Differential effects of ascorbic acid on monocytic cell morphology and protein modification: Shifting from pro-oxidative to antioxidant properties.

In this study, we investigated the properties of ascorbic acid (vitamin C), which is a naturally occurring water-soluble vitamin. Our goal is to evaluate its pro-oxidative and/or antioxidant capabilities. To do this, we initially used a confocal laser scanning microscope (CLSM) to visualize the differentiation pattern in U-937 cells under the treatment of variable concentrations of ascorbic acid. Prior to induction, U-937 cells showed a spherical morphology. After treatment, significant morphological changes were observed in the form of prominent pseudopodia and amoeboid structures. Interestingly, pseudopodia incidences increased with an increase in ascorbic acid concentrations. In addition, our analysis of protein modification using anti-malondialdehyde antibodies showed changes in more than one protein. The findings reveal the link between the differentiation of U-937 cells into macrophages and the protein modifications triggered by the production of reactive oxygen species when U-937 cells are exposed to ascorbic acid. Furthermore, the transformation of ascorbic acid from a pro-oxidative to an antioxidant property is also demonstrated.

Oxidative modification of collagen by malondialdehyde in porcine skin.

Human skin is exposed to various physical and chemical stress factors, which commonly cause the oxidation of lipids and proteins. In this study, azo initiator AAPH [2,2' -azobis(2-methylpropionamidine) dihydrochloride] was employed to initiate lipid peroxidation in porcine skin as an ex vivo model for human skin. We demonstrate that malondialdehyde (MDA), a secondary product of lipid peroxidation, is covalently bound to collagen in the dermis, forming MDA-collagen adducts. The binding of MDA to collagen results in an unfolding of the collagen triple helix, formation of the dimer of α-chains of collagen, and fragmentation of the collagen α-chain. It is proposed here that the MDA is bound to the lysine residues of α-chain collagen, which are involved in electrostatic interaction and hydrogen bonding with the glutamate and aspartate of other α-chains of the triple helix. Our data provide crucial information about the MDA binding topology in the skin, which is necessary to understand better the various types of skin-related diseases and the aging process in the skin under stress.

Formation of free acetaldehydes derived from lipid peroxidation in U937 monocyte-like cells.

Acetaldehyde can be found in human cells as a byproduct of various metabolic pathways, including oxidative processes such as lipid peroxidation. This secondary product of lipid peroxidation plays a role in various pathological processes, leading to various types of civilization diseases. In this study, the formation of free acetaldehyde induced by oxygen-centred radicals was studied in monocyte-like cell line U937. Exposure of U937 cells to peroxyl/alkoxyl radicals induced by azocompound resulted in the formation of free acetaldehyde. Acetaldehyde is formed by the cleavage of fatty acids, which represents the breakdown of fatty acids into smaller fragments initiated by the cyclization of lipid peroxyl radical and ß-scission of lipid alkoxyl radical. The cleavage of fatty acids alters the integrity of the plasma and nuclear membrane, leading to the loss of cell viability. Understanding the pathological processes of acetaldehyde formation is an active area of research with potential implications for preventing and treating various diseases associated with oxidative stress.

Malondialdehyde enhances PsbP protein release during heat stress in Arabidopsis.

Under environmental conditions, plants are exposed to various abiotic and biotic stress factors, which commonly cause the oxidation of lipids and proteins. Lipid peroxidation constantly produces malondialdehyde (MDA), a secondary product of lipid peroxidation, which is covalently bound to proteins forming MDA-protein adducts. The spatial distribution of MDA-protein adducts in Arabidopsis leaves shows that MDA-protein adducts are located in the chloroplasts, uniformly spread out over the thylakoid membrane. At the lumenal side of thylakoid membrane, MDA interacts with PsbP, an extrinsic subunit of the photosystem II (PSII), which is in electrostatic interaction with the PSII core proteins. Under heat stress, when MDA is moderately enhanced, the electrostatic interaction between PsbP and PSII core proteins is weakened, and PsbP with bound MDA is released in the lumen. It is proposed here that the electrophilic MDA is bound to the nucleophilic lysine residues of PsbP, which are involved in electrostatic interactions with the negatively charged glutamate of the PSII core protein. Our data provide crucial information about the MDA binding topology in the higher plant PSII complex, which is necessary to understand better the physiological functions of MDA for plant survival under stress.

