Effect of Garlic Extract on the Erythrocyte as a Simple Model Cell.

Garlic is known to have diverse effects on mammalian cells, being cytotoxic, especially to cancer cells, but also protect against oxidative stress. Mammalian erythrocyte is a simple cell devoid of intracellular organelles, protein synthesis ability, and most signaling pathways. Therefore, examination of the effects of garlic on erythrocytes allows for revealing primary events in the cellular action of garlic extract. In this study, human erythrocytes or erythrocyte membranes were exposed to garlic extract at various dilutions. Hemoglobin oxidation to methemoglobin, increased binding of hemoglobin to the membrane, and formation of Heinz bodies were observed. Garlic extract depleted acid-soluble thiols, especially glutathione, and induced a prooxidative shift in the cellular glutathione redox potential. The extract increased the osmotic fragility of erythrocytes, induced hemolysis, and inhibited hemolysis in isotonic ammonium chloride, indicative of decreased membrane permeability for Cl- and increased the membrane fluidity. Fluorescent probes indicated an increased level of reactive oxygen species and induction of lipid peroxidation, but these results should be interpreted with care since the extract alone induced oxidation of the probes (dichlorodihydrofluorescein diacetate and BODIPY C11). These results demonstrate that garlic extract induces oxidative changes in the erythrocyte, first of all, thiol and hemoglobin oxidation.

Effect of Low Concentration of Nitroxides on SH-SY5Y Cells Transfected with the Tau Protein.

Nitroxides, stable synthetic free radicals, are promising antioxidants, showing many beneficial effects both at the cellular level and in animal studies. However, the cells are usually treated with high millimolar concentrations of nitroxides which are not relevant to the concentrations that could be attained in vivo. This paper aimed to examine the effects of low (≤10 µM) concentrations of three nitroxides, 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO), 4-hydroxy-TEMPO (TEMPOL) and 4-amino-TEMPO (TEMPAMINE), in pure chemical systems and on SH-SY5Y cells transfected with the human tau protein (TAU cells), a model of chronic cellular oxidative stress, and transfected with the empty plasmid (EP cells). All nitroxides were active in antioxidant-activity tests except for the 2,2'-azinobis-(3-ethylbenzthiazolin-6-sulfonate) radical (ABTS•) decolorization assay and reduced Fe3+, inhibited autoxidation of adrenalin and pyrogallol and oxidation of dihydrorhodamine123 by 3-morpholino-sydnonimine SIN-1. TEMPO protected against fluorescein bleaching from hypochlorite, but TEMPAMINE enhanced the bleaching. Nitroxides showed no cytotoxicity and were reduced by the cells to non-paramagnetic derivatives. They decreased the level of reactive oxygen species, depleted glutathione, and increased mitochondrial-membrane potential in both types of cells, and increased lipid peroxidation in TAU cells. These results demonstrate that even at low micromolar concentrations nitroxides can affect the cellular redox equilibrium and other biochemical parameters.

Biomaterial composed of chitosan, riboflavin, and hydroxyapatite for bone tissue regeneration.

Biomaterial engineering approaches involve using a combination of miscellaneous bioactive molecules which may promote cell proliferation and, thus, form a scaffold with the environment that favors the regeneration process. Chitosan, a naturally occurring biodegradable polymer, possess some essential features, i.e., biodegradability, biocompatibility, and in the solid phase good porosity, which may contribute to promote cell adhesion. Moreover, doping of the materials with other biocompounds will create a unique and multifunctional scaffold that will be useful in regenerative medicine. This study is focused on the manufacturing and characterization of composite materials based on chitosan, hydroxyapatite, and riboflavin. The resulting films were fabricated by the casting/solvent evaporation method. Morphological and spectroscopy analyses of the films revealed a porous structure and an interconnection between chitosan and apatite. The composite material showed an inhibitory effect on Staphylococcus aureus and exhibited higher antioxidant activity compared to pure chitosan. In vitro studies on riboflavin showed increased cell proliferation and migration of fibroblasts and osteosarcoma cells, thus demonstrating their potential for bone tissue engineering applications.

