S, N-doped carbon dots-based cisplatin delivery system in adenocarcinoma cells: Spectroscopical and computational approach.

S and N-doped carbon dots (S-CDs and N-CDs) and their cisplatin (cis-Pt) derivatives. (S-CDs@cis-Pt and N-CDs@cis-Pt) were tested on two ovarian cancer cell lines: A2780 and A2780 cells resistant to cis-Pt (A2780R). Several spectroscopic techniques were employed to check S-CDs@cis-Pt and N-CDs@cis-Pt: solid- and solution-state nuclear magnetic resonance, matrix-assisted laser desorption, ionization time-of-flight mass spectrometry, and X-ray photoelectron spectroscopy. In addition, synchrotron-based Fourier Transformed Infrared spectro-microscopy was used to evaluate the biochemical changes in cells after treatment with cis-Pt, S-CDs, N-CDs, or S-CDs@cis-Pt and N-CDs@cis-Pt, respectively. Computational chemistry was applied to establish the model for the most stable bond between S-CDs and N-CDs and cis-Pt. The results revealed the successful modification of S-CDs and N-CDs with cis-Pt and the formation of a stable composite system that can be used for drug delivery to cancer cells and likewise to overcome acquired cis-Pt resistance. Nanoparticle treatment of A2780 and A2780R cells led to the changes in their structure of lipids, proteins, and nucleic acids depending on the treatment. The results showed the S-CDs@cis-Pt and N-CDs@cis-Pt might be used in the combination with cis-Pt to treat the adenocarcinoma, thus having a potential to be further developed as drug delivery systems.

Spatial distribution of apoplastic antioxidative constituents in maize root.

Apoplastic antioxidative constituents (enzymes, primary and secondary metabolites, ROS) from different root zones of hydroponically grown maize (Zea mays L.) were investigated using a noninvasive isolation procedure: filter strip method. Filter strips were placed at specific positions on the root surface: apical zone (tip) and basal zone (base) to absorb apoplastic fluid. Three major classes of low-weight metabolites (organic acids, sugars, and phenolics) have been identified by HPLC-ECD. The longitudinal distribution of sugars and organic acids had the same pattern: higher concentration in the tip than the base, while it was vice versa for phenolics. The specific activities of guaiacol peroxidase, superoxide dismutase, and ascorbate peroxidase were higher in the apoplastic fluid from the root base than the tip, and their different isoforms were separated by isoelectric focusing. Electron paramagnetic resonance (EPR) spectroscopy coupled with the spin-trapping method using DEPMPO showed a persistent generation of hydroxyl radical in the root tip. In vivo EPR imaging of the whole maize root with membrane-permeable and impermeable aminoxyl spin-probes, enabling real-time detection of ROS formation within and outside the membranes, demonstrated ROS accumulation on the root surface, while endodermis and central cylinder were ROS free. For the first time in plant research, 2D EPR images enabled the direct demonstration of site-specific free radical production along the root. Highly sensitive analytical techniques combined with the filter strips, as a non-invasive tool, have increased our knowledge of metabolic processes occurring in the apoplast and their spatial-temporal changes in small regions of the intact root tissue.

Estimation of carbon dots amelioration of copper toxicity in maize studied by synchrotron radiation-FTIR.

Carbon dots are biocompatible and non-toxic nanoparticles with chemical affinity to some heavy metals. Human activities increase soil pollution with copper. Cu is an essential microelement in plants, but excess can induce a harmful effects. In plant response to Cu, the cell wall plays an important role. This study aims to estimate possible amelioration effects of folic acid based CDs on Cu toxicity by studying the intracellular and cell wall compounds in maize (Zea mays L.) roots and leaves after 7 day-treatment in hydroponics. The sub-cellular compartmentalization and bio-macromolecular changes induced by 5 µM Cu applied alone or with CDs (167 and 500 mg/L) were studied using the Synchrotron-based Fourier transformmicro-spectroscopy (SR-FTIR) combined with X-Ray photoelectron spectroscopy (XPS). Cu induced changes in content of cell wall polysaccharides, proteins, and lipids. The XPS detected CDs transport throughout the plants. The Cu/167CDs treatment reduced Cu concentration in the roots, possibly by complexation/trapping between the functional groups on CDs surface and Cu2+. Principal component analysis of FTIR spectra confirmed that Cu/500CDs treatment increased Cu adverse effects in most tissues but alleviated adverse Cu effects on cell wall polysaccharides in the root xylem, and on polysaccharides and proteins in leaf phloem and mesophyll.

