scholarly journals Expression and selective inhibition of the constitutive and inducible forms of human cyclo-oxygenase

1995 ◽  
Vol 305 (2) ◽  
pp. 479-484 ◽  
Author(s):  
J K Gierse ◽  
S D Hauser ◽  
D P Creely ◽  
C Koboldt ◽  
S H Rangwala ◽  
...  
Keyword(s):  
Competitive Inhibition ◽  
Specific Activity ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Selective Inhibition ◽  
Competitive Inhibitor ◽  
Chronic Inflammatory Disease ◽  
Dependent Inactivation ◽  
Inflammatory Drugs ◽  
Cox 1

The enzyme cyclo-oxygenase catalyses the oxygenation of arachidonic acid, leading to the formation of prostaglandins. Recently two forms of cyclo-oxygenase have been described: a constitutive (COX-1) enzyme present in most cells and tissues, and an inducible (COX-2) isoenzyme observed in many cells in response to pro-inflammatory cytokines. Constitutive and inducible forms of human cyclo-oxygenase (hCOX-1 and hCOX-2) were cloned and expressed in insect cells, utilizing a baculovirus expression system. hCOX-1 had a specific activity of 18.8 mumol of O2/mg with a Km of 13.8 microM for arachidonate and Vmax. of 1500 nmol of O2/nmol of enzyme, whereas hCOX-2 had a specific activity of 12.2 mumol of O2/mg with a Km of 8.7 microM for arachidonate and a Vmax. of 1090 nmol of O2/nmol of enzyme. Indomethacin inhibited both hCOX-1 and hCOX-2, whereas NS-398 and Dup-697 selectively inhibited hCOX-2. Both NS-398 and Dup-697 exhibited time-dependent inactivation of hCOX-2, as did indomethacin on both enzymes. The competitive inhibitor of hCOX-1, mefenamic acid, also displayed competitive inhibition of hCOX-2. These results demonstrate the ability to generate selective non-steroidal anti-inflammatory drugs (NSAIDs), which could provide useful improvement therapeutically in the treatment of chronic inflammatory disease.

scholarly journals Cloning and expression of a chick liver glutathione S-transferase CL 3 subunit with the use of a baculovirus expression system

1992 ◽  
Vol 281 (2) ◽  
pp. 545-551 ◽  
Author(s):  
L H Chang ◽  
J Y Fan ◽  
L F Liu ◽  
S P Tsai ◽  
M F Tam
Keyword(s):  
Specific Activity ◽  
Sequence Similarity ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Relative Activity ◽  
Cloning And Expression ◽  
Glutathione S Transferase ◽  
Sds Page ◽  
Glutathione S Transferases ◽  
A Subunit

Glutathione S-transferase CL 3 subunits purified from 1-day-old-chick livers were digested with Achromobacter proteinase I and the resulting fragments were isolated for amino acid sequence analysis. An oligonucleotide probe was constructed accordingly for cDNA library screening. A cDNA clone of 1342 bases, pGCL301, encoding a protein of 26209 Da was isolated and sequenced. Including conservative substitutions, this protein has 75-79% sequence similarity to other Alpha family glutathione S-transferases. The coding sequence of pGCL301 was inserted into a baculovirus vector for infection of Spodoptera frugiperda (SF9) cells. The expressed protein has a high relative activity with ethacrynic acid (47% of the specific activity with 1-chloro-2,4-dinitrobenzene). The enzyme has a subunit molecular mass of 25.2 +/- 1.2 kDa (by SDS/PAGE), a pI of 9.45 and an absorption coefficient A1%1cm of 13.0 +/- 0.5 at 280 nm.

scholarly journals Licofelone in osteoarthritis: is this the awaited drug? a systematic review

2021 ◽  
Vol 10 (5) ◽  
pp. 564
Author(s):  
Vinod Kumar P.
Keyword(s):  
Competitive Inhibitor ◽  
Cartilage Volume ◽  
Joint Disease ◽  
Pain Sensation ◽  
New Methods ◽  
Inflammatory Drugs ◽  
Cox 2 ◽  
Anti Inflammatory ◽  
Cox 1 ◽  
New Strategies

