Paola Patrignani Abstract

The assessment of prostanoids (TXB2, 6-keto-PGF1a, PGD2 and PGE2) generated in isolated or cultured cells in vitro can be directly assessed in media without extraction mainly using immunoassays, i.e. radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), or enzyme immunoassay (EIA). However, the immunoassays have to have some fundamental requisites, such as the use of highly specific and sensitive antibodies. Importantly, the results obtained with immunoassays should be validated by...

Introduction

The potency of acetaminophen, salicylic acid, and aspirin as inhibitors of the prostaglandin H synthases (PGHSs, cyclooxygenases) varies considerably between different cell types. This cellular selectivity is manifested in the human pharmacology of the drugs. For example, all three of these agents exert little or no antiinflamma-tory action at doses that are antipyretic and analgesic. Cellular selectivity is a consequence of the fact that inhibition of the PGHSs by these drugs is regulated by...

Materials

Enzymatic Assays Unless stated otherwise, all reagents are obtained from Sigma-Aldrich, St. Louis, MO. Water is milli-Q purified. 1. COX-1 from sheep seminal vesicles, and recombinant murine COX-2, were purified as described previously (18). 2. A 1.0 M stock solution of Tris buffer, pH 8.1 at 25 C, is prepared by mixing equal volumes of 1.0 M Tris-HCl with 1.0 M Tris base. The stock is filtered through a 0.2 mm CA membrane and stored at 5 C. 3. A 1.0 M stock solution of potassium phosphate...

The Cox1 Cox2 Concept

Most of the biological data suggested that Cox-2 was the predominant inflammatory species, and by implication, the best target for NSAIDs. So was Cox-2 inhibition the true therapeutic modality of NSAIDs If so, Cox-1 inhibition might account for the side effects, such as gastric irritation and depression of platelet aggregation. This was the notion advanced by more than one group (26, 27) which came to be known as the Cox-2-bad Cox-1-good hypothesis. If true, then the obvious corollary was that...

Prostanoids

Biosynthesis Prostanoids i.e. prostaglandin(PG)E2, PGF2a, PGD2, prosta- cyclin(PGI2), and thromboxane(TX)A2 are lipid autacoids which are immediately released outside the cell after intracellular biosynthesis. This occurs probably through the action ofMRP4 ABCC4 (multidrug resistance protein 4 adenosine triphosphate-binding cassettes, subfamily C, member 4), an adenosine triphosphate-dependent export pump for organic anions (1). Prostanoids modulate a wide variety of physiologic and...

Methods

All operations carried out at 4 C unless otherwise noted. 3.1. Wash Cells (Day 1) 1. Suspend frozen cells in 10 volumes homogenization buffer without detergent. (20 g wet weight cells + 200 ml Buffer). 2. Stir until suspended at room temp 1 2 h. 3. Centrifuge 20 min at 10,000 xg (Beckman JA-13 rotor 8,000 rpm). 4. Discard Supernatant (add bleach to 1 to disinfect). 3.2. Solubilisation 1. Resuspend cells in 10 volumes (w v) homogenization buffer. 2. Add decyl-maltoside (C10M) to 1.5 final drop...

COX2 brief overview of the various dilution steps of the standard

Standard number Final concentration pg ml Standard pl Assay buffer pl Dilution 2 2,500 500 of St. 1 500 1 2 3 1,250 500 of St. 2 500 1 2 4 625 500 of St. 3 500 1 2 5 313 500 of St. 4 500 1 2 6 156 500 of St. 5 500 1 2 7 78.1 500 of St. 6 500 1 2 8 39.1 500 of St. 7 500 1 2 7. After centrifugation, different layers should be apparent in Fig. 1. Different cell layers formed after centrifugation. 3. Pipet 3 ml Percoll solution (74 ) in a falcon tube made of polystyrene and put a thin polystyrene...

COX2 purification table

Protein Activity Activity Fold Step (mg) (units) (units mg) purification Yield ( ) Fig. 2. SDS-PAGE of purification steps for recombinant human COX-1 and COX-2. 2 mg total protein were loaded per lane on a 10-20 10 x 10 cm mini-gel and stained with colloidal Coomassie. Fig. 2. SDS-PAGE of purification steps for recombinant human COX-1 and COX-2. 2 mg total protein were loaded per lane on a 10-20 10 x 10 cm mini-gel and stained with colloidal Coomassie. 1. Aliquot into polypropylene tubes and...

Notes

For C18, columns without a reservoir solvents were applied by attaching the columns to a syringe filled with the solvents and consistent pressure is applied. It is difficult to be consistent using this technique and thus variation between samples is very likely. In addition, one can only run up to six columns at a time. A much improved technique is to use columns with reservoirs attached to a manifold (such as the one from Varian Inc.) that attach to a vacuum pump. 2. It is advisable to run a...