Formation of a Purple Product upon the Reaction of ABTS Radicals with Proteins.

The reaction of the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) free radical (ABTS●) with proteins (bovine serum albumin, blood plasma, egg white, erythrocyte membranes, and Bacto Peptone) leads not only to a reduction of ABTS● but also to the appearance of a purple color (absorption maximum at 550-560 nm). The aim of this study was to characterize the formation and explain the nature of the product responsible for the appearance of this color. The purple color co-precipitated with protein, and was diminished by reducing agents. A similar color was generated by tyrosine upon reaction with ABTS●. The most feasible explanation for the color formation is the addiction of ABTS● to proteins' tyrosine residues. The product formation was decreased by nitration of the bovine serum albumin (BSA) tyrosine residues. The formation of the purple product of tyrosine was optimal at pH 6.5. A decrease in pH induced a bathochromic shift of the spectra of the product. The product was not a free radical, as demonstrated by electrom paramagnetic resonance (EPR) spectroscopy. Another byproduct of the reaction of ABTS● with tyrosine and proteins was dityrosine. These byproducts can contribute to the non-stoichiometry of the antioxidant assays with ABTS●. The formation of the purple ABTS adduct may be a useful index of radical addition reactions of protein tyrosine residues.

Zinc-Cobalt Oxide Thin Films: High Curie Temperature Studied by Electron Magnetic Resonance.

The material with a high Curie temperature of cobalt-doped zinc oxide embedded with silver-nanoparticle thin films was studied by electron magnetic resonance. The nanoparticles were synthesized by the homogeneous nucleation technique. Thin films were produced with the pulsed laser deposition method. The main aim of this work was to investigate the effect of Ag nanoparticles on the magnetic properties of the films. Simultaneously, the coexisting Ag0 and Ag2+ centers in zinc oxide structures are shown. A discussion of the signal seen in the low field was conducted. To analyze the temperature dependence of the line parameters, the theory described by Becker was used. The implementation of silver nanoparticles causes a significant shift of the line, and the ferromagnetic properties occur in a wide temperature range with an estimated Curie temperature above 500 K.

The Low-Field Microwave Absorption in EMR Spectra for Ni50-xCoxMn35.5In14.5 Ribbons.

This paper contains a detailed study of low-field microwave absorption, which is observed in EMR spectra registered for a series of Ni50-xCoxMn35.5In14.5 (x=0,3,5) Heusler alloys polycrystalline in situ and annealed at 1173 K ribbons. The LFMA spectra for all ribbons were performed at X-band (∼9.5 GHz), at temperatures below Curie temperature. Additionally, for annealed Ni45Co5Mn35.5In14.5 ribbons, the LFMA signal dependencies of the external magnetic field modulation amplitude, modulation frequency, microwave power and microwave magnetic field phase were registered. These results confirm the resonant character of LFMA. To determine the basic EMR parameters, such as linewidth and resonance field, the experimental data were fitted by the Dyson function. The LFMA signal is satisfactorily matched by the two lines, and the variability of the component lines with temperature is remarkable.

Electron Paramagnetic Resonance Study of PbSe, PbTe, and PbTe:In Semiconductors Obtained by the Pulsed Laser Deposition Method.

The magnetic properties of lead selenide (PbSe) and indium-doped lead telluride (PbTe:In) composites have been studied by using the electron paramagnetic resonance (EPR) technique. The samples were obtained by using the pulsed laser deposition method (PLD). Temperature dependences of the EPR spectra were obtained. The analysis of the temperature dependencies of the integral intensity of the EPR spectra was performed using the Curie-Weiss law. In these materials, the paramagnetic centers of Pb1+ and Pb3+ ions were identified. The results are discussed.

Understanding the effect of structural changes on slow magnetic relaxation in mononuclear octahedral copper(II) complexes.