Detection and characterization of free oxygen radicals induced protein adduct formation in differentiating macrophages.

Reactive oxygen species play a key role in cellular homeostasis and redox signaling at physiological levels, where excessive production affects the function and integrity of macromolecules, specifically proteins. Therefore, it is important to define radical-mediated proteotoxic stress in macrophages and identify target protein to prevent tissue dysfunction. A well employed, THP-1 cell line was utilized as in vitro model to study immune response and herein we employ immuno-spin trapping technique to investigate radical-mediated protein oxidation in macrophages. Hydroxyl radical formation along macrophage differentiation was confirmed by electron paramagnetic resonance along with confocal laser scanning microscopy using hydroxyphenyl fluorescein. Lipid peroxidation product, malondialdehyde, generated under experimental conditions as detected using swallow-tailed perylene derivative fluorescence observed by confocal laser scanning microscopy and high-performance liquid chromatography, respectively. The results obtained from this study warrant further corroboration and study of specific proteins involved in the macrophage activation and their role in inflammations.

Bioactive Compounds and Their Impact on Protein Modification in Human Cells.

Reactive oxygen species (ROS) represent a group of molecules with a signaling role that are involved in regulating human cell proliferation and differentiation. Increased ROS concentrations are often associated with the local nonspecific oxidation of biological macromolecules, especially proteins and lipids. Free radicals, in general, may randomly damage protein molecules through the formation of protein-centered radicals as intermediates that, in turn, decay into several end oxidation products. Malondialdehyde (MDA), a marker of free-radical-mediated lipid oxidation and cell membrane damage, forms adducts with proteins in a nonspecific manner, leading to the loss of their function. In our study, we utilized U-937 cells as a model system to unveil the effect of four selected bioactive compounds (chlorogenic acid, oleuropein, tomatine, and tyrosol) to reduce oxidative stress associated with adduct formation in differentiating cells. The purity of the compounds under study was confirmed by an HPLC analysis. The cellular integrity and changes in the morphology of differentiated U-937 cells were confirmed with confocal microscopy, and no significant toxicity was found in the presence of bioactive compounds. From the Western blot analysis, a reduction in the MDA adduct formation was observed in cells treated with compounds that underlaid the beneficial effects of the compounds tested.

Auxin Metabolite Profiling in Isolated and Intact Plant Nuclei.

The plant nucleus plays an irreplaceable role in cellular control and regulation by auxin (indole-3-acetic acid, IAA) mainly because canonical auxin signaling takes place here. Auxin can enter the nucleus from either the endoplasmic reticulum or cytosol. Therefore, new information about the auxin metabolome (auxinome) in the nucleus can illuminate our understanding of subcellular auxin homeostasis. Different methods of nuclear isolation from various plant tissues have been described previously, but information about auxin metabolite levels in nuclei is still fragmented and insufficient. Herein, we tested several published nucleus isolation protocols based on differential centrifugation or flow cytometry. The optimized sorting protocol leading to promising yield, intactness, and purity was then combined with an ultra-sensitive mass spectrometry analysis. Using this approach, we can present the first complex report on the auxinome of isolated nuclei from cell cultures of Arabidopsis and tobacco. Moreover, our results show dynamic changes in auxin homeostasis at the intranuclear level after treatment of protoplasts with free IAA, or indole as a precursor of auxin biosynthesis. Finally, we can conclude that the methodological procedure combining flow cytometry and mass spectrometry offers new horizons for the study of auxin homeostasis at the subcellular level.

Free Radical-Mediated Protein Radical Formation in Differentiating Monocytes.

Free radical-mediated activation of inflammatory macrophages remains ambiguous with its limitation to study within biological systems. U-937 and HL-60 cell lines serve as a well-defined model system known to differentiate into either macrophages or dendritic cells in response to various chemical stimuli linked with reactive oxygen species (ROS) production. Our present work utilizes phorbol 12-myristate-13-acetate (PMA) as a stimulant, and factors such as concentration and incubation time were considered to achieve optimized differentiation conditions. ROS formation likely hydroxyl radical (HO●) was confirmed by electron paramagnetic resonance (EPR) spectroscopy combined with confocal laser scanning microscopy (CLSM). In particular, U-937 cells were utilized further to identify proteins undergoing oxidation by ROS using anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibodies. Additionally, the expression pattern of NADPH Oxidase 4 (NOX4) in relation to induction with PMA was monitored to correlate the pattern of ROS generated. Utilizing macrophages as a model system, findings from the present study provide a valuable source for expanding the knowledge of differentiation and protein expression dynamics.

