; Schmeiser, Heinz H.; Arlt, Volker M.; Frei, Eva; Stiborová, Marie; Martínek, Václav

    2016-01-01

    Aristolochic acid I (AAI) is a plant drug found in Aristolochia species that causes aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is activated via nitroreduction producing genotoxic N-hydroxyaristolactam, which forms DNA adducts. The major enzymes responsible for the reductive bioactivation of AAI are NAD(P)H:quinone oxidoreductase and cytochromes P450 (CYP) 1A1 and 1A2. Using site-directed mutagenesis we investigated the possible mechanisms of CYP1A1/1A2/1B1-catalyzed AAI nitroreduction. Molecular modelling predicted that the hydroxyl groups of serine122/threonine124 (Ser122/Thr124) amino acids in the CYP1A1/1A2-AAI binary complexes located near to the nitro group of AAI, are mechanistically important as they provide the proton required for the stepwise reduction reaction. In contrast, the closely related CYP1B1 with no hydroxyl group containing residues in its active site is ineffective in catalyzing AAI nitroreduction. In order to construct an experimental model, mutant forms of CYP1A1 and 1A2 were prepared, where Ser122 and Thr124 were replaced by Ala (CYP1A1-S122A) and Val (CYP1A2-T124V), respectively. Similarly, a CYP1B1 mutant was prepared in which Ala133 was replaced by Ser (CYP1B1-A133S). Site-directed mutagenesis was performed using a quickchange approach. Wild and mutated forms of these enzymes were heterologously expressed in Escherichia coli and isolated enzymes characterized using UV-vis spectroscopy to verify correct protein folding. Their catalytic activity was confirmed with CYP1A1, 1A2 and 1B1 marker substrates. Using 32P-postlabelling we determined the efficiency of wild-type and mutant forms of CYP1A1, 1A2, and 1B1 reconstituted with NADPH:CYP oxidoreductase to bioactivate AAI to reactive intermediates forming covalent DNA adducts. The S122A and T124V mutations in CYP1A1 and 1A2, respectively, abolished the efficiency of CYP1A1 and 1A2 enzymes to generate AAI-DNA adducts. In contrast

  20. Active Site Mutations as a Suitable Tool Contributing to Explain a Mechanism of Aristolochic Acid I Nitroreduction by Cytochromes P450 1A1, 1A2 and 1B1.

    PubMed

    Milichovský, Jan; Bárta, František; Schmeiser, Heinz H; Arlt, Volker M; Frei, Eva; Stiborová, Marie; Martínek, Václav

    2016-02-05

    Aristolochic acid I (AAI) is a plant drug found in Aristolochia species that causes aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is activated via nitroreduction producing genotoxic N-hydroxyaristolactam, which forms DNA adducts. The major enzymes responsible for the reductive bioactivation of AAI are quinone oxidoreductase and cytochromes P450 (CYP) 1A1 and 1A2. Using site-directed mutagenesis we investigated the possible mechanisms of CYP1A1/1A2/1B1-catalyzed AAI nitroreduction. Molecular modelling predicted that the hydroxyl groups of serine122/threonine124 (Ser122/Thr124) amino acids in the CYP1A1/1A2-AAI binary complexes located near to the nitro group of AAI, are mechanistically important as they provide the proton required for the stepwise reduction reaction. In contrast, the closely related CYP1B1 with no hydroxyl group containing residues in its active site is ineffective in catalyzing AAI nitroreduction. In order to construct an experimental model, mutant forms of CYP1A1 and 1A2 were prepared, where Ser122 and Thr124 were replaced by Ala (CYP1A1-S122A) and Val (CYP1A2-T124V), respectively. Similarly, a CYP1B1 mutant was prepared in which Ala133 was replaced by Ser (CYP1B1-A133S). Site-directed mutagenesis was performed using a quickchange approach. Wild and mutated forms of these enzymes were heterologously expressed in Escherichia coli and isolated enzymes characterized using UV-vis spectroscopy to verify correct protein folding. Their catalytic activity was confirmed with CYP1A1, 1A2 and 1B1 marker substrates. Using (32)P-postlabelling we determined the efficiency of wild-type and mutant forms of CYP1A1, 1A2, and 1B1 reconstituted with NADPH:CYP oxidoreductase to bioactivate AAI to reactive intermediates forming covalent DNA adducts. The S122A and T124V mutations in CYP1A1 and 1A2, respectively, abolished the efficiency of CYP1A1 and 1A2 enzymes to generate AAI-DNA adducts. In contrast, the

  1. The human sodium-dependent ascorbic acid transporters SLC23A1 and SLC23A2 do not mediate ascorbic acid release in the proximal renal epithelial cell

    PubMed Central

    Eck, Peter; Kwon, Oran; Chen, Shenglin; Mian, Omar; Levine, Mark

    2013-01-01

    Sodium-dependent ascorbic acid membrane transporters SLC23A1 and SLC23A2 mediate ascorbic acid (vitamin C) transport into cells. However, it is unknown how ascorbic acid undergoes cellular release, or efflux. We hypothesized that SLC23A1 and SLC23A2 could serve a dual role, mediating ascorbic acid cellular efflux as well as uptake. Renal reabsorption is required for maintaining systemic vitamin C concentrations. Because efflux from nephron cells is necessary for reabsorption, we studied whether SLC23A1 and SLC23A2 mediate efflux of ascorbic acid in the human renal nephron. We found high gene expression of SLC23A1 but no expression of SLC23A2 in the proximal convoluted and straight tubules of humans. These data rule out SLC23A2 as the ascorbic acid release protein in the renal proximal tubular epithelia cell. We utilized a novel dual transporter-based Xenopus laevis oocyte system to investigate the function of the SLC23A1 protein, and found that no ascorbate release was mediated by SLC23A1. These findings were confirmed in mammalian cells overexpressing SLC23A1. Taken together, the data for SLC23A1 show that it too does not have a role in cellular release of ascorbic acid across the basolateral membrane of the proximal tubular epithelial cell, and that SLC23A1 alone is responsible for ascorbic acid uptake across the apical membrane. These findings reiterate the physiological importance of proper functioning of SLC23A1 in maintaining vitamin C levels for health and disease prevention. The ascorbate efflux mechanism in the proximal tubule of the kidney remains to be characterized. PMID:24400138

  2. Maternal protein restriction during lactation modulated the expression and activity of rat offspring hepatic CYP1A1, CYP1A2, CYP2B1, CYP2B2, and CYP2E1 during development.

    PubMed

    Da Costa, N Meireles; Visoni, S B C; Dos Santos, I L; Barja-Fidalgo, T C; Ribeiro-Pinto, L F

    2016-01-01

    Early nutrition plays a long-term role in the predisposition to chronic diseases and influences the metabolism of several drugs. This may happen through cytochromes P450 (CYPs) regulation, which are the main enzymes responsible for the metabolism of xenobiotics. Here, we analyzed the effects of maternal protein restriction (MPR) on the expression and activity of hepatic offspring's CYPs during 90 days after birth, using Wistar rats as a mammal model. Hepatic CYP1A1, CYP1A2, CYP2B1, CYP2B2 and CYP2E1 mRNA and protein expression, and associated catalytic activities (ECOD, EROD, MROD, BROD, PROD and PNPH) were evaluated in 15-, 30-, 60-, and 90-day-old offspring from dams fed with either a 0% protein (MPR groups) or a standard diet (C groups) during the 10 first days of lactation. Results showed that most CYP genes were induced in 60- and 90-day-old MPR offspring. The inductions detected in MPR60 and MPR90 were of 5.0- and 2.0-fold (CYP1A2), 3.7- and 2.0-fold (CYP2B2) and 9.8- and 5.8- fold (CYP2E1), respectively, and a 3.8-fold increase of CYP2B1 in MPR90. No major alterations were detected in CYP protein expression. The most relevant CYP catalytic activities' alterations were observed in EROD, BROD and PNPH. Nevertheless, they did not follow the same pattern observed for mRNA expression, except for an induction of EROD in MPR90 (3.5-fold) and of PNPH in MPR60 (2.2-fold). Together, these results suggest that MPR during lactation was capable of altering the expression and activity of the hepatic CYP enzymes evaluated in the offspring along development.

  3. Maternal protein restriction during lactation modulated the expression and activity of rat offspring hepatic CYP1A1, CYP1A2, CYP2B1, CYP2B2, and CYP2E1 during development

    PubMed Central

    Da Costa, N. Meireles; Visoni, S.B.C.; Dos Santos, I.L.; Barja-Fidalgo, T.C.; Ribeiro-Pinto, L.F.

    2016-01-01

    Early nutrition plays a long-term role in the predisposition to chronic diseases and influences the metabolism of several drugs. This may happen through cytochromes P450 (CYPs) regulation, which are the main enzymes responsible for the metabolism of xenobiotics. Here, we analyzed the effects of maternal protein restriction (MPR) on the expression and activity of hepatic offspring’s CYPs during 90 days after birth, using Wistar rats as a mammal model. Hepatic CYP1A1, CYP1A2, CYP2B1, CYP2B2 and CYP2E1 mRNA and protein expression, and associated catalytic activities (ECOD, EROD, MROD, BROD, PROD and PNPH) were evaluated in 15-, 30-, 60-, and 90-day-old offspring from dams fed with either a 0% protein (MPR groups) or a standard diet (C groups) during the 10 first days of lactation. Results showed that most CYP genes were induced in 60- and 90-day-old MPR offspring. The inductions detected in MPR60 and MPR90 were of 5.0- and 2.0-fold (CYP1A2), 3.7- and 2.0-fold (CYP2B2) and 9.8- and 5.8– fold (CYP2E1), respectively, and a 3.8-fold increase of CYP2B1 in MPR90. No major alterations were detected in CYP protein expression. The most relevant CYP catalytic activities’ alterations were observed in EROD, BROD and PNPH. Nevertheless, they did not follow the same pattern observed for mRNA expression, except for an induction of EROD in MPR90 (3.5-fold) and of PNPH in MPR60 (2.2-fold). Together, these results suggest that MPR during lactation was capable of altering the expression and activity of the hepatic CYP enzymes evaluated in the offspring along development. PMID:27828666

  4. Combining selectivity and affinity predictions using an integrated Support Vector Machine (SVM) approach: An alternative tool to discriminate between the human adenosine A(2A) and A(3) receptor pyrazolo-triazolo-pyrimidine antagonists binding sites.

    PubMed

    Michielan, Lisa; Bolcato, Chiara; Federico, Stephanie; Cacciari, Barbara; Bacilieri, Magdalena; Klotz, Karl-Norbert; Kachler, Sonja; Pastorin, Giorgia; Cardin, Riccardo; Sperduti, Alessandro; Spalluto, Giampiero; Moro, Stefano

    2009-07-15

    G Protein-coupled receptors (GPCRs) selectivity is an important aspect of drug discovery process, and distinguishing between related receptor subtypes is often the key to therapeutic success. Nowadays, very few valuable computational tools are available for the prediction of receptor subtypes selectivity. In the present study, we present an alternative application of the Support Vector Machine (SVM) and Support Vector Regression (SVR) methodologies to simultaneously describe both A(2A)R versus A(3)R subtypes selectivity profile and the corresponding receptor binding affinities. We have implemented an integrated application of SVM-SVR approach, based on the use of our recently reported autocorrelated molecular descriptors encoding for the Molecular Electrostatic Potential (autoMEP), to simultaneously discriminate A(2A)R versus A(3)R antagonists and to predict their binding affinity to the corresponding receptor subtype of a large dataset of known pyrazolo-triazolo-pyrimidine analogs. To validate our approach, we have synthetized 51 new pyrazolo-triazolo-pyrimidine derivatives anticipating both A(2A)R/A(3)R subtypes selectivity and receptor binding affinity profiles.

  5. Genomic characterization and dynamic methylation of promoter facilitates transcriptional regulation of H2A variants, H2A.1 and H2A.2 in various pathophysiological states of hepatocyte.

    PubMed

    Tyagi, Monica; Reddy, Divya; Gupta, Sanjay

    2017-02-03

    Differential expression of homomorphous variants of H2A family of histone H2A.1 and H2A.2 have been associated with hepatocellular carcinoma and maintenance of undifferentiated state of hepatocyte. However, not much is known about the transcriptional regulation of these H2A variants. The current study revealed the presence of 43bp 5'-regulatory region upstream of translation start site and a 26bp 3' stem loop conserved region for both the H2A.1 and H2A.2 variants. However, alignment of both H2A.1 and H2A.2 5'-untranslated region (UTR) sequences revealed no significant degree of homology between them despite the coding exon being very similar amongst the variants. Further, transient transfection coupled with dual luciferase assay of cloned 5' upstream sequences of H2A.1 and H2A.2 of length 1.2 (-1056 to +144) and 1.379kb (-1160 to +219) from experimentally identified 5'UTR in rat liver cell line (CL38) confirmed their promoter activity. Moreover, in silico analysis revealed a presence of multiple CpG sites interspersed in the cloned promoter of H2A.1 and a CpG island near TSS for H2A.2, suggesting that histone variants transcription might be regulated epigenetically. Indeed, treatment with DNMT and HDAC inhibitors increased the expression of H2A.2 with no significant change in H2A.1 levels. Further, methyl DNA immunoprecipitation coupled with quantitative analysis of DNA methylation using real-time PCR revealed hypo-methylation and hyper-methylation of H2A.1 and H2A.2 respectively in embryonic and HCC compared to control adult liver tissue. Collectively, the data suggests that differential DNA methylation on histone promoters is a dynamic player regulating their expression status in different pathophysiological stages of liver.

  6. HTR-PROTEUS Pebble Bed Experimental Program Cores 1, 1A, 2, and 3: Hexagonal Close Packing with a 1:2 Moderator-to-Fuel Pebble Ratio

    SciTech Connect

    John D. Bess; Barbara H. Dolphin; James W. Sterbentz; Luka Snoj; Igor Lengar; Oliver Köberl

    2013-03-01

    In its deployment as a pebble bed reactor (PBR) critical facility from 1992 to 1996, the PROTEUS facility was designated as HTR-PROTEUS. This experimental program was performed as part of an International Atomic Energy Agency (IAEA) Coordinated Research Project (CRP) on the Validation of Safety Related Physics Calculations for Low Enriched HTGRs. Within this project, critical experiments were conducted for graphite moderated LEU systems to determine core reactivity, flux and power profiles, reaction-rate ratios, the worth of control rods, both in-core and reflector based, the worth of burnable poisons, kinetic parameters, and the effects of moisture ingress on these parameters. Four benchmark experiments were evaluated in this report: Cores 1, 1A, 2, and 3. These core configurations represent the hexagonal close packing (HCP) configurations of the HTR-PROTEUS experiment with a moderator-to-fuel pebble ratio of 1:2. Core 1 represents the only configuration utilizing ZEBRA control rods. Cores 1A, 2, and 3 use withdrawable, hollow, stainless steel control rods. Cores 1 and 1A are similar except for the use of different control rods; Core 1A also has one less layer of pebbles (21 layers instead of 22). Core 2 retains the first 16 layers of pebbles from Cores 1 and 1A and has 16 layers of moderator pebbles stacked above the fueled layers. Core 3 retains the first 17 layers of pebbles but has polyethylene rods inserted between pebbles to simulate water ingress. The additional partial pebble layer (layer 18) for Core 3 was not included as it was used for core operations and not the reported critical configuration. Cores 1, 1A, 2, and 3 were determined to be acceptable benchmark experiments.

