DOCSLIB.ORG

Proteomics Study of the Effect of High-Fat Diet on Rat Liver

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Proteomics Study of the Effect of High-Fat Diet on Rat Liver Downloaded from British Journal of Nutrition (2019), 122, 1062–1072 doi:10.1017/S0007114519001740 © The Authors 2019 https://www.cambridge.org/core Proteomics study of the effect of high-fat diet on rat liver Jian Sang1,2†, Hengxian Qu1,2†, Ruixia Gu1,2, Dawei Chen1,2, Xia Chen1,2, Boxing Yin1,2, Yingping Huang3, 3 3 1,2 Wenbo Xi , Chunlei Wang and Yujun Huang * . IP address: 1College of Food Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225127, People’s Republic of China 2Key Lab of Dairy Biotechnology and Safety Control, Yangzhou, Jiangsu 225127, People’s Republic of China 3Uni-enterprise (China) Holding Ltd., Kunshan, Jiangsu 215300, People’s Republic of China 170.106.35.76 (Submitted 6 March 2019 – Final revision received 9 July 2019 – Accepted 11 July 2019 – First published online 30 September 2019) , on 30 Sep 2021 at 00:24:24 Abstract Excessive intake of high-energy diets is an important cause of most obesity. The intervention of rats with high-fat diet can replicate the ideal animal model for studying the occurrence of human nutritional obesity. Proteomics and bioinformatics analyses can help us to systematically and comprehensively study the effect of high-fat diet on rat liver. In the present study, 4056 proteins were identified in rat liver by using tandem mass tag. A total of 198 proteins were significantly changed, of which 103 were significantly up-regulated and ninety-five were significantly down- regulated. These significant differentially expressed proteins are primarily involved in lipid metabolism and glucose metabolism processes. The , subject to the Cambridge Core terms of use, available at intake of a high-fat diet forces the body to maintain physiological balance by regulating these key protein spots to inhibit fatty acid synthesis, promote fatty acid oxidation and accelerate fatty acid degradation. The present study enriches our understanding of metabolic disorders induced by high-fat diets at the protein level. Key words: High-fat diet: Proteomics: Bioinformatics: Lipid metabolism Changes in the structure of modern diets and excessive intake of an animal model with an obese phenotype similar to human high-fat, high-energy diets cause disorders in the metabolism of obesity(7). The effects of high-fat diet on rat liver protein expres- fats, proteins and carbohydrates. Excess energy is gradually sion were studied by tandem mass tag combined with LC-MS/ accumulated in the body in the form of fat, which leads to MS. Bioinformatics analysis was used to further screen out the obesity(1). After the formation of obesity, it will further induce significant differential proteins that play a key role and to explore insulin resistance (IR), which in turn increases the incidence the mechanism of the body’s response to high-fat diets. https://www.cambridge.org/core/terms of the metabolic syndrome, diabetes, hypertension and CVD(2). The liver plays a vital role in maintaining the body’s carbohydrate, lipid and protein metabolism balance(3). Materials and methods Obesity-related metabolic disorders are manifested in the liver, Animals and treatment causing excessive deposition of fat in the liver cells, which can lead to fatty liver(4). The pathogenesis of obesity is not fully Eighteen healthy male Sprague–Dawley rats which were understood. It is generally believed that obesity is mainly caused 5 weeks old and weighing about 150 g at the start of the experi- by the combination of genetic factors and environmental ment were purchased from Comparative Medical Center of factors(5). In addition, factors such as endocrine, metabolism Yangzhou University, Jiangsu, China. The animal experiments . and central nervous system are