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 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 Antivinculin antibodies in sera of patients with immune thrombocytopenia and in sera of normal subjects

Blood ◽  
1992 ◽  
Vol 79 (1) ◽  
pp. 161-168 ◽  
Author(s):  
Y Tomiyama ◽  
R Kekomaki ◽  
J McFarland ◽  
TJ Kunicki
Keyword(s):  
Immune Thrombocytopenia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Protein ◽  
Normal Subjects ◽  
Enzyme Linked Immunosorbent Assay ◽  
Two Dimensional ◽  
Platelet Protein ◽  
Sds Page ◽  
Significant Difference

Abstract We have characterized a 120-Kd antigen that frequently reacts with serum antibodies from patients with immune thrombocytopenia or normal subjects. Immunoblots made after two-dimensional nonreduced/reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional isoelectric focusing/SDS-PAGE demonstrated that this 120-Kd protein has the same molecular weight under nonreduced or reduced conditions, is not a surface protein, and has an isoelectric point (pl) of 6.4 to 6.5. From these data, one likely candidate is the intracellular platelet protein, vinculin. Monoclonal antivinculin antibody reacts with this 120-Kd protein, and purified human platelet vinculin is bound by antibodies that recognize the 120-Kd protein. Therefore, we conclude that this 120-Kd protein is identical to vinculin. Data obtained from a sensitive enzyme-linked immunosorbent assay demonstrate the presence of naturally occurring antivinculin antibodies in many normal sera. However, the incidence of antivinculin antibodies in patient sera (67%; 55 of 82 sera) is significantly (P less than .01) higher than that in normal sera (40%; 32 of 80 sera), and there is a significant difference (P less than .05) between the mean levels of antivinculin antibodies in patient and normal sera. Whereas the levels of these antibodies in patient and normal sera overlap, 2 of 82 sera from patients with thrombocytopenia express unusually high levels of such antibodies. The pathologic significance of these antibodies remains to be determined.

Heparin Binding Proteins of Black Bengal Buck Semen and Their Correlation with Sperm Characters and Freezability

2019 ◽  
Author(s):  
M Karunakaran ◽  
Vivek C Gajare ◽  
Ajoy Mandal ◽  
Mohan Mondal ◽  
S K Das ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Heparin Binding ◽  
Sds Page ◽  
Significant Difference ◽  
Sperm Characters

This experiment was conducted to study the electrophoretic characters of heparin binding proteins (HBP) of Black Bengal buck semen and their correlation with sperm characters and cryo-survivability. Semen ejaculates (n=20/buck) were collected from nine bucks and in vitro sperm characters were evaluated at collection, after equilibration and after freeze - thawing. HBP were isolated through heparin column and discontinuous Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) was performed to assess molecular weight. Significant difference (plessthan0.01) were observed among the bucks in sperm characters and freezability. Eight protein bands of 17 to 180 kDa in seminal plasma and 7 bands in sperm were found. 180 -136 kDa HBP of seminal plasma and 134-101 kDa HBP of sperm had showed high correlation with in vitro sperm characters. Further studies on identification of these proteins and their correlation with in vivo pregnancy are needed to find their role as marker for buck selection.

scholarly journals Identification of the mRNA and polypeptide subunit for prostaglandin endoperoxide synthase from mouse mastocytoma P-815 cells

1983 ◽  
Vol 212 (1) ◽  
pp. 219-222 ◽  
Author(s):  
M Kondo ◽  
Y Koshihara ◽  
M Kawamura ◽  
S Murota
Keyword(s):  
Polypeptide Chain ◽  
De Novo ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Prostaglandin Endoperoxide ◽  
Single Polypeptide Chain ◽  
Free Translation ◽  
Prostaglandin Endoperoxide Synthase ◽  
Single Polypeptide

Cloned mouse mastocytoma P-815.2-E-6 cells are barely able to synthesize prostaglandins because of a lack of prostaglandin endoperoxide synthase activity. However, the addition of sodium n-butyrate at 1 mM induces synthesis de novo of prostaglandins in this cell line. Employing this system, we could isolate an mRNA for prostaglandin endoperoxide synthase by a combination of cell-free translation and immunoprecipitation. The antibody, prepared in rabbit by injecting purified prostaglandin endoperoxide synthase from bovine vesicular gland, was shown to cross-react with the corresponding enzyme from 2-E-6 cells. The poly(A)-containing mRNA has a sedimentation coefficient of 17S and codes for a single polypeptide chain of Mr 62 000 as estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The Mr of the mouse polypeptide chain appears very similar to that of the purified carbohydrate-free prostaglandin endoperoxide synthase from sheep vesicular gland. These findings are a contribution to the isolation of the gene for prostaglandin endoperoxide synthase.

