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Cell death is an important physiological phenomenon in life. It can be programmed or unprogrammed. Unprogrammed cell death is usually induced by abiotic or biotic stress. Recent studies have shown that many proteins regulate both cell death and immunity in plants. Here, we provide a review on the advances in plant immunity with cell death, especially the molecular regulation and underlying mechanisms of those proteins involved in both cell death and plant immunity. In addition, we discuss potential approaches toward improving plant immunity without compromising plant growth.

With the increasing number of applications of silver nanoparticles (Ag NPs) in consumer products, including food contact applications, it is important to understand potential health effects of ingestion of Ag NPs. The biosafety analysis of Ag NPs in various mammalian cells has been widely studied; however, the influence of the gastrointestinal environment on the physicochemical properties and toxicity of Ag NPs has not been fully addressed. In the present study, we investigated the impact of simulated human gastrointestinal tract (GIT) fluids on the physicochemical properties of Ag NPs and their impact on the in vitro cytotoxicity of Ag NPs in intestinal epithelial (Caco-2) cells. Polyvinylpyrrolidone (PVP)-coated Ag NPs of different sizes (30, 50, 100 nm) were incubated in three GIT fluids that differed in composition and pH (1.6, 5.0, 6.5) and were designed to mimic human gastric fluid in a fasted state (FaSSGF), human intestinal fluid in a fasted state (FaSSIF), and human intestinal fluid in a fed state (FeSSIF). Time-dependent decreases in UV–Vis absorption and increases in dynamic light scattering (DLS) were observed during the three-hour incubation of Ag NPs in GIT fluids. The results of transmission electron microscopy (TEM) coupled with energy dispersive X-ray spectroscopy (EDS) suggested that the Ag NPs aggregated and precipitated as AgCl salts in the presence of FaSSGF and FeSSIF. In the presence of FaSSGF and hydrogen peroxide (H2O2), a particle size- and concentration-dependent generation of hydroxyl radicals ( OH) was detected by electron spin resonance (ESR) spectroscopy. When compared to the effects observed with the incubation of Ag NPs in H2O, incubation of Ag NPs in FaSSGF showed decreased cellular uptake in Caco-2 cells. In contrast, the incubation of Ag NPs in FeSSIF demonstrated increased cellular uptake and cytotoxicity. The results of this study demonstrate that GIT fluids have a significant influence on the physicochemical properties and cytotoxicity of Ag NPs and they provide valuable information that may be used in the safety evaluation of Ag NPs for consumer products.

The promising broad applications of graphene oxide (GO) derivatives in biomedicine have raised concerns about their safety on biological organisms. However, correlations between the physicochemical properties, especially oxidation degree of GOs and their toxicity, and the underlying mechanisms are not well understood. Herein, we evaluated the cytotoxicity of three GO samples with various oxidation degrees on mouse embryo fibroblasts (MEFs). Three samples can be internalized by MEFs observed via transmission electron microscopy (TEM), and were well tolerant by MEFs at lower doses (below 25 mu g/ ml) but significantly toxic at 50 and 100 mu g/ml via Cytell Imaging System. More importantly, as the oxidation degree decreased, GO derivatives led to a higher degree of cytotoxicity and apoptosis. Meanwhile, three GOs stimulated dramatic enhancement in reactive oxygen species (ROS) production in MEFs, where the less oxidized GO produced a higher level of ROS, suggesting the major role of oxidative stress in the oxidation- degree dependent toxicity of GOs. Results from electron spin resonance (ESR) spectrometry showed a strong association of the lower oxidation degree of GOs with their stronger indirect oxidative damage through facilitating H2O2 decomposition into (OH)-O-center dot and higher direct oxidativeabilities on cells. The theoretical simulation revealed the key contributions of carboxyl groups and aromatic domain size of nanosheets to varying the energy barrier of H2O2 decomposition reaction. These systematic explorations in the chemical mechanisms unravel the key physicochemical properties that would lead to the diverse toxic profiles of the GO nanosheets with different oxygenation levels, and offer us new clues in the molecular design of carbon nanomaterials for their safe applications in biomedicine. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