Comparative biochemical characterization of peroxidases (class III) tightly bound to the maize root cell walls and modulation of the enzyme properties as a result of covalent binding.

Comparative biochemical characterization of class III peroxidase activity tightly bound to the cell walls of maize roots was performed. Ionically bound proteins were solubilized from isolated walls by salt washing, and the remaining covalently bound peroxidases were released, either by enzymatic digestion or by a novel alkaline extraction procedure that released covalently bound alkali-resistant peroxidase enzyme. Solubilized fractions, as well as the salt-washed cell wall fragments containing covalently bound proteins, were analyzed for peroxidase activity. Peroxidative and oxidative activities indicated that peroxidase enzymes were predominately associated with walls by ionic interactions, and this fraction differs from the covalently bound one according to molecular weight, isozyme patterns, and biochemical parameters. The effect of covalent binding was evaluated by comparison of the catalytic properties of the enzyme bound to the salt-washed cell wall fragments with the corresponding solubilized and released enzyme. Higher thermal stability, improved resistance to KCN, increased susceptibility to H2O2, stimulated capacity of wall-bound enzyme to oxidize indole-3-acetic acid (IAA) as well as the difference in kinetic parameters between free and bound enzymes point to conformational changes due to covalent binding. Differences in biochemical properties of ionically and covalently bound peroxidases, as well as the modulation of the enzyme properties as a result of covalent binding to the walls, indicate that these two fractions of apoplastic peroxidases play different roles.

Filter strip as a method of choice for apoplastic fluid extraction from maize roots.

Apoplastic fluid was extracted from maize (Zea mays L.) roots using two procedures: collection from the surface of intact plant roots by filter paper strips (AF) or vacuum infiltration and/or centrifugation from excised root segments (AWF). The content of cytoplasmic marker (glucose-6-phosphate, G-6-P) and antioxidative components (enzymes, organic acids, phenolics, sugars, ROS) were compared in the extracts. The results obtained demonstrate that AF was completely free of G-6-P, as opposed to AWF where the cytoplasmic constituent was detected even at mildest centrifugation (200×g). Isoelectric focusing of POD and SOD shows the presence of cytoplasmic isoforms in AWF, and HPLC of sugars and phenolics a much more complex composition of AWF, due to cytoplasmic contamination. Organic acid composition differed in the two extracts, much higher concentrations of malic acid being registered in AF, while oxalic acid due to intracellular contamination being present only in AWF. EPR spectroscopy of DEPMPO spin trap in the extracts showed persistent generation of hydroxyl radical adduct in AF. The results obtained argue in favor of the filter strip method for the root apoplastic fluid extraction, avoiding the problems of cytoplasmic contamination and dilution and enabling concentration measurements in minute regions of the root.

Oxygen regulation of alternative respiration in fungus Phycomyces blakesleeanus: connection with phosphate metabolism.

Environmental changes can often result in oxygen deficiency which influences cellular energy metabolism, but such effects have been insufficiently studied in fungi. The effects of oxygen deprivation on respiration and phosphate metabolites in Phycomyces blakesleeanus were investigated by oxygen electrode and (31)P NMR spectroscopy. Mycelium was incubated in hypoxic and anoxic conditions for 1.5, 3 and 5 h and then reoxygenated. Participation of alternative oxidase (AOX) in total respiration increased gradually in both treatments and after 5 h of anoxia exceeded a value 50% higher than in control. Shortly after reintroduction of oxygen into the system AOX level decreased close to the control level. Oxygen deprivation also caused a reversible decrease of polyphosphate/inorganic phosphate ratio (PPc/Pi), which was strongly correlated with the increase of AOX participation in total respiration. Unexpectedly, ATP content remained almost constant, probably due to the ability of PolyP to sustain energy and phosphate homeostasis of the cell under stress conditions. This was further substantiated by the effects of azide, a cytochrome c oxidase inhibitor, which also decreased PPc/Pi ratio, but to a smaller extent in oxygen deprived than control and reoxygenated specimens.

Cell wall-associated malate dehydrogenase activity from maize roots.

