Oxidation of hydroxylamines to nitroxide spin labels in living cells

In the presence of oxygen, cells can oxidize hydroxylamines, which are the products of the reduction of nitroxides in cells, back to nitroxides. Lipid-soluble hydroxylamines are oxidized much more rapidly than water-soluble ones, and most of this oxidation is inactivated by heat or trichloroacetic acid, indicating that the principal mechanism is enzyme-linked. The rates of oxidation of some lipophilic hydroxylamines are comparable to the rates of reduction of the corresponding nitroxides. Hydroxylamines formed by reduction of aqueous soluble nitroxides are not oxidized by cells, except for slight oxidation of some pyrrolidine derivatives. The latter is due to autoxidation. The kinetics of oxidation of reduced lipid-soluble nitroxides are all first-order with respect to hydroxylamines, regardless of the position of the nitroxide group along the carbon backbone, indicating that the oxidation occurs within the membrane. The oxidation of hydroxylamines in cells in inhibited by cyanide but not by antimycin A or SKF-525A. We also describe an effective method to oxidize hydroxylamines and follow this reaction; the method is based on the use of perdeuterated [15N]Tempone.     

Oxidation of proline by plant mitochondria

Mitochondria isolated from etiolated shoots of corn (Zea mays), wheat (Triticum aestivum), barley (Hordeum vulgare), soybean (Glycine max L. Merr.), and mung bean (Phaseolus aureus) exhibited a proline-dependent O₂ uptake subject to respiratory control. ADP/O ratios with proline as substrate were intermediate between ratios obtained with exogenous NADH and malate + pyruvate as substrates. Isotope studies showed proline metabolism to be dependent on O₂, but not NAD. The major ninhydrin-positive product formed via Δ¹-pyrroline-5-carboxylic acid was glutamate. Mitochondria were capable of further metabolism of glutamate, as radioactive CO₂, organic acids, and aspartate were recovered after [¹⁴C]proline feeding experiments. These results demonstrate the mitochondrial association and O₂ dependence of plant proline metabolism.     

Microtubule reorganization as a response to implementation of NO signals in plant cells

The effects of an exogeneous NO donor, sodium nitroprusside, on the orientation and organization of cortical microtubules in Arabidopsis thaliana root cells expressing GFP-MAP4 were studied in vivo. It was found that sodium nitroprusside treatment (10–500 μM, 24 h) caused the acceleration of primary root growth and enhanced initiation of root hairs in the differentiation zone. The influence of sodium nitroprusside revealed in changes in the orientation and organization of cortical microtubules in different types of cells of A. thaliana root. The most sensitive to sodium nitroprusside exposure were microtubules in epidermal cells of the elongation zone, where native transverse orientation of cortical microtubules turned into random, oblique, or longitudinal relative to the primary root axis. We suggest that NO, as one of the intracellular secondary messengers, triggers cell differentiation by reorientation of cortical microtubules, possibly via tubulin nitrotyrosination.     

Antioxidative activity of hydroxylamines. ESR spectra of radicals derived from hydroxylamines

The antioxidative activity of hydroxylamines was evaluated for the oxidation of tetralin at 61°C and linoleic acid micelles in an aqueous dispersion at 37°C, induced by an azo initiator. The antioxidative efficacy of the hydroxylamines for the oxidation of tetralin was smaller than that of α-tocopherol. However, the hydroxylamines showed more potent antioxidative activity than that of the α-tocopherol against the oxidation of linoleic acid micelles. On the basis of the results of an ESR study and the oxidation product obtained, it is suggested that active position in hydroxylamines depend not only on hydroxyl hydrogen-atom, but also on the allylic hydrogen atom.     