also involved in the pathogenesis conformed the U.S. National Institutes of Health guidelines for https://doi.org/10.1017/S0007114519001740 of obesity(6). the care and use of laboratory animals (NIH publication no. Unlike traditional molecular biology techniques, proteomics 85-23 Rev. 1985) and were approved by the Animal Care enables researchers to define a global overview of protein Committee of the Center for Disease Control and Prevention expression in a specific physical or pathological state. In the (Jiangsu, China). Then, rats were randomly divided into a control present study, rats were treated with a high-fat diet to replicate group (C) and high-fat diet group (HF) (n 9). The control group Abbreviations: GO, gene ontology; HF, high-fat diet group; IR, insulin resistance; KEGG, Kyoto Encyclopedia of Genes and Genomes; UGT, UDP- glucuronosyltransferase. * Corresponding author: Y. Huang, email [email protected] † These authors contributed equally to this work and should be regarded as co-first authors. Downloaded from High-fat diet on rat liver 1063 Table 1. Composition of the diets Filter-aided sample preparation digestion https://www.cambridge.org/core Content Total energy A quantity of 200 μg of proteins for each sample was incorpo- Diet Ingredient (%, w/w) (kJ/100g) rated into 30 μl SDT buffer (4 % SDS, 100 mM dithiothreitol, Low-fat diet Flour 20 690·36 150 mM Tris-HCl, pH 8·0). The detergent, dithiothreitol and other Rice flour 10 low-molecular-weight components were removed using UA Maize 20 buffer (8 M urea, 150 mM Tris-HCl, pH 8·0) by repeated ultrafil- Drum skin 26 μ Soya material 20 tration (microcon units, 10 kDa). Then, 100 l iodoacetamide Fishmeal 2 (100 mM iodoacetamide in UA buffer) was added to block . IP address: Bone flour 2 reduced cysteine residues, and the samples were incubated High-fat diet Lard 10 1548·08 μ Egg powder 10 for 30 min in darkness. The filters were washed with 100 l Cholesterol 1 UA buffer three times and then with 100 μl 100 mM triethylammo- Bile salts 0·2 nium bicarbonate (TEAB) buffer twice. Finally, the protein sus- 170.106.35.76 · Low-fat diet 78 8 pensions were digested with 4 μg trypsin (Promega) in 40 μl TEAB buffer overnight at 37°C, and the resulting peptides were collected as a filtrate. The peptide content was estimated by UV , on was fed a low-fat diet (Table 1). The HF was fed a high-fat diet light spectral density at 280 nm using an extinction coefficient of 30 Sep 2021 at 00:24:24 (Table 1) for 8 weeks. All rats were housed under a 12 h light–12 1·1of0·1 % (g/l) solution which was calculated on the basis of h dark cycle in a controlled room with a temperature of 23°C and the frequency of tryptophan and tyrosine in vertebrate proteins a humidity of 50 % and allowed free access to food and water. After 8 weeks, rats underwent 12 h of fasting prior to being Tandem mass tag labelling anaesthetised and dissected. All rats were euthanised at the μ The peptide mixture (100 g) of each sample was labelled using , subject to the Cambridge Core terms of use, available at anoestrus period, following anaesthesia under 1 % sodium a tandem mass tag reagent according to the manufacturer’s − pentobarbital. Livers were removed and stored at 4 and 80°C instructions (Thermo Fisher Scientific). for subsequent analyses. Peptide fractionation with high pH reversed phase Compliance with ethics guidelines A Pierce high pH reversed-phase fractionation kit (Thermo All institutional and national guidelines for the care and use of Scientific) was used to fractionate tandem mass tag-labelled laboratory animals were followed. digest samples into ten fractions by an increasing acetonitrile step- Haematoxylin–eosin staining gradient elution according to the instructions. Liver samples were fixed in 4 % paraformaldehyde for more than Mass spectrometry 48 h, followed by sectioning after embedding