Glycosylation of chicken haptoglobin: Isolation and characterization of three molecular variants and studies of their distribution in hen plasma before and after turpentine-induced inflammation

1988 ◽  
Vol 66 (3) ◽  
pp. 208-217 ◽  
Author(s):  
Francisco Delers ◽  
Gérard Strecker ◽  
Robert Engler
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Isolation And Characterization ◽  
Molecular Variants ◽  
Hemoglobin Binding ◽  
Significant Difference ◽  
Before And After ◽  
Carbohydrate Unit

Chicken haptoglobin (Hp), a hemoglobin-binding protein isolated from chicken plasma, is composed of three molecular variants that react differently with concanavalin A (ConA). These glycosylation variants of chicken Hp have been isolated by affinity chromatography using Sepharose-bound ConA. They differ in their molecular weight, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Analysis of the glycopeptides obtained after pronase digestion of these variants yielded two types of structures: one, reactive with ConA, corresponded to a biantennary N-linked carbohydrate unit and one, unreactive with ConA, corresponded to a triantennary unit. The strongly ConA-reactive Hp variant bears only two biantennary units and the nonreactive Hp variant bears only two triantennary units; the weakly reactive Hp variant bears equal amounts of both units. The distribution of Hp glycosylation variant does not show any significant difference when obtained from the plasma of laying hens before and after turpentine-induced inflammation.

scholarly journals Assessment of Toxicological Effects of Selected Popular Antidiabetic Drugs in Type II Diabetes Mellitus within Ota, Ogun State, Nigeria

2019 ◽  
pp. 1-10
Author(s):  
Ogunlana Olubanke Olujoke ◽  
Oyebanji Olufisayo Grace ◽  
Ogunlana Oluseyi Ebenezer ◽  
Adekeye Bosede Temitope ◽  
Adeyemi Alaba Oladipupo ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Therapeutic Drug ◽  
Diabetic Patients ◽  
Dietary Control ◽  
Functional Changes ◽  
Ogun State ◽  
Significant Difference ◽  
Liver And Kidney

Aim: The complications associated with diabetes and the new trend of using combination therapy in the management of the disease gave birth to this work, aimed at assessing the hepatotoxic and nephrotoxic effects of selected popularly used antidiabetic medications in type 2 diabetic patients within Ota, Ogun State, Nigeria. Study Design: The participants, diabetic (n=195) and non-diabetic (n=30) were divided into the following groups based on their medications: 1 (Non Diabetic control), 2 (Metformin), 3 (Glimepiride), 4 (Glibenclamide), 5 (Metformin and Glimepiride), 6 (Meformin and Glibenclamide), 7 (Metformin, Glimepiride and Glibenclamide) and 8 (Diabetic Dietary control). Methodology: Serum protein expression profiling, liver and kidney function parameters were assessed in participant’s blood using Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and standard laboratory methods respectively. Results: Glyceamic control within the diabetic groups was 29.23%. Urea concentration was significantly increased (p < 0.05) in groups 5 and 7 compared with groups 1 and 8 while the serum creatinine levels in the different groups showed no significant difference. Activities of alkaline phosphatase and aspartate aminotransferase increased significantly (p < 0.05) in group 5 compared with groups 1 and 8. A low molecular weight protein likely to be Leptin (molecular weight 18 kDa) was over-expressed in all the diabetic groups. Conclusion: This study shows that use of multiple rather than single drugs caused significant functional changes in the liver and kidney. The control of diabetes may best be carried out with dietary control and lifestyle modification as well as good therapeutic drug monitoring for safe assessment of baseline organ function.

scholarly journals A Comparative Study of Distribution of Protein and Cholesterol in Various Fractions of Human Semen from Infertile and Fertile Subjects

2012 ◽  
Vol 3 (3) ◽  
pp. 78-82 ◽  
Author(s):  
MS Srinivas ◽  
Vickram Sundaram ◽  
M Ramesh Pathy ◽  
TB Sridharan
Keyword(s):  
Molecular Weight ◽  
Comparative Study ◽  
Seminal Plasma ◽  
Semen Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Semen ◽  
Exact Function ◽  
Wide Range ◽  
Significant Difference

ABSTRACT Aim To elucidate the concentration of the protein and cholesterol in different fractions of human semen from different infertile categories and comparing them with the fertile group. Materials and methods The human semen was collected from different infertile categories including oligoasthenospermia, asthenospermia, azoospermia, normospermia, oligospermia and fertile group. Immediately after collection, the semen analysis was done as per WHO standard protocols. After that, the semen was centrifuged to get the different fractions. Four main fractions were obtained, (1) spermatozoa, (2) debris or material that precipitates at 12 K rpm for 10 minutes, (3) prostasomes which was precipitated at 20K rpm for 120 minutes, (4) seminal plasma. The protein concentration was done by Lowry's method and cholesterol was estimated by diagnostic kit. Results Sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS PAGE) was run for all the categories of semen samples for their seminal plasma and the fertility associated protein was identified. A significant difference was found in the concentration of proteins in all subfractions when compared between control and infertile categories. Almost 86% of the protein was recovered from soluble fraction. In case of azoospermia, the protein content was very low when compared with fertile group. Seminal plasma proteins were visualized by silver staining. The molecular weight of the protein bands were ranging from 6.5 to 205 kDa. The band with molecular weight around 55 kDa was found to be missing in case of oligoasthenospermia. This particular protein is said to be fertility associated protein. The content of cholesterol for different subfraction of the human semen samples from infertile and fertile samples was compared. A wide range of cholesterol was recovered from prostasomes, that too purified. Conclusion A thrive study have to be done in all the subfractions of the semen irrespective of the category of samples to know the exact function of the each subfractions in terms of protein and cholesterol distribution. How to cite this article Sundaram V, Srinivas MS, Rao KA, Pathy MR, Sridharan TB. A Comparative Study of Distribution of Protein and Cholesterol in Various Fractions of Human Semen from Infertile and Fertile Subjects. Int J Infertility Fetal Med 2012;3(3):78-82.