A broadly applicable theoretical and experimental framework was developed for understanding the photocatalytic mechanism of semiconductors. Using this framework, we found that it is possible to predict the type and reactivity of reactive oxygen species and electrons produced during photoexcitation of semiconductors by comparing the band edge energies of semiconductors with the redox potentials of relevant species. In addition, we could experimentally verify these predictions using electron spin resonance spectroscopy (ESR) with spin trapping and spin labeling techniques. We selected four types of metal sulfides (CdS, ZnS, In2S3, and Bi2S3) to elucidate the applicability of this model system. Using ESR technique, we found that these four sulfides are significantly different in the types of produced reactive oxygen species. When irradiated, ZnS can generate superoxide (O2− ), hydroxyl radicals ( OH), and singlet oxygen (1O2); CdS and In2S3 can produce O2− , while irradiation of Bi2S3 generates none of these reactive oxygen species. These results are correlated with the photocatalytic oxidation and reduction activities of metal sulfide structures.

ZnO micro/nano structures with different morphologies have been prepared by the changing solvents used during their synthesis by solvothermal reaction. Three typical shapes of ZnO structures including hexagonal, bell bottom like and multi-pod formed and were characterized by scanning electron microscopy and X-ray diffraction. Multi pod like ZnO structures exhibited the highest photocatalytic activity toward degradation of methyl orange. Using electron spin resonance spectroscopy coupled with spin trapping techniques, we demonstrate an effective way to identify precisely the generation of hydroxyl radicals, superoxide and singlet oxygen from the irradiated ZnO multi pod structures. The type of reactive oxygen species formed was predictable from the band gap structure of ZnO. These results indicate that the shape of micro-nano structures significantly affects the photocatalytic activity of ZnO, and demonstrate the value of electron spin resonance spectroscopy for characterizing the type of reactive oxygen species formed during photoexcitation of semiconductors.

Many of the chemical and biological effects of silver nanoparticles (Ag NPs) are attributed to the generation of reactive oxygen species (ROS). ESR spectroscopy was used to provide direct evidence for generating ROS during decomposition of H(2)O(2) assisted by Ag NPs. Hydroxyl radical formation was observed under acidic conditions and was accompanied by dissolution of Ag NPs. In contrast, evolution of O(2) was observed in alkaline solutions containing H(2)O(2) and Ag NPs; however, no net dissolution of Ag NPs was observed due to re-reduction of Ag(+) as evidenced by a cyclic reaction. Since H(2)O(2) is a biologically relevant product being continuously generated in cells, these results obtained under conditions mimicking different biological microenvironments may provide insights for finding new biomedical applications for Ag NPs and for risk assessment.

Nano-sized titanium dioxide (TiO(2)) is among the top five widely used nanomaterials for various applications. In this study, we determine the phototoxicity of TiO(2) nanoparticles (nano-TiO(2)) with different molecular sizes and crystal forms (anatase and rutile) in human skin keratinocytes under UVA irradiation. Our results show that all nano-TiO(2) particles caused phototoxicity, as determined by the MTS assay and by cell membrane damage measured by the lactate dehydrogenase (LDH) assay, both of which were UVA dose- and nano-TiO(2) dose-dependent. The smaller the particle size of the nano-TiO(2) the higher the cell damage. The rutile form of nano-TiO(2) showed less phototoxicity than anatase nano-TiO(2). The level of photocytotoxicity and cell membrane damage is mainly dependent on the level of reactive oxygen species (ROS) production. Using polyunsaturated lipids in plasma membranes and human serum albumin as model targets, and employing electron spin resonance (ESR) oximetry and immuno-spin trapping as unique probing methods, we demonstrated that UVA irradiation of nano-TiO(2) can induce significant cell damage, mediated by lipid and protein peroxidation. These overall results suggest that nano-TiO(2) is phototoxic to human skin keratinocytes, and that this phototoxicity is mediated by ROS generated during UVA irradiation.