Current advances in molecular magnetism are aimed at the construction of molecular nanomagnets and spin qubits for their utilization as high-density data storage materials and quantum computers. Mononuclear coordination compounds with low spin values of S = ½ are excellent candidates for this endeavour, but knowledge of their construction via rational design is limited. This particularly applies to the single copper(II) spin center, having been only recently demonstrated to exhibit slow relaxation of magnetisation in the appropriate octahedral environment. We have thus prepared a unique organic scaffold that would allow one to gain in-depth insight into how purposeful structural differences affect the slow magnetic relaxation in monometallic, transition metal complexes. As a proof-of-principle, we demonstrate how one can construct two, structurally very similar complexes with isolated Cu(II) ions in an octahedral ligand environment, the magnetic properties of which differ significantly. The differences in structural symmetry effects and in magnetic relaxation are corroborated with a series of experimental techniques and theoretical approaches, showing how symmetry distortions and crystal packing affect the relaxation behaviour in these isolated Cu(II) systems. Our unique organic platform can be efficiently utilized for the construction of various transition-metal ion systems in the future, effectively providing a model system for investigation of magnetic relaxation via targeted structural distortions.

Cobalt Content Effect on the Magnetic Properties of Ni50-xCoxMn35.5In14.5 Annealed Ribbons.

We present a study of the annealing effect and its influence on magnetic and structural properties for a series of Heusler alloys Ni50-xCoxMn35.5In14.5 (x=0,3,5) prepared in ribbon form. We studied the morphology and composition using scanning electron microscopy (SEM) equipped with an X-ray microanalyzer (EDX). The magnetic properties were determined by two methods: electron magnetic resonance (EMR) and vibrating sample magetometer (VSM). We found that cobalt content in the annealed samples reveals an additional magnetic phase transition at lower temperatures.

EPR Spectra of Sintered Cd1-xCrxTe Powdered Crystals with Various Cr Content.

In this paper, we show a simple method of producing ferromagnetic materials with a Curie temperature above room temperature. The electron paramagnetic resonance (EPR) spectra of Cd1-xCrxTe (0.002 < x < 0.08) were measured with a dependence on temperature (82 K < T < 381 K). Obtained EPR lines were fitted to a Lorentz-shaped curve. The temperature dependencies of the parameters of the EPR lines, such as the peak-to-peak linewidth (Hpp), the intensity (A), as well as the resonance field (Hr), were studied. Ferromagnetism was noticed in samples at high temperatures (near room temperature). For a sample with a nominal concentration of chrome of x = 0.05, a very strong intrinsic magnetic field is observed. The value of the effective gyromagnetic factor for this sample is ge = 30 at T = 240 K. An increase of chrome concentration above x = 0.05 reduces the ferromagnetic properties considerably. Analysis of the temperature dependencies of the integral intensity of EPR spectra was carried out using the Curie-Weiss law and the paramagnetic Curie temperature was obtained.

Influence of Conditioning Temperature on Defects in the Double Al2O3/ZnO Layer Deposited by the ALD Method.

In this work, we present the results of defects analysis concerning ZnO and Al2O3 layers deposited by atomic layer deposition (ALD) technique. The analysis was performed by the X-band electron paramagnetic resonance (EPR) spectroscopy, transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS) methods. The layers were either tested as-deposited or after 30 min heating at 300 °C and 450 °C in Ar atmosphere. TEM and XPS investigations revealed amorphous nature and non-stoichiometry of aluminum oxide even after additional high-temperature treatment. EPR confirmed high number of defect states in Al2O3. For ZnO, we found the as-deposited layer shows ultrafine grains that start to grow when high temperature is applied and that their crystallinity is also improved, resulting in good agreement with XPS results which indicated lower number of defects on the layer surface.

pH-Responsive Redox Nanoparticles Protect SH-SY5Y Cells at Lowered pH in a Cellular Model of Parkinson's Disease.