  7. HTR-PROTEUS Pebble Bed Experimental Program Cores 1, 1A, 2, and 3: Hexagonal Close Packing with a 1:2 Moderator-to-Fuel Pebble Ratio

    SciTech Connect

    John D. Bess; Barbara H. Dolphin; James W. Sterbentz; Luka Snoj; Igor Lengar; Oliver Köberl

    2012-03-01

    In its deployment as a pebble bed reactor (PBR) critical facility from 1992 to 1996, the PROTEUS facility was designated as HTR-PROTEUS. This experimental program was performed as part of an International Atomic Energy Agency (IAEA) Coordinated Research Project (CRP) on the Validation of Safety Related Physics Calculations for Low Enriched HTGRs. Within this project, critical experiments were conducted for graphite moderated LEU systems to determine core reactivity, flux and power profiles, reaction-rate ratios, the worth of control rods, both in-core and reflector based, the worth of burnable poisons, kinetic parameters, and the effects of moisture ingress on these parameters. Four benchmark experiments were evaluated in this report: Cores 1, 1A, 2, and 3. These core configurations represent the hexagonal close packing (HCP) configurations of the HTR-PROTEUS experiment with a moderator-to-fuel pebble ratio of 1:2. Core 1 represents the only configuration utilizing ZEBRA control rods. Cores 1A, 2, and 3 use withdrawable, hollow, stainless steel control rods. Cores 1 and 1A are similar except for the use of different control rods; Core 1A also has one less layer of pebbles (21 layers instead of 22). Core 2 retains the first 16 layers of pebbles from Cores 1 and 1A and has 16 layers of moderator pebbles stacked above the fueled layers. Core 3 retains the first 17 layers of pebbles but has polyethylene rods inserted between pebbles to simulate water ingress. The additional partial pebble layer (layer 18) for Core 3 was not included as it was used for core operations and not the reported critical configuration. Cores 1, 1A, 2, and 3 were determined to be acceptable benchmark experiments.

  8. The PlA1/A2 Polymorphism of Glycoprotein IIIa as a Risk Factor for Myocardial Infarction: A Meta-Analysis

    PubMed Central

    Floyd, Christopher N.; Mustafa, Agnesa; Ferro, Albert

    2014-01-01

    Background The PlA2 polymorphism of glycoprotein IIIa (GPIIIa) has been previously identified as being associated with myocardial infarction (MI), but whether this represents a true association is entirely unclear due to differences in findings from different studies. We performed a meta-analysis to evaluate whether this polymorphism is a risk factor for MI. Methods Electronic databases (MEDLINE and EMBASE) were searched for all articles evaluating genetic polymorphisms of GPIIIa. For studies where acute coronary events were recorded in association with genetic analysis, pooled odds ratios (ORs) were calculated using fixed-effects and random-effects models. The primary outcome measure was MI, and a secondary analysis was also performed for acute coronary syndromes (ACS) more generally. Findings 57 studies were eligible for statistical analysis and included 17,911 cases and 24,584 controls. Carriage of the PlA2 allele was significantly associated with MI (n = 40,692; OR 1.077, 95% CI 1.024–1.132; p = 0.004) but with significant publication bias (p = 0.040). The degree of association with MI increased with decreasing age of subjects (≤45 years old: n = 9,547; OR 1.205, 95% CI 1.067–1.360; p = 0.003) and with adjustment of data for conventional cardiovascular risk factors (n = 12,001; OR 1.240, 95% CI 1.117–1.376; p<0.001). There was a low probability of publication bias for these subgroup analyses (all p<0.05). Conclusions The presence of significant publication bias makes it unclear whether the association between carriage of the PlA2 allele and MI is true for the total population studied. However for younger subjects, the relative absence of conventional cardiovascular risk factors results in a significant association between carriage of the PlA2 allele and MI. PMID:24988220

  9. Effect of UGT1A1, UGT1A3, DIO1 and DIO2 polymorphisms on L-thyroxine doses required for TSH suppression in patients with differentiated thyroid cancer

    PubMed Central

    Santoro, Ana B; Vargens, Daniela D; Barros Filho, Mateus de Camargo; Bulzico, Daniel A; Kowalski, Luiz Paulo; Meirelles, Ricardo M R; Paula, Daniela P; Neves, Ronaldo R S; Pessoa, Cencita N; Struchine, Claudio J; Suarez-Kurtz, Guilherme

    2014-01-01

    Aim To evaluate the impact of genetic polymorphisms in uridine 5′-glucuronosylytansferases UGT1A1 and UGT1A3 and iodothyronine-deiodinases types 1 and 2 on levothyroxine (T4; 3,5,3′,5′-triiodo-L-thyronine) dose requirement for suppression of thyrotropin (TSH) secretion in patients with differentiated thyroid cancer (DTC). Methods Patients (n = 268) submitted to total thyroidectomy and ablation by 131I, under T4 therapy for at least 6 months were recruited in three public institutions in Brazil. Multivariate regression modelling was applied to assess the association of T4 dosing with polymorphisms in UGT1A1 (rs8175347), UGT1A3 (rs3806596 and rs1983023), DIO1 (rs11206244 and rs2235544) and DIO2 (rs225014 and rs12885300), demographic and clinical variables. Results A regression model including UGT1A haplotypes, age, gender, body weight and serum TSH concentration accounted for 39% of the inter-individual variation in the T4 dosage. The association of T4 dose with UGT1A haplotype is attributed to reduced UGT1A1 expression and T4 glucuronidation in liver of carriers of low expression UGT1A1 rs8175347 alleles. The DIO1 and DIO2 genotypes had no influence of T4 dosage. Conclusion UGT1A haplotypes associate with T4 dosage in DTC patients, but the effect accounts for only 2% of the total variability and recommendation of pre-emptive UGT1A genotyping is not warranted. PMID:24910925

  10. Lysosomal-mediated waste clearance in retinal pigment epithelial cells is regulated by CRYBA1/βA3/A1-crystallin via V-ATPase-MTORC1 signaling

    PubMed Central

    Valapala, Mallika; Wilson, Christine; Hose, Stacey; Bhutto, Imran A; Grebe, Rhonda; Dong, Aling; Greenbaum, Seth; Gu, Limin; Sengupta, Samhita; Cano, Marisol; Hackett, Sean; Xu, Guotong; Lutty, Gerard A; Dong, Lijin; Sergeev, Yuri; Handa, James T; Campochiaro, Peter; Wawrousek, Eric; Zigler, Jr, J Samuel; Sinha, Debasish

    2014-01-01

    In phagocytic cells, including the retinal pigment epithelium (RPE), acidic compartments of the endolysosomal system are regulators of both phagocytosis and autophagy, thereby helping to maintain cellular homeostasis. The acidification of the endolysosomal system is modulated by a proton pump, the V-ATPase, but the mechanisms that direct the activity of the V-ATPase remain elusive. We found that in RPE cells, CRYBA1/βA3/A1-crystallin, a lens protein also expressed in RPE, is localized to lysosomes, where it regulates endolysosomal acidification by modulating the V-ATPase, thereby controlling both phagocytosis and autophagy. We demonstrated that CRYBA1 coimmunoprecipitates with the ATP6V0A1/V0-ATPase a1 subunit. Interestingly, in mice when Cryba1 (the gene encoding both the βA3- and βA1-crystallin forms) is knocked out specifically in RPE, V-ATPase activity is decreased and lysosomal pH is elevated, while cathepsin D (CTSD) activity is decreased. Fundus photographs of these Cryba1 conditional knockout (cKO) mice showed scattered lesions by 4 months of age that increased in older mice, with accumulation of lipid-droplets as determined by immunohistochemistry. Transmission electron microscopy (TEM) of cryba1 cKO mice revealed vacuole-like structures with partially degraded cellular organelles, undigested photoreceptor outer segments and accumulation of autophagosomes. Further, following autophagy induction both in vivo and in vitro, phospho-AKT and phospho-RPTOR/Raptor decrease, while pMTOR increases in RPE cells, inhibiting autophagy and AKT-MTORC1 signaling. Impaired lysosomal clearance in the RPE of the cryba1 cKO mice also resulted in abnormalities in retinal function that increased with age, as demonstrated by electroretinography. Our findings suggest that loss of CRYBA1 causes lysosomal dysregulation leading to the impairment of both autophagy and phagocytosis. PMID:24468901

  11. Pyranoflavones: A Group of Small-molecule Probes for Exploring the Active Site Cavities of Cytochrome P450 Enzymes 1A1, 1A2, and 1B1

    PubMed Central

    Liu, Jiawang; Taylor, Shannon F.; Dupart, Patrick S.; Arnold, Corey L.; Sridhar, Jayalakshmi; Jiang, Quan; Wang, Yuji; Skripnikova, Elena V.; Zhao, Ming; Foroozesh, Maryam

    2013-01-01

    Selective inhibition of P450 enzymes is the key to block the conversion of environmental procarcinogens to their carcinogenic metabolites in both animals and humans. To discover highly potent and selective inhibitors of P450s 1A1, 1A2, and 1B1, as well as to investigate active site cavities of these enzymes, 14 novel flavone derivatives were prepared as chemical probes. Fluorimetric enzyme inhibition assays were used to determine the inhibitory activities of these probes towards P450s 1A1, 1A2, 1B1, 2A6, and 2B1. A highly selective P450 1B1 inhibitor, 5-hydroxy-4′-propargyloxyflavone (5H4′FPE) was discovered. Some tested compounds also showed selectivity between P450s 1A1 and 1A2. Alpha-naphthoflavone-like and 5-hydroxyflavone derivatives preferentially inhibited P450 1A2, while beta-naphthoflavone-like flavone derivatives showed selective inhibition of P450 1A1. On the basis of structural analysis, the active site cavity models of P450 enzymes 1A1 and 1A2 were generated, demonstrating a planar long strip cavity and a planar triangular cavity, respectively. PMID:23600958

  12. Solution structure determination of monomeric human IgA2 by X-ray and neutron scattering, analytical ultracentrifugation and constrained modelling: a comparison with monomeric human IgA1.

    PubMed

    Furtado, Patricia B; Whitty, Patrick W; Robertson, Alexis; Eaton, Julian T; Almogren, Adel; Kerr, Michael A; Woof, Jenny M; Perkins, Stephen J

    2004-05-14

    Immunoglobulin A (IgA), the most abundant human immunoglobulin, mediates immune protection at mucosal surfaces as well as in plasma. It exists as two subclasses IgA1 and IgA2, and IgA2 is found in at least two allotypic forms, IgA2m(1) or IgA2m(2). Compared to IgA1, IgA2 has a much shorter hinge region, which joins the two Fab and one Fc fragments. In order to assess its solution structure, monomeric recombinant IgA2m(1) was studied by X-ray and neutron scattering. Its Guinier X-ray radius of gyration R(G) is 5.18 nm and its neutron R(G) is 5.03 nm, both of which are significantly smaller than those for monomeric IgA1 at 6.1-6.2 nm. The distance distribution function P(r)for IgA2m(1) showed a broad peak with a subpeak and gave a maximum dimension of 17 nm, in contrast to the P(r) curve for IgA1, which showed two distinct peaks and a maximum dimension of 21 nm. The sedimentation coefficients of IgA1 and IgA2m(1) were 6.2S and 6.4S, respectively. These data show that the solution structure of IgA2m(1) is significantly more compact than IgA1. The complete monomeric IgA2m(1) structure was modelled using molecular dynamics to generate random IgA2 hinge structures, to which homology models for the Fab and Fc fragments were connected to generate 10,000 full models. A total of 104 compact best-fit IgA2m(1) models gave good curve fits. These best-fit models were modified by linking the two Fab light chains with a disulphide bridge that is found in IgA2m(1), and subjecting these to energy refinement to optimise this linkage. The averaged solution structure of the arrangement of the Fab and Fc fragments in IgA2m(1) was found to be predominantly T-shaped and flexible, but also included Y-shaped structures. The IgA2 models show full steric access to the two FcalphaRI-binding sites at the Calpha2-Calpha3 interdomain region in the Fc fragment. Since previous scattering modelling had shown that IgA1 also possessed a flexible T-shaped solution structure, such a T-shape may be

  13. Earth Observing System/Advanced Microwave Sounding Unit-A (EOS/AMSU-A): Reliability prediction report for module A1 (channels 3 through 15) and module A2 (channels 1 and 2)

    NASA Technical Reports Server (NTRS)

    Geimer, W.

    1995-01-01

    This report documents the final reliability prediction performed on the Earth Observing System/Advanced Microwave Sounding Unit-A (EOS/AMSU-A). The A1 Module contains Channels 3 through 15, and is referred to herein as 'EOS/AMSU-A1'. The A2 Module contains Channels 1 and 2, and is referred herein as 'EOS/AMSU-A2'. The 'specified' figures were obtained from Aerojet Reports 8897-1 and 9116-1. The predicted reliability figure for the EOS/AMSU-A1 meets the specified value and provides a Mean Time Between Failures (MTBF) of 74,390 hours. The predicted reliability figure for the EOS/AMSU-A2 meets the specified value and provides a MTBF of 193,110 hours.

  14. Human natural killer cell microRNA: differential expression of MIR181A1B1 and MIR181A2B2 genes encoding identical mature microRNAs.

    PubMed

    Presnell, S R; Al-Attar, A; Cichocki, F; Miller, J S; Lutz, C T

    2015-01-01

    Natural killer (NK) and T lymphocytes share many properties, yet only NK cells respond rapidly to infection and cancer without pre-activation. We found that few microRNAs (miRNAs) differed significantly between human NK and T cells. Among those miRNAs, miR-181a and miR-181b levels rose during NK cell differentiation. Prior studies indicate that miR-181a and miR-181b are critical for human NK cell development and are co-transcribed from genes on chromosome 1 (MIR181A1B1) and on chromosome 9 (MIR181A2B2). We mapped human MIR181A1B1 and MIR181A2B2 transcription start sites to 78.3 kb and 34.0 kb upstream of the mature miRNAs, generating predominantly unspliced transcripts of 80-127 kb and ~60 kb, respectively. Unlike mouse thymocytes, human T cells expressed both MIR181A1B1 and MIR181A2B2. We tested the hypothesis that NK cells differentially transcribe the two genes during development and in response to immune regulatory cytokines. During NK-cell differentiation, MIR181A2B2 expression rose markedly and exceeded that of MIR181A1B1. TGF-β treatment increased NK-cell MIR181A2B2 transcription, whereas IL-2, IL-15 and IL-12/IL-18 treatments upregulated MIR181A1B1. The MIR181A2B2 promoter was strongly transactivated by SMAD3 and SMAD4 transcription factors, suggesting that TGF-β signaling upregulates MIR181A2B2 expression, at least in part, through SMAD-dependent promoter activation.

