Ultraviolet-B radiation applied to detached peach fruit: A study of free radical generation by EPR spin trapping.

In peaches, phenolic compounds are the major sources of antioxidants, and cyanidin-3-O-glucoside is the main anthocyanin present, above all in the skin. Anthocyanin content has been shown to increase after UV-B irradiation, which may be very harmful for all biological organisms due to the induction of the generation of reactive oxygen species (ROS). Peach fruits (cv. 'Suncrest') were exposed during post-harvest to supplemental ultraviolet-B radiation. A spin-trapping technique was used to monitor the generation of free radicals under UV-B, and 5-(diethoxy-phosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) was used as the spin trap. The flesh of peaches was essentially unaffected by the treatment, whereas the skin was responsive at the end of the treatment, accumulating ascorbate, flavonoids, cyanidin-3-O-glucoside, and showing a higher antioxidant activity. The levels of stable free radicals were also lower at the end of treatment. Carbon-centred radicals contributed the most to the total amounts of free radicals, whereas hydroxyl radicals and oxygen-centred free radicals contributed minimally. The carbon-centred free radical identified was the same as the one obtained after irradiation of authentic cyanidin-3-O-glucoside. During UV-B treatment cyanidin-3-O-glucoside increased and was capable of radicalization protecting the other organic molecules of the cell from oxidation. ROS, among which hydroxyl radicals, were thus maintained to minimal levels. This ability of cyanidin-3-O-glucoside displayed the mechanism underlined the tolerance to UV-B irradiation indicating that shelf life can be prolonged by the presence of anthocyanins. Thus, UV-B technique results a good approach to induce antioxidant production in peach fruits increasing their nutraceutical properties.

Enzymatic and non-enzymatic protective mechanisms in recalcitrant seeds of Araucaria bidwillii subjected to desiccation.

The changes in several antioxidants as well as in the level of C-centered free radicals and thiobarbituric acid reactive substances (TBARS) were studied in seeds of Araucaria bidwillii Hook desiccated to 37%, 28% and 21% moisture content. The lowest-safe moisture content for the seedling establishment was 37%. The embryo, besides double amounts of free radicals, showed higher levels of both enzymatic and non-enzymatic antioxidants than endosperm. Lutein decreased in both organs whereas alpha-tocopherol values were not affected by desiccation. In the embryo at 37% seed moisture content the antioxidant defense system increased giving rise to a decrease in free radicals. Beyond this point, free radicals and TBARS increased in agreement with the umpiring of the ascorbate/glutathione cycle by the decrease in reduced glutathione and glutathione reductase activity (GR, EC 1.6.4.2). At 21% moisture GR decreased. In the endosperm during desiccation, the consumption of ascorbate, total glutathione and lutein prevented the rise in free radicals and TBARS till 28% moisture, at which an increase in oxidized glutathione was also observed.

Structure and dynamics of high molecular weight glutenin subunits of durum wheat (Triticum durum) in water and alcohol solutions studied by electron paramagnetic resonance and circular dichroism spectroscopies.

Complementary information on the structure and dynamics of high molecular weight glutenin subunits (HMW-GS) of durum wheat (Triticum durum) was obtained by means of two spectroscopic techniques. Electron paramagnetic resonance was used to investigate the dynamics of the HMW-GS hydrated with two 2-propanol/water mixtures at temperatures between 268 and 308 K by specific spin labeling of their cysteine residues. Spectra were of a composite type, resulting from two populations of spin labels differing in molecular mobility, both undergoing isotropic rotational diffusion. Diffusional coefficients and populations of the fast- and slow-moving spin labels, determined by an accurate spectral line shape analysis, are discussed as a function of temperature and water content in the solvent systems. Far-UV circular dichroism was employed to provide information on the secondary structure of the HMW-GS in three different solvents [aqueous 50% (v/v) 2-propanol, aqueous 0.1% (v/v) trifluoroacetic acid, and trifluoroethanol]. For the first one, the influence of temperature on HMW-GS structure was also investigated.

Protein dynamics in thylakoids of the desiccation-tolerant plant Boea hygroscopica during dehydration and rehydration.

Plants of Boea hygroscopica F. Muell were dehydrated to 9% relative water content (RWC) by withholding water for 26 d, and afterward the plants were rehydrated. Leaves were taken from control plants after 7, 12, and 26 d from the beginning of dehydration, and after 6 and 48 h from rehydration. The RWC decreased by 80% during dehydration, but the leaves regained RWC with rehydration. Dehydrated plants showed lesser amounts of proteins, lipids, and chlorophyll, all of which increased following rewatering. The lipid-to-protein ratio, which decreased during dehydration, returned to control level after 48 h of rehydration. Thylakoid lipids were more unsaturated when RWC reached the value of 9%. EPR measurements of spin-labeled proteins showed the presence of three different groups of proteins with different mobility in thylakoid membranes. The rotational correlation time of groups 1 and 2 increased with dehydration and decreased upon rehydration, whereas group 3 showed little changes. Desiccation did not cause thylakoid swelling or breakage, but the membrane system assemblage showed changes in thylakoid stacking. After 48 h of rehydration the membrane system recovered completely the organization of the fully hydrated state, showing several well-defined and regularly distributed grana.

Novel priming and crosslinking systems for use with isocyanatomethacrylate dental adhesives.