with paraffin. The paraffin section was stained by routine haematoxylin–eosin HPLC. Each fraction was injected for nanoLC-MS/MS analysis. The peptide mixture was loaded onto a reversed-phase trap col- methods. https://www.cambridge.org/core/terms umn (Thermo Scientific Acclaim PepMap100, 100 μm × 2 cm, nanoViper C18) connected to the C18-reversed-phase analytical Protein extraction and normalisation column (Thermo Scientific Easy Column, 10 cm long, 75 μm μ · SDT buffer (4 % (w/v) SDS, 0·1 MDL-dithiothreitol, 100 mM Tris/ inner diameter, 3 m resin) in buffer A (0 1 % formic acid) HCl, pH 7·6) was added to the liver sample and transferred to 2 and separated with a linear gradient of buffer B (84 % acetonitrile · ml tubes with quartz sand (another 1/4 inch ceramic bead MP and 0 1 % formic acid) at a flow rate of 300 nl/min controlled by 6540-424 for tissue samples). The lysate was homogenised by IntelliFlow technology. · – – an MP homogeniser (24 × 2, 6·0m/s,60s,twice).Thehomog- 1 5 h gradient: 0 55 % buffer B for 80 min, 55 100 % buffer B enate was sonicated and then boiled for 15 min. After centrifuged for 5 min, hold in 100 % buffer B for 5 min. · μ . at 14 000 g for 40 min, the supernatant was filtered with 0 22 m https://doi.org/10.1017/S0007114519001740 filters. The filtrate was quantified with the Bicinchoninic Acid LC-MS/MS analysis. LC-MS/MS analysis was performed on a Q Protein Assay Kit (Bio-Rad). The sample was stored at −80°C. Exactive MS (Thermo Scientific) that was coupled with Easy nLC Equivalent amounts of protein from each of the three different rats (Proxeon Biosystems, now Thermo Fisher Scientific) for 90 min were pooled to generate three protein samples for each group. (determined by project proposal). The MS was operated in pos- itive ion mode. Mass spectometry data were acquired using a data-dependent top-ten method dynamically choosing the most SDS-PAGE separation abundant precursor ions from the survey scan (300–1800 m/z) A quantity of 20 μg of proteins for each sample was mixed with for higher-energy C-trap dissociation (HCD) fragmentation.
Load more

Recommended publications
  • Upregulation of Peroxisome Proliferator-Activated Receptor-Α And
    Upregulation of peroxisome proliferator-activated receptor-α and the lipid metabolism pathway promotes carcinogenesis of ampullary cancer Chih-Yang Wang, Ying-Jui Chao, Yi-Ling Chen, Tzu-Wen Wang, Nam Nhut Phan, Hui-Ping Hsu, Yan-Shen Shan, Ming-Derg Lai 1 Supplementary Table 1. Demographics and clinical outcomes of five patients with ampullary cancer Time of Tumor Time to Age Differentia survival/ Sex Staging size Morphology Recurrence recurrence Condition (years) tion expired (cm) (months) (months) T2N0, 51 F 211 Polypoid Unknown No -- Survived 193 stage Ib T2N0, 2.41.5 58 F Mixed Good Yes 14 Expired 17 stage Ib 0.6 T3N0, 4.53.5 68 M Polypoid Good No -- Survived 162 stage IIA 1.2 T3N0, 66 M 110.8 Ulcerative Good Yes 64 Expired 227 stage IIA T3N0, 60 M 21.81 Mixed Moderate Yes 5.6 Expired 16.7 stage IIA 2 Supplementary Table 2. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of an ampullary cancer microarray using the Database for Annotation, Visualization and Integrated Discovery (DAVID). This table contains only pathways with p values that ranged 0.0001~0.05. KEGG Pathway p value Genes Pentose and 1.50E-04 UGT1A6, CRYL1, UGT1A8, AKR1B1, UGT2B11, UGT2A3, glucuronate UGT2B10, UGT2B7, XYLB interconversions Drug metabolism 1.63E-04 CYP3A4, XDH, UGT1A6, CYP3A5, CES2, CYP3A7, UGT1A8, NAT2, UGT2B11, DPYD, UGT2A3, UGT2B10, UGT2B7 Maturity-onset 2.43E-04 HNF1A, HNF4A, SLC2A2, PKLR, NEUROD1, HNF4G, diabetes of the PDX1, NR5A2, NKX2-2 young Starch and sucrose 6.03E-04 GBA3, UGT1A6, G6PC, UGT1A8, ENPP3, MGAM, SI, metabolism
    [Show full text]
  • Hexokinase 2 Is Targetable for HK1 Negative, HK2 Positive Tumors from a Wide Variety of Tissues of Origin
