DOCSLIB.ORG

Pyrimidine Metabolism in Man. V. the Measurement in Vivo of the Biochemical Effect of Antineoplastic Agents in Animal and Human Subjects

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Pyrimidine Metabolism in Man. V. the Measurement in Vivo of the Biochemical Effect of Antineoplastic Agents in Animal and Human Subjects PYRIMIDINE METABOLISM IN MAN. V. THE MEASUREMENT IN VIVO OF THE BIOCHEMICAL EFFECT OF ANTINEOPLASTIC AGENTS IN ANIMAL AND HUMAN SUBJECTS Mitchell T. Rabkin, … , Myron Lotz, Lloyd H. Smith Jr. J Clin Invest. 1962;41(4):871-883. https://doi.org/10.1172/JCI104544. Research Article Find the latest version: https://jci.me/104544/pdf Journal of Clinical Investigation Vol. 41, No. 4, 1962 PYRIMIDINE METABOLISM IN MAN. V. THE MEASUREMENT IN VIVO OF THE BIOCHEMICAL EFFECT OF ANTINEOPLASTIC AGENTS IN ANIMAL AND HUMAN SUBJECTS * By MITCHELL T. RABKIN, ELIZABETH W. FREDERICK, MYRON LOTZ t AND LLOYD H. SMITH, JR. (From the Departments of Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, Mass.) (Submitted for publication September 25, 1961; accepted October 30, 1961) Many in v'ivo studies have been carried out on facilitated collections. Urine aliquots were counted with purine metabolism in man because of the presence an efficiency of approximately 40 per cent. Orotic acid in the urine was identified by ascending paper chromatog- of a readily available end product, uric acid. In raphy in a propanol: HO: formic acid system (7: 2:1) the absence of a comparable unique end product, through comparison with known standard (Rf approx- no previous over-all measurements of pyrimidine imately 0.40) and also from ultraviolet spectrophotometry metabolism in the intact organism have been re- of acid solutions of the eluted spots (5). ported. Studies have been limited to the excre- In human subjects, after intravenous injection of la- a beled compounds, expired air was collected in meteoro- tion of /8-aminoisobutyric acid, as metabolite of logical balloons. Measured aliquots were passed through thymine (1), the incorporation of pyrimidine pre- 3 N NaOH, which was treated as in the rat assays. cursors into circulating leukocytes (2), the in- Orotic acid-C1' (OA-C4), labeled in either the C-6 or duced urinary excretion of orotic acid and oroti- C-7 (carboxyl) position, was purchased from the New dine (3), and the spontaneous urinary excretion England Nuclear Corporation. D,L-Carbamylaspartic of certain trace pyrimidines (4). In this report acid-C1 (CAA-C"4), labeled in the ureide position, was synthesized as previously described (5). D,L-CAA-C14, a method is described for evaluating pyrimidine labeled in the C-1 position (corresponding to the carboxyl nucleotide synthesis in the intact organism, based carbon of orotic acid), was synthesized by alkaline hy- on the release of carbon dioxide-C14 from car- drolysis of carboxyl-labeled dihydroorotic acid-C1' (5). boxyl-labeled pyrimidine precursors. The useful- Specific activity as utilized was approximately 0.2 /Ac ness of this procedure in defining the onset, per jsmole. Other compounds used in these studies were gifts from degree, and duration of action of certain anti- the following sources: potassium oxonate, Dr. Robert E. metabolites is illustrated by studies in rats, and Handschumacher; 6-azauridine, Dr. Emil Frei, III; 5- in patients with leukemia. fluoroorotic acid and azaserine, Dr. Thomas C. Hall; imidazoleacetic acid hydrochloride, Dr. Howard H. MATERIALS AND METHODS Hiatt; 6-azauracil, Wellcome Research Laboratories; 2-ethylamino-1,3,4-thiadiazole, Lederle Laboratories Divi- Intravenous injections of labeled compounds were given sion of American Cyanamid Co.; Colcemide, Ciba Phar- through indwelling jugular vein catheters to male albino maceutical Products, Inc. 6-Mercaptopurine