Protein S-nitrosation differentially modulates tomato responses to infection by hemi-biotrophic oomycetes of Phytophthora spp.

Regulation of protein function by reversible S-nitrosation, a post-translational modification based on the attachment of nitroso group to cysteine thiols, has emerged among key mechanisms of NO signalling in plant development and stress responses. S-nitrosoglutathione is regarded as the most abundant low-molecular-weight S-nitrosothiol in plants, where its intracellular concentrations are modulated by S-nitrosoglutathione reductase. We analysed modulations of S-nitrosothiols and protein S-nitrosation mediated by S-nitrosoglutathione reductase in cultivated Solanum lycopersicum (susceptible) and wild Solanum habrochaites (resistant genotype) up to 96 h post inoculation (hpi) by two hemibiotrophic oomycetes, Phytophthora infestans and Phytophthora parasitica. S-nitrosoglutathione reductase activity and protein level were decreased by P. infestans and P. parasitica infection in both genotypes, whereas protein S-nitrosothiols were increased by P. infestans infection, particularly at 72 hpi related to pathogen biotrophy-necrotrophy transition. Increased levels of S-nitrosothiols localised in both proximal and distal parts to the infection site, which suggests together with their localisation to vascular bundles a signalling role in systemic responses. S-nitrosation targets in plants infected with P. infestans identified by a proteomic analysis include namely antioxidant and defence proteins, together with important proteins of metabolic, regulatory and structural functions. Ascorbate peroxidase S-nitrosation was observed in both genotypes in parallel to increased enzyme activity and protein level during P. infestans pathogenesis, namely in the susceptible genotype. These results show important regulatory functions of protein S-nitrosation in concerting molecular mechanisms of plant resistance to hemibiotrophic pathogens.

Tocopherol controls D1 amino acid oxidation by oxygen radicals in Photosystem II.

Photosystem II (PSII) is an intrinsic membrane protein complex that functions as a light-driven water:plastoquinone oxidoreductase in oxygenic photosynthesis. Electron transport in PSII is associated with formation of reactive oxygen species (ROS) responsible for oxidative modifications of PSII proteins. In this study, oxidative modifications of the D1 and D2 proteins by the superoxide anion (O2•-) and the hydroxyl (HO•) radicals were studied in WT and a tocopherol cyclase (vte1) mutant, which is deficient in the lipid-soluble antioxidant α-tocopherol. In the absence of this antioxidant, high-resolution tandem mass spectrometry was used to identify oxidation of D1:130E to hydroxyglutamic acid by O2•- at the PheoD1 site. Additionally, D1:246Y was modified to either tyrosine hydroperoxide or dihydroxyphenylalanine by O2•- and HO•, respectively, in the vicinity of the nonheme iron. We propose that α-tocopherol is localized near PheoD1 and the nonheme iron, with its chromanol head exposed to the lipid-water interface. This helps to prevent oxidative modification of the amino acid's hydrogen that is bonded to PheoD1 and the nonheme iron (via bicarbonate), and thus protects electron transport in PSII from ROS damage.

Reactive Oxygen Species Imaging in U937 Cells.

The U937 cell culture is a pro-monocytic, human histiocytic lymphoma cell line. These monocytes can differentiate into either macrophages or dendritic cells (antigen-presenting cells) depending on the initiators. The U937 cells activated in the presence of phorbol 12-myristate 13-acetate (PMA) change their morphology into macrophage-like cells creating pseudopodia and adhering generously. Macrophages are known to produce reactive oxygen species (ROS) mostly during phagocytosis of foreign particles, an important non-specific immune response. Recently, we have focused on the role of hydroxyl radical (HO∙) and provide evidence on its importance for differentiation in U937 cells. Based on electron paramagnetic resonance (EPR) spectroscopy combined with confocal laser scanning microscopy (CLSM), formation of HO∙ was confirmed within the cells undergoing differentiation and/or apoptosis during the PMA treatment. This study aims to increase our knowledge of ROS metabolism in model cell lines used in human research.