Au nanorods @ Pt nanodots core/shell nanostructures, prepared by the Au nanorods (NRs)-mediated growth, exhibit dual functional enzyme-like (peroxidase and oxidase-like) activities. From the viewpoint of enzyme mimics, the relationship between apparent enzyme kinetic parameters and nanomaterials structure is investigated in order to rationally design the catalytic activity. Using peroxidase-like properties of the Au@Pt NRs, the determination of hydrogen peroxide (H2O2) was demonstrated with a limit of detection (LOD) of 4.5 × 10−5 M and a linear range of 4.5 × 10−5–1 × 10−3 M using o-phenylenediamine (OPD) as chromogenic substrate. Furthermore, in combination with highly specific reactions provided by natural enzymes, selective detections of glucose and lipophilic cholesterol were demonstrated with similar LODs and linear ranges. Additionally, owing to the specific oxidase-like activity of the Au@Pt NRs (ascorbate oxidase), interference of ascorbic acid in the detection of glucose could be eliminated. In conclusion, considering the flexibility in the design of nanomatererials, there is a lot of space to improve their activity and explore their potential applications, especially in relatively harsh conditions.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous genotoxic environmental pollutants and potentially pose a health risk to humans. In most if not all cases, PAHs in the environment can be oxidized into their corresponding PAH–diones. This process is considered a detoxification pathway with regard to tumorigenicity. Nevertheless, photo-induced toxicological activity of PAH–diones has not been systematically investigated. In this study, we show that 27 potential environmental PAH–diones induced lipid peroxidation, in a dose (light) response manner, when irradiated with UVA at 7 and 21 J cm −2 . Photoirradiation in the presence of sodium azide, deuterated methanol, or superoxide dismutase revealed that lipid peroxidation is mediated by reactive oxygen species. Electron spin resonance (ESR) spin trapping studies supported this observation. These results suggest that UVA photoirradiation of PAH–diones generates reactive oxygen species and induces lipid peroxidation.

In this paper, we demonstrated that Au nanorods coated with a shell composed of Pt nanodots (Au@Pt nanostructures) exhibited intrinsic oxidase-like, peroxidase-like and catalase-like activity, catalyzing oxygen and hydrogen peroxide reduction and the dismutation decomposition of hydrogen peroxide to produce oxygen. Based on these findings, we established an Au@Pt nanostructures based enzyme linked immunosorbent assay (ELISA) for the detection of mouse interleukin 2 (IL-2). In comparison with natural enzymes, Au@Pt nanostructures have advantages of low cost, easy preparation, better stability, and tunable catalytic activity (compared with HRP), which make them a promising enzyme mimetic candidate and may find potential applications in biocatalysis, bioassays, and nano-biomedicine such as reactive oxygen species (ROS)-related fields (anti-aging and therapeutics for neurodegenerative diseases and cancers).

A combinative method using high-speed counter-current chromatography (HSCCC) and thin layer chromatography (TLC) as an antioxidant autographic assay was developed to separate antioxidant components from the fruits of Psoralea corylifolia. Under the guidance of TLC bioautography, eight compounds including five flavonoids and three coumarins were successfully separated from the fruits of P. corylifolia by HSCCC with an optimized two-phase solvent system, n-hexane-ethyl acetate-methanol-water (1:1.1:1.3:1, v/v/v/v). The separation produced 5.91mg psoralen, 6.26mg isopsoralen, 3.19mg psoralidin, 0.92mg corylifol A, and 2.43mg bavachinin with corresponding purities of 99.5, 99.8, 99.4, 96.4, and 99.0%, as well as three sub-fractions, in a single run from 250mg ethyl acetate fraction of P. corylifolia extract. Following an additional clean-up step by preparative TLC, 0.4mg 8-prenyldaidzein (purity 91.7%), 4.18mg neobavaisoflavone (purity 97.4%) and 4.36mg isobavachalcone (purity 96.8%) were separated from the three individual sub-fractions. The structures of the isolated compounds were identified by (1)H NMR and (13)C NMR. The results of antioxidant activity estimation by electron spin resonance (ESR) method showed that psoralidin was the most active antioxidant with an IC50 value of 44.7microM. This is the first report on simultaneous separation of eight compounds from P. corylifolia by HSCCC.