The damage to SH-SY5Y cells by 6-hydroxydopamine (6-OHDA) is an established cellular model of Parkinson's disease (PD). Redox nanoparticles are a promising tool for therapy, including neurodegenerative diseases. As pH of the brain tissue at sites affected by PD is lowered down to 6.5, we studied the effect of pH-responsive redox nanoparticles (poly(ethylene glycol)-b-poly[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)aminomethylstyrene]), which change their structure in a pH-dependent manner and become active below pH 7 (NRNPs pH), on the viability of SH-SY5Y cells treated with 6-OHDA at pH 6.5 and 7.4. Pretreatment of the cells with NRNPs pH (15-75 µM) prior to the 6-OHDA treatment increased their survival in a concentration-dependent manner at pH 6.5, but not at pH 7.4. Among several parameters studied (ATP and GSH content, the level of reactive oxygen species, mitochondrial potential, mitochondrial mass), only the mitochondrial mass was dose-dependently protected by NRNPs pH at pH 6.5, but not at pH 7.4. These results indicate that the action of NRNPs pH on mitochondria underlies their protective effect in this cellular model of PD. These results may have potential importance for future applications of NRNPs pH in preclinical and perhaps clinical studies.

Nitroxide Radical-Containing Redox Nanoparticles Protect Neuroblastoma SH-SY5Y Cells against 6-Hydroxydopamine Toxicity.

Parkinson's disease (PD) patients can benefit from antioxidant supplementation, and new efficient antioxidants are needed. The aim of this study was to evaluate the protective effect of selected nitroxide-containing redox nanoparticles (NRNPs) in a cellular model of PD. Antioxidant properties of NRNPs were studied in cell-free systems by protection of dihydrorhodamine 123 against oxidation by 3-morpholino-sydnonimine and protection of fluorescein against bleaching by 2,2-azobis(2-amidinopropane) hydrochloride and sodium hypochlorite. Model blood-brain barrier penetration was studied using hCMEC/D3 cells. Human neuroblastoma SH-SY5Y cells, exposed to 6-hydroxydopamine (6-OHDA), were used as an in vitro model of PD. Cells were preexposed to NRNPs or free nitroxides (TEMPO or 4-amino-TEMPO) for 2 h and treated with 6-OHDA for 1 h and 24 h. The reactive oxygen species (ROS) level was estimated with dihydroethidine 123 and Fluorimetric Mitochondrial Superoxide Activity Assay Kit. Glutathione level (GSH) was measured with ortho-phtalaldehyde, ATP by luminometry, changes in mitochondrial membrane potential with JC-1, and mitochondrial mass with 10-Nonyl-Acridine Orange. NRNP1, TEMPO, and 4-amino-TEMPO (25-150 µM) protected SH-SY5Y cells from 6-OHDA-induced viability loss; the protection was much higher for NRNP1 than for free nitroxides. NRNP1 were better antioxidants in vitro and permeated better the model BBB than free nitroxides. Exposure to 6-OHDA decreased the GSH level after 1 h and increased it considerably after 24 h (apparently a compensatory overresponse); NRNPs and free nitroxides prevented this increase. NRNP1 and free nitroxides prevented the decrease in ATP level after 1 h and increased it after 24 h. 6-OHDA increased the intracellular ROS level and mitochondrial superoxide level. Studied antioxidants mostly decreased ROS and superoxide levels. 6-OHDA decreased the mitochondrial potential and mitochondrial mass; both effects were prevented by NRNP1 and nitroxides. These results suggest that the mitochondria are the main site of 6-OHDA-induced cellular damage and demonstrate a protective effect of NRNP1 in a cellular model of PD.

Interaction of Catechins with Human Erythrocytes.

