Albumin is an important plasma antioxidant protein, contributing to protecting mechanisms of cellular and regulatory long-lived proteins. The metal-catalyzed oxidation (MCO) of proteins plays an important role during oxidative stress. In this study, we examine the oxidative modification of albumin using an MCO in vitro system. Mass spectrometry, combined with off-line nano-liquid chromatography, was used to identify modifications in amino acid residues. We have found 106 different residues oxidatively damaged, being the main oxidized residues lysines, cysteines, arginines, prolines, histidines and tyrosines. Besides protein hydroxyl derivatives and oxygen additions, we detected other modifications such as deamidations, carbamylations and specific amino acid oxidative modifications. The oxidative damage preferentially affects particular subdomains of the protein at different time-points. Results suggest the oxidative damage occurs first in exposed regions near cysteine disulfide bridges with residues like methionine, tryptophan, lysine, arginine, tyrosine and proline appearing as oxidatively modified. The damage extended afterwards with further oxidation of cysteine residues involved in disulfide bridges and other residues like histidine, phenylalanine and aspartic acid. The time-course evaluation also shows the number of oxidized residues does not increase linearly, suggesting that oxidative unfolding of albumin occurs through a step-ladder mechanism.
Cardiolipins, a class of mitochondria-specific lipid molecules, is one of the most unusual and ancient phospholipids found in essentially all living species. Typical of mammalian cells is the presence of vulnerable to oxidation polyunsaturated fatty acid resides in CL molecules. The overall role and involvement of cardiolipin oxidation (CLox) products in major intracellular signaling as well as extracellular inflammatory and immune responses have been established. However, identification of individual peroxidized molecular species in the context of their ability to induce specific biological responses has not been yet achieved. This is due, at least in part, to technological difficulties in detection, identification, structural characterization and quantitation of CLox associated with their very low abundance and exquisite diversification. This dictates the need for the development of new methodologies for reliable, sensitive and selective analysis of both CLox. LC-MS-based oxidative lipidomics with high mass accuracy instrumentation as well as new software packages are promising in achieving the goals of expedited and reliable analysis of cardiolipin oxygenated species in biosamples.
Some sea slugs are able to steal functional chloroplasts (kleptoplasts) from their algal food sources, but the role and relevance of photosynthesis to the animal host remain controversial. While some researchers claim that kleptoplasts are slowly digestible ‘snacks’, others advocate that they enhance the overall fitness of sea slugs much more profoundly. Our analysis shows light-dependent incorporation of
13
C and
15
N in the albumen gland and gonadal follicles of the sea slug
Elysia timida
, representing translocation of photosynthates to kleptoplast-free reproductive organs. Long-chain polyunsaturated fatty acids with reported roles in reproduction were produced in the sea slug cells using labelled precursors translocated from the kleptoplasts. Finally, we report reduced fecundity of
E. timida
by limiting kleptoplast photosynthesis. The present study indicates that photosynthesis enhances the reproductive fitness of kleptoplast-bearing sea slugs, confirming the biological relevance of this remarkable association between a metazoan and an algal-derived organelle.
Background: Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) represent the most common primary liver malignancies whose outcome is influenced by the immune response.Methods: In this study, we have functionally characterized, by flow cytometry, circulating myeloid dendritic cells (mDCs) and FcεRI + monocytes in a group of healthy individuals (n = 10) and in a group of patients with HCC (n = 19) and CCA (n = 8), at the time point of the surgical resection (T0) and once the patient had recovered from surgery (T1). Moreover, we proceeded to a more in depth phenotypic characterization of the FcεRI + monocyte subpopulation.Results: A significant decrease in the frequency of TNFα producing FcεRI + monocytes and mDCs in HCC and CCA patients when compared to the group of healthy individuals was observed, and a close association between FcεRI + monocytes and mDCs dysfunction was identified. In addition, the phenotypic characteristics of FcεRI + monocytes from healthy individuals strongly suggest that this population drives to mDCs, which matches with the fact that both populations are functionally affected.Conclusions: The frequency and the function of circulating mDCs and FcεRI + monocytes are affected in both HCC and CCA patients, and FcεRI + monocytes could represent those fated to become mDCs.
Nitrated phospholipids have been recently identified in biological systems and showed to display anti-oxidant and anti-inflammatory potential in models of inflammation in vitro. Here, we have explored the effects of nitrated 1-palmitoyl-2-oleyl-phosphatidyl choline (NO 2-POPC) in cellular models. We have observed that NO 2-POPC, but not POPC, induces cellular changes consisting in cytoskeletal rearrangement and cell shrinking, and ultimately, loss of cell adhesion or impaired cell attachment. NO 2-POPC releases NO in vitro and induces accumulation of NO in cells. Nevertheless, the effects of NO 2-POPC are not superimposable with those of NO donors, which points to distinctive mechanisms of action. Notably, they show a stronger parallelism, although not complete overlap, with the effects of nitrated fatty acids. Interestingly, redistribution of vimentin by NO 2-POPC is attenuated in a C328S mutant, thus indicating that this residue may be a target for direct or indirect modification in NO 2-POPCtreated cells. Additionally, NO 2-POPC interacts with several typical lipoxidation targets in vitro, including vimentin and PPARγ constructs, likely through cysteine residues. Therefore, nitrated phospholipids emerge as potential novel electrophilic lipid mediators with selective actions.
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