Osteoarthritis is a progressive joint disease associated with aging in elderly is, characterized by pain, inflammation, and difficulty in movement. The pathways involved in the progression of this disease remain unclear. The mediators, eicosanoids and leukotrienes are produced by COX-1/COX-2 or 5-LOX. Physicians have always used nonsteroidal anti-inflammatory drugs to treat the pain associated with osteoarthritis. A competitive inhibitor of LOX-5 and COX-2 that has both analgesic and anti-inflammatory activity is licofelone; one of the most promising candidates for the treatment of osteoarthritis is under clinical trial in treatment. A significant reduction in cartilage volume was displayed. A significant improvement over baseline on the WOMAC index and GI tolerance was also observed. Improving the symptoms related pain sensation with a happier life. With new strategies in OA, new methods to target pain, inflammation and movement restrictions would be the ultimate goal in patient satisfaction for the future. Licofelone was found to be effective compared to NSAIDs or coxibs in the treatment of OA. Does this mean this could be the answer?

scholarly journals Effects of selective inhibition of cyclooxygenase and lipooxygenase pathways in follicle rupture and ovulation in the rat

Reproduction ◽  
2006 ◽  
Vol 132 (4) ◽  
pp. 571-577 ◽  
Author(s):  
M Gaytán ◽  
C Bellido ◽  
C Morales ◽  
J E Sánchez-Criado ◽  
F Gaytán
Keyword(s):  
Selective Inhibition ◽  
Spatial Targeting ◽  
Follicle Rupture ◽  
Immature Rats ◽  
Inflammatory Drugs ◽  
Cox 2 ◽  
Anti Inflammatory ◽  
Cox 2 Inhibitors ◽  
Cox 1

Treatment with non-steroidal anti-inflammatory drugs, either non-selective or selective cyclooxygenase-2 (COX-2) inhibitors, consistently impairs ovulation, indicating the essential role of COX-2/prostaglandins in the ovulatory process. Indomethacin, a potent inhibitor of both COX-1 and COX-2, induced several ovulatory alterations, consisting of a decrease in the number of oocytes effectively ovulated, trapping of oocytes inside the luteinized follicle, as well as abnormal follicle rupture at the basolateral sides, with release of the oocyte and follicular fluid to the interstitium. Yet, the precise role of prostaglandins in ovulation and whether some of the ovulatory defects induced by indomethacin are due to interference with additional components of the ovulatory cascade, beyond prostaglandin synthesis, are not completely understood. We have used gonadotrophin-primed immature rats to analyse whether, compared to indomethacin, selective inhibition of COX-2, with or without concomitant inhibition of COX-1, or selective inhibition of the lipooxygenase (LOX) pathway, induce similar ovulatory alterations. Immature rats (27 days of age) were injected PMSG (10 IU), and 48 h later hCG (10 IU) subcutaneously, and different anti-inflammatory drugs. Animals were killed at 21 h after hCG injection. Rats treated with the selective COX-2 inhibitor NS398 (10 mg/kg body weight, (bw)) showed alterations in follicle rupture as those treated with indomethacin (0.5 mg/rat), albeit affecting a lower number of follicles, irrespective of the concomitant inhibition of COX-1 with the selective inhibitor SC560 (10 mg/kg bw). Rats treated with the LOX inhibitor NDGA (300 mg/kg bw) did not show ovulatory alterations. These data indicate that the characteristic alterations of follicle rupture induced by indomethacin, are also induced by selective COX-2 inhibitors, strengthening the contention that prostaglandins play a crucial role in the spatial targeting of follicle rupture at the apex.

