scholarly journals The inhibition of platelet cyclo-oxygenase by aspirin is associated with the acetylation of a 72kDa polypeptide in the intracellular membranes

1984 ◽  
Vol 223 (1) ◽  
pp. 105-111 ◽  
Author(s):  
N Hack ◽  
F Carey ◽  
N Crawford
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Membrane System ◽  
Particulate Material ◽  
Intracellular Membrane ◽  
Membrane Structures ◽  
Major Site ◽  
Intracellular Membranes ◽  
Oxygenase Activity ◽  
Radioactive Labelling

Previous studies by Roth & Majerus [J. Clin. Invest. (1975) 56, 624-632] showed that exposure of platelets to [acetyl-14C]aspirin resulted in the radioactive labelling of three polypeptides, two of which were in the cytosol and not saturable, whilst the third was located in particulate material, and was saturated at 30 microM-aspirin. By using high voltage free flow electrophoresis to separate a platelet mixed membrane fraction into highly purified surface and intracellular membrane subfractions, we have confirmed that the major polypeptide acetylated after exposing whole platelets to [acetyl-14C]aspirin is almost exclusively associated with intracellular membrane structures. We have shown previously that these intracellular membranes are the major site for prostanoid biosynthesis [Carey, Menashi & Crawford (1982) Biochem. J. 204, 847-851] and in the present study the extent of the radioactive labelling correlated well with inhibition of the cyclo-oxygenase activity in these intracellular membranes. In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the [14C]acetylated component, which appears to be a dimer, migrates with a mobility corresponding to 72kDa. Although cyclo-oxygenase is inhibited, there is no discernible radioactive labelling when the platelets are exposed to aromatic-ring-labelled [14C]aspirin. We suggest that the site or sites for aspirin acetylation and cyclo-oxygenase activity are structurally associated in the platelet's intracellular membranes referred to by electron microscopists as the dense tubular membrane system.

scholarly journals Effect of sodium n-butyrate on induction of prostaglandin synthase activity in cloned mastocytoma P-815 2-E-6 cells

1981 ◽  
Vol 194 (1) ◽  
pp. 111-117 ◽  
Author(s):  
Y Koshihara ◽  
M Kawamura ◽  
T Senshu ◽  
S Murota
Keyword(s):  
De Novo ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Short Chain Fatty Acids ◽  
High Concentration ◽  
Sodium Propionate ◽  
Prostaglandin Synthase ◽  
Oxygenase Activity ◽  
Significant Difference ◽  
Maximal Induction

Treatment of a cloned mastocytoma P-815 cell line (2-E-6) with 1 mM-sodium n-butyrate for 40h induced prostaglandin synthase activity and arrested cell growth. The induction of enzyme activity by n-butyrate was not associated with suppression of DNA synthesis, since hydroxyurea had no effect on prostaglandin synthase induction. The effect of sodium n-butyrate was reversible. Experiments with cycloheximide and actinomycin D showed that the induction of prostaglandin synthase activity involved synthesis de novo of protein and RNA. The time of half-maximal induction of the newly synthesized RNA for prostaglandin synthase activity was estimated as about 15h, which is similar to the generation time of the cells. Selective induction of fatty acid cyclo-oxygenase activity by sodium n-butyrate was suggested by the following two pieces of experimental evidence. (1) There was no significant difference between treated and untreated cells in the activities of radioactive prostaglandin H2 conversion into individual prostaglandins. (2) The incorporation of [3H]acetylsalicylic acid into the fraction equivalent to protein of mol.wt. approx. 75000 of sodium n-butyrate-treated cells was higher than that of untreated cells, on analysis of cell particulate fraction by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. A high concentration (5 mM) of sodium propionate also induced prostaglandin synthesis, but other short-chain fatty acids, such as isobutyrate and sodium acetate, had no effect.

scholarly journals Intracellular Membranes: Conserved Mechanisms of Formation and Regulation Throughout Evolution

2020 ◽  
Author(s):  
Jorge Royes Mir ◽  
Valérie Biou ◽  
Nathalie Dautin ◽  
Christophe Tribet ◽  
Bruno Miroux
Keyword(s):  
De Novo ◽  
Structural Data ◽  
Intracellular Membrane ◽  
Phospholipid Biosynthesis ◽  
Membrane Structures ◽  
Intracellular Membranes ◽  
Membrane Morphology ◽  
Mechanisms Of Formation ◽  
And Function ◽  
Alpha Proteobacteria