The effect of allyl isothiocyanate (AITC) on radical scavenging capacity, and fruit decay of blueberries var. Duke (Vaccinium corymbosum L.) was evaluated. Results from this study showed that AITC was effective in retarding blueberry decay during storage at 10 °C. However, AITC-treated fruit decreased the contents of total phenolics and anthocyanins. Compared to control, AITC-treated berries had lower scavenging capacities against radicals of oxygen radical absorbance capacity (pyroxyl radical; ORAC), hydroxyl radical scavenging capacity ( OH) and 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH ), but promoted the accumulation of hydrogen peroxide (H2O2) radicals. The free radical scavenging properties of blueberry fruit with or without AITC treatment were also evaluated by electron spin resonance (ESR). Results of the ESR measurements confirmed that free radical scavenging capacities against OH, DPPH and O 2 − were lower in treated fruit than in control un-treated fruit. The results from this study indicate that AITC does not promote antioxidant property or scavenging of constitutive reactive oxygen species (ROS), but paradoxically generates additional amounts of ROS to inhibit the growth and proliferation of microbial cells, thereby reducing decay in fruit tissue.

Azulene and guaiazulene are popular ingredients in beauty, cosmetic, skin, and body care products. We previously determined that these chemicals are photomutagenic in Salmonella and phototoxic, causing DNA damage in human Jurkat T-cells. In this study we report that photoirradiation of azulene and guaiazulene, respectively, by UVA light at 0–70 J/cm2 in the presence of a lipid, methyl linoleate, resulted in lipid peroxidation in a light dose–responsive manner. When irradiated in the presence of sodium azide or superoxide dismutase, the level of lipid peroxidation decreased, indicating that lipid peroxidation is mediated by free radical and superoxide in particular. In contrast, lipid peroxidation was not enhanced when deuterated methanol was incorporated to the system, which suggests that singlet oxygen is not a predominant photo-induced product. Electron spin resonance (ESR) spin trapping study confirmed that photoirradiation of azulene predominantly generated superoxide and none or very low quantity of singlet oxygen was produced. These results indicate that photoirradiation of azulene and guaiazulene by UVA light generates reactive oxygen species (ROS), and induces lipid peroxidation when irradiation in the presence of a lipid. These results implicate that azulene and guaiazulene are phototoxic when exposed to sunlight.

Nanoparticles have been explored recently as an efficient means of delivering photosensitizers for cancer diagnosis and photodynamic therapy (PDT). Silicon phthalocyanine 4 (Pc4) is currently being clinically tested as a photosensitizer for PDT. Unfortunately, Pc4 aggregates in aqueous solutions, which dramatically reduces its PDT efficacy and therefore limits its clinical application. We have encapsulated Pc4 using silica nanoparticles (Pc4SNP), which not only improved the aqueous solubility, stability, and delivery of the photodynamic drug but also increased its photodynamic efficacy compared to free Pc4 molecules. Pc4SNP generated photo-induced singlet oxygen more efficiently than free Pc4 as measured by chemical probe and EPR trapping techniques. Transmission electron microscopy and dynamic light scattering measurements showed that the size of the particles is in the range of 25-30 nm. Cell viability measurements demonstrated that Pc4SNP was more phototoxic to A375 or B16-F10 melanoma cells than free Pc4. Pc4SNP photodamaged melanoma cells primarily through apoptosis. Irradiation of A375 cells in the presence of Pc4SNP resulted in a significant increase in intracellular protein-derived peroxides, suggesting a Type II (singlet oxygen) mechanism for phototoxicity. More Pc4SNP than free Pc4 was localized in the mitochondria and lysosomes. Our results show that these stable, monodispersed silica nanoparticles may be an effective new formulation for Pc4 in its preclinical and clinical studies. We expect that modifying the surface of silicon nanoparticles encapsulating the photosensitizers with antibodies specific to melanoma cells will lead to even better early diagnosis and targeted treatment of melanoma in the future.

