scholarly journals One step Purification and Characterisation of Abrin Toxin from Abrus Precatorius Seeds

2019 ◽  
Vol 4 (4) ◽  
pp. 231-235
Author(s):  
Swati Banger ◽  
Rita Singh ◽  
Nagesh Tripathi ◽  
Vijai Pal ◽  
Ajay Kumar Goel
Keyword(s):  
Detection System ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Single Step ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plant Toxin ◽  
Abrus Precatorius ◽  
Catalytically Active ◽  
One Step ◽  
A Chain

Abrin is a plant toxin obtained from Abrus precatorius seeds. It belongs to the type II ribosomal inactivating proteins (RIPs) consisting of two chains namely, catalytically active A chain and sugar binding B chain linked by a single disulphide bond.  Due to high toxicity of abrin, its exposure or consumption can lead to serious public health problems. In the present work, we have extracted and purified the abrin toxin from Abrus precatorius seeds. The toxin was purified using a single step anion exchange chromatography. The purified protein was characterized by SDS-PAGE and MALDI- TOF to confirm its purity. The toxicity of purified abrin toxin was also confirmed by injecting the toxin in mice.  The purified protein was further used to raise antibodies in mice and characterized by indirect Enzyme Linked Immunosorbent Assay. The results of present study established the use of single step ion exchange chromatography to purify abrin toxin for further development of its detection system.

Gas-chromatographic determination of cholesterol sulfate in plasma and erythrocytes, for the diagnosis of recessive X-linked ichthyosis.

1983 ◽  
Vol 29 (7) ◽  
pp. 1404-1407 ◽  
Author(s):  
F A Muskiet ◽  
G Jansen ◽  
B G Wolthers ◽  
A Marinkovic-Ilsen ◽  
P C van Voorst Vader
Keyword(s):  
Internal Standard ◽  
Dehydroepiandrosterone Sulfate ◽  
Chromatographic Determination ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Single Step ◽  
Flame Ionization Detection ◽  
Cholesterol Sulfate ◽  
Flame Ionization

Abstract We describe a rapid method for determining cholesterol sulfate in plasma and erythrocytes. After its single-step isolation by means of anion-exchange chromatography cholesterol sulfate is hydrolyzed, trimethylsilylated, and determined by gas chromatography with flame ionization detection. 5 beta-Cholestan-3 alpha-ol sulfate is used as internal standard. The method enables simultaneous determination of dehydroepiandrosterone sulfate in plasma. We applied it for the diagnosis of seven patients with recessive X-linked ichthyosis. Concentrations are given for plasma and erythrocytes from four unaffected relatives of patients with X-linked ichthyosis, a patient with placental sulfatase deficiency, two patients with other types of ichthyoses, and 20 controls. The method may also be of use for the rapid isolation of other organic sulfates from biological material, as illustrated by a comparison of gas chromatograms of urine from a normal pregnant woman and that from a patient with placental sulfatase deficiency.

scholarly journals The quantitative spectrum of inositol phosphate metabolites in avian erythrocytes, analysed by proton n.m.r. and h.p.l.c. with direct isomer detection

1989 ◽  
Vol 264 (2) ◽  
pp. 323-333 ◽  
Author(s):  
T Radenberg ◽  
P Scholz ◽  
G Bergmann ◽  
G W Mayr
Keyword(s):  
Mammalian Cells ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Detection System ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Phosphate Metabolism ◽  
Qualitative And Quantitative ◽  
Inositol Phosphate Metabolism ◽  
Avian Erythrocytes