The Anti-Senescence Activity of Cytokinin Arabinosides in Wheat and Arabidopsis Is Negatively Correlated with Ethylene Production.

Leaf senescence, accompanied by chlorophyll breakdown, chloroplast degradation and inhibition of photosynthesis, can be suppressed by an exogenous application of cytokinins. Two aromatic cytokinin arabinosides (6-benzylamino-9-ß-d-arabinofuranosylpurines; BAPAs), 3-hydroxy- (3OHBAPA) and 3-methoxy- (3MeOBAPA) derivatives, have recently been found to possess high anti-senescence activity. Interestingly, their effect on the maintenance of chlorophyll content and maximal quantum yield of photosystem II (PSII) in detached dark-adapted leaves differed quantitatively in wheat (Triticum aestivum L. cv. Aranka) and Arabidopsis (Arabidopsisthaliana L. (Col-0)). In this work, we have found that the anti-senescence effects of 3OHBAPA and 3MeOBAPA in wheat and Arabidopsis also differ in other parameters, including the maintenance of carotenoid content and chloroplasts, rate of reduction of primary electron acceptor of PSII (QA) as well as electron transport behind QA, and partitioning of absorbed light energy in light-adapted leaves. In wheat, 3OHBAPA had a higher protective effect than 3MeOBAPA, whereas in Arabidopsis, 3MeOBAPA was the more efficient derivative. We have found that the different anti-senescent activity of 3OHBAPA and 3MeOBAPA was coupled to different ethylene production in the treated leaves: the lower the ethylene production, the higher the anti-senescence activity. 3OHBAPA and 3MeOBAPA also efficiently protected the senescing leaves of wheat and Arabidopsis against oxidative damage induced by both H2O2 and high-light treatment, which could also be connected with the low level of ethylene production.

Interplay between antioxidants in response to photooxidative stress in Arabidopsis.

Tocochromanols (tocopherols, tocotrienols and plastochromanol-8), isoprenoid quinone (plastoquinone-9 and plastoquinol-9) and carotenoids (carotenes and xanthophylls), are lipid-soluble antioxidants in the chloroplasts, which play an important defensive role against photooxidative stress in plants. In this study, the interplay between the antioxidant activities of those compounds in excess light stress was analyzed in wild-type (WT) Arabidopsis thaliana and in a tocopherol cyclase mutant (vte1), a homogentisate phytyl transferase mutant (vte2) and a tocopherol cyclase overexpressor (VTE1oex). The results reveal a strategy of cooperation and replacement between α-tocopherol, plastochromanol-8, plastoquinone-9/plastoquinol-9 and zeaxanthin. In the first line of defense (non-radical mechanism), singlet oxygen is either physically or chemically quenched by α-tocopherol; however, when α-tocopherol is consumed, zeaxanthin and plastoquinone-9/plastoquinol-9 can provide alternative protection against singlet oxygen toxicity by functional replacement of α-tocopherol either by zeaxanthin for the physical quenching or by plastoquinone-9/plastoquinol-9 for the chemical quenching. When singlet oxygen escapes this first line of defense, it oxidizes lipids and forms lipid hydroperoxides, which are oxidized to lipid peroxyl radicals by ferric iron. In the second line of defense (radical mechanism), lipid peroxyl radicals are scavenged by α-tocopherol. After its consumption, plastochromanol-8 overtakes this function. We provide a comprehensive description of the reaction pathways underlying the non-radical and radical antioxidant activities of α-tocopherol, carotenoids, plastoquinone-9/plastoquinol-9 and plastochromanol-8. The interplay between the different plastid lipid-soluble antioxidants in the non-radical and the radical mechanism provides step by step insights into protection against photooxidative stress in higher plants.

Differential modulation of S-nitrosoglutathione reductase and reactive nitrogen species in wild and cultivated tomato genotypes during development and powdery mildew infection.