    Journal of Nuclear Medicine, published on August 30, 2018 as doi:10.2967/jnumed.118.212365 Hexokinase 2 Is Targetable for HK1 Negative, HK2 Positive Tumors from a Wide Variety of Tissues of Origin Shili Xu1, Arthur Catapang1, Hanna M. Doh1, Nicholas A. Bayley1, Jason T. Lee1,2,3, Daniel Braas1,4, Thomas G. Graeber1,2,3,4, Harvey R. Herschman1,2,3,5,6* 1Department of Molecular and Medical Pharmacology 2Crump Institute for Molecular Imaging 3Jonsson Comprehensive Cancer Center 4UCLA Metabolomics Center 5Department of Biological Chemistry 6Molecular Biology Institute David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California 90095, USA *Correspondence: Harvey R. Herschman. 341 Boyer Hall, UCLA, 611 Charles E. Young Drive East, Los Angeles CA, 90095. Phone: 310-825-8735. Fax: 310-825-1447. Email: [email protected] First author: Shili Xu, postdoctoral fellow. 329 Boyer Hall, UCLA, 611 Charles E. Young Drive East, Los Angeles CA, 90095. Phone: 310-825-8745. Email: [email protected] Running Title: HK2 Is Targetable for HK1-HK2+ Tumors Financial Disclosure: No potential conflicts of interest relevant to this article exist. Word count: 4997 1 ABSTRACT Although absent in most adult tissues, hexokinase 2 (HK2) is expressed in a majority of tumors and contributes to increased glucose consumption and to in vivo tumor 18F-FDG PET signaling. Methods: Both HK2 knockdown and knockout approaches were used to investigate the role of HK2 in cancer cell proliferation, in vivo xenograft tumor progression and 18F-FDG tumor accumulation. BioProfiler analysis monitored cell culture glucose consumption and lactate production; 18F-FDG PET/CT monitored in vivo tumor glucose accumulation.
    [Show full text]
  • Topic 3.1 Interactions of Xenobiotics with the Steroid Hormone Biosynthesis Pathway*
    Pure Appl. Chem., Vol. 75, Nos. 11–12, pp. 1957–1971, 2003. © 2003 IUPAC Topic 3.1 Interactions of xenobiotics with the steroid hormone biosynthesis pathway* Thomas Sanderson‡ and Martin van den Berg Institute for Risk Assessment Science, Utrecht University, P.O. Box 8017, 3508 TD Utrecht, The Netherlands Abstract: Environmental contaminants can potentially disrupt endocrine processes by inter- fering with the function of enzymes involved in steroid synthesis and metabolism. Such in- terferences may result in reproductive problems, cancers, and toxicities related to (sexual) differentiation, growth, and development. Various known or suspected endocrine disruptors interfere with steroidogenic enzymes. Particular attention has been given to aromatase, the enzyme responsible for the conversion of androgens to estrogens. Studies of the potential for xenobiotics to interfere with steroidogenic enzymes have often involved microsomal frac- tions of steroidogenic tissues from animals exposed in vivo, or in vitro exposures of steroid- ogenic cells in primary culture. Increasingly, immortalized cell lines, such as the H295R human adrenocortical carcinoma cell line are used in the screening of effects of chemicals on steroid synthesis and metabolism. Such bioassay systems are expected to play an increasingly important role in the screening of complex environmental mixtures and individual contami- nants for potential interference with steroidogenic enzymes. However, given the complexities in the steroid synthesis pathways and the biological activities of the hormones, together with the unknown biokinetic properties of these complex mixtures, extrapolation of in vitro effects to in vivo toxicities will not be straightforward and will require further, often in vivo, inves- tigations. INTRODUCTION There is increasing evidence that certain environmental contaminants have the potential to disrupt en- docrine processes, which may result in reproductive problems, certain cancers, and other toxicities re- lated to (sexual) differentiation, growth, and development.