Membrane remodeling and phospholipid biosynthesis are normally tightly regulated to maintain the shape and function of cells. Indeed, different physiological mechanisms ensure a precise coordination between de novo phospholipid biosynthesis and modulation of membrane morphology. Interestingly, the overproduction of certain membrane proteins hijack these regulation networks, leading to the formation of impressive intracellular membrane structures in both prokaryotic and eukaryotic cells. The proteins triggering membrane proliferation share two major common features: 1) they promote the formation of highly curved membrane domains and 2) they lead to an enrichment in anionic, cone-shaped phospholipids (cardiolipin or phosphatidic acid) in the newly formed membranes. Taking into account the available examples of membrane proliferation upon protein overproduction, together with the latest biochemical, biophysical and structural data, we explore the relationship between protein synthesis and membrane biogenesis. We propose a mechanism for the formation of these non-physiological intracellular membranes that shares similarities with natural inner membrane structures found in α-proteobacteria, mitochondria and some viruses-infected cells, pointing towards a conserved feature through evolution. We hope that the information discussed in this review will give a better grasp of the biophysical mechanisms behind physiological and induced intracellular membrane proliferation, inspiring new biotechnological applications.

scholarly journals The anion-transport protein of the human erythrocyte membrane. Studies on fragments produced by pepsin digestion

1979 ◽  
Vol 183 (2) ◽  
pp. 417-427 ◽  
Author(s):  
M J A Tanner ◽  
D G Williams ◽  
D Kyle
Keyword(s):  
Acetic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Anion Transport ◽  
Transport Protein ◽  
Erythrocyte Membranes ◽  
Major Site ◽  
Neutral Ph ◽  
C Terminus ◽  
Peptide Maps

We have studied the fragmentation by pepsin in 1 M-acetic acid of the erythrocyte anion-transport protein in erythrocyte membranes. The location of the fragments obtained was determined by radioiodinating the protein with the use of lactoperoxidase, and identifying the labelled peptides obtained in peptide “maps” of thermolysin digests of the fragments. Three of the fragments were found to be related overlapping products, and shared a common C-terminus. The major site of pepsin cleavage leading to the C-termini of these fragments was shown to be close to the major site of extracellular cleavage of the protein by proteinases active at a neutral pH. Another two fragments were isolated and shown to be derived from the C-terminal portion of the protein. No well-defined large radioactive fragments of the protein were solubilized from the membrane by pepsin in 1 M-acetic acid, the bulk of the radioactivity attributable to the anion transport protein being recovered in very small fragments that could not be resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Our results suggest that the polypeptide chain of the anion-transport protein emerges at the extracellular face of the membrane 8000-13000 daltons on the N-terminal side of the major site of extracellular cleavage of the protein by proteinases that are active at a neutral pH.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

The Recovery of Factor VIII from Fresh-Frozen, Indated and Outdated Human Plasma

1976 ◽  
Vol 36 (01) ◽  
pp. 071-077 ◽  
Author(s):  
Daniel E. Whitman ◽  
Mary Ellen Switzer ◽  
Patrick A. McKee
Keyword(s):  
Factor Viii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
The United States ◽  
Fresh Frozen Plasma ◽  
Red Cross ◽  
Random Samples ◽  
Fresh Frozen ◽  
Factor Viii Concentrates ◽  
Frozen Plasma

SummaryThe availability of factor VIII concentrates is frequently a limitation in the management of classical hemophilia. Such concentrates are prepared from fresh or fresh-frozen plasma. A significant volume of plasma in the United States becomes “indated”, i. e., in contact with red blood cells for 24 hours at 4°, and is therefore not used to prepare factor VIII concentrates. To evaluate this possible resource, partially purified factor VIII was prepared from random samples of fresh-frozen, indated and outdated plasma. The yield of factor VIII protein and procoagulant activity from indated plasma was about the same as that from fresh-frozen plasma. The yield from outdated plasma was substantially less. After further purification, factor VIII from the three sources gave a single subunit band when reduced and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These results indicate that the approximately 287,000 liters of indated plasma processed annually by the American National Red Cross (ANRC) could be used to prepare factor VIII concentrates of good quality. This resource alone could quadruple the supply of factor VIII available for therapy.