was pur- rats, weighing 125 to 150 g. For collection of expired chased from Nutritional Biochemicals Corp. C1402, air was flushed through a closed metabolic cham- ber and the carbon dioxide trapped in 3 N NaOH. Acid- RESULTS ification of aliquots of this solution allowed trapping of the carbon dioxide in Hyamine base and counting in a Validation of the method. In the biosynthetic Packard Tri-Carb liquid scintillation spectrometer at an sequence of pyrimidine nucleotide formation, an efficiency of approximately 45 per cent. Indwelling irreversible decarboxylation occurs in the con- cystostomy catheters were operatively placed in rats for version of orotidine-5'-phosphate (05P) to uri- urinary studies. Sedation with perphenazine (Trilafon, dine-5'-phosphate (UMP) (Figure 1). In a normal or saline Schering), and hydration with hypotonic partially purified system of orotidylic pyrophos- * This work was supported by Grant A-4080, National phorylase and orotidylic decarboxylase from Institutes of Health, United States Public Health Service. yeast, the release of C1402 from carboxyl-labeled Published in part in abstract form (J. clin. Invest. 1961, orotic acid has been shown to be a reliable meas- 40, 1073). t Postdoctoral Research Fellow, National Institute of ure in vitro of pyrimidine nucleotide formation Arthritis and Metabolic Diseases. (6). No evidence has yet been found for specific 871 872 RABKIN, FREDERICK, LOTZ AND SMITH COOH NH COOH - H20 0 0 HCH I I I ,. HIf H2 N-C-O-P-OHi + H2N-CH - O-C HCH COOH ENZ.1 HNh-6H +H20 OH COOH ENZ.2 o CAP L-ASP CAA DHoo ..n +2H \ \ 1 ASPARTATE CARBAMYLTRANSFERASE ENZ. ENZ.3 2 DI HYDROOROTASE ENZ. c ENZ.3 DIHYDROOROTIC DEHYDROGENASE ENZ.4 OROTI DYLI C PYROPHOSPHORYLASE HN/ >CH ENZ.5 OROTIDYLIC DECARBOXYLASE OA | l 05P 0lo \\ /"COOH UMP N 0 0 11 PRPP _/ H zC 11 O /c\-wc m '. HN C HN nr/E NZ. 4 II O ENZ.5 \ C N/ HC HC HCOH I 0 HC OH HC-OHI / P HC 0 HC HC-O- P-OH HC-O-P-OH H I H IH OH FIG. 1. PATHWAY OF URIDINE-5'-PHOSPHATE BIOSYNTHESIS. Abbreviations include: CAP, carbamyl phosphate; L-ASP, L-aspartic acid; CAA, carbamylaspartic acid; DHO, . dihydroorotic acid; OA, orotic acid; PRPP, 5-phosphoribosylpyrophosphate; 05P, orotidine-5'-phosphate (orotidylic acid); UMP, uridine-5'-phosphate.1 decarboxylation of orotidine-5'-phosphate which index of pyrimidine biosynthesis, at least three does not lead to formation of uridine-5'-phos- postulates must be confirmed by experimental phate. Therefore, in the intact organism, the data: 1) C'402 should be released more rapidly recovery of C'402 in the expired air after in- from precursors when the label is in the carbon jection of carboxyl-labeled precursors would be atom destined to become the carboxyl group of expected to reflect in vivo pyrimidine nucleotide 05P than when the isotopic carbon is elsewhere synthesis. To support this hypothesis and to in the molecule; 2) antimetabolites whose action validate the specificity of the procedure as an has been demonstrated in vitro to inhibit UMP synthesis should interfere with the respiratory 1Classification numbers and systematic names of the excretion of C1402 from carboxyl-labeled pre- enzymes are as follows: aspartate carbamyltransferase, cursors given in vivo; 3) antimetabolites whose 2.1.3.2, carbamoylphosphate:L-aspartate carbamoyl trans- action is not directed toward pyrimidine bio- ferase; dihydroorotase, 3.5.2.3, 4,5 - L - dihydroorotate amidohydrolase; dihydroorotic dehydrogenase, 1.3.3.1, synthesis should not "nonspecifically" interfere 4,5-L-dihydroorotate: 02 oxidoreductase; orotidylic py- with the metabolism of the test compounds. rophosphorylase, 2.4.2.10, orotidine-5'-phosphate: pyro- 1) Degradative enzymatic systems have been phosphate phosphoribosyltransferase; orotidylic decar- described in mammalian preparations