Isolated cell walls from maize (Zea mays L.) roots exhibited ionically and covalently bound NAD-specific malate dehydrogenase activity. The enzyme catalyses a rapid reduction of oxaloacetate and much slower oxidation of malate. The kinetic and regulatory properties of the cell wall enzyme solubilized with 1M NaCl were different from those published for soluble, mitochondrial or plasma membrane malate dehydrogenase with respect to their ATP, Pi, and pH dependence. Isoelectric focusing of ionically-bound proteins and specific staining for malate dehydrogenase revealed characteristic isoforms present in cell wall isolate, different from those present in plasma membranes and crude homogenate. Much greater activity of cell wall-associated malate dehydrogenase was detected in the intensively growing lateral roots compared to primary root with decreased growth rates. Presence of Zn(2+) and Cu(2+) in the assay medium inhibited the activity of the wall-associated malate dehydrogenase. Exposure of maize plants to excess concentrations of Zn(2+) and Cu(2+) in the hydroponic solution inhibited lateral root growth, decreased malate dehydrogenase activity and changed isoform profiles. The results presented show that cell wall malate dehydrogenase is truly a wall-bound enzyme, and not an artefact of cytoplasmic contamination, involved in the developmental processes, and detoxification of heavy metals.

The effects of manganese and copper in vitro and in vivo on peroxidase catalytic cycles.

Here we present the results of in vitro and in vivo studies of the influence of Mn²+ and Cu²+ on the peroxidative and oxidative catalytic functions of class III peroxidase. Complex peroxidase catalysis by intermediates generated in the reaction was analyzed by utilizing the activating effect of Mn²+ and the inhibitory effect of Cu²+ on the oxidative reaction in vitro. p-Coumaric acid was used as an enzyme substrate in the peroxidative reaction and as a cofactor in the oxidative reaction. In order to correlate the observed in vitro effects with the in vivo situation, we exposed maize plants to excess concentrations of Mn²+ and Cu²+ in the hydroponic solutions. Copper severely arrested plant growth, while manganese exerted no significant effect. The effects on peroxidase activity and isoforms profile of root soluble and cell wall bound fractions were studied. Inhibition of the peroxidase oxidative function by copper was reversible, localized in the cell wall, and accompanied by disappearance of some and appearance of new cationic isoforms. Copper-mediated changes were suppressed by the presence of manganese, although Mn²+ treatment per se did not affect the activity of the peroxidase enzyme. The results on the peroxidase activity in maize roots grown with excess Mn²+ and Cu²+ point to the coupling between the oxidative cycle, root growth and different peroxidase isoforms.

Outwardly rectifying anionic channel from the plasma membrane of the fungus Phycomyces blakesleeanus.

In the present report, by using a patch clamp technique, we provide, to our knowledge, the first detailed description of an anionic channel from filamentous fungi. The characterized channel, an outwardly rectifying anionic channel (ORAC), is the most prominent feature of the cell membrane of the fungus Phycomyces blakesleeanus in the absence of energizing substrates. The unitary conductance of the channel is 11.3 +/- 0.4 pS. It is characterized by a strong voltage dependence of the open-channel probability (zdelta; the gating charge is 2.1 +/- 0.1), and the channel is activated by depolarization. The values of the time constants for voltage-induced activation and deactivation of 28 +/- 3 ms for tau(a) and 39 +/- 9 ms for tau(d) show that the ORAC is characterized by fast activation/deactivation kinetics. The ORAC shows strong selectivity for anions over cations and weak selectivity among anions, with a selectivity sequence of I(-) >or= NO(3)(-) > Br(-) > Cl(-) > SO(4)(2-) = 4.8 > 4.4 > 2.2 > 1 > 0.55, which corresponds to Eisenman series 1. The channel is characterized by two open and two closed states, with dominant long open (tau(o2) = 35.0 +/- 3.9 ms) and long closed (tau(c2) = 166 +/- 28 ms) states occupying 63% +/- 8% and 79% +/- 3% of total open and closed times, respectively. The ORAC is insensitive to anthracene-9-carboxylic acid (<200 microM), but 2 mM malate reversibly inhibits 59% +/- 12% of the channel activity. Based on the electrophysiological properties of the channel, we propose that the ORAC plays a role in anion accumulation and in membrane potential regulation through local membrane depolarization.

Effectiveness of phenoxyl radicals generated by peroxidase/H2O2-catalyzed oxidation of caffeate, ferulate, and p-coumarate in cooxidation of ascorbate and NADH.