Tamoxifen is a powerful drug used to treat breast cancer patients, and more than 500,000 women in the U.S. are being treated with this drug. In our study, tamoxifen is found to be photomutagenic in Salmonella typhimurium TA102 at concentrations as low as 0.08 μM and reaches maximum photomutagenicity at 0.4 μM under a light dose equivalent to 20 min sunlight. These concentrations are comparable to the plasma tamoxifen concentration of 0.4–3 μM for patients undergoing tamoxifen therapy. The toxicity seems to be the result of DNA damage and/or lipid peroxidation caused by light irradiation of tamoxifen. The DNA damage caused by irradiation of ФX174 DNA in the presence of tamoxifen appears to be formation of DNA–tamoxifen covalent adducts, not single strand/double strand cleavages, and there is no oxygen involvement. This is confirmed by EPR experiments that carbon-centerd radicals are formed by light irradiation of tamoxifen and there is no singlet oxygen formation. Although superoxide radical is formed, it is not involved in DNA damage.

The preventive and therapeutic effects of a major component of catechins of green tea, epigallocatechin-3-gallate (EGCG), on prostate cancer have been demonstrated in many studies. It is well known that metal ions are necessary for human health, but an imbalance in metal ions metabolism can lead to many diseases including prostate cancer. Understanding the interactions of EGCG with metal ions might elucidate its mechanism in preventing and curing prostate cancer. The present study focused on the effects of Cd(2+) and EGCG on the growth of androgen-insensitive prostate cancer cell PC-3 investigated by MTT assay, the effects of EGCG and Cd(2+) on absorption of Cd(2+) and Zn(2+) by PC-3 cells were detected by atomic absorption spectroscopy (AAS), and the interactions of EGCG with Cd(2+) were determined by distribution coefficient and UV-Vis spectroscopy detection. The results showed that Cd(2+) suppressed viability of PC-3 cells in concentration- and time-dependent manner, and EGCG enhanced the effect of Cd(2+) on PC-3 cells. EGCG was shown to decrease the absorption Cd(2+) and increase the absorption of Zn(2+) by PC-3 cells, while the effects of Cd(2+) on the absorption of Cd(2+) and Zn(2+) were opposite to that of EGCG. In the presence of both EGCG and Cd(2+), absorption of Cd(2+) and Zn(2+) by PC-3 cells was dependent on concentrations of EGCG, Cd(2+) and its order of addition. Results from the distribution coefficient determination and UV-Vis spectroscopy analysis indicated that Cd(2+) might affect conformation of EGCG, while no complex of EGCG with Cd(2+) was observed in the system.

Aloe vera whole leaf extracts are incorporated into a wide variety of topically applied commercial products. Aloe vera whole leaf extracts may contain anthraquinones, which have been shown to generate reactive oxygen species in the presence of ultraviolet A (UVA) light. Exposure to UVA light alone can also generate reactive oxygen species and is associated with photo-damaged and photo-aged skin in humans. This paper examines the photochemical properties of two Aloe vera whole leaf extracts that differed in their anthraquinone content. In the presence of methyl linoleate, the UVA irradiation of Aloe vera leaf extracts induced lipid peroxidation. The amounts of lipid peroxides formed were higher in the Aloe vera leaf extract that contained lower amounts of anthraquinones. Superoxide dismutase and sodium azide inhibited and deuterium oxide enhanced the formation of lipid peroxides, suggesting that singlet oxygen and superoxide were involved in the mechanism. Spin trapping electron spin resonance (ESR) spectroscopy was used to investigate the generation of free radicals by the UVA photo-irradiated Aloe vera plant extracts. ESR measurements indicated that the UVA photo-irradiation of Aloe vera plant extracts produced carbon-centered free radicals. These results suggest that humans exposed to products that contain Aloe vera whole leaf extracts may have enhanced sensitivity to ultraviolet light.