which re- boxylase, 4.1.1.23, orotidine-5'-phosphate carboxy-lyase. lease precursors without (Report of the Commission on Enzymes of the Inter- CO2 from pyrimidine national Union of Biochemistry, New York, Pergamon prior formation of pyrimidine nucleotides (7, 8). Press, 1961.) The release of C1402 by such catabolic pathways IN VIVO STUDIES OF PYRIMIDINE METABOLISM 873 would be comparable from carboxyl- or noncar- OROTIC ACID (RAT) boxyl-labeled precursors. In contrast, a more rapid release of C'40, from a carboxyl-labeled 5C 3- ~~~~~~~~~0 precursor should reflect a pathway of selective de- zC'N HN CH such as that represented by oro- 40 carboxylation, CARBOXYL C tidylic decarboxylase (enzyme 5, Figure 1). In LABELED / * Figure 2, a comparison is presented of the rates 1.3 30 H 0 at which the rat expired C1402 after the intra- 20 venous injection of 1.67 Mmoles per 100 g body .k RING HN CH~ LABELED H.. weight of carboxyl-labeled or C-6-labeled orotic 10 acid (OA-C14). Approximately 50 per cent of ~~H~~~ the isotope administered as carboxyl-labeled OA- 0 10 20 30 440 50 60 C'4 appeared in the expired air in 1 hour; another MINUTES 37 per cent was excreted in the urine. Intra- FIG. 2. CUMULATIVE RECOVERY OF EXPIRED C"02 AFTER venous injection of C-6-labeled OA-C14 resulted INJECTION OF CARBOXYL- OR RING-LABELED OROTIC ACID-C4 in the respiratory recovery of less than 3 per IN THE RAT. 1.67 ,moles per 100 g carboxyl-labeled OA- cent of the administered radioactivity within 1 C" (upper curve, average of 6 experiments) and C-6- labeled OA-C" (lower curve, average of 3 experiments) hour, but the excretion of similar amounts (32 injected intravenously. TABLE I Cumulative recovery of C'4 from orotic acid-C'4 in rats cretion was approximately 47 per cent of the administered isotope. The fraction which repre- Excretory product 20 min 30 min 60 min sented the D-isomer was not determined. in the rat From OA-C'4 In summary, in vivo studies using carboxyl-labeled demon- Expired C'402 Mean 28.8% 35.0% 49.9% selectively labeled pyrimidine precursors N 17 23 6 strated a sharp contrast in the fate of the car- SD i 6.4% i 6.9% contrast Urinary OA-C'4 34.6% 37.2% boxyl- and noncarboxyl-carbons. This supports the assumption that the measured re- From ring (C-6) labeled OA-C'4 lease of C140, from the carboxyl carbon of these Expired C1402 0.5% 1.0% 2.8% Urinary OA-C'4 30.7% 31.9% precursors is a reflection of the formation of UMP, the parent compound of pyrimidine nucleo- tides.
Load more

Recommended publications
  • CHEM 461 to CHEM 462 Mod 2013-10-16
    CALIFORNIA STATE UNIVERSITY CHANNEL ISLANDS COURSE MODIFICATION PROPOSAL Courses must be submitted by October 15, 2013, and finalized by the end of the fall semester to make the next catalog (2014-15) production DATE (CHANGE DATE EACH TIME REVISED): 10/14/2013; REV 11.13.13 PROGRAM AREA(S) : CHEM Directions: All of sections of this form must be completed for course modifications. Use YELLOWED areas to enter data. All documents are stand alone sources of course information. 1. Indicate Changes and Justification for Each. [Mark an X by all change areas that apply then please follow-up your X’s with justification(s) for each marked item. Be as brief as possible but, use as much space as necessary.] x Course title Course Content Prefix/suffix Course Learning Outcomes x Course number References x Units GE Staffing formula and enrollment limits Other x Prerequisites/Corequisites Reactivate Course x Catalog description Mode of Instruction Justification: Modified CHEM 462 has a new number, since 461 will become Biochemistry I lab. CHEM 462 will also separate classroom and lab component, allowing for greater flexibility on the part of the students. Classroom and laboratory content for the modified CHEM 462. And the new lab class, CHEM 463, will consist of the same lab content as the original CHEM 461. Removes the pre-requisite course CHEM 305, Computer applications in Chemistry, as unnecessary for CHEM 462. Catalog description altered to remove reference to lab fee. 2. Course Information. [Follow accepted catalog format.] (Add additional prefixes