  15. Structural and Kinetic Basis of Steroid 17α,20-Lyase Activity in Teleost Fish Cytochrome P450 17A1 and Its Absence in Cytochrome P450 17A2*

    PubMed Central

    Pallan, Pradeep S.; Nagy, Leslie D.; Lei, Li; Gonzalez, Eric; Kramlinger, Valerie M.; Azumaya, Caleigh M.; Wawrzak, Zdzislaw; Waterman, Michael R.; Guengerich, F. Peter; Egli, Martin

    2015-01-01

    Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals, a single enzyme, P450 17A1, catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s; zebrafish P450 17A1 catalyzes both 17α-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17α-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the two-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17α-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1 but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and with Prog for P450 17A2. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116–119) showed only a few differences near the active site, despite only ∼50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternative ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity. PMID:25533464

  16. Glucuronidation of OTS167 in Humans Is Catalyzed by UDP-Glucuronosyltransferases UGT1A1, UGT1A3, UGT1A8, and UGT1A10

    PubMed Central

    Ramírez, Jacqueline; Mirkov, Snezana; House, Larry K.

    2015-01-01

    OTS167 is a potent maternal embryonic leucine zipper kinase inhibitor undergoing clinical testing as antineoplastic agent. We aimed to identify the UDP-glucuronosyltransferases (UGTs) involved in OTS167 metabolism, study the relationship between UGT genetic polymorphisms and hepatic OTS167 glucuronidation, and investigate the inhibitory potential of OTS167 on UGTs. Formation of a single OTS167-glucuronide (OTS167-G) was observed in pooled human liver (HLM) (Km = 3.4 ± 0.2 µM), intestinal microsomes (HIM) (Km = 1.7 ± 0.1 µM), and UGTs. UGT1A1 (64 µl/min/mg) and UGT1A8 (72 µl/min/mg) exhibited the highest intrinsic clearances (CLint) for OTS167, followed by UGT1A3 (51 µl/min/mg) and UGT1A10 (47 µl/min/mg); UGT1A9 was a minor contributor. OTS167 glucuronidation in HLM was highly correlated with thyroxine glucuronidation (r = 0.91, P < 0.0001), SN-38 glucuronidation (r = 0.79, P < 0.0001), and UGT1A1 mRNA (r = 0.72, P < 0.0001). Nilotinib (UGT1A1 inhibitor) and emodin (UGT1A8 and UGT1A10 inhibitor) exhibited the highest inhibitory effects on OTS167-G formation in HLM (68%) and HIM (47%). We hypothesize that OTS167-G is an N-glucuronide according to mass spectrometry. A significant association was found between rs6706232 and reduced OTS167-G formation (P = 0.03). No or weak UGT inhibition (range: 0–21%) was observed using clinically relevant OTS167 concentrations (0.4–2 µM). We conclude that UGT1A1 and UGT1A3 are the main UGTs responsible for hepatic formation of OTS167-G. Intestinal UGT1A1, UGT1A8, and UGT1A10 may contribute to first-pass OTS167 metabolism after oral administration. PMID:25870101

  17. MRCI study on the spectroscopic parameters and molecular constants of the X1Σ+, a3Σ+, A1Π and C1Σ- electronic states of the SiO molecule.

    PubMed

    Shi, Deheng; Li, Wentao; Sun, Jinfeng; Zhu, Zunlue

    2012-02-15

    The potential energy curves (PECs) of the X(1)Σ(+), a(3)Σ(+), A(1)Π and C(1)Σ(-) electronic states of the SiO molecule are studied using an ab initio quantum chemical method. The calculations have been made employing the complete active space self-consistent field (CASSCF) method, which is followed by the valence internally contracted multireference configuration interaction (MRCI) approach in combination with several correlation-consistent basis sets. The effect on the PECs by the core-valence correlation and relativistic corrections is included. The way to consider the relativistic correction is to use the third-order Douglas-Kroll Hamiltonian approximation. The core-valence correlation correction is carried out with the cc-pCVQZ basis set, and the relativistic correction is performed at the level of the cc-pVQZ basis set. To obtain more reliable results, the PECs determined by the MRCI calculations are also corrected for size-extensivity errors by means of the Davidson modification (MRCI+Q). The PECs of these electronic states are extrapolated to the complete basis set limit by the total-energy extrapolation scheme. Employing these PECs, the spectroscopic parameters are calculated and compared with those reported in the literature. With these PECs determined by the MRCI+Q/CV+DK+56 calculations, by solving the radial Schrödinger equation of nuclear motion, 110 vibrational states for the X(1)Σ(+), 69 for the a(3)Σ(+), 54 for the A(1)Π and 67 for the C(1)Σ(-) electronic state are predicted when the rotational quantum number J equals zero. The vibrational manifolds of the first 20 vibrational states are reported and compared with the available RKR data for each electronic state. On the whole, as expected, the most accurate spectroscopic parameters and molecular constants of the SiO molecule are obtained by the MRCI+Q/CV+DK+56 calculations. And the present molecular constants of the a(3)Σ(+), C(1)Σ(-) and A(1)Π electronic states determined by the MRCI

  18. Sequencing and characterization of mixed function monooxygenase genes CYP1A1 and CYP1A2 of Mink (Mustela vison) to facilitate study of dioxin-like compounds

    SciTech Connect

    Zhang Xiaowei; Moore, Jeremy N.; Newsted, John L.; Hecker, Markus Zwiernik, Matthew J.; Jones, Paul D.; Bursian, Steven J.

    2009-02-01

    As part of an ongoing effort to understand aryl hydrocarbon receptor (AhR) mediated toxicity in mink, cDNAs encoding for CYP1A1 and the CYP1A2 mixed function monooxygenases were cloned and characterized. In addition, the effects of selected dibenzofurans on the expression of these genes and the presence of their respective proteins (P4501A) were investigated, and then correlated with the catalytic activities of these proteins as measured by ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-deethylase (MROD) activities. The predicted protein sequences for CYP1A1 and CYP1A2 comprise 517 and 512 amino acid residues, respectively. The phylogenetic analysis of the mink CYP1As with protein sequences of other mammals revealed high sequence homology with sea otter, seals and the dog, with amino acid identities ranging from 89 to 95% for CYP1A1 and 81 to 93% for CYP1A2. Since exposure to both 2,3,7,8-Tetrachlorodibenzofuran (TCDF) and 2,3,4,7,8-Pentachlorodibenzofuran (PeCDF) resulted in dose-dependent increases of CYP1A1 mRNA, CYP1A2 mRNA and CYP1A protein levels an underlying AhR-mediated mechanism is suggested. The up-regulation of CYP1A mRNA in liver was more consistent to the sum adipose TEQ concentration than to the liver TEQ concentration in minks treated with TCDF or PeCDF. The result suggested that the hepatic-sequestered fraction of PeCDF was biologically inactive to the induction of CYP1A1 and CYP1A2.

  19. Structural and kinetic basis of steroid 17α,20-lyase activity in teleost fish cytochrome P450 17A1 and its absence in cytochrome P450 17A2.

    PubMed

    Pallan, Pradeep S; Nagy, Leslie D; Lei, Li; Gonzalez, Eric; Kramlinger, Valerie M; Azumaya, Caleigh M; Wawrzak, Zdzislaw; Waterman, Michael R; Guengerich, F Peter; Egli, Martin

    2015-02-06

    Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals, a single enzyme, P450 17A1, catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s; zebrafish P450 17A1 catalyzes both 17α-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17α-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the two-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17α-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1 but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and with Prog for P450 17A2. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116-119) showed only a few differences near the active site, despite only ∼50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternative ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Effect of polyunsaturated fatty acids ω-3 on the induction of activity and expression of CYP1A1 and CYP1A2 genes in the liver of rats under the influence of indole-3-carbinol.

    PubMed

    Kravchenko, L V; Tutel'yan, V A; Trusov, N V; Guseva, G V; Aksenov, I V

    2014-01-01

    Supplementation of the ration with eicosapentaenoic and docosahexaenoic ω-3 polyunsaturated fatty acids (PUFA) in doses of 0.3 and 1 g/kg body weight for 4 weeks had no effect on ethoxyresorufin O-dealkylase (EROD) activity and expression of the CYP1A1 gene in male Wistar rats, but caused a dose-dependent increase in methoxyresorufin O-dealkylase (MROD) activity of CYP1A2 (by 28 and 73%, respectively) without significant changes in CYP1A2 mRNA expression. ω-3 PUFA had no effect on the indole-3-carbinol-induced (20 mg/kg body weight over the last 7 days of the experiment) EROD activity and expression of CYP1A1 mRNA. The indole-3-carbinol-induced MROD activity was shown to increase by 6.2 times in rats not receiving ω-3 PUFA and only by 3.9 and 2.7 times in animals receiving ω-3 PUFA. The indole-3-carbinol-induced expression of CYP1A2 mRNA slightly increased in animals receiving ω-3 PUFA. Our results suggest that the effect of ω-3 PUFA on the induced and basal activity of CYP1A2 is not related to modulation of CYP1A2 gene expression.

  1. Ethanol and 4-methylpyrazole increase DNA adduct formation of furfuryl alcohol in FVB/N wild-type mice and in mice expressing human sulfotransferases 1A1/1A2.

    PubMed

    Sachse, Benjamin; Meinl, Walter; Glatt, Hansruedi; Monien, Bernhard H

    2016-03-01

    Furfuryl alcohol (FFA) is a carcinogenic food contaminant, which is formed by acid- and heat-catalyzed degradation of fructose and glucose. The activation by sulfotransferases (SULTs) yields a DNA reactive and mutagenic sulfate ester. The most prominent DNA adduct, N(2)-((furan-2-yl)methyl)-2'-deoxyguanosine (N(2)-MF-dG), was detected in FFA-treated mice and also in human tissue samples. The dominant pathway of FFA detoxification is the oxidation via alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs). The activity of these enzymes may be greatly altered in the presence of inhibitors or competitive substrates. Here, we investigated the impact of ethanol and the ADH inhibitor 4-methylpyrazole (4MP) on the DNA adduct formation by FFA in wild-type and in humanized mice that were transgenic for human SULT1A1/1A2 and deficient in the mouse (m) Sult1a1 and Sult1d1 genes (h1A1/1A2/1a1(-)/1d1(-)). The administration of FFA alone led to hepatic adduct levels of 4.5 N(2)-MF-dG/10(8) nucleosides and 33.6 N(2)-MF-dG/10(8) nucleosides in male and female wild-type mice, respectively, and of 19.6 N(2)-MF-dG/10(8) nucleosides and 95.4 N(2)-MF-dG/10(8) nucleosides in male and female h1A1/1A2/1a1(-)/1d1(-) mice. The coadministration of 1.6g ethanol/kg body weight increased N(2)-MF-dG levels by 2.3-fold in male and by 1.7-fold in female wild-type mice and by 2.5-fold in male and by 1.5-fold in female h1A1/1A2/1a1(-)/1d1(-) mice. The coadministration of 100mg 4MP/kg body weight had a similar effect on the adduct levels. These findings indicate that modulators of the oxidative metabolism, e.g. the drug 4MP or consumption of alcoholic beverages, may increase the genotoxic effects of FFA also in humans. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. Integral cross sections for the direct excitation of the A 3 (sigma) u +, B 3 (pi) g, W 3 (delta) u, B' 3 (sigma) u -, a' 1 (sigma) u -, a 1 (pi) g, w 1 (delta) u, and C 3 (pi) u electronic states in

    NASA Technical Reports Server (NTRS)

    Johnson, P. V.; Malone, C. P.; Kanik, I.

    2005-01-01

    Integral cross sections for electron impact excitation out of the ground state (X 1(sigma)g +) to the A 3(sigma)u +, B 3(pi)g, W 3(delta)u, B' 3(sigma)u -, a' 1(sigma)u -, a 1(pi)g, w 1(delta)u, and states in N2 are reported at incident energies ranging between 10 and 100 eV. These data have been derived by integrating differential cross sections previously reported by this group. New differential cross section measurements for the a 1(pi)g state at 200 eV are also presented to extend the range of the reported integral cross sections for this state, which is responsible for the emissions of the Lyman-Birge-Hopfield band system (a 1(pi)g (rightwards arrow) X 1(sigma)g +). The present results are compared and critically evaluated against existing cross sec In general, the present cross sections are smaller than previous results at low impact energies from threshold through the excitation function peak regions. These lower cross sections have potentially significant implications on our understanding of UV emissions in the atmospheres of Earth and Titan.

  3. Integral cross sections for the direct excitation of the A 3 (sigma) u +, B 3 (pi) g, W 3 (delta) u, B' 3 (sigma) u -, a' 1 (sigma) u -, a 1 (pi) g, w 1 (delta) u, and C 3 (pi) u electronic states in

    NASA Technical Reports Server (NTRS)

    Johnson, P. V.; Malone, C. P.; Kanik, I.

    2005-01-01

    Integral cross sections for electron impact excitation out of the ground state (X 1(sigma)g +) to the A 3(sigma)u +, B 3(pi)g, W 3(delta)u, B' 3(sigma)u -, a' 1(sigma)u -, a 1(pi)g, w 1(delta)u, and states in N2 are reported at incident energies ranging between 10 and 100 eV. These data have been derived by integrating differential cross sections previously reported by this group. New differential cross section measurements for the a 1(pi)g state at 200 eV are also presented to extend the range of the reported integral cross sections for this state, which is responsible for the emissions of the Lyman-Birge-Hopfield band system (a 1(pi)g (rightwards arrow) X 1(sigma)g +). The present results are compared and critically evaluated against existing cross sec In general, the present cross sections are smaller than previous results at low impact energies from threshold through the excitation function peak regions. These lower cross sections have potentially significant implications on our understanding of UV emissions in the atmospheres of Earth and Titan.

  4. Design, synthesis and evaluation of the inhibitory selectivity of novel trans-resveratrol analogues on human recombinant CYP1A1, CYP1A2 and CYP1B1.

