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Relationship between AOPP (Advanced Oxidation Protein Products) and bovine neutrophils "in vitro": AOPP production by neutrophils and AOPP effects on neutrophils ROS production and viability
Full text linkThe aim of the present study was to evaluate the relationship between AOPP (Advanced oxidation protein products) and bovine neutrophils 'in vitro'. For this purpose AOPP were produced "in vitro" by oxidizing bovine serum albumin with HOCl (hypochlorous acid) and bovine neutrophils were isolated from whole blood of dairy cattles. AOPP-BSA were incubated with freshly isolated bovine neutrophils, unstimulated and stimulated with PMA a strong activator of the respiratory burst. Neutrophils ROS production and viability were measured by luminol-amplified chemiluminescence and by MTT and lactate dehydrogenase assays (LDH), respectively. Results obtained have shown that AOPP-BSA are able to reduce significantly ROS production of PMA stimulated neutrophil and their viability measured by MTT assay, no cell lysis was detected by LDH assay. On the basis of these results, our work has studied if AOPP are able to trigger apoptotic events. For this purpose, caspase 8-9-3 and DNA laddering were used as markers in order to discriminate between the 'intrinsic' and the 'extrinsic' pathway of apoptosis. The results obtained showed that un-stimulated bovine neutrophils incubated with AOPP-BSA show a higher but not significant production of active caspase 8 in comparison with the incubation with BSA. Also caspase 3 display an increase, but not significant, in un-stimulated neutrophils after 6 hours of incubation with AOPP-BSA, respects the incubation with BSA. No differences were obtained for caspase 9 and for DNA laddering. Therefore, in these experimental conditions is possible to conclude that the 'intrinsic' pathway of apoptosis was not involved in the reduced functionality of neutrophils or in their reduced viability, but bovine neutrophils incubated with AOPP-BSA seem to be "accompanied" to the early phases of the 'extrinsic' pathways of apoptosis.
In addition, the present work wanted to evaluate the capacity of triggered neutrophils to generate AOPP in vitro. BSA was incubated with un-stimulated and PMA-stimulated bovine neutrophils for 1-2-3 hours and the production of specific markers of protein oxidation such as AOPP, dityrosines and carbonyls was assessed. BSA incubated with stimulated neutrophils presents a significant higher level of AOPP and dityrosines respects the incubation with un-stimulated neutrophils. Carbonyls don't seem to be produced in these condition, at least at the beginning of the incubation. In parallel, BSA incubated with the same concentration of HOCl produced by PMA-stimulated neutrophils, for 1-2-3 hours, presents a higher level of AOPP, dityrosines and carbonyls. Therefore, it's possible to conclude that bovine neutrophils are able to oxidize BSA in vitro and generate chemical and structural modification such as AOPP and dityrosines, in the experimental condition used. However, carbonyls seem to be a non-specific indicator of neutrophils-mediated protein oxidation. The direct exposure of BSA to HOCl couldn't fully mimic the complex events leading to BSA oxidation and AOPP production by activated 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)
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
Production 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
Advanced oxidation protein products are generated by bovine neutrophils and inhibit free radical production in vitro
No full textDespite the recognised importance of oxidative stress in the health and immune function of dairy cows, protein oxidation markers have been poorly studied in this species. The current study aimed to characterise markers of protein oxidation generated by activated bovine neutrophils and investigate the biological effects of advanced oxidation protein products (AOPP) on bovine neutrophils. Markers of protein oxidation (AOPP, dityrosines and carbonyls) were measured in culture medium containing bovine serum albumin (BSA) exposed to neutrophils. The effect of AOPP-BSA on generation of reactive oxygen species (ROS) was assessed by chemiluminescence. Activation of caspases-3, -8 and -9 and the presence of DNA
laddering were used as apoptosis markers.
Greater amounts of AOPP were generated by phorbol myristate acetate (PMA)-activated than non-activated neutrophils (1.46 ± 0.13 vs. 0.75 ± 0.13 nmol/mg protein, respectively; P < 0.05). Activated neutrophils and hypochlorous acid generated slightly different patterns of oxidized protein markers. Exposure to AOPP-BSA did not stimulate ROS production. Activated neutrophils generated a lesser amount of ROS when incubated with AOPP-BSA (P < 0.001). Activation with PMA induced a loss of viable neutrophils
after 3 h, which was greater with AOPP-BSA incubation (P < 0.05). Detectable amounts of active caspases-3, -8 and -9 were found in nearly all samples but differences in caspase activation or DNA laddering were not observed comparing treatment groups. Apoptosis was unlikely to be responsible for the greater loss of PMA-activated neutrophils cultured in AOPP-BSA and it is possible that primary necrosis occurred.
The results suggest that accumulation of oxidized proteins at an inflammatory site might result in a progressive reduction of neutrophil viability
The inflammatory status but not the physiological phase affects plasma advanced oxidized protein product (AOPP) concentration in dairy cow.
No full textAdvanced oxidized 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 upon chlorinated agents related to neutrophil and monocyte activation, and is thus considered a good indicator of inflammatory response. In addition, AOPP can activate both human monocytes and neutrophils in vitro. The observation that AOPP were higher in plasma of cows affected by embryonic loss in late pregnancy raised our interest in the biological role of AOPP in dairy cattle. The aim of this work was to study the effect of the physiological phase on plasma AOPP concentrations and to assess any possible relationship between AOPP increase and inflammatory status in dairy cows. Plasma concentrations of AOPP, total proteins (TP), haptoglobin (Hp), and WBC counts were determined in 40 healthy dairy cows in different physiological phases (weeks 3-1 before parturition, and weeks 1, 4-6, 16-20 and more than 24 after parturition), and in 23 animals with inflammatory processes such as endometritis, mastitis and lameness. Unhealthy cows were first classified into groups of Acute Inflammation (11 subjects; clinical signs present for less than 3 days) and Chronic Inflammation (12 subjects; clinical signs observed for 3 or more days), respectively; both groups were compared with healthy cows in weeks 1 to 24 after parturition. In healthy animals, plasma Hp concentrations were significantly affected by the physiological phase, plasma levels being higher in week 1 after parturition (P<0.05), as opposed to AOPP. Plasma concentrations of Hp and the AOPP/TP ratio were significantly higher in the Acute Inflammation than in the Chronic Inflammation group (P<0.05), and healthy cows (P<0.001). Finally, animals were allocated on the basis of plasma Hp concentrations into Control (Hp<0.4 mg/ml) and Acute Phase (Hp≥0.4 mg/ml) groups. Plasma AOPP concentrations were significantly higher (P<0.05) in the Acute Phase group, and a significant positive correlation (R2=0.430; P<0.001) between plasma AOPP and Hp was observed. Our results support the hypothesis that AOPP can be used in the bovine as indicators of oxidative stress related to the inflammatory response. As plasma AOPP concentrations seemed unaffected around parturition, they may represent a good marker of the inflammatory status in the post-partum and early pregnancy phases in dairy cows. However, more research is needed to identify the physiological threshold of AOPP concentrations and to assess factors that may influence AOPP concentrations in body fluids
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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