    [Show full text]
  • Analysis of Porcine Adipose Tissue Transcriptome Reveals Differences In
    Analysis of porcine adipose tissue transcriptome reveals differences in de novo fatty acid synthesis in pigs with divergent muscle fatty acid composition Jordi Corominas, Yuliaxis Ramayo-Caldas, Anna Puig-Oliveras, Jordi Estelle Fabrellas, Anna Castello, Estefania Alves, Ramona N. Pena, Maria Ballester, Josep M. Folch To cite this version: Jordi Corominas, Yuliaxis Ramayo-Caldas, Anna Puig-Oliveras, Jordi Estelle Fabrellas, Anna Castello, et al.. Analysis of porcine adipose tissue transcriptome reveals differences in de novo fatty acid synthesis in pigs with divergent muscle fatty acid composition. BMC Genomics, BioMed Central, 2013, 14, 10.1186/1471-2164-14-843. hal-01193819 HAL Id: hal-01193819 https://hal.archives-ouvertes.fr/hal-01193819 Submitted on 29 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Corominas et al. BMC Genomics 2013, 14:843 http://www.biomedcentral.com/1471-2164/14/843 RESEARCH ARTICLE Open Access Analysis of porcine adipose tissue transcriptome reveals differences in de novo fatty acid synthesis in pigs with divergent muscle fatty acid composition Jordi Corominas1,2*, Yuliaxis Ramayo-Caldas1,2, Anna Puig-Oliveras1,2, Jordi Estellé3,4,5, Anna Castelló1, Estefania Alves6, Ramona N Pena7, Maria Ballester1,2 and Josep M Folch1,2 Abstract Background: In pigs, adipose tissue is one of the principal organs involved in the regulation of lipid metabolism.
    [Show full text]
  • Identifying Nucleic Acid-Associated Proteins in Mycobacterium Smegmatis by Mass Spectrometry-Based Proteomics Nastassja L
    Kriel et al. BMC Molecular and Cell Biology (2020) 21:19 BMC Molecular and https://doi.org/10.1186/s12860-020-00261-6 Cell Biology RESEARCH ARTICLE Open Access Identifying nucleic acid-associated proteins in Mycobacterium smegmatis by mass spectrometry-based proteomics Nastassja L. Kriel1*, Tiaan Heunis1,2, Samantha L. Sampson1, Nico C. Gey van Pittius1, Monique J. Williams1,3† and Robin M. Warren1† Abstract Background: Transcriptional responses required to maintain cellular homeostasis or to adapt to environmental stress, is in part mediated by several nucleic-acid associated proteins. In this study, we sought to establish an affinity purification-mass spectrometry (AP-MS) approach that would enable the collective identification of nucleic acid- associated proteins in mycobacteria. We hypothesized that targeting the RNA polymerase complex through affinity purification would allow for the identification of RNA- and DNA-associated proteins that not only maintain the bacterial chromosome but also enable transcription and translation. Results: AP-MS analysis of the RNA polymerase β-subunit cross-linked to nucleic acids identified 275 putative nucleic acid-associated proteins in the model organism Mycobacterium smegmatis under standard culturing conditions. The AP-MS approach successfully identified proteins that are known to make up the RNA polymerase complex, as well as several other known RNA polymerase complex-associated proteins such as a DNA polymerase, sigma factors, transcriptional regulators, and helicases. Gene ontology enrichment analysis of the identified proteins revealed that this approach selected for proteins with GO terms associated with nucleic acids and cellular metabolism. Importantly, we identified several proteins of unknown function not previously known to be associated with nucleic acids.