    PubMed

    Mikstacka, Renata; Rimando, Agnes M; Dutkiewicz, Zbigniew; Stefański, Tomasz; Sobiak, Stanisław

    2012-09-01

    A series of trans-stilbene derivatives containing 4'-methylthio substituent were synthesized and evaluated for inhibitory activities on human recombinant cytochrome P450(s): CYP1A1, CYP1A2, and CYP1B1. CYP1A2-related metabolism of stilbene derivatives was estimated by using NADPH oxidation assay. Additionally, for CYP1A2 and CYP1B1 molecular docking analysis was carried out to provide information on enzyme-ligand interactions and putative site of metabolism. 3,4,5-Trimethoxy-4'-methylthio-trans-stilbene, an analogue of DMU-212 (3,4,5,4'-tetramethoxy-trans-stilbene) was an effective inhibitor of all CYP1 enzymes. On the other hand, 2,3,4-trimethoxy-4'-methylthio-trans-stilbene, appeared to be the most selective inhibitor of the isozymes CYP1A1 and CYP1B1, displaying extremely low affinity towards CYP1A2. Molecular modeling suggested that the most probable binding poses of the methylthiostilbene derivatives in CYP1A2 active sites are those with the methylthio substituent directed towards the heme iron. Products of CYP1A2-catalyzed oxidation of 2,4,5-trimethoxy-4'-methylthiostilbene and 3,4,5-trimethoxy-4'-methylthiostilbene were identified as monohydroxylated compounds. Other studied derivatives appeared to be poor substrates of CYP1A2. Structure-activity relationship analysis rendered better understanding of the mechanism of action of xenobiotic-metabolizing enzymes crucial at the early stage of carcinogenesis. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Rovibrational dynamics of the strontium molecule in the A(1)Σ(u)+, c(3)Π(u), and a(3)Σ(u)+ manifold from state-of-the-art ab initio calculations.

    PubMed

    Skomorowski, Wojciech; Pawłowski, Filip; Koch, Christiane P; Moszynski, Robert

    2012-05-21

    State-of-the-art ab initio techniques have been applied to compute the potential energy curves for the electronic states in the A(1)Σ(u)(+), c(3)Π(u), and a(3)Σ(u)(+) manifold of the strontium dimer, the spin-orbit and nonadiabatic coupling matrix elements between the states in the manifold, and the electric transition dipole moment from the ground X(1)Σ(g)(+) to the nonrelativistic and relativistic states in the A+c+a manifold. The potential energy curves and transition moments were obtained with the linear response (equation of motion) coupled cluster method limited to single, double, and linear triple excitations for the potentials and limited to single and double excitations for the transition moments. The spin-orbit and nonadiabatic coupling matrix elements were computed with the multireference configuration interaction method limited to single and double excitations. Our results for the nonrelativistic and relativistic (spin-orbit coupled) potentials deviate substantially from recent ab initio calculations. The potential energy curve for the spectroscopically active (1)0(u)(+) state is in quantitative agreement with the empirical potential fitted to high-resolution Fourier transform spectra [A. Stein, H. Knöckel, and E. Tiemann, Eur. Phys. J. D 64, 227 (2011)]. The computed ab initio points were fitted to physically sound analytical expressions, and used in converged coupled channel calculations of the rovibrational energy levels in the A+c+a manifold and line strengths for the A(1)Σ(u)(+)←X(1)Σ(g (+) transitions. Positions and lifetimes of quasi-bound Feshbach resonances lying above the (1)S(0) + (3)P(1) dissociation limit were also obtained. Our results reproduce (semi)quantitatively the experimental data observed thus far. Predictions for on-going and future experiments are also reported.

  6. TSU-16, (Z)-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone, is a potent activator of aryl hydrocarbon receptor and increases CYP1A1 and CYP1A2 expression in human hepatocytes.

    PubMed

    Matsuoka-Kawano, Kazuaki; Yoshinari, Kouichi; Nagayama, Sekio; Yamazoe, Yasushi

    2010-04-15

    (Z)-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone (TSU-16), is a potent anti-angiogenic agent that inhibits the tyrosine kinase of vascular endothelial growth factor receptor-2. In clinical trials with daily or twice weekly intravenous administration of TSU-16, its increased clearance was observed. To understand the mechanism underlying this observation, we have investigated the TSU-16-mediated regulation of cytochrome P450 expression. In human hepatocytes, TSU-16 increased mRNA levels of CYP1A1 and CYP1A2, but not CYP2B6 and CYP3A4. The extent of increase and profiles of the time-dependent changes in CYP1A1 and CYP1A2 mRNA levels after TSU-16 treatment were similar to those after treatment with 3-methylcholanthrene (3MC), a well-known activator of the aryl hydrocarbon receptor (AhR). In reporter assays using a plasmid construct that contained the human CYP1A1 5'-flanking region including the region crucial for the AhR-dependent transcription of both human CYP1A1 and CYP1A2, TSU-16 treatment increased reporter activities to an extent similar to that obtained with 3MC. Treatment of HepG2 cells and human hepatocytes with AhR-targeting siRNA suppressed the increase in both mRNA levels and CYP1A activities after treatment with TSU-16 as well as after that with omeprazole or 3MC. TSU-16 also time-dependently reduced cellular AhR protein levels in HepG2 cells to a similar extent with 3MC treatment. Furthermore, we demonstrated that unlabeled TSU-16 and 3MC but not omeprazole completely inhibited the specific binding of [(3)H]-3MC to mouse Hepa1c1c7 cytosol, suggesting TSU-16 as an AhR ligand. In conclusion, our present results suggest that TSU-16 binds to and activates AhR to enhance the expression of both human CYP1A1 and CYP1A2. Because TSU-16 is metabolized mainly by CYP1A2, its increased clearance after repeated dosing may be attributed to the enhanced expression of hepatic CYP1A2.

  7. Identification and analysis of the maize P700 chlorophyll a apoproteins PSI-A1 and PSI-A2 by high pressure liquid chromatography analysis and partial sequence determination.

    PubMed

    Fish, L E; Bogorad, L

    1986-06-25

    We recently described a pair of partially homologous maize chloroplast genes, one of which was shown to code for an apoprotein of the P700 chlorophyll a complex of photosystem I (Fish, L.E., Kück, U., and Bogorad, L. (1985) J. Biol. Chem. 260, 1413-1421). Two chlorophyll-free apoprotein bands from maize chlorophyll-protein complex I (CPI) can be resolved on lithium dodecyl sulfate (LDS)-urea polyacrylamide gels. Proteins in both bands react with antibodies prepared against CPI, but antibodies prepared against two synthetic peptides corresponding to predicted sequences of PSI-A1 react only with the upper band. The presence of products of the two genes, ps1A1 and ps1A2, in CPI was verified by analysis of cyanogen bromide (CNBr) fragments of the lower apoprotein band obtained from LDS-urea polyacrylamide gels by reverse-phase high pressure liquid chromatography. Amino-terminal sequencing of five CNBr fragments indicates that the lower band contains a product of the ps1A2 gene. The possibility of extensive processing was investigated because the apparent molecular masses of the maize CPI proteins are about 58-70 kDa on LDS-polyacrylamide gels rather than the predicted sizes of about 83 kDa. Antibodies against a synthetic peptide corresponding to a predicted sequence in PSI-A1 were used to determine that the amino-terminal end of PSI-A1 is intact beyond about position 52. The amino-terminal CNBr fragment of PSI-A2 was identified by sequencing, indicating that the amino-terminal end of PSI-A2 is not processed. The carboxyl-terminal CNBr fragment of PSI-A2 was also identified by sequencing. These results indicate that the PSI-A1 and PSI-A2 polypeptides are not extensively processed, although some processing at the carboxyl-terminal end has not been ruled out.

  8. Hall Effect Thruster Interactions Data From the Russian Express-A2 and Express-A3 Satellites. Part 3; Acquire Express-A3 SPT-100 Based Propulsion Subsystem and Other Subsystem Flight Operation TM-Data for the Period of June 24, 2000 to and Including September 30, 2000, Task 30

    NASA Technical Reports Server (NTRS)

    Sitnikova, N.; Volkov, D.; Maximov, I.; Petrusevich, V.; Allen, D.

    2003-01-01

    This 12-part report documents the data obtained from various sensor measurements taken aboard the Russian Express-A2 and Express-A3 spacecraft in Geosynchronous Earth Orbit (GEO). These GEO communications satellites, which were designed and built by NPO Prikladnoy Mekhaniki (NPO PM) of Zheleznogorsk, Russia, utilize Hall thruster propulsion systems for north-south and east-west stationkeeping and as of June 2002, were still operating at 80 E. and 11 W., respectively. Express-A2 was launched on March 12, 2000, while Express-A3 was launched on June 24, 2000. The diagnostic equipment from which these data were taken includes electric field strength sensors, ion current and energy sensors, and pressure sensors. The diagnostics and the Hall thruster propulsion systems are described in detail along with lists of tabular data from those diagnostics and propulsion system and other satellite systems. Space Power, Inc., now part of Pratt & Whitney's Chemical Systems Division, under contract NAS3 99151 to the NASA Glenn Research Center, obtained these data over several periods from March 12, 2000, through September 30, 2001. Each of the 12 individual reports describe, in detail, the propulsion systems as well as the diagnostic sensors utilized. Finally, parts 11 and 12 include the requirements to which NPO PM prepared and delivered these data.

  9. Utility of Nicotiana tabacum cell suspension cultures expressing human CYP1A1, CYP1A2 and CYP3A4 to study the oxidative metabolism of the herbicide 14C-fluometuron.

    PubMed

    Breuer, Maren Anne; Schmidt, Burkhard; Schuphan, Ingolf

    2009-01-01

    The metabolism and biotransformation of the (14)C-labeled phenylurea herbicide fluometuron was examined using tobacco cell suspension cultures transformed separately with human cyp1a1, cyp1a2 and cyp3a4, and corresponding non-transformed cultures in order to screen and predict metabolic patterns. Experimental parameters modified were concentration of (14)C-fluometuron, incubation period, and additional application of inhibitor carbaryl. Media and cell extracts were analyzed by radio-TLC and radio-HPLC, isolated metabolites by LC-MS, and non-extractable residues by combustion. During 48 hours, the CYP1A1 expressing cultures metabolized 90.0 % of applied fluometuron, while the non-transgenic controls transformed 67.0 %. The CYP1A2 expressing cultures exhibited highest rates (95.1 %), CYP3A4 expressing cultures lowest rates (43.0 %). The primary metabolites identified were mono-demethyl (main metabolite in controls) and di-demethyl fluometuron (mainly in CYP1A2 cultures), besides a non-identified primary product (mainly in CYP1A1 cultures); metabolic profiles differed distinctly among cultures. After addition of carbaryl, rates of fluometuron decreased noticeably in controls and not in CYP3A4 expressing cultures. This may indicate inhibition of endogenous tobacco P450s involved in fluometuron metabolism but not of CYP3A4. Additionally, the P450-transgenic cultures proved to be valuable tools to produce large amounts of metabolites for thorough identification.

  10. Sequence variations of ABCB1, SLC6A2, SLC6A3, SLC6A4, CREB1, CRHR1 and NTRK2: association with major depression and antidepressant response in Mexican-Americans.

    PubMed

    Dong, C; Wong, M-L; Licinio, J

    2009-12-01

    We studied seven genes that reflect events relevant to antidepressant action at four sequential levels: (1) entry into the brain, (2) binding to monoaminergic transporters, and (3) distal effects at the transcription level, resulting in (4) changes in neurotrophin and neuropeptide receptors. Those genes are ATP-binding cassette subfamily B member 1 (ABCB1), the noradrenaline, dopamine, and serotonin transporters (SLC6A2, SLC6A3 and SLC6A4), cyclic AMP-responsive element binding protein 1 (CREB1), corticotropin-releasing hormone receptor 1 (CRHR1) and neurotrophic tyrosine kinase type 2 receptor (NTRK2). Sequence variability for those genes was obtained in exonic and flanking regions. A total of 56 280 000 bp across were sequenced in 536 unrelated Mexican Americans from Los Angeles (264 controls and 272 major depressive disorder (MDD)). We detected in those individuals 419 single nucleotide polymorphisms (SNPs); the nucleotide diversity was 0.00054 + or - 0.0001. Of those, a total of 204 novel SNPs were identified, corresponding to 49% of all previously reported SNPs in those genes: 72 were in untranslated regions, 19 were in coding sequences of which 7 were non-synonymous, 86 were intronic and 27 were in upstream/downstream regions. Several SNPs or haplotypes in ABCB1, SLC6A2, SLC6A3, SLC6A4, CREB1 and NTRK2 were associated with MDD, and in ABCB1, SLC6A2 and NTRK2 with antidepressant response. After controlling for age, gender and baseline 21-item Hamilton Depression Rating Scale (HAM-D21) score, as well as correcting for multiple testing, the relative reduction of HAM-D21 score remained significantly associated with two NTRK2-coding SNPs (rs2289657 and rs56142442) and the haplotype CAG at rs2289658 (splice site), rs2289657 and rs2289656. Further studies in larger independent samples will be needed to confirm these associations. Our data indicate that extensive assessment of sequence variability may contribute to increase understanding of disease susceptibility and

  11. Tissue- and cell type-specific expression of cytochrome P450 1A1 and cytochrome P450 1A2 mRNA in the mouse localized in situ hybridization.

    PubMed

    Dey, A; Jones, J E; Nebert, D W

    1999-08-01

    We used in situ hybridization to examine organ- and cell type-specific constitutive and 3-methylcholanthrene (3MC)-inducible cytochrome P450 (CYP)1A1 and CYP1A2 mRNA expression in various tissues of the C57BL/6N mouse. In situ hybridization was carried out 10 hr after the mice had received intraperitoneal 3MC, or vehicle alone. We detected levels of 3MC-induced CYP1A1 mRNA in: liver (centrilobular, more so than periportal, regions); lung (Clara Type II cells much more than Type I epithelial cells); brain, especially endothelial cells lining the vascular surface of the choroid plexus; the digestive tract (duodenum > jejunum > ileum > colon > esophagus > stomach--in particular, the villous epithelium, plus cells surrounding glands in the lamina propria); renal corpuscles of the kidney; the ovary (medulla more so than cortex); and the endothelial cells of blood vessels throughout the animal. Constitutive CYP1A1 mRNA was not detectable by in situ hybridization in any of these tissues. In contrast, constitutive CYP1A2 mRNA was measurable in liver, and 3MC-inducible CYP1A2 mRNA was observed only in liver, lung, and duodenum (having cell-type locations similar to those of CYP1A1); the other above-mentioned tissues were negative for CYP1A2 mRNA. These data demonstrate the striking differences in tissue- and cell type-specific expression between the two members of the mouse Cypla subfamily. Because of the ubiquitous nature of 3MC-inducible CYP1A1 throughout the animal rather than just "portals of entry," these results support our hypothesis that CYP1A1, induced by particular endogenous signals in various tissues and cell types, might participate in one or more critical life processes--in addition to its well-established role of metabolism of polycyclic hydrocarbons, certain drugs, and other environmental pollutants.

  12. Analytical evaluation of the ADAMS(™) A1c HA 8180 thalassemia mode high-pressure liquid chromatography analyser for the measurement of HbA2 and HbF.

