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Lipid membrane turnover and myelin repair play a central role in diseases and lesions of the central nervous system (CNS). The aim of the present study was to analyze lipid composition changes due to inflammatory conditions. We measured the fatty acid (FA) composition in erythrocytes (RBCs) and spinal cord tissue (gas chromatography) derived from mice affected by experimental allergic encephalomyelitis (EAE) in acute and remission phases; cholesterol membrane content (Filipin) and GM1 membrane assembly (CT-B) in EAE mouse RBCs, and in cultured neurons, oligodendroglial cells and macrophages exposed to inflammatory challenges. During the EAE acute phase, the RBC membrane showed a reduction in polyunsaturated FAs (PUFAs) and an increase in saturated FAs (SFAs) and the omega-6/omega-3 ratios, followed by a restoration to control levels in the remission phase in parallel with an increase in monounsaturated fatty acid residues. A decrease in PUFAs was also shown in the spinal cord. CT-B staining decreased and Filipin staining increased in RBCs during acute EAE, as well as in cultured macrophages, neurons and oligodendrocyte precursor cells exposed to inflammatory challenges. This regulation in lipid content suggests an increased cell membrane rigidity during the inflammatory phase of EAE and supports the investigation of peripheral cell membrane lipids as possible biomarkers for CNS lipid membrane concentration and assembly. [ABSTRACT FROM AUTHOR]

Oxygen is important for lipid metabolism, being involved in both enzymatic transformations and oxidative reactivity, and is particularly influent when genetic diseases impair the repair machinery of the cells, such as described for Cockayne syndrome (CS). We used two cellular models of transformed fibroblasts defective for CSA and CSB genes and their normal counterparts, grown for 24 h under various oxygen tensions (hyperoxic 21%, physioxic 5% and hypoxic 1%) to examine the fatty acid-based membrane remodeling by GC analysis of fatty acid methyl esters derived from membrane phospholipids. Overall, we first distinguished differences due to oxygen tensions: (a) hyperoxia induced a general boost of desaturase enzymatic activity in both normal and defective CSA and CSB cell lines, increasing monounsaturated fatty acids (MUFA), whereas polyunsaturated fatty acids (PUFA) did not undergo oxidative consumption; (b) hypoxia slowed down desaturase activities, mostly in CSA cell lines and defective CSB, causing saturated fatty acids (SFA) to increase, whereas PUFA levels diminished, suggesting their involvement in hypoxia-related signaling. CSB-deprived cells are the most sensitive to oxidation and CSA-deprived cells are the most sensitive to the radical-based formation of trans fatty acids (TFA). The results point to the need to finely differentiate biological targets connected to genetic impairments and, consequently, suggest the better definition of cell protection and treatments through accurate molecular profiling that includes membrane lipidomes. [ABSTRACT FROM AUTHOR]

The importance of sapienic acid (6c-16:1), a monounsaturated fatty acid of the n-10 family formed from palmitic acid by delta-6 desaturase, and of its metabolism to 8c-18:1 and sebaleic acid (5c,8c-18:2) has been recently assessed in cancer. Data are lacking on the association between signaling cascades and exposure to sapienic acid comparing cell lines of the same cancer type. We used 50 μM sapienic acid supplementation, a non-toxic concentration, to cultivate MCF-7 and 2 triple-negative breast cancer cells (TNBC), MDA-MB-231 and BT-20. We followed up for three hours regarding membrane fatty acid remodeling by fatty acid-based membrane lipidome analysis and expression/phosphorylation of EGFR (epithelial growth factor receptor), mTOR (mammalian target of rapamycin) and AKT (protein kinase B) by Western blotting as an oncogenic signaling cascade. Results evidenced consistent differences among the three cell lines in the metabolism of n-10 fatty acids and signaling. Here, a new scenario is proposed for the role of sapienic acid: one based on changes in membrane composition and properties, and the other based on changes in expression/activation of growth factors and signaling cascades. This knowledge can indicate additional players and synergies in breast cancer cell metabolism, inspiring translational applications of tailored membrane lipid strategies to assist pharmacological interventions. [ABSTRACT FROM AUTHOR]

Oxygen-Dependent Accumulation of Purine DNA Lesions in Cockayne Syndrome Cells. The originally published article [[1]] contains an error in the depiction of the structures of adenine derivatives in Figure A1 (Appendix A). Reference 1 Krokidis M.G., D'Errico M., Pascucci B., Parlanti E., Masi A., Ferreri C., Chatgilialoglu C. Oxygen-Dependent Accumulation of Purine DNA Lesions in Cockayne Syndrome Cells. [Extracted from the article]