    [Show full text]
  • THE INFLUENCE of INADEQUATE WATER SUPPLY on METABOLISM in BIOLOGICAL SYSTEMS with EMPHASIS on PROTEIN SYNTHESIS and NUCLEIC ACID ME':Rabolism
    TijE INFLU~NCE or INADEQU.4\TE WA,TE;It SUPPLY ON ME'l'ABOLISM IN B;I:OLOGICAL SYSTEMS by S. H. WEST Profes~or Qf Agronomy University of Floriqq PUBLICATION NO.9 of the FLORID.4\ WATER RESOURCES RESEARCH CENTER RES~ARCH PROJECT TECHNICAL COMPLETION REPORT OWRR Projeot Number A~008~fLA Annual Allot~ent Agreement Numbers 14-01 1"0001 .. 1077 (1968) 14 .. 01 or-POQ1.,.1628 (l9q9) 14 ... 31 .. 0001-3009 (1910) ~eport SUblll'l .. tt~dl August 26, 1970 The wQrk upon whic::n thiq r~~ort 'is based was suppo!"t~d in part by funds provideclby t;:he l)pitedStats$ pepartment of the Interior, Office ot Water ResourCes Research, as auth0ri~ed under the Water R~sources Research Act of 1964, TABLE OF CONTENTS Page ABSTRACT • ,. • • • • II • • 'f • • • • , • , • II • ~ • ,. • • • • • • • III ~ • • , • • " • • • ~ • ". • • • 1 P"R.O.)ECT ~Utv1t1A RY • III ••••• ~ • II • ~ ••••••••• ,. ,. • " , , II • II II " III •••• II • • 2 INTRODUCTlON . II • " '1/ ... ~ ........... I II II ••••• I II •• II • II , ••• II • " " II • 3 INITIAL RESEARCH PLAN AND RESULTS .......• ",.... ....... 4 CHARACTER,IZATION OF RNA ACCUMULATED IN DROUGHT ••••.•••• 5 :PROTEIN SYN1HESIS ALTERED BY DROUGHT •••• , •• ~ • ~ •• , •• , • • • 7 LITERA TURE C I TED ., •••••••..... , .••.• , ••..•.•.• "....... 1 1 ABSTRACT THE INFLUENCE OF INADEQUATE WATER SUPPLY ON METABOLISM IN BIOLOGICAL SYSTEMS WITH EMPHASIS ON PROTEIN SYNTHESIS AND NUCLEIC ACID ME':rABOLISM Data have been obtained that show the effect of drought on growth itself and how this reduction in growth may be a result of specific changes in total protein produc­ tion, nucleic acid metabolism and on functional activity of a fraction of nucleic acids. While the drcught treatments decreased total protein by only 40 percent, growth was re­ duced 80 percent.
    [Show full text]
  • A Path Towards SARS-Cov-2 Attenuation: Metabolic Pressure on CTP Synthesis Rules the Virus Evolution
    bioRxiv preprint doi: https://doi.org/10.1101/2020.06.20.162933; this version posted June 21, 2020. 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-ND 4.0 International license. 1 A path towards SARS-CoV-2 attenuation: metabolic pressure on CTP synthesis rules the virus evolution Zhihua Ou1,2, Christos Ouzounis3, Daxi Wang1,2, Wanying Sun1,2,4, Junhua Li1,2, Weijun Chen2,5*, Philippe Marlière6, Antoine Danchin7,8* 1. BGI-Shenzhen, Shenzhen 518083, China. 2. Shenzhen Key Laboratory of Unknown Pathogen Identification, BGI-Shenzhen, Shenzhen 518083, China. 3. Biological Computation and Process Laboratory, Centre for Research and Technology Hellas, Chemical Process and Energy Resources Institute, Thessalonica 57001, Greece 4. BGI Education Center, University of Chinese Academy of Sciences, Shenzhen, 518083, China. 5. BGI PathoGenesis Pharmaceutical Technology, BGI-Shenzhen, Shenzhen, China. 6. TESSSI, The European Syndicate of Synthetic Scientists and Industrialists, 81 rue Réaumur, 75002, Paris, France 7. Kodikos Labs, Institut Cochin, 24, rue du Faubourg Saint-Jacques Paris 75014, France. 8. School of Biomedical Sciences, Li KaShing Faculty of Medicine, Hong Kong University, 21 Sassoon Road, Pokfulam, Hong Kong. * To whom correspondence should be addressed Tel: +331 4441 2551; Fax: +331 4441 2559 E-mail: [email protected] Correspondence may also be addressed to [email protected] Keywords ABCE1; cytoophidia; innate immunity; Maxwell’s demon; Nsp1; phosphoribosyltransferase; queuine bioRxiv preprint doi: https://doi.org/10.1101/2020.06.20.162933; this version posted June 21, 2020.