    PubMed

    Urrechaga, E

    2016-12-01

    ADAMS(™) A1cHA-8180T is a HPLC system; within 3.5 min, it quantifies HbF, HbA2 , and HbA0 and flags abnormal peaks. We evaluate its analytical performance for routine estimation of HbA2 and HbF, and critical tests were performed for identifying β-thalassemia carriers. Trueness imprecision, carry over, linearity, and effect of anemia were evaluated according to ICLH, ICLS, or manufacture's guidelines. Comparison (ADAMS(™) A1c HA-8160T) was performed by running 400 samples from healthy subjects, 30 alpha and 80 beta carriers (range: 1.9-5.7 %). Trueness - HbA2 2.7 %, bias 0.81 %; HbA2 5.8 %, bias 0.38 %. HbA2 4.0% is not affected by Hb in the range 221-40 g/L. Carry over was negligible. Within run: normal control - CV 1.5 %, high control - CV 0.9 %.Within laboratory: normal control - total CV% 1.59%; high control - 0.92 %. Linearity - y = 1.034x - 0.17, R(2 ) = 0.998 (range: 2.8-4.8%).Method comparison - y = 0.93x + 0.22, R(2)  = 0.997. HbF imprecision CVs between 0.66 and 1.24% and trueness between 0 and 2.8%. Linearity - y = 1.088x - 0.27, R(2)  = 0.999 (0.1-5.7%). ADAMS(™) A1c HA-8180T provides a rapid and reliable separation of HbA2 . The measurement is accurate and reproducible, which is needed because of the slight difference between normal and pathological values. The gap in HbA2 values between normal subjects and β-thalassemia carriers makes this an appropriate method for rapid screening for carriers. © 2016 John Wiley & Sons Ltd.

  13. A-3 Test Stand

    NASA Image and Video Library

    2012-06-08

    A tethered Stennis Space Center employee climbs an A-3 Test Stand ladded June 8, 2012, against the backdrop of the A-2 and B-1/B-2 stands. The new A-3 Test Stand will enable simulated high-altitude testing of next-generation rocket engines.

  14. A-3 Test Stand

    NASA Image and Video Library

    2012-06-08

    A tethered Stennis Space Center employee climbs an A-3 Test Stand ladder June 8, 2012, against the backdrop of the A-2 and B-1/B-2 stands. The new A-3 Test Stand will enable simulated high-altitude testing of next-generation rocket engines.

  15. Organic cation transporter 3 (OCT3/SLC22A3) and multidrug and toxin extrusion 1 (MATE1/SLC47A1) transporter in the placenta and fetal tissues: expression profile and fetus protective role at different stages of gestation.

    PubMed

    Ahmadimoghaddam, Davoud; Zemankova, Lenka; Nachtigal, Petr; Dolezelova, Eva; Neumanova, Zuzana; Cerveny, Lukas; Ceckova, Martina; Kacerovský, Marian; Micuda, Stanislav; Staud, Frantisek

    2013-03-01

    In our previous study, we described synchronized activity of organic cation transporter 3 (OCT3/SLC22A3) and multidrug and toxin extrusion 1 (MATE1/SLC47A1) transporter in the passage of organic cations across the rat placenta and the role of these transporters in fetal defense; in this study, we hypothesized that changes in placental levels of OCT3 and MATE1 throughout gestation might affect the fetal protection and detoxification. Using quantitative RT-PCR, Western blot analysis, and immunohistochemistry, we were able to detect Oct3/OCT3 and Mate1/MATE1 expression in the rat placenta as early as on Gestation Day (gd) 12 with increasing tendency toward the end of pregnancy. Comparing first versus third trimester human placenta, we observed stable expression of OCT1 and decreasing expression of OCT2 and OCT3 isoforms. Contrary to the current literature, we were able to detect also MATE1/MATE2 isoforms in the human placenta, however, with considerable inter- and intraindividual variability. Using infusion of 1-methyl-4-phenylpyridinium (MPP(+)), a substrate of OCT and MATE transporters, into pregnant dams, we investigated the protective function of the placenta against organic cations at different gds. The highest amount of MPP(+) reached the fetus on gd 12 while from gd 15 onward, maternal-to-fetal transport of MPP(+) decreased significantly. We conclude that increased expression of placental OCT3 and MATE1 along with general maturation of the placental tissues results in significantly lower transport of MPP(+) from mother to fetus. In contrast, decreasing expression of OCT3 and MATE1 in human placenta indicates these transporters may play a role in fetal protection preferentially at earlier stages of gestation.

  16. Effects of peppermint tea consumption on the activities of CYP1A2, CYP2A6, Xanthine Oxidase, N-acetyltranferase-2 and UDP-glucuronosyltransferases-1A1/1A6 in healthy volunteers.

    PubMed

    Begas, Elias; Tsioutsiouliti, Athanasia; Kouvaras, Evangelos; Haroutounian, Serkos A; Kasiotis, Konstantinos M; Kouretas, Dimitrios; Asprodini, Eftihia

    2017-02-01

    Peppermint leaves are widely used for the symptomatic treatment of digestive disorders. Previous studies have shown significant effects of its natural products on human enzyme activity; however, there is no study available concerning the effects of peppermint tea on metabolizing enzymes in humans. Aim of the present study was to investigate the effect of peppermint tea on CYP1A2, CYP2A6, Xanthine Oxidase (XO), N-acetyltranferase-2 (NAT2) and UDP-glucuronosyltransferases-1A1/1A6 (UGT1A1/1A6) activities in healthy subjects. Four males and five females consumed peppermint tea (2 g of dry leaves/200 mL water, twice daily) for six days. CYP1A2, CYP2A6, XO, NAT2 and UGT1A1/1A6 activities were determined before and at the end of the study period, using the following caffeine and paracetamol metabolic ratios: CYP1A2: 17MX/137MX (saliva) and (AFMU+1MU+1MX)/17MU (urine); CYP2A6: 17MU/(17MU + 17MX), XO: 1MU/(1MU+1MX), NAT2, AFMU/(AFMU+1MU+1MX) and UGT1A1/1A6 glucuronidated/total paracetamol, all determined in urine. NAT2 metabolic ratio was significantly reduced following peppermint consumption (0.15 ± 0.13 vs 0.14 ± 0.13; p < 0.05). CYP1A2 urine and saliva indices were reduced, yet not significantly, following peppermint consumption (urine: 3.17 ± 1.08 vs 2.91 ± 0.76, saliva: 0.56 ± 0.12 vs 0.50 ± 0.12; p > 0.05). Peppermint had no influence on CYP2A6, XO and UGT1A1/1A6 indices. Daily ingestion of peppermint tea may alter pharmacokinetics of clinically administered drugs and promote cancer chemoprevention through NAT2 inhibition. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Loss of Interdependent Binding by the FoxO1 and FoxA1/A2 Forkhead Transcription Factors Culminates in Perturbation of Active Chromatin Marks and Binding of Transcriptional Regulators at Insulin-sensitive Genes.

    PubMed

    Yalley, Akua; Schill, Daniel; Hatta, Mitsutoki; Johnson, Nicole; Cirillo, Lisa Ann

    2016-04-15

    FoxO1 binds to insulin response elements located in the promoters of insulin-like growth factor-binding protein 1 (IGFBP1) and glucose-6-phosphatase (G6Pase), activating their expression. Insulin-mediated phosphorylation of FoxO1 promotes cytoplasmic translocation, inhibiting FoxO1-mediated transactivation. We have previously demonstrated that FoxO1 opens and remodels chromatin assembled from the IGFBP1 promoter via a highly conserved winged helix motif. This finding, which established FoxO1 as a "pioneer" factor, suggested a model whereby FoxO1 chromatin remodeling at regulatory targets facilitates binding and recruitment of additional regulatory factors. However, the impact of FoxO1 phosphorylation on its ability to bind chromatin and the effect of FoxO1 loss on recruitment of neighboring transcription factors at its regulatory targets in liver chromatin is unknown. In this study, we demonstrate that an amino acid substitution that mimics insulin-mediated phosphorylation of a serine in the winged helix DNA binding motif curtails FoxO1 nucleosome binding. We also demonstrate that shRNA-mediated loss of FoxO1 binding to the IGFBP1 and G6Pase promoters in HepG2 cells significantly reduces binding of RNA polymerase II and the pioneer factors FoxA1/A2. Knockdown of FoxA1 similarly reduced binding of RNA polymerase II and FoxO1. Reduction in acetylation of histone H3 Lys-27 accompanies loss of FoxO1 and FoxA1/A2 binding. Interdependent binding of FoxO1 and FoxA1/A2 possibly entails cooperative binding because FoxO1 and FoxA1/A2 facilitate one another's binding to IGFPB1 promoter DNA. These results illustrate how transcription factors can nucleate transcriptional events in chromatin in response to signaling events and suggest a model for regulation of hepatic glucose metabolism through interdependent FoxO/FoxA binding.

  18. STAT5a promotes the transcription of mature mmu-miR-135a in 3T3-L1 cells by binding to both miR-135a-1 and miR-135a-2 promoter elements.

    PubMed

    Wei, Xiajie; Cheng, Xiaoyan; Peng, Yongdong; Zheng, Rong; Chai, Jin; Jiang, Siwen

    2016-08-01

    Despite extensive research on the role of miR-135a in biological processes, very little attention has been paid to the regulation of its transcription. We have previously reported that miR-135a suppresses 3T3-L1 preadipocyte differentiation and adipogenesis by directly targeting the adenomatous polyposis coli (APC) gene and activating the canonical Wnt/β-catenin signaling pathway, but the regulatory elements that regulate the expression of the two isoforms of miR-135a (miR-135a-1 and miR-135a-2) remain poorly understood. Here, by using deletion analysis, we predicted two binding sites (-874/-856 and -2020/-2002) for the transcription factor Signal Transducers and Activators of Transcription 5a (STAT5a) within the core promoters of miR-135a-1 and miR-135a-2 (-1128/-556 and -2264/-1773), and the subsequent site-directed mutagenesis indicated that the two STAT5a binding sites regulated the activity of the miR-135a-1 and miR-135a-2 promoters. The binding of STAT5a to the miR-135a-1/2 core promoters in vitro and in cell culture was identified by electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) assays. Overexpression and RNAi knockdown of STAT5a showed that the transcription factor regulated the endogenous miR-135a expression. Additionally, The expression time frame of STAT5a and APC indicated a potential negative feedback between them. In sum, the overall results from this study indicate that STAT5a regulates miR-135a transcription by binding to both miR-135a-1 and miR135a-2 promoter elements and the findings provide novel insights into the molecular regulatory mechanisms of miR-135a during adipogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Cytochrome b(5) shifts oxidation of the anticancer drug ellipticine by cytochromes P450 1A1 and 1A2 from its detoxication to activation, thereby modulating its pharmacological efficacy.

    PubMed

    Kotrbová, Věra; Mrázová, Barbora; Moserová, Michaela; Martínek, Václav; Hodek, Petr; Hudeček, Jiří; Frei, Eva; Stiborová, Marie

    2011-09-15

    Ellipticine is a pro-drug, whose activation is dependent on its oxidation by cytochromes P450 (CYP) and peroxidases. Cytochrome b(5) alters the ratio of ellipticine metabolites formed by isolated reconstituted CYP1A1 and 1A2, favoring formation of 12-hydroxy- and 13-hydroxyellipticine metabolites implicated in ellipticine-DNA adduct formation, at the expense of 9-hydroxy- and 7-hydroxyellipticine that are detoxication products. Cytochrome b(5) enhances the production of 12-hydroxy and 13-hydroxyellipticine. The change in metabolite ratio results in an increased formation of covalent ellipticine-DNA adducts, one of the DNA-damaging mechanisms of ellipticine antitumor action. This finding explains previous apparent discrepancies found with isolated enzymes and in vivo, where CYP1A enzymatic activation correlated with ellipticine-DNA-adduct levels while isolated CYP1A1 or 1A2 in reconstituted systems were much less effective than CYP3A4. The effect of cytochrome b(5) might be even more pronounced in vivo, since, as we show here, ellipticine increases levels of cytochrome b(5) in rat liver. Our results demonstrate that both the native 3D structure of cytochrome b(5) and the presence of the heme as an electron transfer agent in this protein enable a shift in ellipticine metabolites formed by CYP1A1/2. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Changes in HbA1c levels and body mass index after successful decompression surgery in patients with type 2 diabetes mellitus and lumbar spinal stenosis: results of a 2-year follow-up study.

    PubMed

    Kim, Kyoung-Tae; Cho, Dae-Chul; Sung, Joo-Kyung; Kim, Chi Heon; Kang, Hyun; Kim, Du Hwan

    2017-02-01

    Lumbar spinal stenosis (LSS) can hinder a patient's physical activity, which in turn can impair glucose tolerance and body weight regulation in patients with type 2 diabetes mellitus (DM-2). Therefore, successful lumbar surgery could facilitate glycemic control and body weight regulation. This study aimed to evaluate the effects of postoperative improvement in physical activity on body mass index (BMI) and hemoglobin A1c (HbA1c) level in patients with LSS and DM-2 over a 2-year follow-up period. Prospective longitudinal observational study. Patients with LSS and DM-2. Visual analogue scale (VAS) scores for back pain and leg pain, Oswestry Disability Index (ODI) scores, Japanese Orthopaedic Association (JOA) scores, JOA Back Pain Evaluation Questionnaire (JOABPEQ) sections, BMI, and blood analysis for HbA1c were carried out. A total of 119 patients were enrolled for analysis of the effect of successful decompression surgery on changes in HbA1c levels and BMI. The VAS score, ODI score, JOA score, JOABPEQ, BMI, HbA1c were reassessed at 6 months, 1 year, and 2 years after surgery. Additionally, correlations between changes in HbA1c and changes in the ODI, JOA, JOABPEQs, and BMI were analyzed. The overall values of HbA1c before and at 6 months, 1 year, and 2 years after the surgery were 7.08±0.94%, 6.58±0.87%, 6.59±0.79%, and 6.59±0.79%, respectively (p-values; 6 months: .024; 1 year: .021; 2 years: .038). In the not well-controlled sugar (non-WCS) group (preoperative HbA1c>6.5%), the difference between pre- and postoperative HbA1c was highly statistically significant (p<.01). The overweight group (preoperative BMI≥25) showed statistically significant BMI reduction in the second year after surgery (p=.034). The postoperative HbA1c changes are strongly correlated with the improvements of ODI, JOA, and JOABPEQ after surgery. The present study demonstrates that in patients with DM-2 and LSS, successful lumbar surgery may facilitate glycemic control by enabling an

  1. Ex vivo biomechanical comparison of a 3.5 mm locking compression plate applied cranially and a 2.7 mm locking compression plate applied medially in a gap model of the distal aspect of the canine radius.

