Validation of protein carbonyl measurement: a multi-centre study.

Protein carbonyls are widely analysed as a measure of protein oxidation. Several different methods exist for their determination. A previous study had described orders of magnitude variance that existed when protein carbonyls were analysed in a single laboratory by ELISA using different commercial kits. We have further explored the potential causes of variance in carbonyl analysis in a ring study. A soluble protein fraction was prepared from rat liver and exposed to 0, 5 and 15min of UV irradiation. Lyophilised preparations were distributed to six different laboratories that routinely undertook protein carbonyl analysis across Europe. ELISA and Western blotting techniques detected an increase in protein carbonyl formation between 0 and 5min of UV irradiation irrespective of method used. After irradiation for 15min, less oxidation was detected by half of the laboratories than after 5min irradiation. Three of the four ELISA carbonyl results fell within 95% confidence intervals. Likely errors in calculating absolute carbonyl values may be attributed to differences in standardisation. Out of up to 88 proteins identified as containing carbonyl groups after tryptic cleavage of irradiated and control liver proteins, only seven were common in all three liver preparations. Lysine and arginine residues modified by carbonyls are likely to be resistant to tryptic proteolysis. Use of a cocktail of proteases may increase the recovery of oxidised peptides. In conclusion, standardisation is critical for carbonyl analysis and heavily oxidised proteins may not be effectively analysed by any existing technique.

Developing accurate models of the human airways.

OBJECTIVES: Particle delivery to the airways is an attractive prospect for many potential therapeutics, including vaccines. Developing strategies for inhalation of particles provides a targeted, controlled and non-invasive delivery route but, as with all novel therapeutics, in vitro and in vivo testing are needed prior to clinical use. Whilst advanced vaccine testing demands the use of animal models to address safety issues, the production of robust in vitro cellular models would take account of the ethical framework known as the 3Rs (Replacement, Reduction and Refinement of animal use), by permitting initial screening of potential candidates prior to animal use. There is thus a need for relevant, realistic in vitro models of the human airways. KEY FINDINGS: Our laboratory has designed and characterised a multi-cellular model of human airways that takes account of the conditions in the airways and recapitulates many salient features, including the epithelial barrier and mucus secretion. SUMMARY: Our human pulmonary models recreate many of the obstacles to successful pulmonary delivery of particles and therefore represent a valid test platform for screening compounds and delivery systems.

Redox regulation of protein damage in plasma.

The presence and concentrations of modified proteins circulating in plasma depend on rates of protein synthesis, modification and clearance. In early studies, the proteins most frequently analysed for damage were those which were more abundant in plasma (e.g. albumin and immunoglobulins) which exist at up to 10 orders of magnitude higher concentrations than other plasma proteins e.g. cytokines. However, advances in analytical techniques using mass spectrometry and immuno-affinity purification methods, have facilitated analysis of less abundant, modified proteins and the nature of modifications at specific sites is now being characterised. The damaging reactive species that cause protein modifications in plasma principally arise from reactive oxygen species (ROS) produced by NADPH oxidases (NOX), nitric oxide synthases (NOS) and oxygenase activities; reactive nitrogen species (RNS) from myeloperoxidase (MPO) and NOS activities; and hypochlorous acid from MPO. Secondary damage to proteins may be caused by oxidized lipids and glucose autooxidation. In this review, we focus on redox regulatory control of those enzymes and processes which control protein maturation during synthesis, produce reactive species, repair and remove damaged plasma proteins. We have highlighted the potential for alterations in the extracellular redox compartment to regulate intracellular redox state and, conversely, for intracellular oxidative stress to alter the cellular secretome and composition of extracellular vesicles. Through secreted, redox-active regulatory molecules, changes in redox state may be transmitted to distant sites.

Use of multiple drains after mastectomy is associated with more patient discomfort and longer postoperative stay.

BACKGROUND: Seromas constitute a common complication following surgery for breast cancer, and closed drainage is used routinely to reduce its incidence. The aim of this study was to evaluate the influence of number of drains on patient discomfort, seroma formation, and hospital stay during the immediate postoperative period after mastectomy for breast cancer. PATIENTS AND METHODS: Based on a retrospective review of our clinical database, 110 consecutive patients from January 2004 through January 2006 who had undergone a mastectomy and axillary clearance for breast cancer were sent a simple postal questionnaire for collection of data. RESULTS: A total of 70 patients responded (all women; mean age, 69.4 +/- 11.4 years). Twenty-seven patients (38.57%) had 3 drains implanted unilaterally, 24 (34.28%) had 2, and 19 (27.14%) had 1 drain. They were divided into 2 groups: the first group with 1 drain (19 patients) and the other with 2 or 3 drains (51 patients). Median postoperative hospital stay was 2 days (range, 1-8 days); patients with 1 drain had a significantly shorter postoperative hospital stay (median, 2 days [range, 1-4 days] vs. 2 days [range, 1-8 days]; Mann-Whitney U test, P = .02). A total of 15 patients (21.43%) complained of a seroma. There was no difference in seroma rates between groups. Patients who had a single drain implanted had a significantly lower rate of discomfort (median, 2 [range, 1-5] vs. 3 [range, 1-7]; Mann-Whitney U test; P = .04). CONCLUSION: The number of drains used after a mastectomy for breast cancer did not significantly affect the rate or amount of seromas in this study, but the use of a single drain after mastectomy was significantly associated with less discomfort and shorter postoperative hospital stay.

In vivo vitamin C supplementation increases phosphoinositol transfer protein expression in peripheral blood mononuclear cells from healthy individuals.

Ascorbate can act as both a reducing and oxidising agent in vitro depending on its environment. It can modulate the intracellular redox environment of cells and therefore is predicted to modulate thiol-dependent cell signalling and gene expression pathways. Using proteomic analysis of vitamin C-treated T cells in vitro, we have previously reported changes in expression of five functional protein groups associated with signalling, carbohydrate metabolism, apoptosis, transcription and immune function. The increased expression of the signalling molecule phosphatidylinositol transfer protein (PITP) was also confirmed using Western blotting. Herein, we have compared protein changes elicited by ascorbate in vitro, with the effect of ascorbate on plasma potassium levels, on peripheral blood mononuclear cell (PBMC) apoptosis and PITP expression, in patients supplemented with vitamin C (0-2 g/d) for up to 10 weeks to investigate whether in vitro model systems are predictive of in vivo effects. PITP varied in expression widely between subjects at all time-points analysed but was increased by supplementation with 2 g ascorbate/d after 5 and 10 weeks. No effects on plasma potassium levels were observed in supplemented subjects despite a reduction of K+ channel proteins in ascorbate-treated T cells in vitro. Similarly, no effect of vitamin C supplementation on PBMC apoptosis was observed, whilst ascorbate decreased expression of caspase 3 recruitment domain protein in vitro. These data provide one of the first demonstrations that proteomics may be valuable in developing predictive markers of nutrient effects in vivo and may identify novel pathways for studying mechanisms of action in vivo.