The rate of ascorbate and nicotinamide adenine dinucleotide plus hydrogen (NADH) cooxidation (i.e., their nonenzymic oxidation by peroxidase/H2O2-generated phenoxyl radicals of three hydroxycinnamates: caffeate, ferulate and p-coumarate) was studied in vitro. The reactions initiated by different sources of peroxidase (EC 1.11.1.7) [isolates from soybean (Glycine max L.) seed coat, maize (Zea mays L.) root-cell wall, and commercial horseradish peroxidase] were monitored. Native electrophoresis of samples and specific staining for peroxidase activity revealed various isoforms in each of the three enzyme sources. The peroxidase sources differed both in the rate of H2O2-dependent hydroxycinnamate oxidation and in the order of affinity for the phenolic substrates. The three hydroxycinnamates did not differ in their ability to cooxidize ascorbate, whereas NADH cooxidation was affected by substitution of the phenolic ring. Thus, p-coumarate was more efficient than caffeate in NADH cooxidation, with ferulate not being effective at all. Metal ions (Zn2+ and Al3+) inhibited the reaction of peroxidase with p-coumarate and affected the cooxidation rate of ascorbate and the peroxidase reaction in the same manner with all substrates used. However, inhibition of p-coumarate oxidation by metal ions did not affect NADH cooxidation rate. We propose that both the ascorbate and NADH cooxidation systems can function as mechanisms to scavenge H2O2 and regenerate phenolics in different cellular compartments, thus contributing to protection from oxidative damage.

Monosaccharide-H2O2 reactions as a source of glycolate and their stimulation by hydroxyl radicals.

An analysis of the H(2)O(2)-induced breakdown and transformation of different keto-monosaccharides at physiological concentrations reveals that glycolate and other short-chained carbohydrates and organic acids are produced. Depletion of monosaccharides and glycolate synthesis occurs at increased rates as the length of the carbohydrate chain is decreased, and is significantly increased in the presence of trace amounts of Fe(2+) ions (10 microM). Rates of monosaccharide depletion (initial concentration of 3 mM) observed were up to 1.55 mmol h(-1) in the case of fructose, and 2.59 mmol h(-1) in the case of dihydroxyacetone, depending upon pH, H(2)O(2) concentration, temperature and the presence or absence of catalytic amounts of Fe(2+). Glycolate was produced by dihydroxyacetone cleavage at rates up to 0.45 mmol h(-1) in the absence, and up to 1.88 mmol h(-1) in the presence of Fe(2+) ions (pH 8). Besides glycolate, other sugars (ribose, glyceraldehyde, glucose), glucitol (sorbitol) and organic acids (formic and 2-oxogluconic acid) were produced in such H(2)O(2)-induced reactions with fructose or dihydroxyacetone. EPR measurements demonstrated the participation of the OH radical, especially at higher pH. Presence of metal ions at higher pH values, resulting in increased glycolate synthesis, was accompanied by enhanced hydroxyl radical generation. Observed changes in intensity of DEPMPO-OH signals recorded from dihydroxyacetone and fructose reactions demonstrate a strong correlation with changes in glycolate yield, suggesting that OH radical formation enhances glycolate synthesis. The results presented suggest that different mechanisms are responsible for the cleavage or other reactions (isomerisation, auto- or free-radical-mediated oxidation) of keto-monosaccharides depending of experimental conditions.

Species-dependent changes in stomatal sensitivity to abscisic acid mediated by external pH.

The direct effects of pH changes and/or abscisic acid (ABA) on stomatal aperture were examined in epidermal strips of Commelina communis L. and Arabidopsis thaliana. Stomata were initially opened at pH 7 or pH 5. The stomatal closure induced by changes in external pH and/or ABA (10 microM or 10 nM) was monitored using video microscopy and quantified in terms of changes in stomatal area using image analysis software. Measurements of aperture area enabled stomatal responses and, in particular, small changes in stomatal area to be quantified reliably. Both plant species exhibited a biphasic closure response to ABA: an initial phase of rapid stomatal closure, followed by a second, more prolonged, phase during which stomata closure proceeded at a slower rate. Changes in stomatal sensitivity to ABA were also observed. Comparison of these effects between C. communis and A. thaliana demonstrate that this differential sensitivity of stomata to ABA is species-dependent, as well as being dependent on the pH of the extracellular environment.

The coexistence of the oxidative and reductive systems in roots: the role of plasma membranes.

Different components of the plasma membrane bound and associated redox system, which participate in the energy transfer from the predominantly reducing intercellular environment to the extracellular oxidizing environment, are reviewed. Special attention is given to plant root cells. An analysis of the plasma membrane-associated redox components, such as the cytochromes, quinones, and different types of oxidoreductases (dehydrogenases, oxidases, peroxidases, and superoxide dismutases), is made, as well as their coupling with naturally occurring extracellular substrates, such as oxygen and its reactive forms, phenols, ascorbate, nitrate, ferric ion, and organic acids. The participation of different free radical species in most of the plasma membrane-bound redox reactions is documented.

Nonenzymatic reaction of dihydroxyacetone with hydrogen peroxide enhanced via a Fenton reaction.