Purification and Properties of Adenosine Kinase from Rat Liver: Separation from Deoxyadenosine Kinase Activity

1985 ◽  
Vol 40 (1-2) ◽  
pp. 34-41 ◽  
Author(s):  
Alicja K. Drabikowska ◽  
Lidia Halec ◽  
David Shugar
Keyword(s):  
Rat Liver ◽  
Competitive Inhibition ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Residual Activity ◽  
Deae Cellulose ◽  
Intrinsic Property ◽  
Competitive Inhibitor ◽  
Adenosine Kinase ◽  
Initial Ph

Ion exchange and affinity chromatography techniques, similar to those previously reported for purification of adenosine kinase from human placenta, were applied to purification of rat liver adenosine kinase. The enzyme, purified 400-fold in 41% yield, was homogeneous on SDS- polyacrylamide gel electrophoresis, with a molecular weight of 52000. It specific activity, 18 μmol/min/mg protein, is the highest hitherto reported for this enzyme from mammalian sources. Chromatography on DEAE-cellulose removed about 98% of the phosphorylating activity towards 2′-deoxyadenosine present in the initial pH-treated liver extract. The final preparation exhibited only minimal activity (~ 1.5%) under optimal conditions (pH 7.5) vs- 2′-deoxy- adenosine, the lowest yet reported for such a preparation, with a Km of 670 μᴍ, as compared to 0.3 μᴍ for adenosine. The residual activity towards deoxyadenosine is considered an intrinsic property of the purified adenosine kinase and, in fact, phosphorylation of adenosine was inhibited competitively by deoxyadenosine, with a of 70 μᴍ. Competitive inhibition was also exhibited by cordycepin (3′-deoxyadenosine) with a Ki of 150 μᴍ. A more potent competitive inhibitor was tubercidin, the Ki for which was 1.9 μᴍ.

A three-step kinetic mechanism for selective inhibition of cyclo-oxygenase-2 by diarylheterocyclic inhibitors

2001 ◽  
Vol 357 (3) ◽  
pp. 709-718 ◽  
Author(s):  
Mark C. WALKER ◽  
Ravi G. KURUMBAIL ◽  
James R. KIEFER ◽  
Kirby T. MORELAND ◽  
Carol M. KOBOLDT ◽  
...  
Keyword(s):  
Selective Inhibition ◽  
Kinetic Mechanism ◽  
Time Dependent ◽  
Irreversible Inhibition ◽  
Inhibitory Mechanisms ◽  
Dependent Inhibition ◽  
Inflammatory Drugs ◽  
Cox 2 ◽  
Time Dependent Inhibition ◽  
Cox 1

Cyclo-oxygenase (COX) enzymes are the targets for non-steroidal anti-inflammatory drugs (NSAIDs). These drugs demonstrate a variety of inhibitory mechanisms, which include simple competitive, as well as slow binding and irreversible inhibition. In general, most NSAIDs inhibit COX-1 and −2 by similar mechanisms. A unique class of diarylheterocyclic inhibitors has been developed that is highly selective for COX-2 by virtue of distinct inhibitory mechanisms for each isoenzyme. Several of these inhibitors, with varying selectivity, have been utilized to probe the mechanisms of COX inhibition. Results from analysis of both steady-state and time-dependent inhibition were compared. A generalized mechanism for inhibition, consisting of three sequential reversible steps, can account for the various types of kinetic behaviour observed with these inhibitors.

Kinetic Aspects of the Interaction of Blood Clotting Enzymes

1968 ◽  
Vol 19 (03/04) ◽  
pp. 364-367 ◽  
Author(s):  
H. C Hemker ◽  
P. W Hemker
Keyword(s):  
Enzyme Kinetics ◽  
Blood Clotting ◽  
Competitive Inhibition ◽  
Competitive Inhibitor ◽  
Kinetics Of

SummaryThe enzyme kinetics of competitive inhibition under conditions prevailing in clotting tests are developed and a method is given to measure relative amounts of a competitive inhibitor by means of the t — D plot.