Satellite cells say NO to radiation

Skeletal muscles are commonly exposed to radiation for diagnostic procedures and the treatment of cancers and heterotopic bone formation. Few studies have considered the impact of clinical doses of radiation on the ability of satellite cells (myogenic stem cells) to proliferate, differentiate and contribute to recovering/maintaining muscle mass. The primary objective of this study was to determine whether the proliferation of irradiated satellite cells could be rescued by manipulating NO levels via pharmacological approaches and mechanical stretch (which is known to increase NO levels). We used both SNP (NO donor) and PTIO (NO scavenger) to manipulate NO levels in satellite cells. We observed that SNP was highly effective in rescuing the proliferation of irradiated satellite cells, especially at doses less than 5 Gy. The potential importance of NO was further illustrated by the effects of PTIO, which completely inhibited the rescue effect of SNP. Mechanical cyclic stretch was found to produce significant increases in NO levels of irradiated satellite cells, and this was associated with a robust increase in satellite cell proliferation. The effects of both radiation and NO on two key myogenic regulatory factors (MyoD and myogenin) were also explored. Irradiation of satellite cells produced a significant increase in both MyoD and myogenin, effects that were mitigated by manipulating NO levels via SNP. Given the central role of myogenic regulatory factors in the proliferation and differentiation of satellite cells, the findings of the current study underscore the need to more fully understand the relationship between radiation, NO and the functionality of satellite cells.     

Antioxidative activity of hydroxylamines. ESR spectra of radicals derived from hydroxylamines.

The antioxidative activity of hydroxylamines was evaluated for the oxidation of tetralin at 61 degrees C and linoleic acid micelles in an aqueous dispersion at 37 degrees C, induced by an azo initiator. The antioxidative efficacy of the hydroxylamines for the oxidation of tetralin was smaller than that of alpha-tocopherol. However, the hydroxylamines showed more potent antioxidative activity than that of the alpha-tocopherol against the oxidation of linoleic acid micelles. On the basis of the results of an ESR study and the oxidation product obtained, it is suggested that active position in hydroxylamines depend not only on hydroxyl hydrogen-atom, but also on the allylic hydrogen atom.     

Differential protection by nitroxides and hydroxylamines to radiation-induced and metal ion-catalyzed oxidative damage

Modulation of radiation- and metal ion-catalyzed oxidative-induced damage using plasmid DNA, genomic DNA, and cell survival, by three nitroxides and their corresponding hydroxylamines, were examined. The antioxidant property of each compound was independently determined by reacting supercoiled DNA with copper II/1,10-phenanthroline complex fueled by the products of hypoxanthine/xanthine oxidase (HX/XO) and noting the protective effect as assessed by agarose gel electrophoresis. The nitroxides and their corresponding hydroxylamines protected approximately to the same degree (33-47% relaxed form) when compared to 76.7% relaxed form in the absence of protectors. Likewise, protection by both the nitroxide and corresponding hydroxylamine were observed for Chinese hamster V79 cells exposed to hydrogen peroxide. In contrast, when plasmid DNA damage was induced by ionizing radiation (100 Gy), only nitroxides (10 mM) provide protection (32.4-38.5% relaxed form) when compared to radiation alone or in the presence of hydroxylamines (10 mM) (79.8% relaxed form). Nitroxide protection was concentration dependent. Radiation cell survival studies and DNA double-strand break (DBS) assessment (pulse field electrophoresis) showed that only the nitroxide protected or prevented damage, respectively. Collectively, the results show that nitroxides and hydroxylamines protect equally against the damage mediated by oxidants generated by the metal ion-catalyzed Haber-Weiss reaction, but only nitroxides protect against radiation damage, suggesting that nitroxides may more readily react with intermediate radical species produced by radiation than hydroxylamines.     

Inhibition of enzymes of polyamine biosynthesis by substrate-like O-substituted hydroxylamines

The biogenic amines spermine, spermidine, and putrescine are essential factors of cell growth and differentiation. To inhibit pyridoxal-5"-phosphate dependent ornithine decarboxylase and pyruvate dependent S-adenosylmethionine decarboxylase, key enzymes of polyamine biosynthesis, a system of substrate-like O-substituted hydroxylamines is suggested. The best of these compounds were active at nanomolar concentrations. High potency and specificity of this type of inhibitors are discussed in terms of structural similarity of E–I and E–S complexes.     

Oxidation of reduced nicotinamide adenine dinucleotide phosphate by plant mitochondria.