scholarly journals Human platelet fibrinogen: purification and hemostatic properties

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 808-815 ◽  
Author(s):  
TJ Kunicki ◽  
PJ Newman ◽  
DL Amrani ◽  
MW Mosesson
Keyword(s):  
Platelet Aggregation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Calcium Ionophore ◽  
Fluid Phase ◽  
Calcium Ionophore A23187 ◽  
Plasma Fibrinogen ◽  
Ionophore A23187 ◽  
Significant Difference

Abstract Conditions were developed in which 80% to 90% of platelet fibrinogen could be routinely purified in nondegraded form from the fluid phase of platelet suspensions stimulated with the calcium ionophore, A23187, in the presence of calcium, leupeptin, and prostaglandin E1. Fibrinogen was separated from other released proteins by chromatography on diethylaminoethanol (DEAE)-cellulose using a continuous pH and ionic strength gradient. Purified platelet fibrinogen, greater than 98% homogeneous by immunoelectrophoresis and sodium-dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), consisted of intact A alpha, B beta and gamma A chains, but not gamma' chains, and was 95% to 96% clottable. Platelet fibrinogen was shown to compete for the binding of radiolabeled plasma fibrinogen to ADP-activated platelets in a manner identical to that of unlabeled plasma fibrinogen itself. Also, at equivalent protein concentrations, platelet and plasma fibrinogens supported platelet aggregation to an equivalent extent. Based upon these results, we conclude that there is no significant difference between platelet and plasma fibrinogen with respect to their size, their clottability, their affinity for the activated platelet fibrinogen receptor, or their capacity to support subsequent platelet aggregation.

scholarly journals Dephosphorylation of Phytate by Using theAspergillus niger Phytase with a High Affinity for Phytate

1999 ◽  
Vol 65 (10) ◽  
pp. 4682-4684 ◽  
Author(s):  
Tadashi Nagashima ◽  
Tatsuya Tange ◽  
Hideharu Anazawa
Keyword(s):  
Phytic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Aspergillus Ficuum ◽  
Michaelis Constant ◽  
High Affinity ◽  
Significant Difference

ABSTRACT A phytase (EC 3.1.3.8 ) with a high affinity for phytic acid was found in Aspergillus niger SK-57 and purified to homogeneity in four steps by using ion-exchange chromatography (two types), gel filtration, and chromatofocusing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme gave a single stained band at a molecular mass of approximately 60 kDa. The Michaelis constant of the enzyme for phytic acid (18.7 ± 4.6 μM) was statistically analyzed. In regard to the orthophosphate released from phytic acid, a significant difference between a lowKm phytase from A. niger SK-57 and a high Km phytase from Aspergillus ficuum was recognized.

De novo protein synthesis and protein phosphorylation during anoxia and recovery in the red-eared turtle

1993 ◽  
Vol 265 (6) ◽  
pp. R1380-R1386 ◽  
Author(s):  
S. P. Brooks ◽  
K. B. Storey
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Protein Phosphorylation ◽  
De Novo ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Total Radioactivity ◽  
Relative Molecular Mass ◽  
De Novo Protein Synthesis

Changes in de novo protein synthesis and protein phosphorylation were monitored during anoxia and recovery in the red-eared slider Trachemys (= Pseudemys) scripta elegans. Time courses of 35S-radiolabeled methionine incorporation into acid-precipitable material showed an increase up to 5 h postinjection and remained constant after this time. Comparison of the total and acid-precipitable 35S label incorporation into tissues from 20-h control, anoxic, and recovering animals showed differences between these groups: total radioactivity in brain was 2.9-fold lower in recovering turtles, whereas protein-associated radioactivity was 2.4-fold higher in anoxic liver, 2.3-fold lower in recovering skeletal muscle, and 3.7-fold lower in recovering brain tissue. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radiolabeled proteins showed the existence of a newly synthesized protein band (relative molecular mass = 72 kDa) that was apparent only in 20-h recovering liver and skeletal muscle. Use of 32P labeling to monitor changes in protein phosphorylation patterns during anoxia revealed 1.6-, 1.4-, and 1.5-fold increases in 32P incorporation in anoxic brain, heart, and liver, respectively. Changes in protein phosphorylation were localized to the plasma membrane and cytosolic fractions in brain and to the cytosolic fraction in liver.

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