The spectrum of inositol phosphate isomers present in avian erythrocytes was investigated in qualitative and quantitative terms. Inositol phosphates were isolated in micromolar quantities from turkey blood by anion-exchange chromatography on Q-Sepharose and subjected to proton n.m.r. and h.p.l.c. analysis. We employed a h.p.l.c. technique with a novel, recently described complexometric post-column detection system, called ‘metal-dye detection’ [Mayr (1988) Biochem. J. 254, 585-591], which enabled us to identify non-radioactively labelled inositol phosphate isomers and to determine their masses. The results indicate that avian erythrocytes contain the same inositol phosphate isomers as mammalian cells. Denoted by the ‘lowest-locant rule’ [NC-IUB Recommendations (1988) Biochem. J. 258, 1-2] irrespective of true enantiomerism, these are Ins(1,4)P2, Ins(1,6)P2, Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, Ins(1,3,4,5,6)P5, and InsP6. Furthermore, we identified two inositol trisphosphate isomers hitherto not described for mammalian cells, namely Ins(1,5,6)P3 and Ins(2,4,5)P3. The possible position of these two isomers in inositol phosphate metabolism and implications resulting from absolute abundances of inositol phosphates are discussed.

scholarly journals The Role of Distinctive Sphingolipids in the Inflammatory and Apoptotic Effects of Electronegative LDL on Monocytes

Biomolecules ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 300 ◽  
Author(s):  
Puig ◽  
Estruch ◽  
Jin ◽  
Sanchez-Quesada ◽  
Benitez
Keyword(s):  
Enzyme Inhibitors ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cytokine Release ◽  
Monocyte Apoptosis ◽  
Apoptotic Effects

Electronegative low-density lipoprotein (LDL(−)) is a minor LDL subfraction that is present in blood with inflammatory and apoptotic effects. We aimed to evaluate the role of sphingolipids ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) in the LDL(−)-induced effect on monocytes. Total LDL was subfractioned into native LDL and LDL(−) by anion-exchange chromatography and their sphingolipid content evaluated by mass spectrometry. LDL subfractions were incubated with monocytes in the presence or absence of enzyme inhibitors: chlorpromazine (CPZ), d-erythro-2-(N-myristoyl amino)-1-phenyl-1-propanol (MAPP), and N,N-dimethylsphingosine (DMS), which inhibit Cer, Sph, and S1P generation, respectively. After incubation, we evaluated cytokine release by enzyme-linked immunosorbent assay (ELISA) and apoptosis by flow cytometry. LDL(−) had an increased content in Cer and Sph compared to LDL(+). LDL(−)-induced cytokine release from cultured monocytes was inhibited by CPZ and MAPP, whereas DMS had no effect. LDL(−) promoted monocyte apoptosis, which was inhibited by CPZ, but increased with the addition of DMS. LDL enriched with Sph increased cytokine release in monocytes, and when enriched with Cer, reproduced both the apoptotic and inflammatory effects of LDL(−). These observations indicate that Cer content contributes to the inflammatory and apoptotic effects of LDL(−) on monocytes, whereas Sph plays a more important role in LDL(−)-induced inflammation, and S1P counteracts apoptosis.

Oxidant stress stimulates mucin secretion and PLC in airway epithelium via a nitric oxide-dependent mechanism

1996 ◽  
Vol 271 (5) ◽  
pp. L854-L861 ◽  
Author(s):  
D. T. Wright ◽  
B. M. Fischer ◽  
C. Li ◽  
L. G. Rochelle ◽  
N. J. Akley ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Oxidant Stress ◽  
Bronchial Epithelium ◽  
Anion Exchange Chromatography ◽  
Airway Epithelial Cells ◽  
Exchange Chromatography ◽  
Enzyme Linked Immunosorbent Assay ◽  
Airway Epithelial ◽  
Mucin Secretion ◽  
Transformed Cell Line