Five phenolic acids commonly present in wheat grain and fractions were examined and compared for their radical-scavenging properties and chelating capacities. The free radical-scavenging properties were evaluated against 2,2-diphenyl-1-picryhydrazyl radical (DPPH ), radical cation (ABTS +), peroxide radical anion ( O 2 - ) and hydroxyl radical (HO ), whereas the chelating properties were evaluated against Fe (II) and Cu (II) using spectrophotometric and electron spin resonance measurements. These phenolic acids differed in their abilities to react with and quench HO , O 2 - , ABTS + and DPPH , as well as their capacities to form chelating complexes with transition metals. 4-Hydroxybenzoic acid had neither free radical-scavenging nor chelating activity under the experimental conditions. Strong structure–activity relationships were observed in the present study. Both substituents on the phenyl ring and the conjugated carbon skeleton may influence the antioxidant properties of phenolic acids. The presence of an additional methoxyl group in the ortho position of the hydroxyl group showed a strong influence on the chelating property of phenolic acids and their radical-scavenging capacity against O 2 - , ABTS + and DPPH , but not on their HO -scavenging activity.

Catechins, which exist in green tea, are excellent antioxidants and metals enhance their pro-oxidative character. In this study, we investigated damage to the cell cytoplasmic membrane of the androgen-insensitive prostate cancer cell PC-3 from rearrangement of EGCG (epigallocatechin gallate), the main bioactive component of catechins. In a cell culture system containing Cu2+, the generation of free radicals from EGCG rearrangement was measured. Electron microscopy, flow cytometry, gel electrophoresis, and ESR measurements showed that the cytoplasmic membrane of PC-3 cells was disrupted in this culture system and that the death of most cells followed within a few minutes. Hydroxyl radicals, detected in the ESR experiment using the spin trapping method, may cause damage to the cytoplasmic membrane. Analysis of F-12 medium after 6 h, by HPLC, showed small amounts of EGCG when PC-3 cells were absent, and no detectable levels when PC-3 cells were present. In addition to the concentration and order in which EGCG and Cu2+ are added to the culture medium, oxides, polymers, or some compounds integrated with the cytoplasmic membrane of the PC-3 cells themselves may have a key role in damage to the PC-3 cell cytoplasmic membrane.

We have previously demonstrated that American ginseng berry extract exhibited significant protection against oxidant-mediated injury in cardiomyocytes. To extend this work, we sought to investigate the antioxidant effects of Re, a protopanaxatriols-type and single chemical integrant present in American ginseng berry extract, using the same chick cardiomyocyte model of oxidant injury as well as ESR spectroscopy in a cell-free chemical system. In cells exposed to 2 h of H2O2 (0.5 mM), pretreatment with Re (0.05, 0.1, or 0.5 mg/ml for 2 h) significantly attenuated 2',7'-dichlorofluorescein (DCF) fluorescence by 51% (from 1345+/-67 to 658+/-46 a.u., P

The mechanism of resistance of cancer cells to the anticancer drug cisplatin is not fully understood. Using cisplatin-sensitive KB-3-1 and -resistant KCP-20 cells, we found that the resistant cells have higher membrane potential, as determined by membrane potential sensing oxonol dye. Electron spin resonance and fluorescence polarization studies revealed that the resistant cells have more "fluid" plasma membranes than the sensitive cells. Because of this observed difference in membrane "fluidity," we attempted modification of the plasma membrane fluidity by the incorporation of heptadecanoic acid into KB-3-1 and KCP-20 cell membranes. We found that such treatment resulted in increased heptadecanoic acid content and increased fluidity in the plasma membranes of both cell types, and also resulted in increased cisplatin resistance in the KCP-20 cells. This finding is in accord with our results, which showed that the cisplatin-resistant KCP-20 cells have more fluid membranes than the cisplatin-sensitive KB-3-1 cells. It remains to be determined whether the observed differences in biophysical status and/or fatty acid composition alone, or the secondary effect of these differences on the structure or function of some transmembrane protein(s), is the reason for increased cisplatin resistance.