    PubMed

    Uhl, Justin M; Kapatkin, Amy S; Garcia, Tanya C; Stover, Susan M

    2013-10-01

    To compare a medially applied 2.7 mm locking compression plate (LCP) to a cranially applied 3.5 mm LCP in a cadaveric distal radial fracture gap model. In vitro mechanical testing of paired cadaveric limbs Paired radii (n = 8) stabilized with either a 2.7 mm LCP medially or a 3.5 mm LCP cranially. Simulated distal radial comminuted fractures were created and stabilized with an LCP plate on the cranial surface in 1 limb, and on the medial surface in the contralateral limb. Gap stiffness, gap strain, and failure properties were compared between cranial and medial plate positions. Limb constructs were axially loaded, cyclically through 4 conditions that allowed mediolateral or craniocaudal bending at walk and trot loads, before monotonic failure loading. The effects of plate position on mechanical variables were assessed using paired t-tests. Gap stiffness was greater for cranial plate constructs than medial plate constructs for axial loading with mediolateral bending, but lower with craniocaudal bending. However, in loading that facilitated craniocaudal bending the medial plate construct also had bending apparent in the mediolateral direction. Gap strains for the different conditions followed similar trends as stiffness. Cranial plate constructs had significantly higher monotonic stiffness, yield, and failure loads. The larger, cranially applied LCP was biomechanically superior to the smaller, medially applied LCP in our distal radial fracture gap model, however the medial plate was superior to the cranial plate in cyclic loading allowing craniocaudal bending. © Copyright 2013 by The American College of Veterinary Surgeons.

  2. Hall Effect Thruster Interactions Data From the Russian Express-A2 and Express-A3 Satellites. Acquire Express-A2 SPT-100 Based Propulsion Subsystem and Other Subsystem Flight Operation TM-Data for the Period of March 12, 2000 to and Including June 15, 2000, Task 29

    NASA Technical Reports Server (NTRS)

    Sitnikova, N.; Volkov, D.; Maximov, I.; Petrusevich, V.; Allen, D.

    2003-01-01

    This 12-part report documents the data obtained from various sensor measurements taken aboard the Russian Express-A2 and Express-A3 spacecraft in Geosynchronous Earth Orbit (GEO). These GEO communications satellites, which were designed and built by NPO Prikladnoy Mekhaniki (NPO PM) of Zheleznogorsk, Russia, utilize Hall thruster propulsion systems for north-south and east-west stationkeeping and as of June 2002, were still operating at 80 E. and 11 W., respectively. Express-A2 was launched on March 12, 2000, while Express-A3 was launched on June 24, 2000. The diagnostic equipment from which these data were taken includes electric field strength sensors, ion current and energy sensors, and pressure sensors. The diagnostics and the Hall thruster propulsion systems are described in detail along with lists of tabular data from those diagnostics and propulsion system and other satellite systems. Space Power, Inc., now part of Pratt & Whitney s Chemical Systems Division, under contract NAS3 99151 to the NASA Glenn Research Center, obtained these data over several periods from March 12, 2000, through September 30, 2001. Each of the 12 individual reports describe, in detail, the propulsion systems as well as the diagnostic sensors utilized. Finally, parts 11 and 12 include the requirements to which NPO PM prepared and delivered these data.

  3. Antifungal activity of the primycin complex and its main components A1, A2 and C1 on a Candida albicans clinical isolate, and their effects on the dynamic plasma membrane changes.

    PubMed

    Virág, Eszter; Belagyi, Joseph; Kocsubé, Sándor; Vágvölgyi, Csaba; Pesti, Miklós

    2013-02-01

    The in vitro antifungal activities of the macrolide lactone antibiotic complex primycin (PC) and its main components, A1 (50%), A2 (7.3%) and C1 (13%), against the opportunistic pathogenic fungus Candida albicans 33erg(+)were determined by microdilution testing. The MIC(100) (the minimal concentration required for 100% growth inhibition) values found, A2 (2 μg ml(-1)), PC (32 μg ml(-1)), A1 (32 μg ml(-1)) and C1 (64 μg ml(-1)), suggested that the biological activity of PC is highly dependent on the proportions of its constituents. In vivo measurements of the biophysical properties of plasma membranes were carried out by electron paramagnetic resonance (EPR) spectroscopic methods, using the spin probe 5-(4,4-dimethyloxazolidine-N-oxyl)stearic acid. Conventional EPR measurements demonstrated altered phase transition temperatures (Tm) of the plasma membrane of strain 33erg(+) as a consequence of treatment with PC or its constituents: for cells treated with 128 μg ml(-1) PC, A1, A2 or C1 for 15 min, Tm was 17, 21, 14.5 and 15 °C, respectively; that is significantly higher than the Tm of untreated cells, 12 °C. The molecular motions of the near-surface hydrophobic region of the plasma membrane, estimated by saturation transfer EPR spectroscopy, reflected changes in the membrane phases after the treatment. Two physiological membrane phases were detected in control samples: liquid-ordered and liquid-disordered, characterized by molecular movements ∼10(-6)-10(-8) s and ≥10(-9) s. The cells treated with the investigated compounds showed the strong presence of a non-physiological gel phase additional to the above phases, characterized by movements ≤10(-5) s.

  4. Differential inducing effect of benzo[a]pyrene on gene expression and enzyme activity of cytochromes P450 1A1 and 1A2 in Sprague-Dawley and Wistar rats.

    PubMed

    Floreani, Maura; Gabbia, Daniela; Barbierato, Massimo; DE Martin, Sara; Palatini, Pietro

    2012-01-01

    The objective of this study was to compare RT-PCR, Western blot and determination of enzyme activity in the assessment of the induction of cytochromes P450 (CYPs) 1A1 and 1A2 by benzo[a]pyrene (BaP) in Sprague-Dawley and Wistar rats. Inhibition studies and kinetic analyses confirmed literature data indicating that methoxyresorufin is a specific CYP1A2 substrate in both uninduced and BaP-treated rats, whereas ethoxyresorufin is a specific CYP1A1 substrate only in BaP-treated rats. BaP treatment increased mRNA and protein expressions of both CYP1A enzymes to a greater extent in Wistar than Sprague-Dawley rats. It consistently caused a higher increase in mRNA and protein expression of the aryl hydrocarbon receptor in the former rats. By contrast, CYP1A2 enzyme activity was much more markedly increased in Sprague-Dawley than Wistar rats and CYP1A1 activity was induced to similar levels. A BaP-induced increase in the turnover number of CYP1A enzymes in Sprague-Dawley rats, relative to Wistar rats, may provide a plausible explanation for the differential effect of BaP on gene expression and enzyme activity. These results have methodological implications, since they show that RT-PCR and Western blot may not provide a quantitative measure of induction of CYP1A activity, which is the actual measure of the change in CYP1A-mediated metabolism.

  5. Altered expression of adenosine A1 and A2A receptors in the carotid body and nucleus tractus solitarius of adult male and female rats following neonatal caffeine treatment.

    PubMed

    Bairam, Aida; Joseph, Vincent; Lajeunesse, Yves; Kinkead, Richard

    2009-09-01

    Neonatal caffeine treatment (adenosine receptor antagonist, 15 mg/kg/day, between postnatal days 3 and 12) affects respiratory patterns in adult male but not female rats as shown by an increase in the respiratory frequency in the early phase of response to hypoxia and an increase in the tidal volume in the late phase of response. Here, we tested the hypothesis that these changes are correlated with modified expression of adenosine receptors in the chemoreflex pathway. Carotid bodies, nucleus tractus solitarii, and superior cervical ganglia were collected from 3-month-old male and female rats that were either naive (not manipulated during the neonatal period) or treated with caffeine (NCT) or water (NWT) between postnatal days 3 and 12 by gavage. Western blot analysis was used to assess the expression of adenosine A(1) and A(2A) receptors and tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis. In male rats, there was a 37% increase in the level of A(2A) receptor and a 17% decrease in tyrosine hydroxylase in the carotid body of NCT (p<0.001) as compared to NWT rats. In the nucleus tractus solitarius, we found a 13% and 19% decrease in A(1) receptor expression in NWT and NCT rats (p<0.01), respectively, compared to naive rats. In the superior cervical ganglion, there was no change in A(1) receptor, A(2A) receptor, and tyrosine hydroxylase expression. In female rats, the only changes observed were decreases of 12% and 15% in A(1) receptor levels in the nucleus tractus solitarius of NWT and NCT rats (p<0.01), respectively, compared to naive rats. We conclude that NCT induces long-term changes in the adenosine receptor system. These changes may partially explain the modifications of the respiratory pattern induced by NCT in adults. The increased expression of the adenosine A(2A) receptor (specific to male rats), combined with the decreased tyrosine hydroxylase expression in the carotid body, suggests that NCT affects adenosine-dopamine interactions

  6. Binding of β4γ5 by Adenosine A1 and A2A Receptors Determined by Stable Isotope Labeling with Amino Acids in Cell Culture and Mass Spectrometry†

    PubMed Central

    Wang, Dora Bigler; Sherman, Nicholas E.; Shannon, John D.; Leonhardt, Susan A.; Mayeenuddin, Linnia H.; Yeager, Mark; McIntire, William E.

    2011-01-01

    Characterization of G protein βγ dimer isoform expression in different cellular contexts has been impeded by low levels of protein expression, broad isoform heterogeneity, and antibodies of limited specificity, sensitivity or availability. As a new approach, we used quantitative mass spectrometry to characterize native βγ dimers associated with adenosine A1:αi1 and adenosine A2A:αS receptor fusion proteins expressed in HEK-293 cells. Cells expressing A1:αi1 were cultured in media containing [13C6] Arg and [13C6] Lys, and βγ labeled with heavy isotopes purified. Heavy βγ was combined with either recombinant βγ purified from Sf9 cells, βγ purified from the A2A:αS expressed in HEK-293 cells cultured in standard media, or an enriched βγ fraction from HEK-293 cells. Samples were separated by SDS-PAGE, and protein bands containing β and γ were excised, digested with trypsin, separated by HPLC and isotope ratios analyzed by mass spectrometry. Three β isoforms, β1, β2 and β4, and seven γ isoforms, γ2, γ4, γ5, γ7, γ10, γ11 and γ12 were identified in the analysis. β1 and γ5 were most abundant in the enriched βγ fraction, and this βγ profile was generally mirrored in the fusion proteins. However, both A2A:αS and A1:αi1 bound more β4 and γ5 compared to the enriched βγ fraction; also, more β4 was associated with A2A:αS than A1:αi1. Both fusion proteins also contained less γ2, γ10 and γ12 than the enriched βγ fraction. These results suggest that preferences for particular βγ isoforms may be driven in part by structural motifs common to adenosine receptor family members. PMID:21128647

  7. Promiscuous Recognition of a Trypanosoma cruzi CD8+ T Cell Epitope among HLA-A2, HLA-A24 and HLA-A1 Supertypes in Chagasic Patients

    PubMed Central

    Guzmán, Fanny; Rosas, Fernando; Thomas, M. Carmen; López, Manuel Carlos; González, John Mario; Cuéllar, Adriana; Puerta, Concepción J.

    2016-01-01

    Background TcTLE is a nonamer peptide from Trypanosoma cruzi KMP-11 protein that is conserved among different parasite strains and that is presented by different HLA-A molecules from the A2 supertype. Because peptides presented by several major histocompatibility complex (MHC) supertypes are potential targets for immunotherapy, the aim of this study was to determine whether MHC molecules other than the A2 supertype present the TcTLE peptide. Methodology/Principal Findings From 36 HLA-A2-negative chagasic patients, the HLA-A genotypes of twenty-eight patients with CD8+ T cells that recognized the TcTLE peptide using tetramer (twenty) or functional (eight) assays, were determined. SSP-PCR was used to identify the A locus and the allelic variants. Flow cytometry was used to analyze the frequency of TcTLE-specific CD8+ T cells, and their functional activity (IFN-γ, TNFα, IL-2, perforin, granzyme and CD107a/b production) was induced by exposure to the TcTLE peptide. All patients tested had TcTLE-specific CD8+ T cells with frequencies ranging from 0.07–0.37%. Interestingly, seven of the twenty-eight patients had HLA-A homozygous alleles: A*24 (5 patients), A*23 (1 patient) and A*01 (1 patient), which belong to the A24 and A1 supertypes. In the remaining 21 patients with HLA-A heterozygous alleles, the most prominent alleles were A24 and A68. The most common allele sub-type was A*2402 (sixteen patients), which belongs to the A24 supertype, followed by A*6802 (six patients) from the A2 supertype. Additionally, the A*3002/A*3201 alleles from the A1 supertype were detected in one patient. All patients presented CD8+ T cells producing at least one cytokine after TcTLE peptide stimulation. Conclusion/Significance These results show that TcTLE is a promiscuous peptide that is presented by the A24 and A1 supertypes, in addition to the A2 supertype, suggesting its potential as a target for immunotherapy. PMID:26974162

  8. Beyond Donor-Acceptor (D-A) Approach: Structure-Optoelectronic Properties-Organic Photovoltaic Performance Correlation in New D-A1 -D-A2 Low-Bandgap Conjugated Polymers.

    PubMed

    Chochos, Christos L; Drakopoulou, Sofia; Katsouras, Athanasios; Squeo, Benedetta M; Sprau, Christian; Colsmann, Alexander; Gregoriou, Vasilis G; Cando, Alex-Palma; Allard, Sybille; Scherf, Ullrich; Gasparini, Nicola; Kazerouni, Negar; Ameri, Tayebeh; Brabec, Christoph J; Avgeropoulos, Apostolos

    2017-04-01

    Low-bandgap near-infrared polymers are usually synthesized using the common donor-acceptor (D-A) approach. However, recently polymer chemists are introducing more complex chemical concepts for better fine tuning of their optoelectronic properties. Usually these studies are limited to one or two polymer examples in each case study so far, though. In this study, the dependence of optoelectronic and macroscopic (device performance) properties in a series of six new D-A1 -D-A2 low bandgap semiconducting polymers is reported for the first time. Correlation between the chemical structure of single-component polymer films and their optoelectronic properties has been achieved in terms of absorption maxima, optical bandgap, ionization potential, and electron affinity. Preliminary organic photovoltaic results based on blends of the D-A1 -D-A2 polymers as the electron donor mixed with the fullerene derivative [6,6]-phenyl-C71 -butyric acid methyl ester demonstrate power conversion efficiencies close to 4% with short-circuit current densities (J sc ) of around 11 mA cm(-2) , high fill factors up to 0.70, and high open-circuit voltages (V oc s) of 0.70 V. All the devices are fabricated in an inverted architecture with the photoactive layer processed in air with doctor blade technique, showing the compatibility with roll-to-roll large-scale manufacturing processes.

  9. Effects of milk containing only A2 beta casein versus milk containing both A1 and A2 beta casein proteins on gastrointestinal physiology, symptoms of discomfort, and cognitive behavior of people with self-reported intolerance to traditional cows' milk.