    [Show full text]
  • Gene Expression Signature for Biliary Atresia and a Role for Interleukin-8
    SUPPLEMENTARY “PATIENTS AND METHODS” “Gene expression signature for biliary atresia and a role for Interleukin-8 in pathogenesis of experimental disease” Bessho K, et al. PATIENTS AND METHODS Patients. Liver biopsies, serum samples and clinical data were obtained from infants with cholestasis enrolled into a prospective study (ClinicalTrials.gov Identifier: NCT00061828) of the NIDDK-funded Childhood Liver Disease Research and Education Network (www.childrennetwork.org) or from infants evaluated at Cincinnati Children’s Hospital Medical Center. For subjects with biliary atresia (BA), liver biopsies were obtained from 64 infants during the preoperative workup or at the time of intraoperative cholangiogram, with ages ranging from 22-169 days after birth (Supplementary Table 7). For subjects with intrahepatic cholestasis (serving as diseased controls, and referred to as non- BA), liver biopsy samples were obtained percutaneously or intraoperatively from 14 infants at the time of diagnostic evaluation, with ages ranging from 19-189 days. Their diagnosis were Alagille syndrome (N=1), multidrug resistance protein-3 deficiency (N=2), alpha-1-antitrypsin deficiency (N=2) and cholestasis with unknown etiology (N=9) (Supplementary Table 7). Representative liver biopsy photomicrographs are shown in Supplementary Figure 6A-D. A third group of normal controls (NC) consisted of liver biopsy samples obtained from 7 deceased-donor children aged 22-42 months as described previously (1). This group serves as a reference cohort, with the median levels of gene expression used to normalize gene expression across all patients in the BA and non-BA groups. This greatly facilitates the visual identification of key differences in gene expression levels between BA and non-BA groups.
    [Show full text]
  • Supplementary Table 1
    Supplementary Table 1. 492 genes are unique to 0 h post-heat timepoint. The name, p-value, fold change, location and family of each gene are indicated. Genes were filtered for an absolute value log2 ration 1.5 and a significance value of p ≤ 0.05. Symbol p-value Log Gene Name Location Family Ratio ABCA13 1.87E-02 3.292 ATP-binding cassette, sub-family unknown transporter A (ABC1), member 13 ABCB1 1.93E-02 −1.819 ATP-binding cassette, sub-family Plasma transporter B (MDR/TAP), member 1 Membrane ABCC3 2.83E-02 2.016 ATP-binding cassette, sub-family Plasma transporter C (CFTR/MRP), member 3 Membrane ABHD6 7.79E-03 −2.717 abhydrolase domain containing 6 Cytoplasm enzyme ACAT1 4.10E-02 3.009 acetyl-CoA acetyltransferase 1 Cytoplasm enzyme ACBD4 2.66E-03 1.722 acyl-CoA binding domain unknown other containing 4 ACSL5 1.86E-02 −2.876 acyl-CoA synthetase long-chain Cytoplasm enzyme family member 5 ADAM23 3.33E-02 −3.008 ADAM metallopeptidase domain Plasma peptidase 23 Membrane ADAM29 5.58E-03 3.463 ADAM metallopeptidase domain Plasma peptidase 29 Membrane ADAMTS17 2.67E-04 3.051 ADAM metallopeptidase with Extracellular other thrombospondin type 1 motif, 17 Space ADCYAP1R1 1.20E-02 1.848 adenylate cyclase activating Plasma G-protein polypeptide 1 (pituitary) receptor Membrane coupled type I receptor ADH6 (includes 4.02E-02 −1.845 alcohol dehydrogenase 6 (class Cytoplasm enzyme EG:130) V) AHSA2 1.54E-04 −1.6 AHA1, activator of heat shock unknown other 90kDa protein ATPase homolog 2 (yeast) AK5 3.32E-02 1.658 adenylate kinase 5 Cytoplasm kinase AK7
    [Show full text]
  • Downloaded from Here
    bioRxiv preprint doi: https://doi.org/10.1101/017566; this version posted November 19, 2015. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 1 Testing for ancient selection using cross-population allele 2 frequency differentiation 1;∗ 3 Fernando Racimo 4 1 Department of Integrative Biology, University of California, Berkeley, CA, USA 5 ∗ E-mail: [email protected] 6 1 Abstract 7 A powerful way to detect selection in a population is by modeling local allele frequency changes in a 8 particular region of the genome under scenarios of selection and neutrality, and finding which model is 9 most compatible with the data. Chen et al. [2010] developed a composite likelihood method called XP- 10 CLR that uses an outgroup population to detect departures from neutrality which could be compatible 11 with hard or soft sweeps, at linked sites near a beneficial allele. However, this method is most sensitive 12 to recent selection and may miss selective events that happened a long time ago. To overcome this, 13 we developed an extension of XP-CLR that jointly models the behavior of a selected allele in a three- 14 population tree. Our method - called 3P-CLR - outperforms XP-CLR when testing for selection that 15 occurred before two populations split from each other, and can distinguish between those events and 16 events that occurred specifically in each of the populations after the split.