Electron spin resonance (ESR)1 spin-label oximetry and spin trapping techniques have been used to study the effect of fumonisin B1 (FB1), an amphipathic mycotoxin on lipid peroxidation in egg yolk phosphatidylcholine (EYPC) bilayers. In the study of the interaction between FB1 and lipid bilayers our results show that fumonisin disturbs the ordering of membranes, enhances oxygen transport in membranes, and also increases membrane permeability. In our model system, lipid peroxidations were initiated by extended incubation of the liposomes, or by inducing Fe2+ ions, UV illumination of H2O2 or ultrasound irradiation. As an indication of the rates of lipid oxidation in EYPC, the consumption of molecular oxygen was studied by monitoring the oxygen concentration in the aqueous phases of the liposomes. Lipid-derived free radicals generated during the oxidation process were measured by a spin trapping method. The incorporation of FB1 in the test samples made the membranes highly susceptible to oxidation. Our results provide the first evidence that the fumonisins appear to increase the rate of oxidation, promote the free radical intermediate production and accelerate the chain reactions associated with lipid peroxidation. The disruption of membrane structure, the enlargement of the relative oxygen diffusion–concentration products, as well as the enhancement effects on membrane permeability, thus provide additional insights into potential mechanisms by which the fumonisins could enhance oxidative stress and cell damage.

The semiconductor TiO2 is known to have photobiological activity in prokaryotic and eukaryotic cells. Applications of this photobiological activity have been suggested including sterilization of waste water and phototherapy of malignant cells. Here, several model and cellular systems were used to study the mechanism of photocatalysis by TiO2. Treatment of TiO2 (anatase, 0.45 microns), suspended in water containing a spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), with UV radiation (320 nm) resulted in an electron spin resonance (ESR) signal characteristic of the hydroxyl radical. Irradiation of solutions containing calf thymus DNA and TiO2 with UVA (320-400 nm) radiation resulted in hydroxylation of guanine bases. The degree of hydroxylation was dependent on both UVA fluence and amount of TiO2 in suspension. Human skin fibroblasts, preincubated 18 h with 10 micrograms/cm2 TiO2 and then UVA-irradiated (0-58 KJ/m2), showed dose dependent photocytoxicity. RNA, isolated from similarly treated fibroblasts, contained significant levels of photooxidation, measured as hydroxylation of guanine bases. However, no oxidative damage was detectable in cellular DNA. These results suggest that nucleic acids are a potential target for photooxidative damage sensitized by TiO2, and support the view that TiO2 photocatalyzes free radical formation.

Electron-electron double-resonance (ELDOR) experiments on nitroxide-radical-spin-labeled liposomes have been performed using a loop-gap resonator. The signal-to-noise ratio expressed on a molarity basis is 20-fold over the best that has been achieved using a bimodal cavity. This improvement permits ELDOR experiments on spin-labeled plasma membranes of intact cells, as illustrated by a prototype experiment on red blood cells labeled with stearic acid spin label. Moreover, 20 times greater pumping energy density at the sample is achievable for a given incident pump power, permitting ELDOR experiments on less readily saturated systems. Pump and observing frequencies are introduced directly into the loop-gap resonator, which has a relatively low Q , and the pump electron paramagnetic resonance signal is isolated from the receiver using a high Q trap microwave filter.