Biochemical Chgaracterization of Recombinant Baculovirus 373 K/E p53 Protein

2018 ◽  
Vol 9 (03) ◽  
pp. 20204-20223
Author(s):  
Maghsoudi, Hossein ◽  
U Pati
Keyword(s):  
Dna Binding ◽  
P53 Protein ◽  
Expression System ◽  
Gel Filtration ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Cross Linking ◽  
Mobility Shift ◽  
Dna Complexes ◽  
P53 Antibodies

In this study, we expressed and purified the recombinant baculovirus 373 K/E p53 protein in a baculovirus expression system to characterize this mutant and compare it with wild type p53. Gel- filtration chromatography and chemical cross-linking experiments indicated that purified recombinant baculovirus 373 K/E p53 protein assembles into multimeric forms ranging from tetramers to polymers. Gel-mobility shift assays and protein-DNA cross-linking studies demonstrated that the recombinant protein binds, to a consensus DNA target as a dimer but that additional p53 mutant molecules may then associate with the preformed p53-dimer-DNA complexes to form a larger p53_DNA complexes. These observations suggest that the p53 mutant tetramers and polymers that forms the minimal p53 mutant complex in solution dissociated upon DNA binding to form p53 mutant dimmer DNA complexes. The DNA binding activity of this mutant was then investigated using electrophoretic mobility shift assays as well as supershift assay with anti-p53 antibodies. Binding of the anti-p53 antibody PAb421to the oligomerization promoting domain on p53 stimulated the sequential formation of both the p53_dimer DNA and larger p53-DNA complexes

scholarly journals Expression of functional G protein beta gamma dimers of defined subunit composition using a baculovirus expression system.

1992 ◽  
Vol 267 (19) ◽  
pp. 13123-13126 ◽  
Author(s):  
S.G. Graber ◽  
R.A. Figler ◽  
V.K. Kalman-Maltese ◽  
J.D. Robishaw ◽  
J.C. Garrison
Keyword(s):  
G Protein ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Subunit Composition ◽  
Baculovirus Expression

scholarly journals Gastric Acid-Related Diseases: Focus on Esomeprazole

2010 ◽  
Vol 2 ◽  
pp. CMT.S4500
Author(s):  
B. Al-Judaibi ◽  
N. Chande ◽  
G.K. Dresser ◽  
N. Sultan ◽  
J.C. Gregor
Keyword(s):  
Duodenal Ulcer ◽  
Helicobacter Pylori ◽  
Symptom Relief ◽  
Competitive Inhibitor ◽  
Gastric Ph ◽  
Disease Treatment ◽  
Ulcer Disease ◽  
Zollinger Ellison Syndrome ◽  
Prevention And Treatment ◽  
Inflammatory Drugs

Esomeprazole (S-omeprazole) is a single optical enantiomer proton-pump inhibitor (PPI) approved for the management of gastro-oesophageal reflux disease, the prevention and treatment of Non-Steroidal Anti-Inflammatory Drugs (NSAID) associated gastric ulcer disease, treatment of duodenal ulcer disease associated with Helicobacter pylori infection, and the treatment of Zollinger-Ellison syndrome. Esomeprazole has been shown to be safe and effective during pregnancy and was introduced to the market in 2001. PPI therapy may interact with clopidogrel by cytocrome 2C19. Clopidogrel is a prodrug which is partially activated by cytochrome 2C19 and esomeprazole is a competitive inhibitor of 2C19. Esomeprazole is more effective than other PPIs in controlling esophageal and gastric pH, but efficacy in symptom relief is less clear.

scholarly journals Production, processing and partial purification of functional G protein βγ subunits in baculovirus-infected insect cells

1992 ◽  
Vol 286 (3) ◽  
pp. 677-680 ◽  
Author(s):  
J D Robishaw ◽  
V K Kalman ◽  
K L Proulx
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Insect Cells ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Partial Purification ◽  
Baculovirus Expression ◽  
Gamma Subunits ◽  
Infected Insect ◽  
Beta 2

As a result of the inability to resolve the heterogeneous mixture of G protein beta gamma subunits present in tissues, it has not been possible to compare different beta gamma subunits of the G proteins in terms of their proposed roles in receptor-effector coupling. This study was undertaken to establish the utility of the baculovirus expression system in producing homogeneous beta gamma subunits of defined composition for the comparative analysis of these subunits in reconstitution systems. In this study we report the expression, and appropriate post-translational processing, of recombinant beta 2, gamma 2 and gamma 3 subunits. In addition, we show that the recombinant beta gamma subunits can be readily purified, and can functionally interact with the alpha subunits of the G proteins.

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