Equine trypsin: purification and development of a radio-immunoassay

Shock is accompanied by generalised splanchnic hypoperfusion, and splanchnic organs like the pancreas can be damaged, as shown in animal experimental models and in humans, by the presence of high plasma concentrations of trypsin and other pancreatic enzymes. In order to design a radioimmunoassay technique (RIA) for the measurement of equine trypsin-like immunoreactivity (TLI) in biological fluids, trypsin was purified (with purity greater than or equal to 96 %) from the equine pancreas by extraction in an acid medium, ammonium sulfate precipitations, gel filtration chromatography and, after activation of trypsinogen into trypsin, affinity chromatography. Gel polyacrylamide electrophoresis showed a monomeric enzyme with a molecular weight of 27 kDa. The purified equine trypsin served for the immunisation of rabbits in order to obtain a specific antiserum, and the labelled antigen was prepared by iodination of equine trypsin with I-125. The RIA was based on the binding of the antigen to the antibody followed by the separation of the antigen-antibody complex by immunoprecipitation in the presence of sheep anti-rabbit gammaglobulins and the assay of the radioactivity in the precipitate. The RIA showed good sensitivity, specificity, precision, accuracy and reproducibility. The reference mean value of TLI in the plasma of healthy horses (n = 20) was 30.01 +/- 6.84 ng/mL (upper confidence limit 50.52 ng/mL; p < 0.01). Three horses with non strangulating intestinal obstruction without shock showed TLI values within normal limits whereas 5 of 7 horses with strangulation obstruction showed TLI levels above the upper confidence limit. Further studies using the RIA and the enzymatic assay should be performed in order to confirm the role of the pancreas in equine intestinal obstruction

Enhancement of prostacyclin generation by cicletanine (an in vivo investigation).

The effect of cicletanine, an antihypertensive drug, was studied in two in vivo models. It was demonstrated that the drug markedly affected the response of intravenously administered arachidonic acid in the rabbit. In this model a marked increase of 6-oxo-PGF1 alpha was observed when challenged by the intravenous injection of 50 micrograms/kg b.w. of arachidonic acid. In the rat model used for the study of platelet-vessel wall interaction, it was demonstrated that the inhibition of prostacyclin synthetase could be offset by cicletanine. These results indicate that the drug modulates the generation of prostacyclin and as such is capable of affecting the peripheral resistances which determine the level of the blood pressure.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Plasma trypsin level in horses suffering from acute intestinal obstruction

peer reviewedGastrointestinal disorders in horses leading to endotoxic shock could have further consequences on other splanchnic organs such as the pancreas, as can be seen in humans suffering from septic shock. In this study, the range of enzymatically active trypsin (EAT) in healthy horses was established and is similar to the range observed in healthy humans. EAT values were determined in horses with acute abdominal crises on admission as well as during anaesthesia and in the postoperative phase. A significant increase in plasma EAT was found in 59% of the horses with surgical colic when compared to our established reference range. Significantly higher values were found in severe shock cases. When separated in groups according to the duration of colic before referral, significantly higher EAT values were observed in the non-survivor group compared to the survivor group of colics of short duration. EAT plasma values increased significantly during the postoperative phase, and were significantly higher in small intestine obstructions than in large bowel disorders. In human medicine, hypovolaemic or septic shock patients show an increase in pancreatic proteases. Splanchnic hypoperfusion during shock could lead to pancreatic damage resulting in trypsin liberation into the peritoneal space and an increase in plasma levels. Trypsin is able to activate inflammatory cascades and leucocytes and could play a role in multiple organ failure. Further studies are needed to evaluate the implications of changes in plasma trypsin in the disease process of equine acute abdomen and to demonstrate possible pancreatic damage

In vitro evaluation of glutathione peroxidase (GPx)-like activity and antioxidant properties of some Ebselen analogues