    [Show full text]
  • NUCLEOTIDE METABOLISM Mark Rush
    NUCLEOTIDE METABOLISM Mark Rush Nucleotides serve various metabolic functions. For example, they are: • Substrates (building blocks) for nucleic acid biosynthesis and repair, • The main storage form of “high energy phosphate”, • Components of many “so-called” co-enzymes (NAD, NADP, FAD, CoA), • Components of many activated metabolic intermediates (such as UDPG, SAM), • Major allosteric effectors (such as AMP, ADP, ATP, GTP), • Major second messengers (such as 3',5' cAMP), and • Precursors for the biosynthesis of a variety of important compounds (such as biopterin and histidine). Nucleotide biochemistry can be treated both as an aspect of nitrogen metabolism, along with such compounds as amino acids and porphyrins, and as an aspect of nucleic acid metabolism. When the focus is on the biosynthesis and degradation of nucleotides, in other words on their turnover, the treatment is similar to that of other nitrogenous compounds. When the focus is on the role of nucleotides in overall nucleic acid metabolism, the treatment is included in molecular biology. Both aspects will be considered here with the major emphasis directed toward relating defects in nucleotide turnover to either metabolic diseases or chemotherapy. I. Nomenclature (pages 11 and 12). II. Overall metabolic pathways (page 4). 1) PRPP synthetase. 2) Nucleoside phosphorylases. 3) Phosphoribosyl transferases. nucleotides - 1 III. Biosynthesis of purines and pyrimidines (pages 5, 6, 8). A minimum amount of time will be spent discussing these pathways in lecture. Please examine them carefully in the text and note that: 1) Purine synthesis begins at the nucleotide level, while pyrimidine synthesis does not. 2) Both syntheses are regulated at their committed steps.
    [Show full text]
  • Nucleic Acid Metabolism in Regenerating Rat Liver I. the Rate of Deoxyribonucleic Acid Synthesis in Vivo1
    Nucleic Acid Metabolism in Regenerating Rat Liver I. The Rate of Deoxyribonucleic Acid Synthesis in Vivo1 LISELOTTEI.HECHTANDVANR. POTTER (McArdie Memorial Laboratory, University of Wisconsin, Madison 6, Wis.) An ideal system for examining the relationship MATERIALS AND METHODS between ribonucleic acid (UNA) and deoxyribo- Partial hepatectomy was performed by the method of nucleic acid (DNA) metabolism in animals would Higgins and Anderson (18) on male albino rats1 weighing 180 be a tissue where synchronous cell division occurs. to 185 gm. The animals were fasted for 16-18 hours before the Isotopie tracer studies (9, 22, 25) and determina operation and fed ad libitum after the operation. At the indi cated times each animal received a single intraperitoneal in tion of the DNA content per nucleus in the liver jection of 1 mg. (5.75 pinoles) of orotic acid-6-C14which con cells of partially hepatectomized rats (25, 26, 83) tained 4.1 X 10* counts/min/mg. Following the injection, have suggested that in animals the most feasible some animals were kept in metabolism cages to facilitate col approach to this situation is by the use of re lection of urine and respiratory CO2. The animals were killed by decapitation. The liver was perfused in situ with ice-cold generating liver. The observations that incorpora 0.25 H sucrose containing 0.00018 M CaCl2, excised, and tion of isotopically labeled precursors into DNA weighed. is correlated with the occurrence of cell division Preparation of cellfractions.'—Theliver was forced through (5,17,30) and that isotopes are retained extensive a plastic mincer, and a 10 per cent homogenate of the liver was ly in the DNA of mitotically inactive and active prepared in 0.25 Msucrose + 0.00018 u CaCl2 (19) with the use cells (2, 4, 6,10-12,16, 31, 32) indicate that DNA of a Potter-Elvehjem glass homogenizer.