    PubMed

    Jianqin, Sun; Leiming, Xu; Lu, Xia; Yelland, Gregory W; Ni, Jiayi; Clarke, Andrew J

    2016-04-02

    Cows' milk generally contains two types of β-casein, A1 and A2 types. Digestion of A1 type can yield the peptide β-casomorphin-7, which is implicated in adverse gastrointestinal effects of milk consumption, some of which resemble those in lactose intolerance. This study aimed to compare the effects of milk containing A1 β-casein with those of milk containing only A2 β-casein on inflammation, symptoms of post-dairy digestive discomfort (PD3), and cognitive processing in subjects with self-reported lactose intolerance. Forty-five Han Chinese subjects participated in this double-blind, randomized, 2 × 2 crossover trial and consumed milk containing both β-casein types or milk containing only A2 β-casein. Each treatment period was 14 days with a 14-day washout period at baseline and between treatment periods. Outcomes included PD3, gastrointestinal function (measured by smart pill), Subtle Cognitive Impairment Test (SCIT), serum/fecal laboratory biomarkers, and adverse events. Compared with milk containing only A2 β-casein, the consumption of milk containing both β-casein types was associated with significantly greater PD3 symptoms; higher concentrations of inflammation-related biomarkers and β-casomorphin-7; longer gastrointestinal transit times and lower levels of short-chain fatty acids; and increased response time and error rate on the SCIT. Consumption of milk containing both β-casein types was associated with worsening of PD3 symptoms relative to baseline in lactose tolerant and lactose intolerant subjects. Consumption of milk containing only A2 β-casein did not aggravate PD3 symptoms relative to baseline (i.e., after washout of dairy products) in lactose tolerant and intolerant subjects. Consumption of milk containing A1 β-casein was associated with increased gastrointestinal inflammation, worsening of PD3 symptoms, delayed transit, and decreased cognitive processing speed and accuracy. Because elimination of A1 β-casein attenuated these effects

  10. Spectral Modification and Catalytic Inhibition of Human Cytochromes P450 1A1, 1A2, 1B1, 2A6 and 2A13 by Four Chemopreventive Organoselenium Compounds

    PubMed Central

    Shimada, Tsutomu; Murayama, Norie; Tanaka, Katsuhiro; Takenaka, Shigeo; Guengerich, F. Peter; Yamazaki, Hiroshi; Komori, Masayuki

    2011-01-01

    Several organoselenium compounds including benzyl selenocyanate (BSC), 1,2-phenylenebis(methylene)selenocyanate (o-XSC), 1,3-phenylenebis(methylene)selenocyanate (m-XSC), and 1,4-phenylenebis(methylene)selenocyanate (p-XSC) have been shown to prevent cancers caused by polycyclic aromatic hydrocarbons (PAHs) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in experimental animals; these chemical carcinogens are activated by human P450 1 and 2A family enzymes, respectively, to carcinogenic metabolites. In this study, we examined whether these selenium compounds interact with and inhibit human P450 1 and 2A enzymes in vitro. Four organoselenium compounds induced Reverse Type I binding spectra with P450 1A1, 1A2, and 1B1 and Type I binding spectra with P450 2A6 and 2A13. The spectral dissociation constants (Ks) for the interaction of P450 1B1 with these chemicals were 3.6–5.7 µM; the values were lower than those with seen with P450 1A1 (19–30 µM) or 1A2 (6.3–13 µM). The Ks values for Type I binding of P450 2A13 with m-XSC and BSC were both 0.20 µM; the values were very low compared to the interaction of P450 2A6 with m-XSC (5.7 µM) and BSC (2.0 µM). Four selenium compounds directly inhibited 7-ethoxyresorufin O-deethylation activities catalyzed by P450 1A1, 1A2, and 1B1 with IC50 values <1.0 µM, except for the inhibition of P450 1A2 by BSC (1.3 µM). Coumarin 7-hydroxylation activities of P450 2A13 were more inhibited by four selenium compounds than those of P450 2A6, with IC50 values of 0.22–1.4 µM for P450 2A13 and 2.4–6.2 µM for P450 2A6. Molecular docking studies of the interaction of four organoselenium compounds with human P450 enzymes suggest that these chemicals can be docked into the active sites of these human P450 enzymes and that the sites of the selenocyanate functional groups of these chemicals differ between the P450 1 and 2A family enzymes. PMID:21732699

  11. Marker Assisted Transfer of Two Powdery Mildew Resistance Genes PmTb7A.1 and PmTb7A.2 from Triticum boeoticum (Boiss.) to Triticum aestivum (L.)

    PubMed Central

    Elkot, Ahmed Fawzy Abdelnaby; Chhuneja, Parveen; Kaur, Satinder; Saluja, Manny; Keller, Beat; Singh, Kuldeep

    2015-01-01

    Powdery mildew (PM), caused by Blumeria graminis f. sp. tritici, is one of the important wheat diseases, worldwide. Two PM resistance genes, designated as PmTb7A.1 and PmTb7A.2, were identified in T. boeoticum acc. pau5088 and mapped on chromosome 7AL approximately 48cM apart. Two resistance gene analogue (RGA)-STS markers Ta7AL-4556232 and 7AL-4426363 were identified to be linked to the PmTb7A.1 and PmTb7A.2, at a distance of 0.6cM and 6.0cM, respectively. In the present study, following marker assisted selection (MAS), the two genes were transferred to T. aestivum using T. durum as bridging species. As many as 12,317 florets of F1 of the cross T. durum /T. boeoticum were pollinated with T. aestivum lines PBW343-IL and PBW621 to produce 61 and 65 seeds, respectively, of three-way F1. The resulting F1s of the cross T. durum/T. boeoticum//T. aestivum were screened with marker flanking both the PM resistance genes PmTb7A.1 and PmTb7A.2 (foreground selection) and the selected plants were backcrossed to generate BC1F1. Marker assisted selection was carried both in BC1F1 and the BC2F1 generations. Introgression of alien chromatin in BC2F1 plants varied from 15.4 - 62.9 percent. Out of more than 110 BC2F1 plants showing introgression for markers linked to the two PM resistance genes, 40 agronomically desirable plants were selected for background selection for the carrier chromosome to identify the plants with minimum of the alien introgression. Cytological analysis showed that most plants have chromosome number ranging from 40-42. The BC2F2 plants homozygous for the two genes have been identified. These will be crossed to generate lines combining both the PM resistance genes but with minimal of the alien introgression. The PM resistance gene PmTb7A.1 maps in a region very close to Sr22, a stem rust resistance gene effective against the race Ug99. Analysis of selected plants with markers linked to Sr22 showed introgression of Sr22 from T. boeoticum in several BC2F1 plants

  12. Genome Sequencing of Giardia lamblia Genotypes A2 and B Isolates (DH and GS) and Comparative Analysis with the Genomes of Genotypes A1 and E (WB and Pig)

    PubMed Central

    Adam, Rodney D.; Dahlstrom, Eric W.; Martens, Craig A.; Bruno, Daniel P.; Barbian, Kent D.; Ricklefs, Stacy M.; Hernandez, Matthew M.; Narla, Nirmala P.; Patel, Rima B.; Porcella, Stephen F.; Nash, Theodore E.

    2013-01-01

    Giardia lamblia (syn G. intestinalis, G. duodenalis) is the most common pathogenic intestinal parasite of humans worldwide and is a frequent cause of endemic and epidemic diarrhea. G. lamblia is divided into eight genotypes (A–H) which infect a wide range of mammals and humans, but human infections are caused by Genotypes A and B. To unambiguously determine the relationship among genotypes, we sequenced GS and DH (Genotypes B and A2) to high depth coverage and compared the assemblies with the nearly completed WB genome and draft sequencing surveys of Genotypes E (P15; pig isolate) and B (GS; human isolate). Our results identified DH as the smallest Giardia genome sequenced to date, while GS is the largest. Our open reading frame analyses and phylogenetic analyses showed that GS was more distant from the other three genomes than any of the other three were from each other. Whole-genome comparisons of DH_A2 and GS_B with the optically mapped WB_A1 demonstrated substantial synteny across all five chromosomes but also included a number of rearrangements, inversions, and chromosomal translocations that were more common toward the chromosome ends. However, the WB_A1/GS_B alignment demonstrated only about 70% sequence identity across the syntenic regions. Our findings add to information presented in previous reports suggesting that GS is a different species of Giardia as supported by the degree of genomic diversity, coding capacity, heterozygosity, phylogenetic distance, and known biological differences from WB_A1 and other G. lamblia genotypes. PMID:24307482

  13. Genome sequencing of Giardia lamblia genotypes A2 and B isolates (DH and GS) and comparative analysis with the genomes of genotypes A1 and E (WB and Pig).

    PubMed

    Adam, Rodney D; Dahlstrom, Eric W; Martens, Craig A; Bruno, Daniel P; Barbian, Kent D; Ricklefs, Stacy M; Hernandez, Matthew M; Narla, Nirmala P; Patel, Rima B; Porcella, Stephen F; Nash, Theodore E

    2013-01-01

    Giardia lamblia (syn G. intestinalis, G. duodenalis) is the most common pathogenic intestinal parasite of humans worldwide and is a frequent cause of endemic and epidemic diarrhea. G. lamblia is divided into eight genotypes (A-H) which infect a wide range of mammals and humans, but human infections are caused by Genotypes A and B. To unambiguously determine the relationship among genotypes, we sequenced GS and DH (Genotypes B and A2) to high depth coverage and compared the assemblies with the nearly completed WB genome and draft sequencing surveys of Genotypes E (P15; pig isolate) and B (GS; human isolate). Our results identified DH as the smallest Giardia genome sequenced to date, while GS is the largest. Our open reading frame analyses and phylogenetic analyses showed that GS was more distant from the other three genomes than any of the other three were from each other. Whole-genome comparisons of DH_A2 and GS_B with the optically mapped WB_A1 demonstrated substantial synteny across all five chromosomes but also included a number of rearrangements, inversions, and chromosomal translocations that were more common toward the chromosome ends. However, the WB_A1/GS_B alignment demonstrated only about 70% sequence identity across the syntenic regions. Our findings add to information presented in previous reports suggesting that GS is a different species of Giardia as supported by the degree of genomic diversity, coding capacity, heterozygosity, phylogenetic distance, and known biological differences from WB_A1 and other G. lamblia genotypes.

  14. Structure-Function Relationships of Inhibition of Human Cytochromes P450 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 Flavonoid Derivatives

    PubMed Central

    Shimada, Tsutomu; Tanaka, Katsuhiro; Takenaka, Shigeo; Murayama, Norie; Martin, Martha V.; Foroozesh, Maryam K.; Yamazaki, Hiroshi; Guengerich, F. Peter; Komori, Masayuki

    2010-01-01

    Structure-function relationships for inhibition of human cytochrome P450s (P450s) 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 flavonoid derivatives were studied. Thirty-two of the 33 flavonoids tested produced Reverse Type I binding spectra with P450 1B1, and the potencies of binding were correlated with the abilities to inhibit 7-ethoxyresorufin O-deethylation activity. The presence of a hydroxyl group in flavones, e.g. 3-, 5-, and 7-monohydroxy- and 5,7-dihydroxyflavone, decreased the 50% inhibition concentration (IC50) of P450 1B1 from 0.6 µM to 0.09, 0.21, 0.25, and 0.27 µM, respectively, and 3,5,7-trihydroxyflavone (galangin) was the most potent, with an IC50 of 0.003 µM. The introduction of a 4’-methoxy- or 3’,4’-dimethoxy group into 5,7-dihydroxyflavone yielded other active inhibitors of P450 1B1 with IC50 values of 0.014 and 0.019 µM, respectively. The above hydroxyl- and/or methoxy-groups in flavone molecules also increased the inhibition activity with P450 1A1 but not always towards P450 1A2, where 3-, 5-, or 7-hydroxyflavone, and 4’-methoxy-5,7-dihydroxyflavone were less inhibitory than flavone itself. P450 2C9 was more inhibited by 7-hydroxy-,5,7-dihydroxy-, and 3,5,7-trihydroxyflavones than by flavone but was weakly inhibited by 3-and 5-hydroxyflavone. Flavone and several other flavonoids produced Type I binding spectra with P450 3A4, but such binding was not always related to the inhibitiory activities towards P450 3A4. These results indicate that there are different mechanisms of inhibition for P450s 1A1, 1A2, 1B1, 2C9, and 3A4 by various flavonoid derivatives and that the number and position of hydroxyl and/or methoxy groups highly influence the inhibitory actions of flavonoids towards these enzymes. Molecular docking studies suggest that there are different mechanisms involved in the interaction of various flavonoids with the active site of P450s, thus causing differences in inhibition of these P450 catalytic activities by flavonoids. PMID

  15. Structure-function relationships of inhibition of human cytochromes P450 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 flavonoid derivatives.

    PubMed

    Shimada, Tsutomu; Tanaka, Katsuhiro; Takenaka, Shigeo; Murayama, Norie; Martin, Martha V; Foroozesh, Maryam K; Yamazaki, Hiroshi; Guengerich, F Peter; Komori, Masayuki

    2010-12-20

    Structure-function relationships for the inhibition of human cytochrome P450s (P450s) 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 flavonoid derivatives were studied. Thirty-two of the 33 flavonoids tested produced reverse type I binding spectra with P450 1B1, and the potencies of binding were correlated with the abilities to inhibit 7-ethoxyresorufin O-deethylation activity. The presence of a hydroxyl group in flavones, for example, 3-, 5-, and 7-monohydroxy- and 5,7-dihydroxyflavone, decreased the 50% inhibition concentration (IC50) of P450 1B1 from 0.6 μM to 0.09, 0.21, 0.25, and 0.27 μM, respectively, and 3,5,7-trihydroxyflavone (galangin) was the most potent, with an IC50 of 0.003 μM. The introduction of a 4'-methoxy- or 3',4'-dimethoxy group into 5,7-dihydroxyflavone yielded other active inhibitors of P450 1B1 with IC50 values of 0.014 and 0.019 μM, respectively. The above hydroxyl and/or methoxy groups in flavone molecules also increased the inhibition activity with P450 1A1 but not always toward P450 1A2, where 3-, 5-, or 7-hydroxyflavone and 4'-methoxy-5,7-dihydroxyflavone were less inhibitory than flavone itself. P450 2C9 was more inhibited by 7-hydroxy-, 5,7-dihydroxy-, and 3,5,7-trihydroxyflavones than by flavone but was weakly inhibited by 3- and 5-hydroxyflavone. Flavone and several other flavonoids produced type I binding spectra with P450 3A4, but such binding was not always related to the inhibitiory activities toward P450 3A4. These results indicate that there are different mechanisms of inhibition for P450s 1A1, 1A2, 1B1, 2C9, and 3A4 by various flavonoid derivatives and that the number and position of hydroxyl and/or methoxy groups highly influence the inhibitory actions of flavonoids toward these enzymes. Molecular docking studies suggest that there are different mechanisms involved in the interaction of various flavonoids with the active site of P450s, thus causing differences in inhibition of these P450 catalytic activities by flavonoids.

  16. Elemental abundance analyses with DAO spectrograms. XXVII. The superficially normal stars theta And (A2 IV), epsilon Del (B6 III), epsilon Aqr (A1.5 V), and iota And (B9 V)

    NASA Astrophysics Data System (ADS)

    Kocer, D.; Adelman, S. J.; Caliskan, H.; Gulliver, A. F.; Gokmen Tektunali, H.