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

1982 ◽  
Vol 47 (01) ◽  
pp. 014-018 ◽  
Author(s):  
H Sumi ◽  
N Toki ◽  
S Takasugi ◽  
S Maehara ◽  
M Maruyama ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Plasmin Activity ◽  
Molecular Weights ◽  
Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

Identifikasi Daging Tikus Pada Produk Baso Dengan Metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

2018 ◽  
Vol 26 (2) ◽  
pp. 058
Author(s):  
Anna P. Roswiem ◽  
Triayu Septiani
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Sds Page

<em>Bahan<strong> </strong>baku untuk membuat baso adalah daging hewan, pada umumnya dari daging sapi, ayam, ikan dan babi. Di beberapa daerah di Indonesia terjadi kasus baso tikus. Tujuan penelitian ini adalah menguji ada tidaknya kandungan daging tikus pada produk baso yang dijual di pasar Cempaka Putih-Kecamatan Kramat Jakarta Pusat dan di pedagang baso atau mie baso di sekitar kampus Universitas YARSI Jakarta. Daging adalah protein salah satu metode untuk mengidentifikasi protein adalah metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE).<strong> </strong>Hasil penelitian menunjukkan bahwa dari 6 sampel baso terindikasi ada 2 sampel baso dengan nomor 1 dan 5 yang dibuat dari campuran daging sapi dan tikus; ada 1 sampel baso dengan nomor 6 yang terbuat dari daging tikus; dan 2 sampel baso dengan nomor 2 dan 3 yang terbuat dari campuran sapi  dan babi, dan hanya 1 sampel baso dengan nomor sampel 4 yang benar-benar terbuat dari daging sapi.</em>

Majra Honey Abrogated the Normal and Cancer Cells Proliferation Inhibition by Juniperus procera Extract and Extract/Honey Generated AgNPs

2020 ◽  
Vol 20 (8) ◽  
pp. 970-981
Author(s):  
Hamed A. Ghramh ◽  
Essam H. Ibrahim ◽  
Mona Kilnay
Keyword(s):  
Anticancer Activity ◽  
Cancer Cells ◽  
Biological Activities ◽  
Sugar Content ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bioactive Molecules ◽  
Sds Page ◽  
Splenic Cells ◽  
Juniperus Procera

Background: Juniperus procera and Majra honey are well-known as a folk medicine in many countries. Objectives: This work aimed to study the immunomodulatory effects after mixing Majra honey, J. procera water leaves extract and silver Nanoparticles (AgNPs) on immune or cancer cells. Methods: Juniperus procera water leaves extract and 20% Majra honey were prepared. Both the extract and honey were used separately to synthesize AgNPs. AgNPs were characterized using UV/Vis spectrophotometry and electron microscopy. Bioactive molecules in honey and the extract were explored using Fourier Transform Infrared (FT-IR) spectroscopy. Protein profile of honey was explored using Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) and honey sugar content was determined using High- Performance Liquid Chromatography (HPLC). Biological activities of honey and the extract were tested. Results: The results demonstrated the ability of the extract/honey to produce AgNPs in a spherical shape. The extract/honey contained many functional groups. SDS-PAGE of Majra honey showed many protein bands. HPLC revealed honey is of good quality and no external additives are added to it. The extract and extract+ AgNPs inhibited the growth of normal rat splenic cells while honey stimulated it. The extract+honey turned stimulatory to the splenic cells’ growth and significantly diminished the inhibitory potential of the extract containing AgNPs. Both the extract and honey have antimicrobial activities, this potential increased in the presence of AgNPs. Honey and Honey+AgNPs inhibited HepG2 cancer cell proliferation while Hela cell growth inhibited only with honey+AgNPs. Conclusion: Both honey and the extract have antibacterial and immunomodulatory potentials as well as the power to produce AgNPs. Majra honey alone showed anticancer activity against HepGe2 cells, but not against Hela cells, and when contained AgNPs had anticancer activity on both cell lines. Mixing of Majra honey with J. procera extract showed characterized immunomodulatory potentials that can be described as immunostimulant.

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