An analysis of the reaction of dihydroxyacetone with hydrogen peroxide was performed using HPLC with electrochemical and spectrophotometric detection and EPR spectroscopy of the reactants and products. Glycolate production in such a reaction has been documented, and the glycolate yield analyzed at different pH values. The participation of hydroxyl free radical is shown with DEPMPO spin trap. Two types of mechanisms are proposed, one at higher pH values and pronounced participation of free radicals (enhanced by the presence of metal ions and Fenton reaction) and the other at lower pH values, with decreased free radical production and lower glycolate yield.

Characterization of enzymatically synthesized diferulate.

Horseradish peroxidase was used to synthesize diferulates by a procedure in which ethyl ferulate was used as substrate. Four different forms were obtained, of which two dominant were the 5-5' and 8-5' diferulate. Fluorescence emission spectra of the diferulates (excited at 284 nm) indicate that they contain two chromophores, as opposed to the substrate molecule. Fluorescence excitation spectra with emission at 417 nm further demonstrate the difference between the synthesized diferulates and starting substrates.

A new model system for investigation of ionic channels in filamentous fungi: evidence for existence of two K+-permeable ionic channels in Phycomyces blakesleeanus.

A technique was developed to obtain viable cytoplasmic droplets, enabling the formation of a gigaohm seal. Such cytoplasmic droplets can be used for characterization of plasma membrane ion channels from filamentous fungi by patch-clamp technique. Two K(+) ionic channels are characterized with a conductance of 43 pS and 74 pS.

Ascorbic acid and the oxidative processes in pea root cell wall isolates: characterization by fluorescence and EPR spectroscopy.

A comparative fluorescence and oxygen radical-sensitive spin trap EPR spectroscopic study of isolated cell walls (with proteins or deproteinated), in the presence and absence of ascorbate and H(2)O(2) is presented. Fluorescence spectra indicate the presence of at least two fluorophores, one degraded and the other synthesized after reduction or oxidation, indicating phenol di/polymerization. DEPMPO spin trap measurements show that isolated cell walls are capable of oxygen-dependent hydroxyl radical generation in the absence of NADH or other reductants, ascorbate addition, or deproteination of the cell wall abolishing the signal due to hydroxyl radicals.

Fluorescent properties of spinach leaf plasma membranes and chloroplast envelopes.

Plasma membranes and chloroplast envelopes were isolated from green spinach leaves, and emission and excitation spectra recorded in aqueous solution at 25 degrees C. Chloroplast envelopes excited by 420 nm showed strong emission peaks at 635 and 680 nm that came from chlorophyll precursors present only in these membranes. Upon UV excitation, both plasma membranes and chloroplast envelopes exhibited emission peak at 420 nm originating from pterins and 520 nm due to flavins. Oxidation of the membranes increased both the emission and excitation fluorescence intensity.

The mutual effect of extracellular Ca2+, abscisic acid, and pH on the rate of stomatal closure.

An analysis of the rate of stomatal closure in epidermal strips of Commelina communis L. induced by different calcium concentrations (10 nM to 1 mM) demonstrated a biphasic dependency, the initial process of closure being more calcium dependent. This dependency was more evident at pH 7 than pH 5 of the bathing medium. Addition of low concentrations of abscisic acid (10 nM) induced a broadening of the Ca(2) dependency, while 10 microM abscisic acid increased the rate of stomatal closure in the whole Ca(2+) range and to a large extent abolished the calcium-induced effects. Such results indicate an interaction of external protons, Ca(2+), and abscisic acid, and possible competition for the membrane associated binding sites of the mechanism(s) responsible for the regulation of stomatal closure.

Oxygen radicals produced by plant plasma membranes: an EPR spin-trap study.

Plant plasma membranes are known to produce superoxide radicals, while the production of the hydroxyl radical, previously detected in complex plant tissues, is thought to occur in the cell wall. The mechanism of production of superoxide radicals by plant plasma membranes is, however, under dispute. It is shown, using electron paramagnetic resonance spectroscopy with a 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide spin-trap capable of differentiating between radical species, that isolated purified plasma membranes from maize roots produce hydroxyl radicals besides superoxide radicals. The results argue in favour of superoxide production through an oxygen and diphenylene iodonium-sensitive, NADH-dependent superoxide synthase mechanism, as well as through other unidentified mechanism(s). The hydroxyl radical is produced by an oxygen-insensitive, NADH-stimulated mechanism, which is enhanced in membranes in which the superoxide synthase is incapacitated by substrate removal or inhibition.