Rate equations describing the time dependence of population differences of the five allowed transitions in an 14N15N spin-label pair problem are set up. Included in the formulation are the three Heisenberg exchange rate constants and different nitrogen nuclear spin-lattice relaxation rates, electron spin-lattice relaxation rates, and populations for the 14N and 15N moieties. Using matrix algebra, stationary and time-dependent solutions are obtained in a unified theoretical framework. The calculations apply to stationary and pulse electron-electron double resonance and to saturation-recovery ESR. Particular emphasis is placed on short pulse initial excitation, where the transverse relaxation processes are sufficiently slow that only the population difference of the irradiated transition departs significantly from Boltzmann equilibrium during the excitation.

Human plasma fibronectin is a dimer consisting of two subunits; each contains two cryptic thiol groups that were selectively labeled with an 15N,2H-maleimide spin label. Previous studies using conventional X-band electron spin resonance (ESR) methods showed that the spectrum of the labeled protein displays a single strongly immobilized component with an effective rotational correlation time of approximately 17 ns, suggesting that the physical environments of the two labeled sites per chain are indistinguishable. Here we have used saturation-recovery ESR to measure directly electron spin-lattice relaxation time (T1) of the labeled protein in solution at 27 degrees C. Interestingly, the time evolution of the signal was found to be biphasic, which was deconvoluted into two T1 values of 1.37 and 4.53 microseconds. Thus, the two spin-labeled sulfhydryl sites of plasma fibronectin (Fn), being similar in rates of rotational diffusion, differ by a factor of 3.2 in T1. Parallel experiments using various fibronectin fragments showed that the 1.37-microseconds component is associated with the label attached onto the thiol located in between the DNA-binding and the cell-binding domains, and the 4.53-microseconds component is associated with the label attached onto the thiol located within the carboxyl-terminal fibrin-binding domain. The data suggest that the saturation-recovery ESR is a useful method for differentiating multiple spin-labeled sites on macromolecules in which the labels undergo similar rates of rotational motion.

Electron-electron double resonance (ELDOR) and saturation-recovery spectroscopy employing 14N:15N stearic acid spin-label pairs have been used to study the effects of cholesterol on lateral diffusion and vertical fluctuations in lipid bilayers. The 14N:15N continuous wave electron-electron double resonance (CW ELDOR) theory has been developed using rate equations based on the relaxation model. The collision frequency between 14N-16 doxyl stearate and 15N-16 doxyl stearate, WHex (16:16), is indicative of lateral diffusion of the spin probes, while the collision frequency between 14N-16 doxyl stearate and 15N-5 doxyl stearate, WHex (16:5), provides information on vertical fluctuations of the 14N-16 doxyl stearate spin probe toward the membrane surface. Our results show that: (a) cholesterol decreases the electron spin-lattice relaxation time Tle of 14N-16 doxyl stearate spin label in dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylcholine (egg PC). (b) Cholesterol increases the biomolecular collision frequency WHex (16:16) and decreases WHex (16:5), suggesting that incorporation of cholesterol significantly orders the part of the bilayer that it occupies and disorders the interior region of the bilayer. (c) Alkyl chain unsaturation of the host lipid moderates the effect of cholesterol on both vertical fluctuations and lateral diffusion of 14N-16 doxyl stearate. And (d), there are marked differences in the effects of cholesterol on lateral diffusion and vertical fluctuations between 0–30 mol% and 30–50 mol% of cholesterol that suggest an inhomogeneous distribution of cholesterol in the membrane.

Electron-electron double resonance (ELDOR) techniques employing [14N], [15N] 16-Doxylstearate spin-label pairs have been used to measure the lateral diffusion constant, D, of lipids in the surface membrane of intact human blood platelets. For freshly prepared platelets, D is 1.0 X 10(-8) cm2/s at 37 degrees C and for platelets stored for 3 d at room temperature under accepted routine blood bank conditions, D is 2.6 X 10(-8) cm2/s at 37 degrees C. This is the first time that D in the surface membrane of platelets is reported. The marked increase in D for stored platelets may be attributed at least partly to loss of cholesterol during storage, suggesting a correlation between lipid lateral diffusion and cholesterol levels in cell membranes.