Four analogues of Ebselen were synthesized and their glutathione peroxidase activity and antioxidant property evaluated and compared to Ebselen. Among the studied compounds, only diselenide [3] exhibited both glutathione peroxidase activity and radical-scavenging capability. Compounds [3] and [4] showed a strong inhibitory effect (53% and 43%, respectively) on the lipid peroxidation of linoleic acid compared to Ebselen and selenide derivatives ([1] and [2]) which were less active (28%, 26% and 18% inhibition, respectively). A concentration-dependent inhibitory effect was also found in the model of the formation of ABTS*+ radical cation: 65% and 89% inhibition for compound [3] at 10(-4) M and 5 x 10(-5) M, respectively, and 68% and 90% for compound [4], compared to 14% and 52% inhibition for Ebselen and the diselenides [1] and [2] (29%, 46% and 45%, 68%, respectively). By EPR spin trapping technique, the following inhibitory profile of the Ebselen analogues was observed towards the formation of thiyl radicals: Ebselen = [3]>[1]>[2]>[4]. Studies with compound [3] are in progress on oxidative stress cell models

Resveratrol and curcumin reduce the respiratory burst of Chlamydia-primed THP-1 cells

The intracellular bacterium Chlamydia pneumoniae is involved in the inflammation process of atherosclerosis. We previously demonstrated that C. pneumonia infected monocytes (THP-1 cells) responded to stimulation by an increased respiratory burst linked to an increased NADPH oxidase (NOX) activity. We now tested agents acting on the assembly of the NOX subunits or on protein kinase C, a trigger of NOX activity. Apocynin, resveratrol, rutin, quercetin, curcumin, and tocopherols were tested. The cells were pre-incubated with Chlamydia and the agent for 19 h, and then stimulated with phorbol myristate acetate. The NOX activity was monitored by measuring the hydrogen peroxide production. Resveratrol and curcumin (10(-4)-10(-6) M) were better inhibitors than apocynin. alpha-Tocopherol was inactive, and gamma-tocopherol inhibitor at 10(-4) M only. Quercetin was inactive, and rutin a moderate but significant inhibitor. The inhibition by resveratrol was increased by 10(-6) M rutin or quercetin. Resveratrol and curcumin thus appeared to be interesting for atherosclerosis treatment

Propofol reacts with peroxynitrite to form phenoxyl radicals. Demonstration by ESR

Peroxynitrite (ONOO-), resulting from the reaction of nitric oxide with superoxide anion, is a powerful oxidant produced in activated macrophages, during ischemia-reperfusion processes as well as in neurodegenerative disorders. This study investigated the reaction of the anesthetic agent propofol (PPF) with ONOO-, using electron spin resonance (ESR) and UV-visible spectrometry. Peroxynitrite was synthetized either from acidified hydrogen peroxide and nitrite, or from sodium azide and ozone. The addition of ONOO- to PPF in alkaline solution (pH 12) allowed to detect a, short lifetime, ESR signal corresponding to a phenoxyl radical. This finding was confirmed by a UV-visible study, resulting in the appearance of 427 nm peak and the disappearance of the peak located at 239 nm. The 291 nm peak remained unchanged. The identification of the end-product of the reaction of PPF with ONOO- needs further investigations

How hyperhydric shoots try to survive

peer reviewedaudience: researche

Potential Antioxidant properties of Aceclofenac and its Metabolites: Investigation on an in vitro model

Introduction: Recent studies have shown that some steroidal anti-inflammatory drugs (NSAIDs) could exert their actions by multifactorial processes. Among them, the potential antioxidant activity of certain NSAIDs towards various reactive oxygen species (ROS) is often suggested and could have pharmacological relevance. Objective: This study was designed to assess the potential antioxidant properties (IC50 values) of aceclofenac and its metabolites (4’OH-aceclofenanc and diclofenac) on three different systems of ROS production, using chemiluminescence (CL) technique with luminal and electron spin resonance (ESR) spin trapping. Material and Methods: Isolated human PMNs (1x106 cells) were activated with 5x10-7 M PMA in the presence of luminal (CL assays) with or without drug addition. For spin trapping experiments, 100 mM DMPO, a radical trapping agent, was added to the reaction milieu containing 6x106 cells/ml. For free-cell experiments, the Fenton’s reagent was used for generation of ·OH and xanthine/xanthine-oxidase system for O2-radicals. The NaOCl-induced CL, amplified by luminal, was used to test the drug effects on HOCl. Results: On the model of PMA-activated PMNs, 4’OH-aceclofenac exhibited the best antioxidant profile (IC50 = 10 µM) while the effect of the parent drug was less pronounced (IC50 = 100 µM). Diclofenac did not inhibit CL response even at the high dose of 1 mM. Quite similar results were obtained on the NaOCl-induced chemiluminescence, where the efficacy of the drug was as follows: 4’HO-ACE (25 µM) > ACE (1 mM) > DICLO (no effect at 1 mM). By ESR technique, 4’HO-ACE also showed an inhibitory effect (501 µM) on the ROS production by PMA-activated PMN as well as on the ·OH production, while ACE (IC50 = 100 µM) was less efficient and DICLO (IC50 = 1 mM) without significant effect. These findings indicate that beside its anti-inflammatory effects, aceclofenac acts as an antioxidant, at least in part, by the way of its metabolite especially 4’HO-ACE