    [Show full text]
  • Current Status on Genome–Metabolome-Wide Associations: an Opportunity in Nutrition Research
    Genes Nutr (2013) 8:19–27 DOI 10.1007/s12263-012-0313-7 REVIEW Current status on genome–metabolome-wide associations: an opportunity in nutrition research Ivan Montoliu • Ulrich Genick • Mirko Ledda • Sebastiano Collino • Franc¸ois-Pierre Martin • Johannes le Coutre • Serge Rezzi Received: 15 February 2012 / Accepted: 2 August 2012 / Published online: 16 October 2012 Ó Springer-Verlag 2012 Abstract Genome-wide association studies (GWASs) impact on the homeostatic concentrations of specific have become a very important tool to address the genetic metabolites. A particularly interesting aspect of this work origin of phenotypic variability, in particular associated takes into account interactions of environment and lifestyle with diseases. Nevertheless, these types of studies provide with the genome and how this interaction translates into limited information about disease etiology and the molec- changes in the metabolome. For instance, the role of PY- ular mechanisms involved. Recently, the incorporation of ROXD2 in trimethylamine metabolism points to an inter- metabolomics into the analysis has offered novel oppor- action between host and microbiome genomes (host/ tunities for a better understanding of disease-related met- microbiota). Often, these findings reveal metabolic dere- abolic deregulation. The pattern emerging from this work is gulations, which could eventually be tuned with a nutri- that gene-driven changes in metabolism are prevalent and tional intervention. Here we review the development of that common genetic variations can have a profound gene–metabolism association studies from a single-gene/ single-metabolite to a genome-wide/metabolome-wide approach and highlight the conceptual changes associated Ivan Montoliu and Ulrich Genick contributed equally to this work.
    [Show full text]
  • Regulation of Cytochrome P450 (CYP) Genes by Nuclear Receptors Paavo HONKAKOSKI*1 and Masahiko NEGISHI† *Department of Pharmaceutics, University of Kuopio, P
    Biochem. J. (2000) 347, 321–337 (Printed in Great Britain) 321 REVIEW ARTICLE Regulation of cytochrome P450 (CYP) genes by nuclear receptors Paavo HONKAKOSKI*1 and Masahiko NEGISHI† *Department of Pharmaceutics, University of Kuopio, P. O. Box 1627, FIN-70211 Kuopio, Finland, and †Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, U.S.A. Members of the nuclear-receptor superfamily mediate crucial homoeostasis. This review summarizes recent findings that in- physiological functions by regulating the synthesis of their target dicate that major classes of CYP genes are selectively regulated genes. Nuclear receptors are usually activated by ligand binding. by certain ligand-activated nuclear receptors, thus creating tightly Cytochrome P450 (CYP) isoforms often catalyse both formation controlled networks. and degradation of these ligands. CYPs also metabolize many exogenous compounds, some of which may act as activators of Key words: endobiotic metabolism, gene expression, gene tran- nuclear receptors and disruptors of endocrine and cellular scription, ligand-activated, xenobiotic metabolism. INTRODUCTION sex-, tissue- and development-specific expression patterns which are controlled by hormones or growth factors [16], suggesting Overview of the cytochrome P450 (CYP) superfamily that these CYPs may have critical roles, not only in elimination CYPs constitute a superfamily of haem-thiolate proteins present of endobiotic signalling molecules, but also in their production in prokaryotes and throughout the eukaryotes. CYPs act as [17]. Data from CYP gene disruptions and natural mutations mono-oxygenases, with functions ranging from the synthesis and support this view (see e.g. [18,19]). degradation of endogenous steroid hormones, vitamins and fatty Other mammalian CYPs have a prominent role in biosynthetic acid derivatives (‘endobiotics’) to the metabolism of foreign pathways.
