Reduced phospholipase A2 activity is not accompanied by reduced arachidonic acid release

Arachidonic acid release in cells highly over expressing cytosolic phospholipase A2 has been attributed to mitogen-activated protein kinase phosphorylation of cytosolic phospholipase A2 on serine-505. To investigate the role of cytosolic phospholipase A2 in cellular physiology, we attempted to inhibit cytosolic phospholipase A2 in the intact cell employing an antisense RNA strategy. Swiss 3T3 cells were stably transfected with an antisense cytosolic phospholipase A2 expression vector. A clone of cells with reduced immunodetectable cytosolic phospholipase A2, compared to a vector transfected cell line, was identified by Western blotting and a corresponding decrease in phospholipase A2 activity was confirmed by enzymatic assay in cell free extracts. However, arachidonic acid release from intact cells in response to agonists was not different between antisense and control cell lines. Thus, arachidonic acid release in intact cells with decreased cytosolic phospholipase A2 activity is likely to be modulated by rate limiting factors that are extrinsic to cytosolic phospholipase A2.

Proteomic analysis reveals oxidative stress response as the main adaptative physiological mechanism in cows under different production systems

Three groups of cows representing three ranges of welfare in the production system were included in the study: two groups of Bruna dels Pirineus beef cattle maintained under different management systems (good and semiferal conditions) and a group of Alberes cows, a breed that lives in the mountains (hardest conditions).

In order to identify new stress/welfare biomarkers, serum from Bruna cows living in both environments was subjected to DIGE labelling, two-dimensional electrophoresis and MALDI-MS or ion trap MS. Identification was achieved for 15 proteins, which mainly belonged to three biological functions, the oxidative stress pathway (glutathione peroxidase (GPx) and paraoxonase (PON-1)), the acute phase protein family (Heremans Schmid glycoprotein alpha2 (α2-HSG)) and the complement system.

Biological validation included the Alberes breed. GPx and PON-1 were validated by an enzymatic assay and found to be higher and lower, respectively, in cows living in hard conditions. α2-HSG was validated by ELISA and found to be reduced in hard conditions. Other biomarkers of the redox status were also altered by living conditions: protein carbonyl content, superoxide dismutase (SOD) and glutathione reductase (GR).

Our results show that changes in the redox system are the main adaptation of cows living in challenging environmental conditions. This article is part of a Special Issue entitled: “Farm animal proteomics”.

LIQUID-CHROMATOGRAPHY THERMOSPRAY MASS-SPECTROMETRIC ASSAY FOR TRENBOLONE IN BOVINE BILE AND FECES

A liquid chromatography-thermospray mass spectrometric assay was developed and validated to confirm the presence of illegal residues of the synthetic androgenic growth promoter, trenbolone acetate, in cattle. The assay was specific for 17alpha-trenbolone, the major bovine metabolite of trenbolone acetate. Methods were developed for the determination of 17alpha-trenbolone in both bile and faeces, the most appropriate matrices for the control of trenbolone acetate abuse. The clean-up.procedure developed relied on enzymatic hydrolysis, followed by sequential liquid-liquid and liquid-solid extraction. The extracts were then subjected to immunoaffinity chromatography. 17alpha-Trenbolone was detected by selected ion monitoring at m/z 271 using positive ion thermospray ionisation. The limit of detection was approximately 0.5 ng/g in faeces and 0.5 ng/ml in bile.

An assay for angiotensin-converting enzyme using capillary zone electrophoresis

A sensitive and rapid method was developed for angiotensin-converting enzyme (ACE) activity determination by capillary zone electrophoresis. Hippuryl-View the MathML source-histidyl-View the MathML source-leucine, a synthetic tripeptide, was used as the ACE-specific substrate. Capillary zone electrophoresis was employed to separate the products of the enzymatic reaction and the ACE activity was determined by quantification of hippuric acid, a result of the enzymatic reaction on the tripeptide. The capillary electrophoresis was performed in a 27 cm × 75 μm i.d. fused-silica capillary using 200 mM boric acid–borate buffer (pH 9.0) as a run buffer with an applied voltage of 8.1 kV at a capillary temperature of 23°C. The electrophoresis was monitored at 228 nm. Each electrophoretic run requires only a nanoliter of the enzymatic reactant solution, at only 6 min, rendering a powerful tool for the ACE assay.

Development of a rapid colorimetric time-kill assay for determining the in vitro activity of ceftazidime and tobramycin in combination against Pseudomonas aeruginosa

It is standard clinical practice to use a combination of two or more antimicrobial agents to treat an infection caused by Pseudonionas aeruginosa. The antibiotic combinations are usually selected empirically with methods to determine the antimicrobial effect of the combination such as the time-kill assay rarely used as they are time-consuming and labour intensive to perforin. Here, we report a modified time-kill assay, based on the reduction of the tetrazolium salt, 2,3-bis[2-methyloxy-4-nitro-5-sulfopheny1]-2H-tetrazolium-5-carboxanilide (XTT), that allows simple, inexpensive and more rapid determination of the in vitro activity of antibiotic combinations against P aeruginosa. The assay was used to determine the in vitro activity of ceftazidime and tobramycin in combination against P. aertiginosa isolates from cystic fibrosis patients and the results obtained compared with those from conventional viable count time-kill assays. There was good agreement in interpretation of results obtained by the XTT and conventional viable count assays, with similar growth curves apparent and the most effective concentration combinations determined by both methods identical for all isolates tested. The XTT assay clearly indicated whether an antibiotic combination had a synergistic, indifferent or antagonistic effect and could, therefore, provide a useful method for rapidly determining the activity of a large number of antibiotic combinations against clinical isolates. (C) 2004 Elsevier B.V. All rights reserved.

A comparative assessment of potential components of partial disease resistance to Fusarium head blight detected in a detached leaf assay of wheat, barley and oats.

The relative resistance of 15 winter barley, three winter wheat and three winter oat cultivars on the UK recommended list 2003 and two spring wheat cultivars on the Irish 2003 recommended list were evaluated using Microdochium nivale in detached leaf assays to further understand components of partial disease resistance (PDR) and Fusarium head blight (FHB) resistance across cereal species. Barley cultivars showed incubation periods comparable to, and latent periods longer than the most FHB resistant Irish and UK wheat cultivars evaluated. In addition, lesions on barley differed from those on wheat as they were not visibly chlorotic when placed over a light box until sporulation occurred, in contrast to wheat cultivars where chlorosis of the infected area occurred when lesions first developed. The pattern of delayed chlorosis of the infected leaf tissue and longer latent periods indicate that resistances are expressed in barley after the incubation period is observed, and that these temporarily arrest the development of mycelium and sporulation. Incubation periods were longer for oats compared to barley or wheat cultivars. However, oat cultivars differed from both wheat and barley in that mycelial growth was observed before obvious tissue damage was detected under macroscopic examination, indicating tolerance of infection rather than inhibition of pathogen development, and morphology of sporodochia differed, appearing less well developed and being much less abundant. Longer latent periods have previously been related to greater FHB resistance in wheat. The present results suggest the longer latent periods of barley and oat cultivars, than wheat, are likely to play a role in overall FHB resistance if under the same genetic control as PDR components expressed in the head. However the limited range of incubation and latent periods observed within barley and oat cultivars evaluated was in contrast with wheat where incubation and latent periods were shorter and more variable among genotypes. The significance of the various combinations of PDR components detected in the detached leaf assay as components of FHB resistance in each crop requires further investigation, particularly with regard to the apparent tolerance of infection in oats and necrosis in barley, after the incubation period is observed, associated with retardation of mycelial growth and sporulation.