    [Show full text]
  • RNA Synthesizing Activities and Glucocorticoid Binding Capacities in Thymus Cells During Rat Development
    Pediat. Res. 11: 705-709 (1977) Dexamethasone RNA synthesis glucocorticoid receptor protein thymus RNA Synthesizing Activities and Glucocorticoid Binding Capacities in Thymus Cells during Rat Development K. LJpp<3 n AND N. VANDERMEULEN Universitiits-Kinderklinik, Marburg, West Germany Summary steroid-resistant ce·lls have little or no hormone binding capaci­ ties. In view of this functional role we studied the dexametha­ This investigation was designed to study specific glucocorti­ sone binding capacity of the cytoplasmic fraction and of nuclei of coid binding to cytoplasmic fraction and nuclei of thymus cells thymus cells during rat development. Changes in hormone bind­ during rat development and to find out whether these data can ing should be correlated to an altered response of the tissue. be correlated to changes of RNA synthesizing activity of nuclei Therefore, we measured nuclear RNA synthesizing activity and and cytoplasm. The dexamethasone binding capacity of cyto­ also the DNA-dependent RNA polymerase Illa in cytoplasm plasm rose rapidly in rats weighing up to 125 g and decreased that controls synthesis of tRNA (7), and the inhibition of RNA significantly in animals weighing more than 160 g. Hormone synthesis by dexa[llethasone in whole cells and purified nuclei binding to nuclei revealed similar but less pronounced changes. during rat development. RNA synthesizing activity of nuclei measured by [3H]uridi~e incorporation in vitro decreased from 57 ± 4.6 dpm/µg DNA m young to 23 ± 2.2 dpm/µg DNA in adult rats. RNA synthesiz­ MATERIALS AND METHODS ing activity of the cytoplasmic fraction fell 21.6% and that of Han-Wistar rats were obtained from the Institut fiir Versuchs­ purified polymerase Illa fell 27 .8% during development.
    [Show full text]
  • Purine and Pyrimidine Metabolism in Human Epidermis* Jean De Bersaques, Md
    THE JOURNAL OP INVESTIGATIVE DERMATOLOGY Vol. 4s, No. Z Copyright 1957 by The Williams & Wilkins Co. Fri nte,1 in U.S.A. PURINE AND PYRIMIDINE METABOLISM IN HUMAN EPIDERMIS* JEAN DE BERSAQUES, MD. The continuous cellular renewal occurring inthine, which contained 5% impurity, and for uric the epidermis requires a very active synthesisacid, which consisted of 3 main components. The reaction was stopped after 1—2 hours in- and breakdown of nuclear and cytoplasmiecubation at 37° and the products were spotted on nucleic acids. Data on the enzyme systemsWhatman 1 filter paper sheets. According to the participating in these metabolic processes arereaction products expected, a choice was made of rather fragmentary (1—9) and some are, inat least 2 among the following solvents, all used terms of biochemical time, in need of up- in ascending direction: 1. isoamyl alcohol—5% Na2HPO4 (1:1), dating. In some other publications (10—18), 2. water-saturated n-butanol, the presence and concentration of various in- 3. distilled water, termediate products is given. 4. 80% formic acid—n-hutanol——n-propanol— In this paper, we tried to collect and supple- acetone—30% trichloro-aeetic acid (5:8:4: ment these data by investigating the presence 5:3), 5. n-butanol——4% boric acid (43:7), or absence in epidermis of enzyme systems 6. isobutyrie acid—water—ammonia 0.880—ver- that have been described in other tissues. sene 0.1M(500:279:21:8), This first investigation was a qualitative one, 7. upper phase of ethyl acetate—water—formic and some limitations were set by practical acid (12:7:1), 8.
    [Show full text]
  • Nitrogen-Stimulated Orotic Acid Synthesis and Nucleotide Imbalance1
    [CANCER RESEARCH (SUPPL.) 52. 2082s-2084s. April I. 1992] Nitrogen-stimulated Orotic Acid Synthesis and Nucleotide Imbalance1 Willard J. Visek2 University of Illinois, College of Medicine, Urbana, Illinois 61801 Abstract bound to the inner mitochondria! membrane. The cytoplasmic enzymes reside in two separate multifunctional complexes. One Orotic acid, first discovered in ruminant milk, is an intermediate in contains carbamoyl phosphate synthetase II, aspartate trans- the pyrimidine biosynthesis pathway of animal cells. Its synthesis is carbamylase, and dihydroorotase, whereas the other includes initiated by the formation of carbamoyl phosphate (CP) in the cytoplasm, orotate phosphoribosyl transferase and orotodine-5"-phosphate with ammonia derived from glutamine. Ureotelic species also form CP in the first step of urea synthesis in liver mitochondria. For that, ammonia decarboxylase (2, 3). A deficiency of the latter two enzyme is derived from tissue fluid. When there is insufficient capacity for activities results in accumulation of orotate and a profound rise detoxifying the load of ammonia presented for urea synthesis, CP leaves in its excretion in the urine, a condition known as hereditary the mitochondria and enters the pyrimidine pathway, where orotic acid orotic aciduria (4). This bifunctional protein complex with its biosynthesis is stimulated, orotic acid excretion in urine then increases. two enzyme activities is also referred to as UMP synthase. Orotic acid synthesis is abnormally high with hereditary deficiencies of A severe deficiency of UMP synthase elevates urinary orotic urea-cycle enzymes or uridine monophosphate synthase. It is also ele acid excretion in humans to 1500 mg/day, compared with the vated by ammonia intoxication and during feeding of diets high in protein, usual 2.5 mg/day.