Are COX-2 Inhibitors Active on Intracellular Oxidative Processes? A Study on In Vitro and Cellular Models

In the last years, there has been an increasing interest of using cyclooxygenase-2 (COX-2) inhibitors to treat the inflammatory pain and chronic inflammatory diseases such as osteoarthritis and rheumatoid arthritis. The beneficial effects were to avoid the secondary adverse effects such as bleeding and gastric irritation, generally observed with aspirin and conventional NSAIDs. COX-1 is constitutively expressed in most tissues and involved in the regulation of normal homeostatic functions, while COX-2 is not detected in most tissues but induced by inflammatory stimuli. These outcomes motivated the commercial development of selective COX-2 inhibitors. Recent data suggested that the COX-2 enzyme can be expressed within atherosclerotic lesions and could play a crucial role in various types of cancers, by the way of its activity on the ROS production, gene transcription and prostaglandin (PGE2) production. Consequently, the COX-2 enzyme has become a real target for the study of various classes of compounds and specially the possible additional properties of COX-2 inhibitors. We and other groups have already investigated the pro or antioxidant profile of conventional NSAIDs and some COX-2 inhibitors. With the recent withdrawal of two compounds of the coxib’s family (rofecoxib and celecoxib), for adverse cardiovascular events, concerns regarding the safety of all COX-2 inhibitors have been raised. To answer to these concerns, different approaches were developed by studying on in vitro models, the potential inhibiting-or-stimulating activities on oxidative phenomena of new drugs with already recognized therapeutic effects. Preliminary data obtained with COX-2 inhibitors showed a moderate inhibiting effect on the intracellular oxidant processes and others a stimulating activity. New hypotheses for the treatment of inflammation are now suggested for compounds like nimesulide and its analogous, which are selective towards COX-2 with little activity on COX-1. Here, we reported the in vitro effects of some Cox-2 inhibitors, in comparison with traditional drugs (ibuprofen, diclofenac and aceclofenac) by using two cellular models: a human lung type II alveolar cell line (A549) and a human promonocyte cell line (THP-1). The direct interactions between the drugs and ROS were also investigated in cell-free systems

Oxidant activity of rabbit synoviocytes (HIG-82) demonstrated by oxymetry and ethylene production.

We are interested in a possible role of synoviocytes in the ROS production implicated in osteoarthritis, therefore we studied the response of a rabbit synoviocyte cell line (HIG-82) to variable oxygen tensions and the oxidant activity of these cells in response to stimuli. Synoviocytes were cultured at 5 and 21 % O2, their O2 consumption (cellular respiration, monitored with Clark electrode) was measured at 21% O2 and after anoxia, before and after stimulation with phorbol myristate acetate (PMA), and their oxidant response to PMA stimulation was quantified by measuring ethylene (gas chromatography) released when the substrate, alpha-keto-gamma-methylbutyric acid, is oxidised by the ROS produced by the cells. Cell growth was faster at 21 % O2 than at 5% O2, and microscopic observation revealed 2 cell populations: a few small round cells in suspension and many adherent cells. By oxymetry, we observed that a 106 synoviocytes suspension in 2 ml completely consumed O2 within 15 min, that anoxia (7 min) slightly slowed the respiration rate down and that PMA stimulation increased O2 consumption (150 % increase). The oxidant activity (ethylene production) of the cells was stimulated by PMA in a dose-dependent manner (10-9 to 10-7M) but the cell response was highly variable (from 150 to 1500 % increase) and was largely reduced by diphenyliodonium, an inhibitor of NADPH-oxidase and NO-synthase. The capacity to produce free radical species was confirmed for the small round cells by detection of an electron paramagnetic resonance (EPR) signal after stimulation. These results thus demonstrate a sensibility to O2 and an oxidant activity of synoviocytes at least related to ROS production by NADPH-oxidase activity