Mitochondria isolated from various plant tissues (leaves, etiolated shoots and hypocotyls, and stem tubers) oxidize exogenous NADPH with respiratory control values and ADP:O ratios similar to those obtained with exogenous NADH as substrate. In all the mitochondria investigated, the electron-transfer inhibitors rotenone and amytal each had the same effect on the oxidation of NADPH as they had on the oxidation of NADH. The oxidation of exogenous NADPH by white potato tuber mitochondria was much more sensitive to inhibition by citrate or ethylene glycol bis-(beta-aminoethyl ether)-N,N-tetraacetic acid than was the oxidation of NADH. Mitochondria isolated from aged beetroot slices showed an increased capacity for the oxidation of exogenous NADH (compared with mitochondria from fresh tissue) but no such increase in the capacity to oxidize exogenous NADPH. These results suggest that exogenous NADPH and NADH are oxidized via different flavoproteins in plant mitochondria.     

Inactivation of aphidicolin by plant cells

Plant cells are endowed with an aphidicolin inactivating activity. Data on cultured cells show that the rate of inactivation depends on the cell type, Daucus carota cells being the most effective among the other tested materials (Oryza sativa and Nicotiana plumbaginifolia). Also germinating seedling of Haplopappus gracilis and of Citrullus vulgaris inactivate aphidicolin. Inactivation, which may lead to unexpected results when a prolonged incubation with the drug is required, as in the case of the induction of synchrony of the cell cycle by aphidicolin, can be controlled by appropriately choosing the experimental conditions.     

Biodegradation of polychlorinated biphenyls by plant cells

The PCB biodegradative ability of plant cells cultivated in vitro in media containing a mixture of PCB congeners, Delor 103, is demonstrated. For experiments we used submerged cultures of Armoracia rusticana, Solanum aviculare, Atropa bella-donna, transformed hairy root or embryogenic cultures of Solanum nigrum. Transformation of PCB was followed by gas chromatography after cultivations of the above-mentioned cultures with Delor 103 (10 mg 100 ml⁻¹). The overall PCB metabolizing capability and also degradation of individual congeners greatly differed from strain to strain. The highest capability to metabolize PCB was assayed with differentiated cultures of Solanum nigrum. Beside the capability of PCB degradation, total peroxidase activity in the medium and the cell extract was also followed. Differentiated or hairy root cultures exhibiting higher degradation abilities of PCB also showed increase of peroxidase activities.     

Glycosylation of sesamol by cultured plant cells

The glycosylation of sesamol was investigated using cultured cells of Nicotiana tabacum and Eucalyptus perriniana. The cultured suspension cells of N. tabacum converted sesamol into its β-glucoside (7%) as well as the disaccharide, sesamyl 6-O-(β-D-glucopyranosyl)-β-D-glucopyranoside (β-gentiobioside, 30%). On the other hand, sesamyl 6-O-(α-L-rhamnopyranosyl)-β-D-glucopyranoside (β-rutinoside, 56%), together with the β-glucoside (3%), was produced when sesamol was incubated with suspension cells of E. perriniana.     

Specific perception of ergosterol by plant cells

Ergosterol (a fungal membrane component) induced modification of proton fluxes and membrane hyperpolarization in motor cells of Mimosa pudica. These reactions appear specific since they were not induced by the other sterols tested. A specific desensitization was observed, since cells did not react to a second ergosterol application. Exposed at first to other sterols, cells remained reactive to ergosterol. Comparatively, chitosan (a fungal wall component with known elicitor properties) triggered a membrane depolarization and also induced specific desensitization. This comparative study shows that ergosterol and chitosan are distinctly perceived by plant cells and induced different early events at the plasma membrane level.     

Imaging plant cells by two-photon excitation

Summary. Along the past recent years, two-photon excitation (TPE) microscopy has moved from the realms of technical curiosity to be a standard application in many advanced cell biology laboratories. The growing body of literature covered in this review points out the obvious advantages of TPE over any other imaging method based on fluorescence, clearly improving signal-to-noise ratio and thick-tissue penetration and showing added potential for vital imaging. Like any new technology that has to gain its own space, TPE microscopy is still going through the growing pains in which reproducible protocols, probes, and applications are scarce. Yet, the published reports and unpublished results covered in this review point out that TPE can eventually accommodate most available protocols and probes, most of the times with evident advantages. Further, the potential for plant sciences is obvious, as plant cells possess many absorbing molecules and structures and are routinely more opaque than tissues of other organisms. Since prices make it one of the most expensive microscopies, TPE is coming slow to be a generalised technology, but enough data are emerging to establish it as a method with no alternative for some objectives.Correspondence and reprints: Instituto Gulbenkian de Ciência. 2780-156 Oeiras, Portugal.