Nitric oxide plays an important role in the pathogenesis of Pseudoidium neolycopersici, the causative agent of tomato powdery mildew. S-nitrosoglutathione reductase, the key enzyme of S-nitrosothiol homeostasis, was investigated during plant development and following infection in three genotypes of Solanum spp. differing in their resistance to P. neolycopersici. Levels and localization of reactive nitrogen species (RNS) including NO, S-nitrosoglutathione (GSNO) and peroxynitrite were studied together with protein nitration and the activity of nitrate reductase (NR). GSNOR expression profiles and enzyme activities were modulated during plant development and important differences among Solanum spp. genotypes were observed, accompanied by modulation of NO, GSNO, peroxynitrite and nitrated proteins levels. GSNOR was down-regulated in infected plants, with exception of resistant S. habrochaites early after inoculation. Modulations of GSNOR activities in response to pathogen infection were found also on the systemic level in leaves above and below the inoculation site. Infection strongly increased NR activity and gene expression in resistant S. habrochaites in contrast to susceptible S. lycopersicum. Obtained data confirm the key role of GSNOR and modulations of RNS during plant development under normal conditions and point to their involvement in molecular mechanisms of tomato responses to biotrophic pathogens on local and systemic levels.

Nuclear Disposition of Alien Chromosome Introgressions into Wheat and Rye Using 3D-FISH.

During interphase, the chromosomes of eukaryotes decondense and they occupy distinct regions of the nucleus, called chromosome domains or chromosome territories (CTs). In plants, the Rabl's configuration, with telomeres at one pole of nucleus and centromeres at the other, appears to be common, at least in plants with large genomes. It is unclear whether individual chromosomes of plants adopt defined, genetically determined addresses within the nucleus, as is the case in mammals. In this study, the nuclear disposition of alien rye and barley chromosomes and chromosome arm introgressions into wheat while using 3D-FISH in various somatic tissues was analyzed. All of the introgressed chromosomes showed Rabl's orientation, but their relative positions in the nuclei were less clear. While in most cases pairs of introgressed chromosomes occupied discrete positions, their association (proximity) along their entire lengths was rare, and partial association only marginally more frequent. This arrangement is relatively stable in various tissues and during various stages of the cell cycle. On the other hand, the length of a chromosome arm appears to play a role in its positioning in a nucleus: shorter chromosomes or chromosome arms tend to be located closer to the centre of the nucleus, while longer arms are more often positioned at the nuclear periphery.

Characterization of Protein Radicals in Arabidopsis.

Oxidative modification of proteins in photosystem II (PSII) exposed to high light has been studied for a few decades, but the characterization of protein radicals formed by protein oxidation is largely unknown. Protein oxidation is induced by the direct reaction of proteins with reactive oxygen species known to form highly reactive protein radicals comprising carbon-centered (alkyl) and oxygen-centered (peroxyl and alkoxyl) radicals. In this study, protein radicals were monitored in Arabidopsis exposed to high light by immuno-spin trapping technique based on the detection of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) nitrone adducts using the anti-DMPO antibody. Protein radicals were imaged in Arabidopsis leaves and chloroplasts by confocal laser scanning microscopy using fluorescein conjugated with the anti-DMPO antibody. Characterization of protein radicals by standard blotting techniques using PSII protein specific antibodies shows that protein radicals are formed on D1, D2, CP43, CP47, and Lhcb3 proteins. Protein oxidation reflected by the appearance/disappearance of the protein bands reveals that formation of protein radicals was associated with protein fragmentation (cleavage of the D1 peptide bonds) and aggregation (cross-linking with another PSII subunits). Characterization of protein radical formation is important for better understating of the mechanism of oxidative modification of PSII proteins under high light.

Instability of Alien Chromosome Introgressions in Wheat Associated with Improper Positioning in the Nucleus.

Alien introgressions introduce beneficial alleles into existing crops and hence, are widely used in plant breeding. Generally, introgressed alien chromosomes show reduced meiotic pairing relative to the host genome, and may be eliminated over generations. Reduced pairing appears to result from a failure of some telomeres of alien chromosomes to incorporate into the leptotene bouquet at the onset of meiosis, thereby preventing chiasmate pairing. In this study, we analysed somatic nuclei of rye introgressions in wheat using 3D-FISH and found that while introgressed rye chromosomes or chromosome arms occupied discrete positions in the Rabl's orientation similar to chromosomes of the wheat host, their telomeres frequently occupied positions away from the nuclear periphery. The frequencies of such abnormal telomere positioning were similar to the frequencies of out-of-bouquet telomere positioning at leptotene, and of pairing failure at metaphase I. This study indicates that improper positioning of alien chromosomes that leads to reduced pairing is not a strictly meiotic event but rather a consequence of a more systemic problem. Improper positioning in the nuclei probably impacts the ability of introgressed chromosomes to migrate into the telomere bouquet at the onset of meiosis, preventing synapsis and chiasma establishment, and leading to their gradual elimination over generations.