    [Show full text]
  • Purine and Pyrimidine Metabolism by N. ZOLLNER, Department Of
    Proc. Nuti. Soc. (1982), 41,329 329 Purine and pyrimidine metabolism By N. ZOLLNER,Department of Medicine, University of Munchen, West Germany Purines and pyrimidines are essential constituents of animal and plant cells and are contained in various compounds. It is interesting to consider that some of these compounds are very stable, e.g. DNA, while others are rapidly turned over, e.g. ATP. In birds and reptiles uric acid also serves to excrete nitrogen. The aim of this paper is to give a short review of purine and pyrimidine metabolism and to describe in some detail aspects important to the field of nutrition, with emphasis placed on work done in vitro and in man. Purines The most important structure in purine biochemistry is the nucleotide consisting of a purine base, ribose or deoxyribose, and phosphoric acid. The most important purine bases are adenine, guanine, hypoxanthine and xanthine. The ribosides of all of them are known to occur in metabolism. Adenine and guanine themselves are usually not found in the tissues of mammals, but free hypoxanthine and xanthine are intermediates in the degradation of purines. Uric acid is a divalent acid, but the second dissociation constant is so small that at around pH 7 only the monobasic salts are formed. These are sparingly soluble in the body fluids. Peters & van Slyke (1946) have calculated a maximum solubility of 6.5 mg/Ioo ml (as uric acid) in plasma. Gout and uric acid nephropathies are due to this low solubility. Nucleosides are pentose-glycosides containing ribose or deoxyribose. Normally the linkage is with atom 9, but nucleosides with atom 3 do occur.
    [Show full text]
  • Omics-Based Analyses Revealed Metabolic Responses of Clostridium Acetobutylicum to Lignocellulose-Derived Inhibitors Furfural, F
    Liu et al. Biotechnol Biofuels (2019) 12:101 https://doi.org/10.1186/s13068-019-1440-9 Biotechnology for Biofuels RESEARCH Open Access Omics-based analyses revealed metabolic responses of Clostridium acetobutylicum to lignocellulose-derived inhibitors furfural, formic acid and phenol stress for butanol fermentation Huanhuan Liu1,2, Jing Zhang1,2, Jian Yuan3, Xiaolong Jiang3, Lingyan Jiang3, Guang Zhao4, Di Huang3* and Bin Liu3* Abstract Background: Clostridium acetobutylicum is a model fermentative anaerobe for consolidated bioprocessing of ligno- cellulose hydrolysates into acetone–butanol–ethanol (ABE). However, the main inhibitors (acids, furans and phenols) ubiquitous in lignocellulose hydrolysates strictly limit the conversion efciency. Thus, it is essential to understand the underlying mechanisms of lignocellulose hydrolysate inhibitors to identify key industrial bottlenecks that undermine efcient biofuel production. The recently developed omics strategy for intracellular metabolites and protein quantif- cation now allow for the in-depth mapping of strain metabolism and thereby enable the resolution of the underlying mechanisms. Results: The toxicity of the main inhibitors in lignocellulose hydrolysates against C. acetobutylicum and ABE produc- tion was systematically investigated, and the changes in intracellular metabolism were analyzed by metabolomics and proteomics. The toxicity of the main lignocellulose hydrolysate inhibitors at the same dose was ranked as fol- lows: formic acid > phenol > furfural. Metabolomic analysis based on weighted gene coexpression network analysis (WGCNA) revealed that the three inhibitors triggered the stringent response of C. acetobutylicum. Proteomic analysis based on peptide mass spectrometry (MS) supported the above results and provided more comprehensive conclu- sions. Under the stress of three inhibitors, the metabolites and key enzymes/proteins involved in glycolysis, reductive tricarboxylic acid (TCA) cycle, acetone–butanol synthesis and redox metabolism were lower than those in the control group.
    [Show full text]