    [Show full text]
  • X-Ray Fluorescence Analysis Method Röntgenfluoreszenz-Analyseverfahren Procédé D’Analyse Par Rayons X Fluorescents
    (19) & (11) EP 2 084 519 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: G01N 23/223 (2006.01) G01T 1/36 (2006.01) 01.08.2012 Bulletin 2012/31 C12Q 1/00 (2006.01) (21) Application number: 07874491.9 (86) International application number: PCT/US2007/021888 (22) Date of filing: 10.10.2007 (87) International publication number: WO 2008/127291 (23.10.2008 Gazette 2008/43) (54) X-RAY FLUORESCENCE ANALYSIS METHOD RÖNTGENFLUORESZENZ-ANALYSEVERFAHREN PROCÉDÉ D’ANALYSE PAR RAYONS X FLUORESCENTS (84) Designated Contracting States: • BURRELL, Anthony, K. AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Los Alamos, NM 87544 (US) HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR (74) Representative: Albrecht, Thomas Kraus & Weisert (30) Priority: 10.10.2006 US 850594 P Patent- und Rechtsanwälte Thomas-Wimmer-Ring 15 (43) Date of publication of application: 80539 München (DE) 05.08.2009 Bulletin 2009/32 (56) References cited: (60) Divisional application: JP-A- 2001 289 802 US-A1- 2003 027 129 12164870.3 US-A1- 2003 027 129 US-A1- 2004 004 183 US-A1- 2004 017 884 US-A1- 2004 017 884 (73) Proprietors: US-A1- 2004 093 526 US-A1- 2004 235 059 • Los Alamos National Security, LLC US-A1- 2004 235 059 US-A1- 2005 011 818 Los Alamos, NM 87545 (US) US-A1- 2005 011 818 US-B1- 6 329 209 • Caldera Pharmaceuticals, INC. US-B2- 6 719 147 Los Alamos, NM 87544 (US) • GOLDIN E M ET AL: "Quantitation of antibody (72) Inventors: binding to cell surface antigens by X-ray • BIRNBAUM, Eva, R.
    [Show full text]
  • NAT8 Antibody (Center) Affinity Purified Rabbit Polyclonal Antibody (Pab) Catalog # Ap4957c
    10320 Camino Santa Fe, Suite G San Diego, CA 92121 Tel: 858.875.1900 Fax: 858.622.0609 NAT8 Antibody (Center) Affinity Purified Rabbit Polyclonal Antibody (Pab) Catalog # AP4957c Specification NAT8 Antibody (Center) - Product Information Application WB, IHC-P,E Primary Accession Q9UHE5 Other Accession Q9UHF3 Reactivity Human Host Rabbit Clonality Polyclonal Isotype Rabbit Ig Antigen Region 110-138 NAT8 Antibody (Center) - Additional Information Gene ID 9027 Western blot analysis of NAT8 Antibody Other Names (Center) (Cat. #AP4957c) in ZR-75-1 cell line N-acetyltransferase 8, 231-, lysates (35ug/lane). NAT8 (arrow) was Acetyltransferase 2, ATase2, Camello-like detected using the purified Pab. protein 1, Cysteinyl-conjugate N-acetyltransferase, CCNAT, NAT8 (<a href ="http://www.genenames.org/cgi-bin/gene_ symbol_report?hgnc_id=18069" target="_blank">HGNC:18069</a>) Target/Specificity This NAT8 antibody is generated from rabbits immunized with a KLH conjugated synthetic peptide between 110-138 amino acids from the Central region of human NAT8. Dilution WB~~1:1000 IHC-P~~1:50~100 NAT8 Antibody (Center) (Cat. #AP4957c) IHC analysis in formalin fixed and paraffin Format embedded mouse kidney tissue followed by Purified polyclonal antibody supplied in PBS peroxidase conjugation of the secondary with 0.09% (W/V) sodium azide. This antibody and DAB staining. This data antibody is purified through a protein A demonstrates the use of the NAT8 Antibody column, followed by peptide affinity (Center) for immunohistochemistry. Clinical purification. relevance has not been evaluated. Storage Maintain refrigerated at 2-8°C for up to 2 NAT8 Antibody (Center) - Background weeks. For long term storage store at -20°C in small aliquots to prevent freeze-thaw This protein, isolated using the differential cycles.