New fluorescently labeled auxins exhibit promising anti-auxin activity.

The plant hormone auxin is a key player in the regulation of plant growth and development. Despite numerous studies devoted to understanding its role in a wide spectrum of physiological processes, full appreciation of its function is linked to a comprehensive determination of its spatio-temporal distribution, which plays a crucial role in its mode of action. Conjugation of fluorescent tracers to plant hormones enables sensitive and specific visualization of their subcellular and tissue-specific localization and transport in planta, which represents a powerful tool for plant physiology. However, to date, only a few fluorescently labeled auxins have been developed. We report the synthesis of four novel fluorescently labeled derivatives of indole-3-acetic acid (IAA) in the form of a conjugate with a nitrobenzoxadiazole (NBD) fluorophore together with validation of their biological activity. These compounds, unlike other previously reported auxins fluorescently labeled at N1 position (nitrogen of the indole ring), do not possess auxin activity but rather show dose-dependent inhibition of auxin-induced effects, such as primary root growth inhibition, root hair growth and the auxin reporter DR5::GUS expression. Moreover, the study demonstrates the importance of the character of the linker and optimal choice of the labeling site in the preparation of fluorescently labeled auxins as important variables influencing their biological activity and fluorescent properties.

Organic radical imaging in plants: Focus on protein radicals.

Biomolecule (lipid and protein) oxidation products formed in plant cells exposed to photooxidative stress play a crucial role in the retrograde signaling and oxidative damage. The oxidation of biomolecules initiated by reactive oxygen species is associated with formation of organic (alkyl, peroxyl and alkoxyl) radicals. Currently, there is no selective and sensitive technique available for the detection of organic radicals in plant cells. Here, based on the analogy with animal cells, immuno-spin trapping using spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to image organic radicals in Arabidopsis leaves exposed to high light. Using antibody raised against the DMPO nitrone adduct conjugated with the fluorescein isothiocyanate, organic radicals were imaged by confocal laser scanning microscopy. Organic radicals are formed predominantly in the chloroplasts located at the periphery of the cells and distributed uniformly throughout the grana stack. Characterization of protein radicals by standard immunological techniques using anti-DMPO antibody shows protein bands with apparent molecular weights of 32 and 34 kDa assigned to D1 and D2 proteins and two protein bands below the D1/D2 band with apparent molecular weights of 23 and 18 kDa and four protein bands above the D1/D2 band with apparent molecular weights of 41, 43, 55 and 68 kDa. In summary, imaging of organic radicals by immuno-spin trapping represents selective and sensitive technique for the detection of organic radicals that might help to clarify mechanistic aspects on the role of organic radicals in the retrograde signaling and oxidative damage in plant cell.

Reactive Oxygen Species as a Response to Wounding: In Vivo Imaging in Arabidopsis thaliana.

Mechanical injury or wounding in plants can be attributed to abiotic or/and biotic causes. Subsequent defense responses are either local, i.e. within or in the close vicinity of affected tissue, or systemic, i.e. at distant plant organs. Stress stimuli activate a plethora of early and late reactions, from electric signals induced within seconds upon injury, oxidative burst within minutes, and slightly slower changes in hormone levels or expression of defense-related genes, to later cell wall reinforcement by polysaccharides deposition, or accumulation of proteinase inhibitors and hydrolytic enzymes. In the current study, we focused on the production of reactive oxygen species (ROS) in wounded Arabidopsis leaves. Based on fluorescence imaging, we provide experimental evidence that ROS [superoxide anion radical (O2 •-) and singlet oxygen (1O2)] are produced following wounding. As a consequence, oxidation of biomolecules is induced, predominantly of polyunsaturated fatty acid, which leads to the formation of reactive intermediate products and electronically excited species.