Reactive oxygen species (ROS) have been implicated in the pathogenesis of a wide variety of respiratory diseases. We investigated mechanisms of ROS-induced mucin secretion by guinea pig tracheal epithelial (GPTE) cells in primary culture, and ROS-induced activation of the second messenger-producing enzyme phospholipase C (PLC), in GPTE cells and in a virally transformed cell line (BEAS-2B) derived from human bronchial epithelium. Mucin secretion was measured by a monoclonal antibody-based enzyme-linked immunosorbent assay, and PLC activation was assessed by anion exchange chromatography. ROS generated enzymatically by xanthine oxidase (XO, 500 microM) in the presence of purine (500 microM) enhanced release of mucin by GPTE cells and activated PLC in GPTE and BEAS cells. Hypersecretion of mucin and activation of PLC in response to purine + XO appeared to occur via an intracellular pathway(s) dependent on endogenously produced nitric oxide and possibly intracellularly generated oxidants. Both responses could be blocked or attenuated by preincubation of the cells with NG-monomethyl-L-arginine, an inhibitor of the enzyme nitric oxide synthase, or with dimethylthiourea, a compound that can react with a variety of intracellular oxidant species. Reactive nitrogen species generated chemically also stimulated secretion of mucin and activated PLC via a mechanism dependent (at least in part) on intracellular oxidant-mediated process(es). The results suggest that intracellularly generated radical species of nitrogen and oxygen may be important modulators of the response of airway epithelial cells to external oxidant stress.

Purification and Amino Acid Sequence of Fructose-1,6-bisphosphate Aldolase from the Electric Organ of Electrophorus electricus (L.)

2006 ◽  
Vol 61 (11-12) ◽  
pp. 884-888
Author(s):  
Salvatore G. De-Simone ◽  
Christiane M. Cardoso de Salles ◽  
Celia M. Batistae Silva ◽  
Aida Hassón-Voloch
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gel Filtration ◽  
Electric Organ ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
High Homology ◽  
Electrophorus Electricus ◽  
One Step ◽  
Fructose 1,6 Bisphosphate

Abstract A soluble fructose-1,6-bisphosphate aldolase enzyme has been purified 50.2-fold (2.36%) at the homogeneity from the electric organ of Electrophorus electricus by one step of DEAE- 52 anion exchange chromatography followed by Superose-12 gel filtration-FPLC. Like other aldolase enzymes the E. electricus protein is a dimer with two identical subunits of 45 kDa. The N-terminal (20 residues) revealed a high homology with S. aurata (75%, goldfish), R. ratus and M. musculus (mouse, 80%) enzymes.

STRUCTURAL AND FUNCTIONAL COMPARISON OF THROMBOSPONDIN FROM PLATELETS, ENDOTHELIAL CELLS AND FIBROBLASTS

1987 ◽  
Author(s):  
P Clezardin ◽  
J L McGregor
Keyword(s):  
Endothelial Cells ◽  
Silver Staining ◽  
Solid Phase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Enzyme Linked Immunosorbent Assay ◽  
Proteolytic Fragment ◽  
Reducing Conditions ◽  
Wide Range

Thrombospondin (TBSP) is a 450 kDa glycoprotein secreted by a wide range of cells including platelets, endothelial cells and fibroblasts. Using non-denaturating conditions, we recently reported that platelet TBSP was structurally different from endothelial and fibroblast TBSP (P. Clezardin et al., Eur. J. Biochem., 1986, 159, 569-579). The aim of this study was to compare the structure of TBSP purified from platelets, endothelial cells and fibroblasts using denaturating conditions. Moreover, the interaction of fibrinogen with these three forms of TBSP was also investigated. TBSPs were first purified by heparin-Sepharose and immunoaffinity chromatography followed by Mono O anion-exchange chromatography on a FPLC system. Thermolysin digests of purified TBSPs were analysed by SDS-polyacrylamide gel electrophoresis under reducing conditions and the subsequent electrophoresed proteolytic fragments identified by Coomassie and silver staining. The interaction of undigested and digested TBSPs with solid-phase adsorbed fibrinogen was investigated by enzyme-linked immunosorbent assay using an anti-TBSP monoclonal antibody (P10). when using Coomassie staining, a 70 kDa proteolytic fragment of thermolysin-treated platelet TBSP was absent from the endothelial and fibroblast TBSP digests. Moreover, a 18 kDa fragment from thermolysin-treated endothelial and fibroblast TBSP was undetectable in the platelet TBSP digest when using silver staining on SDS-polyacrylamide gels. The binding of undigested TBSPs to solid-phase adsorbed fibrinogen was different from that obtained with digested TBSPs. These results indicate that the observed structural differences might induce functional differences between platelet and the two other forms of TBSP.

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