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Advanced oxidation protein products (AOPP) inhibit bovine neutrophil activity in vitro.
No full textAdvanced oxidation protein products (AOPP) are novel markers of protein oxidation that were first described and characterized in plasma of uremic patients. Their formation is mainly dependent from chlorinated agents related to neutrophil and monocyte activation, thus being also considered a good indicator of the inflammatory response. In addition, AOPP can activate both human neutrophils and monocytes in vitro. The observation that AOPP were higher in plasma of cows affected by late embryonic loss raised our interest in the biological role of AOPP in dairy cattle. The aim of this work was to study the effects of AOPP on bovine neutrophil activation in vitro. Standard AOPP-BSA was produced by oxidizing bovine serum albumin (BSA) with hypochlorous acid. Bovine neutrophils were isolated by Ficoll gradient from peripheral blood obtained from healthy dairy heifers aged 10-13 months (N=10). The effects of either BSA or AOPP-BSA in the culture medium were tested in neutrophils either stimulated or unstimulated with phorbol myristate acetate (PMA,1 ug/ml) over a 3-hour period. PMA-stimulated and unstimulated neutrophils incubated without proteins in the culture medium were used as the control. Myeloperoxidase (MPO)-dependent formation of ROS was measured by chemiluminescence using luminol as substrate during the whole incubation period at 5-minute intervals, and results were analysed as the area under the curve (AUC). Parallel experiments were conducted to assess cell viability using both LDH and MTT assays. AOPP-BSA failed to trigger any response in the unstimulated bovine neutrophils. The ROS generation in PMA-stimulated was significantly lower (P<0.05) in neutrophils cultured with AOPP-BSA (AUC: 4860±35 CPS*min) in comparison to those cultured with both BSA (AUC: 14528±53) and without protein in the culture medium (AUC:7342±48 CPS*min). Cell viability, measured by the MTT assay, was significantly lower in PMA-stimulated neutrophils incubated with AOPP-BSA (P<0.05). Potential effects of BSA and AOPP-BSA on luminol reaction were studied in a cell-free system, by activating the MPO enzyme with increasing concentration of hydrogen peroxide in presence of either BSA or AOPP-BSA. The AUC observed in presence of BSA (13004±70 CPS*min) was not significantly different from that measured in presence of AOPP-BSA (14993±65 CPS*min).The results of this study suggested that AOPP-BSA inhibits the response of bovine neutrophil to PMA activation possibly by affecting cell viability. Moreover, it is noteworthy to underline that the bovine neutrophil response to AOPP-BSA observed in this study was totally different than that reported for the human neutrophils
Characterization of advanced oxidation protein products (AOPP) production by bovine neutrophils
No full textAdvanced oxidation protein products (AOPP) are novel markers of protein oxidation first characterized in uremic patients and related to hypochlorous acid (HOCl) production by activated neutrophils. To establish how oxidants contribute to the pathology of inflammation it is important to identify physiologically relevant targets of oxidation and to develop specific biomarkers to quantify the oxidative damage. Here, we characterized the relative reactivity of HOCl (0-25 mM) and cumene hydroperoxyde (CuOOH; 0-100 mM) with bovine serum albumin (BSA), bovine gamma globulins (bG) and whole bovine plasma. HOCl concentrations higher than 25 mM induced a massive protein denaturation, bG being the most susceptible. AOPP levels significantly increased in a dose-dependent manner in both HOCl-treated BSA and bG (P<0.01), whereas AOPP levels in plasma were significantly higher than the unexposed control only at 25mM HOCl concentration (P<0.05). The AOPP yield was lower when CuOOH was the oxidant agent. The AOPP levels were significantly higher (P<0.01) than the unexposed control when BSA was treated with 10 mM CuOOH, and bG and plasma were treated with 100 mM CuOOH. A significant (P<0.05) dose-dependent dityrosine and carbonyl group formation was observed in both BSA and bG exposed to HOCl. A significant dose-dependent dityrosine production was induced by CuOOH in BSA (P<0.01), bG (P<0.001) and whole plasma (P<0.05). Strong positive correlations were observed between AOPP and dityrosine in HOCl-treated BSA (r=0.977, P<0.001), bG (r=0.962, P<0.001) and plasma (r=0.811, P<0.01). Correlations between AOPP and dityrosine were weaker CuOOH-treated BSA (r=0.572, P<0.05), bG (r=0.812, P<0.01) and plasma (r=0.672, P<0.05)
Modification of exploratory behaviour and LH release in female mice induced by exposure to male MUP.
No full textB-casokinin 10 (YGG peptide) modulates proliferation and cytokine expression in bovine peripheral blood lymphocytes.
No full textPeptides contained in milk and fermented milk as a potential source of regulators of bioactivities.
No full textCortisol and dehydroepiandrosterone levels in dogs (Canis familiaris) of different gender and age.
No full textProduction and characterization of the advanced oxidation protein products (AOPP) in bovine plasma proteins exposed to either hypochlorous acid or cumene hydroperoxyde.