Cockayne Syndrome (CS) is an autosomal recessive neurodegenerative premature aging disorder associated with defects in nucleotide excision repair (NER). Cells from CS patients, with mutations in CSA or CSB genes, present elevated levels of reactive oxygen species (ROS) and are defective in the repair of a variety of oxidatively generated DNA lesions. In this study, six purine lesions were ascertained in wild type (wt) CSA, defective CSA, wtCSB and defective CSB-transformed fibroblasts under different oxygen tensions (hyperoxic 21%, physioxic 5% and hypoxic 1%). In particular, the four 5′,8-cyclopurine (cPu) and the two 8-oxo-purine (8-oxo-Pu) lesions were accurately quantified by LC-MS/MS analysis using isotopomeric internal standards after an enzymatic digestion procedure. cPu levels were found comparable to 8-oxo-Pu in all cases (3–6 lesions/106 nucleotides), slightly increasing on going from hyperoxia to physioxia to hypoxia. Moreover, higher levels of four cPu were observed under hypoxia in both CSA and CSB-defective cells as compared to normal counterparts, along with a significant enhancement of 8-oxo-Pu. These findings revealed that exposure to different oxygen tensions induced oxidative DNA damage in CS cells, repairable by NER or base excision repair (BER) pathways. In NER-defective CS patients, these results support the hypothesis that the clinical neurological features might be connected to the accumulation of cPu. Moreover, the elimination of dysfunctional mitochondria in CS cells is associated with a reduction in the oxidative DNA damage. [ABSTRACT FROM AUTHOR]

Xeroderma Pigmentosum (XP) is a DNA repair disease characterized by nucleotide excision repair (NER) malfunction, leading to photosensitivity and increased incidence of skin malignancies. The role of XP-A in NER pathways has been well studied while discrepancies associated with ROS levels and the role of radical species between normal and deficient XPA cell lines have been observed. Using liquid chromatography tandem mass spectrometry we have determined the four 5',8-cyclopurines (cPu) lesions (i.e., 5′R-cdG, 5′S-cdG, 5′R-cdA and 5′S-cdA), 8-oxo-dA and 8-oxo-dG in wt (EUE-pBD650) and XPA-deficient (EUE-siXPA) human embryonic epithelial cell lines, under different oxygen tension (hyperoxic 21%, physioxic 5% and hypoxic 1%). The levels of Fe and Cu were also measured. The main findings of our study were: (i) the total amount of cPu (1.82–2.52 lesions/106 nucleotides) is the same order of magnitude as 8-oxo-Pu (3.10–4.11 lesions/106 nucleotides) in both cell types, (ii) the four cPu levels are similar in hyperoxic and physioxic conditions for both wt and deficient cell lines, whereas 8-oxo-Pu increases in all cases, (iii) both wt and deficient cell lines accumulated high levels of cPu under hypoxic compared to physioxic conditions, whereas the 8-oxo-Pu levels show an opposite trend, (iv) the diastereoisomeric ratios 5′R/5′S are independent of oxygen concentration being 0.29 for cdG and 2.69 for cdA for EUE-pBD650 (wt) and 0.32 for cdG and 2.94 for cdA for EUE-siXPA (deficient), (v) in deficient cell lines Fe levels were significantly higher. The data show for the first time the connection of oxygen concentration in cells with different DNA repair ability and the levels of different DNA lesions highlighting the significance of cPu. Membrane lipidomic data at 21% O2 indicated differences in the fatty acid contents between wild type and deficient cells, envisaging functional effects on membranes associated with the different repair capabilities, to be further investigated. [ABSTRACT FROM AUTHOR]

Purine 5′,8-cyclo-2′-deoxynucleosides (cPu) are tandem-type lesions observed among the DNA purine modifications and identified in mammalian cellular DNA in vivo. These lesions can be present in two diasteroisomeric forms, 5′R and 5′S, for each 2′-deoxyadenosine and 2′-deoxyguanosine moiety. They are generated exclusively by hydroxyl radical attack to 2′-deoxyribose units generating C5′ radicals, followed by cyclization with the C8 position of the purine base. This review describes the main recent achievements in the preparation of the cPu molecular library for analytical and DNA synthesis applications for the studies of the enzymatic recognition and repair mechanisms, their impact on transcription and genetic instability, quantitative determination of the levels of lesions in various types of cells and animal model systems, and relationships between the levels of lesions and human health, disease, and aging, as well as the defining of the detection limits and quantification protocols. [ABSTRACT FROM AUTHOR]

5',8-Cyclo-2'-deoxyadenosine (cdA), in the 5'R and 5'Sdiastereomeric forms, are typical non strand-break oxidative DNA lesions, induced by hydroxyl radicals, with emerging importance as a molecular marker. These lesions are exclusively repaired by the nucleotide excision repair (NER) mechanism with a low efficiency, thus readily accumulating in the genome. Poly(ADP-ribose) polymerase1 (PARP1) acts as an early responder to DNA damage and plays a key role as a nick sensor in the maintenance of the integrity of the genome by recognizing nicked DNA. So far, it was unknown whether the two diastereomeric cdA lesions could induce specific PARP1 binding. Here, we provide the first evidence of PARP1 to selectively recognize the diastereomeric lesions of 5'S-cdA and 5'R-cdA in vitro as compared to deoxyadenosine in model DNA substrates (23-mers) by using circular dichroism, fluorescence spectroscopy, immunoblotting analysis, and gel mobility shift assay. Several features of the recognition of the damaged and undamaged oligonucleotides by PARP1 were characterized. Remarkably, PARP1 exhibits different affinities in binding to a double strand (ds) oligonucleotide, which incorporates cdA lesions in R and S diastereomeric form. In particular, PARP1 proved to bind oligonucleotides, including a 5'S-cdA, with a higher affinity constant for the 5'S lesion in a model of ds DNA than 5'R-cdA, showing different recognition patterns, also compared with undamaged dA. This new finding highlights the ability of PARP1 to recognize and differentiate the distorted DNA backbone in a biomimetic system caused by different diastereomeric forms of a cdA lesion. [ABSTRACT FROM AUTHOR]