    [Show full text]
  • Supplementary Data
    SUPPLEMENTARY DOCUMENT Survival transcriptome in the coenzyme Q10 deficiency syndrome is acquired by epigenetic modifications: a modelling study for human coenzyme Q10 deficiencies. Daniel J. M. Fernández-Ayala1,5, Ignacio Guerra1,5, Sandra Jiménez-Gancedo1,5, Maria V. Cascajo1,5, Angela Gavilán1,5, Salvatore DiMauro2, Michio Hirano2, Paz Briones3,5, Rafael Artuch4,5, Rafael de Cabo6, Leonardo Salviati7,8 and Plácido Navas1,5,8 1Centro Andaluz de Biología del Desarrollo (CABD-CSIC), Universidad Pablo Olavide, Seville, Spain 2Department of Neurology, Columbia University Medical Center, New York, NY, USA 3Instituto de Bioquímica Clínica, Corporació Sanitaria Clínic, Barcelona, Spain 4Department of Clinical Biochemistry, Hospital Sant Joan de Déu, Barcelona, Spain 5CIBERER, Instituto de Salud Carlos III, Spain 6Laboratory of Experimental Gerontology, National Institute on Aging, NIH, Baltimore, MD 21224, USA 7Clinical Genetics Unit, Department of Woman and Child Health, University of Padova, Italy 8 Senior authorship is shared by LS and PN Corresponding author Plácido Navas Tel (+34)954349381; Fax (+34)954349376 e-mail: [email protected] 1 SUPPLEMENTARY DATA Identification of genes, biological process and pathways regulated in CoQ10 deficiency. Data analysis for each gene in the array compared the signal of each patient’s fibroblast mRNA with the signal of each control fibroblast mRNA, over which the statistical analyses were performed. MAS5 algorithm and SAM software were performed in order to select those probe sets with significant differences in gene expression. Approximately from 6% to 11% of the probe sets present in the array were picked with a significant fold change higher than 1.5, while the false discovery rate (FDR) was maintained lower than 5%, the maximum value to consider changes as significant [1].
    [Show full text]
  • Product Datasheet NAT8 Overexpression
    Product Datasheet NAT8 Overexpression Lysate NBL1-13491 Unit Size: 0.1 mg Store at -80C. Avoid freeze-thaw cycles. Protocols, Publications, Related Products, Reviews, Research Tools and Images at: www.novusbio.com/NBL1-13491 Updated 3/17/2020 v.20.1 Earn rewards for product reviews and publications. Submit a publication at www.novusbio.com/publications Submit a review at www.novusbio.com/reviews/destination/NBL1-13491 Page 1 of 2 v.20.1 Updated 3/17/2020 NBL1-13491 NAT8 Overexpression Lysate Product Information Unit Size 0.1 mg Concentration The exact concentration of the protein of interest cannot be determined for overexpression lysates. Please contact technical support for more information. Storage Store at -80C. Avoid freeze-thaw cycles. Buffer RIPA buffer Target Molecular Weight 25.4 kDa Product Description Description Transient overexpression lysate of N-acetyltransferase 8 (GCN5-related, putative) (NAT8) The lysate was created in HEK293T cells, using Plasmid ID RC203157 and based on accession number NM_003960. The protein contains a C-MYC/DDK Tag. Gene ID 9027 Gene Symbol NAT8 Species Human Notes HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.This product is manufactured by and sold under license from OriGene Technologies and its use is limited solely for research purposes.
    [Show full text]