No full textAdvanced oxidation protein products (AOPP) are novel markers of protein oxidation described in plasma of uremic patients and related to neutrophil activation. The observation that AOPP were higher in plasma of cows affected by late embryonic loss raised our interest in their biological role in cattle. Chlorinating agents, such as HOCl, induce protein modifications, which could be used as biomarkers of pathological events. In this study, we aimed to characterise bovine serum albumin (BSA; concentration) chlorination by bovine neutrophils, and to measure the AOPP production. BSA was incubated for 1, 2 and 3 hours with neutrophils isolated from healthy heifers (N=7), either activated or not activated with PMA. HOCl production was significantly higher in PMA-activated (400.5 ± 36.3 moles*hour) than in not-activated cells (126.8 ± 19.2 moles*hour; P<0.05). In parallel experiments (N=5), BSA was exposed to the same HOCl concentration observed in activated neutrophils over the same time-intervals. Levels of chloramines, dityrosines and carbonyls were significantly higher (P<0.05) when BSA was incubated with PMA-activated neutrophils or HOCl. Not-activated neutrophils produced a tangible amount of chloramines and dityrosines, likely attributable to basal HOCl release. PMA-activated cells generated significantly less (P<0.05) di-tyrosines (16.9 ± 1.1 nmol/mg*hour) and carbonyls (852,845 ± 299,271 OD unit*hour) than HOCl alone (di-tyrosines: 30.2 ± 1.5 nmol/mg*hour; carbonyls: 2,020,282 ± 726,031 OD unit*hour). In conclusion, bovine neutrophils produce enough HOCl to generate AOPP “in vitro”. However, the use of HOCl alone cannot fully mimic the complex events leading to BSA chlorination and AOPP production by activated neutrophils
Ontogenesi della risposta interrenalica allo stress nell'orata (Sparus aurata) e nell'ombrina (Umbrina cirrosa)
No full textEmbryonic mortality and plasma advanced oxidation protein products (AOPP) increase in dairy cows
No full textIntroduction
In dairy cows, embryonic mortality (EM) within day 42 post insemination brings fertilization rate about 50% and represents a major limitation to fertility (Mann and Lamming, 1999).
The causes of embryonic mortality are still poorly understood. Several papers indicate reactive oxygen species (ROS) and oxidative stress (OS) having a role in pathophysiology of mammal reproduction during (Fujii et al, 2005).
Accumulation of free radicals can occur in dairy cows exposed to metabolic stress or adverse environmental conditions (heat stress). If ROS generation exceeds body’s antioxidant production capacity, OS may develop (Castillo et al, 2005). In dairy cows, OS has been associated with several pathological conditions, which in turn may lead to a declined fertility (Miller et al, 1993).
Excessive ROS production can cause macromolecule damages (Fujii et al, 2005). ROS-mediated protein oxidation is complex and may generate multiple products. Oxidized proteins are often functionally inactive, can be more or, on the opposite, less susceptible to proteases leading to accumulation. They may activate the immune reaction and be the target of autoantibodies (Dean et al, 1997).
Advanced oxidation protein products (AOPP) are proteins damaged by OS formed mainly by chlorinated oxidants resulting from myeloperoxidase activity. In humans, they are responsible of induction of proinflammatory activities and cytokines, and increase in the circulation in some pathological situations. Moreover, they are referred to as markers of OS and neutrophil activation (Kalusova et al., 2005).
The aim of this work was to detect possible relationships between the artificial isemination (AI) outcome and AOPP as an indicator of protein oxidation to study the implication of protein oxidation in EM.
Materials and methods
This study was carried out in northern Italy over a ten months period. A total of 157 AIs were studied in 69 dairy cows. Blood samples were collected on the day of AI (d 0) and on days 15, 28, 35, 45, and 60 after AI.
Plasma concentrations of AOPP, glutathione (GSH), pregnancy-associated glycoprotein (PAG), malonyldehaldide (MDA), total proteins (TP), albumins (A), globulins (G) and urea were measured. Whey progesterone (P4) was analysed in samples taken every 3-4 days from day 15 post-partum to day 45 after each positive AI. Examination of whey P4 profiles was used to assess the beginning of ovarian activity.
The AI outcome was classified ex post in negative (AI-), positive (AI+) and EM. The diagnosis of EM was performed by the simultaneous examination of P4 in whey and PAG in maternal plasma.
Data were analysed using the general linear model procedures of SPSS (SPSS 15.0, 2005).
Results and discussion
The diagnosis of EM performed by the analysis of maternal plasma PAG was sensitive in detecting EM occurring after day 25 (Zoli et al., 1992). Therefore, return to oestrus before day 24 was classified as negative AI (AI-). In this study, 9 EM, 89 AI- and 49 AI+ were observed.
Plasma AOPP concentrations were significantly higher in EM (P<0.01; fig.1). On the contrary, plasma MDA and GSH were not affected by the AI outcome. However, AOPP showed weak but significant correlations with both MDA (R=0.323, P<0.001) and GSH (R=0.096, P<0.05), suggesting that AOPP may be used as a marker of OS. Although GSH is the most abundant non-protein thiol in mammalian cells and a good indicator of the blood oxidative scavenging capacity, it is also an important source of storage and transport of cysteine (Wu et al. 2004). Thus, total plasma GSH is likely more related to metabolic adaptations, such as sulphur amino acid sparing, rather than to oxidative stress and for this reason the relationship between AOPP and GSH is not very strong.
Plasma TP, A and G were not affected by the AI outcome, but they increased significantly during the post partum (P<0.01). Plasma urea was significantly affected by the AI outcome (P<0.01; fig.1), and it increased steadily throughout the post partum (P<0.05).
TP and A were significantly higher in cows beginning the ovarian activity before day 42 post partum (respectively P<0.05 and P<0.001).
In this study, we report for the first time the variations of AOPP in parallel with those of circulating proteins in the cow plasma. Plasma AOPP variations are independent from those of TP, A and G, suggesting a greater degree of protein oxidation in case of EM.
Since AOPP are considered marker of both OS and inflammation (Kalusova et al., 2005), it is possible that higher levels of AOPP observed in EM may reflect inflammatory events at uterine levels that can alter embryo development.
The data gathered in this study seem to indicate an association between EM and protein oxidation, which warrant further investigation.
Acknowledgements
Work supported by the Italian Ministry of University and Scientific Research (PRIN 2005).
References
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