The aim of this study was to characterize the interaction of chosen catechins ((+)-catechin, (-)-epigallocatechin (EGC), and (-)-epigallocatechin gallate (EGCG)) with human erythrocytes and their protective effects against oxidative damage of erythrocytes. Uptake of the catechins by erythrocytes was studied by fluorimetry, their interaction with erythrocyte membrane was probed by changes in erythrocyte osmotic fragility and in membrane fluidity evaluated with spin labels, while protection against oxidative damage was assessed by protection against hemolysis induced by permanganate and protection of erythrocyte membranes against lipid peroxidation and protein thiol group oxidation. Catechin uptake was similar for all the compounds studied. Accumulation of catechins in the erythrocyte membrane was demonstrated by the catechin-induced increase in osmotic resistance and rigidification of the erythrocyte membrane detected by spin labels 5-doxyl stearic acid and 16-doxyl stearic acid. (-)-Epigallocatechin and EGCG inhibited erythrocyte acetylcholinesterase (mixed-type inhibition). Catechins protected erythrocytes against permanganate-induced hemolysis, oxidation of erythrocyte protein thiol groups, as well as membrane lipid peroxidation. These results contribute to the knowledge of the beneficial effects of catechins present in plant-derived food and beverages.

Dietary antioxidants as a source of hydrogen peroxide.

Studies of 54 antioxidants revealed that 27 of them, mainly polyphenols, generated hydrogen peroxide (H2O2) when added to Dulbecco's modified Eagle's medium (DMEM), other media used for culture of mammalian and yeast cells and phosphate-buffered saline. The most active antioxidants were: propyl gallate (PG), (-)-epigallocatechin gallate (EGCG) and quercetin (Q). Chelex treatment and iron chelators decreased H2O2 generation suggesting that transition metal ions catalyze antioxidant autoxidation and H2O2 production. Green tea also generated H2O2; tea prepared on tap water generated significantly more H2O2 than tea prepared on deionized water. Ascorbic acid decreased H2O2 production although it generated H2O2 itself, in the absence of other additives. Lemon added to the tea significantly reduced generation of H2O2. Hydrogen peroxide generated in the medium contributed to the cytotoxicity of PG, EGCG and Q to human prostate carcinoma DU-145 cells, since catalase increased the survival of the cells subjected to these compounds in vitro.

Tempo-phosphate as an ESR tool to study phosphate transport.

TEMPO-phosphate has been introduced as a phosphate analogue to study phosphate transport in erythrocytes. The nitroxide is reduced intracellularly upon entering the cells, the membrane transport being the rate-limiting step of the loss of ESR signal. The use of TEMPO-phosphate is convenient and avoids the hazard of radioactivity. We studied the inhibition of TEMPO-phosphate transport to human erythrocytes by various compounds. DIDS and SITS, inhibitors of Band 3, inhibited the TEMPO-phosphate transport. 1-cyano-4-hydroxycinnamic acid, inhibitor of monocarboxylate transporters, did not affect the permeation of TEMPO-phosphate. The transport of TEMPO-phosphate was inhibited by various polyphenols, especially curcumin, naringin, quercetin, luteolin and kaempferol. Interestingly, 3-bromopyruvic acid, an alkylating agent and potential anticancer agent, induced an apparent enhancement of TEMPO-phosphate transport into erythrocytes.

Glycation of bovine serum albumin by ascorbate in vitro: Possible contribution of the ascorbyl radical?

Ascorbic acid (AA) has been reported to be both pro-and antiglycating agent. In vitro, mainly proglycating effects of AA have been observed. We studied the glycation of bovine serum albumin (BSA) induced by AA in vitro. BSA glycation was accompanied by oxidative modifications, in agreement with the idea of glycoxidation. Glycation was inhibited by antioxidants including polyphenols and accelerated by 2,​2'-​azobis-​2-​methyl-​propanimidamide and superoxide dismutase. Nitroxides, known to oxidize AA, did not inhibit BSA glycation. A good correlation was observed between the steady-state level of the ascorbyl radical in BSA samples incubated with AA and additives and the extent of glycation. On this basis we propose that ascorbyl radical, in addition to further products of AA oxidation, may initiate protein glycation.