    2003-08-01

    The superficially normal stars theta And (A2 V), epsilon Del (B6 III), epsilon Aqr (A1.5 V), and iota And (B9 V), which have rotationally broadened line profiles, are analyzed in a manner consistent with previous studies of this series using 2.4 Åmm-1 spectrograms obtained with CCD detectors and S/N >=200. Their variable radial velocities strongly suggest they are spectroscopic binaries. As no evidence is seen for lines of their companions they are analyzed as single stars. Their derived abundances are generally near solar. But those for theta And suggest that it is possibly a fast rotating Am star. Table 5 is only available in electronic form at the CDS via anonymous ftp to cdsarc.u-strasbg.fr (130.79.128.5) or via http://cdsweb.u-strasbg.fr/cgi-bin/qcat?J/A+A/406/975

  17. Induction of CYP1A1, CYP1A2, CYP1B1, increased oxidative stress and inflammation in the lung and liver tissues of rats exposed to incense smoke.

    PubMed

    Hussain, Tajamul; Al-Attas, Omar S; Al-Daghri, Nasser M; Mohammed, Arif A; De Rosas, Edgard; Ibrahim, Shebl; Vinodson, Benjamin; Ansari, Mohammed G; El-Din, Khaled I Alam

    2014-06-01

    Incense smoke is increasingly being recognized as a potential environmental contaminant and is linked to malignant and non-malignant respiratory diseases. The detoxification of environmental contaminants including polycyclic aromatic hydrocarbons (PAHs) involves the induction of cytochrome P-450 family enzymes (CYPs) by PAHs. However, the detoxification of PAHs also results in the generation of reactive and unstable intermediary metabolites which are implicated in the oxidative stress, DNA damage, and inflammation. It is unclear whether CYPs are similarly induced by incense smoke, which incidentally contains substantial amounts of PAHs. Here, we examined the impact of long-term incense smoke exposure on the induction of CYPs in male Wister Albino rats. Incense smoke exposure significantly induced the expression of CYP1A1, CYP1A2, and CYP1B1 mRNAs in both lung and liver tissues. The extent of CYP1A1 and CYP1B1 induction was significantly higher in the liver compared to that in the lung, while that of CYP1A2 was greater in the lung than in liver. Incense smoke exposure also increased malondialdehyde and reduced glutathione levels in lung and liver tissues, and the catalase activity in the liver tissues to significant levels. Furthermore incense smoke exposure led to a marked increase in TNF-α and IL-4 levels. The data demonstrate for the first time the capacity of incense smoke to induce CYP1 family enzymes in the target and non-target tissues. Induction of CYPs increased oxidative stress and inflammation appear to be intimately linked to promote the carcinogenesis and health complications in people chronically exposed to incense smoke.

  18. Comparable Efficacy of a 1-L PEG and Ascorbic Acid Solution Administered with Bisacodyl versus a 2-L PEG and Ascorbic Acid Solution for Colonoscopy Preparation: A Prospective, Randomized and Investigator-Blinded Trial

    PubMed Central

    Im, Jong Pil; Kim, Su Hwan; Koh, Seong-Joon; Kim, Byeong Gwan; Lee, Kook Lae; Kim, Sang Gyun; Kim, Joo Sung; Jung, Hyun Chae

    2016-01-01

    Background Two liters of polyethylene glycol (PEG) solution administered with ascorbic acid (Asc) can provide efficacy similar to that of a 4-L PEG solution for colonoscopy preparation. In addition, oral bisacodyl (Bis) has been shown to reduce the volume of PEG needed for a bowel preparation with comparable efficacy. This study aimed to compare the efficacy, tolerability and safety of a 2-L PEG solution mixed with Asc versus the combination of Bis, Asc and a 1-L PEG solution. Methods This was a prospective, randomized, multi-centre, single-blind, non-inferiority trial. Participants who were scheduled for colonoscopy were included and randomized to receive either 2-L PEG and Asc (2L PEG/Asc group) or 1-L PEG, Asc and 20 mg Bis (1L PEG/Asc + Bis group). The quality of bowel preparation was assessed using the Boston Bowel Preparation Scale. Data regarding tolerance, compliance and adverse events were also gathered. Results A total of 187 participants were analyzed; 96 were allocated to the 2L PEG/Asc group and 91 to the 1L PEG/Asc + Bis group. Bowel preparation was adequate in 87.5% (84/96) of patients in the 2L PEG/Asc group and 94.5% of the 1L PEG/Asc + Bis group (86/91, p = 0.10). There was no significant difference between the two groups with respect to compliance, tolerability or safety. The patients allocated to the 1L PEG/Asc + Bis group expressed more willingness to repeat the procedure than patients in the 2L PEG/Asc group (p = 0.01). Conclusions Bowel preparation with Bis and a 1-L PEG/Asc solution is as effective, well-tolerated, and safe as a 2-L PEG/Asc solution. Trial Registration ClinicalTrials.gov NCT 01745835; Clinical Research Information Service (CRiS) KCT0000708 PMID:27588943

  19. Hepatic foci in rats after diethylnitrosamine initiation and 2,3,7,8-tetrachlorodibenzo-p-dioxin promotion: evaluation of a quantitative two-cell model and of CYP 1A1/1A2 as a dosimeter.

    PubMed

    Conolly, R B; Andersen, M E

    1997-10-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent hepatic tumor promoter in female rats. We used a quantitative, stochastic initiation-promotion model based on R. B. Conolly and J. S. Kimbell (Toxicol. Appl. Pharmacol. 124, 284-295, 1994) to analyze initiation-promotion results from a previously published study (H. C. Pitot et al., Carcinogenesis 8, 1491-1499, 1987) within the context of a negative selection model of tumor promotion. In this model, two types of initiated cells (called A and B cells) are produced by DEN initiation. Visually excellent correspondence between model predictions and data (i.e., foci/cm3 liver and percentage of liver occupied by foci) are obtained when TCDD is described as having dose-responsive effects on division and death (apoptotic) rates of these two cell types. For A cells, both the division and the death rates increase while the difference between division and apoptotic rates decreases. For B cells, the difference between division and apoptotic rates increases, primarily due to a decrease in the apoptotic rate. We also linked these alterations in cell kinetics to a pharmacokinetic model for TCDD incorporating a five subcompartment model of the liver acinus with induction of CYP1A1 and 1A2 genes in the subcompartments. Alterations in A cell kinetics correlate with effects of TCDD in the region most sensitive to induction (subcompartment 5-centrilobular region); B cell dynamics correlate with induction in subcompartments 3-5 (centrilobular and mid-zonal regions). In summary, these modeling exercises show that (1) the two-cell model, without presuming effects of TCDD on the mutation rate of normal hepatocytes, reproduces the data of Pitot et al. (1987) and (2) induction of CYP1A1/1A2 in different regions of the hepatic acinus can be used as a general correlate of these presumed changes in cell growth kinetics.

  20. The environmental pollutant and carcinogen 3-nitrobenzanthrone and its human metabolite 3-aminobenzanthrone are potent inducers of rat hepatic cytochromes P450 1A1 and -1A2 and NAD(P)H:quinone oxidoreductase.

    PubMed

    Stiborová, Marie; Dracínská, Helena; Hájková, Jana; Kaderábková, Pavla; Frei, Eva; Schmeiser, Heinz H; Soucek, Pavel; Phillips, David H; Arlt, Volker M

    2006-08-01

    3-Nitrobenzanthrone (3-NBA), a suspected human carcinogen occurring in diesel exhaust and air pollution, and its human metabolite 3-aminobenzanthrone (3-ABA) were investigated for their ability to induce biotransformation enzymes in rat liver and the influence of such induction on DNA adduct formation by the compounds. Rats were treated (i.p.) with 0.4, 4, or 40 mg/kg body weight 3-NBA or 3-ABA. When hepatic cytosolic fractions from rats treated with 40 mg/kg body weight 3-NBA or 3-ABA were incubated with 3-NBA, DNA adduct formation, measured by 32P-postlabeling analysis, was 10-fold higher in incubations with cytosols from pretreated rats than with controls. The increase in 3-NBA-derived DNA adduct formation corresponded to a dose-dependent increase in protein levels and enzymatic activity of NAD(P)H:quinone oxidoreductase (NQO1). NQO1 is the major enzyme reducing 3-NBA in human and rat livers. Incubations of 3-ABA with hepatic microsomes of rats treated with 3-NBA or 3-ABA (40 mg/kg body weight) led to as much as a 12-fold increase in 3-ABA-derived DNA adduct formation compared with controls. The observed stimulation of DNA adduct formation by both compounds was attributed to their potential to induce protein expression and enzymatic activity of cytochromes P450 1A1 and/or -1A2 (CYP1A1/2), the major enzymes responsible for 3-ABA activation in human and rat livers. Collectively, these results demonstrate for the first time, to our knowledge, that by inducing hepatic NQO1 and CYP1A1/2, both 3-NBA and 3-ABA increase the enzymatic activation of these two compounds to reactive DNA adduct-forming species, thereby enhancing their own genotoxic potential.

  1. A-3 Cleared Site

    NASA Image and Video Library

    2007-06-18

    Work to clear the site for the A-3 Test Stand progresses quickly, as seen in this photo taken June 18 from atop the A-1 Test Stand. The next step in construction at 19-acre site will be the arrival of fill dirt in mid-July, followed by pilings and piling caps.

  2. A-3 Cleared Site

    NASA Technical Reports Server (NTRS)

    2007-01-01

    Work to clear the site for the A-3 Test Stand progresses quickly, as seen in this photo taken June 18 from atop the A-1 Test Stand. The next step in construction at 19-acre site will be the arrival of fill dirt in mid-July, followed by pilings and piling caps.

  3. The influence of genetic polymorphisms in Ahr, CYP1A1, CYP1A2, CYP1B1, GST M1, GST T1 and UGT1A1 on urine 1-hydroxypyrene glucuronide concentrations in healthy subjects from Rio Grande do Sul, Brazil.

    PubMed

    Abnet, Christian C; Fagundes, Renato B; Strickland, Paul T; Kamangar, Farin; Roth, Mark J; Taylor, Philip R; Dawsey, Sanford M

    2007-01-01

    Polymorphisms in genes encoding polycyclic aromatic hydrocarbon (PAH) metabolizing enzymes may alter metabolism of these carcinogens and contribute to inter-individual difference in urine concentrations. We investigated the influence of genetic polymorphism on PAH metabolism in urine from 199 healthy subjects from Southern Brazil. We measured urine 1-hydroxypyrene glucuronide (1-OHPG) concentrations using immunoaffinity chromatography and synchronous fluorescence spectroscopy and genotyped subjects using standard methods. Genetic variants in CYP1B1 (rs1056827, rs1800440, rs10012) were strongly associated with urine 1-OHPG with P-values < 0.010. Variants in aryl hydrocarbon receptor (Ahr) (rs4986826), CYP1A1 (rs1799814) and CYP1A2 (rs2069514) were also, although less strongly, associated with changes in urine 1-OHPG concentrations. These variants had P-values of 0.074, 0.040 and 0.025, respectively. The median urine 1-OHPG concentrations (pmol/ml) in the homozygous wild-type and homozygous variants for CYP1B1 (rs10012) and the Ahr, CYP1A1 and CYP1A2 variants listed above were 2.16 and 0.10, 2.16 and 0.41, 2.03 and 0.46, 2.19 and 2.79, respectively. We found no effect of deletions in GST M1 or GST T1, or different alleles of UGT1A1*28. Adjusting for age, sex, place of residence, tobacco smoke exposure, maté drinking, cachaça and barbeque preparation had only a minor impact on the associations. A model containing just exposure variables had an r2 of 0.21; a model with single genotypes for Ahr, CYP1A1, CYP1A2 and CYP1B1 had an r2 of 0.10; and a model combining both exposure and genotype information had a total r2 of 0.33. Our results suggest that CYP1B1 genotypes are strongly associated with urine 1-OHPG concentrations in this population.

  4. Genotoxicity of three food processing contaminants in transgenic mice expressing human sulfotransferases 1A1 and 1A2 as assessed by the in vivo alkaline single cell gel electrophoresis assay.

    PubMed

    Høie, Anja Hortemo; Svendsen, Camilla; Brunborg, Gunnar; Glatt, Hansruedi; Alexander, Jan; Meinl, Walter; Husøy, Trine

    2015-10-01

    The food processing contaminants 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 5-hydroxymethylfurfural (HMF) and 2,5 dimethylfuran (DMF) are potentially both mutagenic and carcinogenic in vitro and/or in vivo, although data on DMF is lacking. The PHIP metabolite N-hydroxy-PhIP and HMF are bioactivated by sulfotransferases (SULTs). The substrate specificity and tissue distribution of SULTs differs between species. A single oral dose of PhIP, HMF or DMF was administered to wild-type (wt) mice and mice expressing human SULT1A1/1A2 (hSULT mice). DNA damage was studied using the in vivo alkaline single cell gel electrophoresis (SCGE) assay. No effects were detected in wt mice. In the hSULT mice, PhIP and HMF exposure increased the levels of DNA damage in the liver and kidney, respectively. DMF was not found to be genotoxic. The observation of increased DNA damage in hSULT mice compared with wt mice supports the role of human SULTs in the bioactivation of N-hydroxy-PhIP and HMF in vivo.

  5. Genotoxicity of three food processing contaminants in transgenic mice expressing human sulfotransferases 1A1 and 1A2 as assessed by the in vivo alkaline single cell gel electrophoresis assay

    PubMed Central

    Høie, Anja Hortemo; Svendsen, Camilla; Brunborg, Gunnar; Glatt, Hansruedi; Alexander, Jan; Meinl, Walter

    2015-01-01

    The food processing contaminants 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP), 5‐hydroxymethylfurfural (HMF) and 2,5 dimethylfuran (DMF) are potentially both mutagenic and carcinogenic in vitro and/or in vivo, although data on DMF is lacking. The PHIP metabolite N‐hydroxy‐PhIP and HMF are bioactivated by sulfotransferases (SULTs). The substrate specificity and tissue distribution of SULTs differs between species. A single oral dose of PhIP, HMF or DMF was administered to wild‐type (wt) mice and mice expressing human SULT1A1/1A2 (hSULT mice). DNA damage was studied using the in vivo alkaline single cell gel electrophoresis (SCGE) assay. No effects were detected in wt mice. In the hSULT mice, PhIP and HMF exposure increased the levels of DNA damage in the liver and kidney, respectively. DMF was not found to be genotoxic. The observation of increased DNA damage in hSULT mice compared with wt mice supports the role of human SULTs in the bioactivation of N‐hydroxy‐PhIP and HMF in vivo. Environ. Mol. Mutagen. 56:709–714, 2015. © 2015 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc. PMID:26270892

  6. The aryl hydrocarbon receptor-interacting protein (AIP) is required for dioxin-induced hepatotoxicity but not for the induction of the Cyp1a1 and Cyp1a2 genes.

    PubMed

    Nukaya, Manabu; Lin, Bernice C; Glover, Edward; Moran, Susan M; Kennedy, Gregory D; Bradfield, Christopher A

    2010-11-12

    The aryl hydrocarbon receptor (A