OBJECTIVES: (a) to design, formulate and evaluate prototype primers and a crosslinking agent for use with isocyanatomethacrylate-based comonomer adhesives and (b) to establish correlations between bond strength and solubility parameter differences between the adhesives and etched dentin, and the permeability coefficients of the adhesives. METHODS: Equimolar mixtures of 2-isocyanatoethyl methacrylate (IEM) and a methacrylate comonomer were formulated with tri-n-butyl borane oxide (TBBO) as the free radical initiator to have cure times of 6-10 min. Shear bond strengths to dentin were determined for each adhesive mixture (n = 7) using standard testing protocols. Shear bond strengths for the three systems were also determined after application of "reactive primers" to the dentin surface. The "reactive primers" contained 10-20 parts by weight of the respective comonomer mixture and 3.5 parts by weight TBBO in acetone. Solubility parameters difference values (delta delta) and permeability coefficients (P) were approximated for each adhesive system and correlated with shear bond strength values. Additionally, a crosslinking agent was prepared by bulk reaction of an equimolar mixture containing IEM and a methacrylate comonomer. The effects of crosslinker addition on: (a) the setting time of IEM; and (b) the setting times and initiator requirements of selected IEM/comonomer mixtures were determined. RESULTS: Shear bond strength values (MPa): IEM/HEMA 13.6 +/- 2.0 (no primer), 20.1 +/- 2.0 (with primer); IEM/HETMA 9.3 +/- 3.3 (no primer), 20.8 +/- 8.1 (with primer); IEM/AAEMA 13.6 +/- 1.9 (no primer), 17.3 +/- 3.2 (with primer). Also, approximated permeability coefficients showed a significant correlation (r = +0.867, p < 0.001) with shear bond strength values. Crosslinker addition studies with IEM/4-META: (a) at 5-9 mol% reduced the setting time of IEM polymerization by 79%; and (b) at 6 mol% reduced initiator level requirements 60-70% to achieve a comparable setting time, and decreased setting times by ca. 75% for a given initiator level with selected IEM/methacrylate adhesive systems. SIGNIFICANCE: The shear bond strengths of isocyanatomethacrylate-based dental adhesives can be enhanced by using reactive primers; their setting times and initiator requirements can be improved using a dimethacrylate crosslinker. Approximated permeability coefficients may be useful as indicators of bonding performance for dentin adhesive systems.

Degradation of gluten by proteases from dry and germinating wheat (Triticum durum) seeds: An in vitro approach to storage protein mobilization.

Vital gluten was used as an ideal substrate to investigate the role of some proteases in storage protein degradation. Aspartic proteinase and carboxypeptidase were identified as endogenous enzymes adsorbed on gluten and their optimum pH values determined. SDS-PAGE of soluble products released by gluten digestion revealed that the activity of these proteases plays a minor role in protein mobilization, whereas cysteine proteinase, purified from wheat seeds at the fourth day of germination, is extremely effective, producing a remarkable protein degradation in short times. Synergistic effects of aspartic and cysteine proteinase were not observed. Spin labeling of the sulfhydryl groups of gluten proteins enabled a comparative EPR investigation of the consequences of proteolytic degradation on gluten elasticity. It was found that storage protein mobilization brings a loss of elasticity to the polymeric network of gluten, which is particularly marked when the hydrolysis is performed by cysteine proteinase.

Aging, free radicals, and antioxidants in wheat seeds.

Free radical oxidative attack is considered a major cause of disruption and deteriorative changes observed in aged seeds. Antioxidant defense mechanisms may remove potentially damaging molecular species, and carotenoids may act as radical scavengers. The content of lutein, the major carotenoid in wheat seeds, was determined in the flours. It showed a rapid decrease during seed aging. In addition, the content of free radicals in glutens made from flours of wheat seeds after long-term storage was studied. The concentration of radicals appeared to be age dependent, because the highest content of radicals was detected between 13 and 15 years of aging over 36 years of storage. Specific spin labeling of the sulfhydryl groups of gluten proteins enabled comparative EPR studies of the rigidity of the protein chains. A progressive stiffening of polymeric gluten with seed storage was found.

Design, formulation, and evaluation of isocyanatoacrylate copolymer dental adhesives.

Experiments have recently been completed to explore the development of isocyanatoacrylate copolymers as new dental adhesives. A main goal of this work was to test the utility of solubility parameter differences between the candidate adhesives and etched dentin as a predictor of relative bond strength. All candidate adhesive mixtures contained 2-isocyanatoethyl methacrylate (IEM), a selected amount of tri-n-butylborane oxide (TBBO) initiator, and one of 13 methacrylate comonomers. Reactivity ratios were computed for comonomer pairs as indicators of relative reactivity. The concentration of TBBO was optimized for each comonomer mixture to obtain working times of 2-6 min and setting times of 6-10 min. The solubility parameter difference Deltadelta (J/cm(3))(1/2) was calculated for each test mixture with respect to an etched dentin substrate, as an approximation of wetting ability. Using standard techniques for shear bond strength evaluation, mean shear bond strength values ranging between 7-15.5 MPa were obtained for comonomer adhesives in bonding Z-100 composite to treated dentin. Shear bond strength values showed a good correlation (r = -0.612, P

Superoxide and hydroxyl radical generation, and superoxide dismutase in PSII membrane fragments from wheat.

Illumination of photosystem II (PSII) membrane fragments of wheat (Triticum durum Desf. cv. Adamello) gave rise to both O2*- and *OH radicals adducts of the novel spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO). With time, *OH became predominant displaying the conversion of O2*- into *OH. An intrinsic activity of superoxide dismutase (SOD) was found in PSII. Photoreduction of nitroblue tetrazolium (NBT) by PSII membrane fragments was induced by the addition of sodium azide and hydrogen peroxide. Western blotting of PSII proteins showed that a 29 kDa protein was recognised by an antibody against chloroplastic Fe-SOD from water lily. An increased formation rate of O2*- was observed in damaged PSII where the SOD activity decreased following a treatment with a free radical-generating system. Damage in PSII consisted also in a decrease in chlorophyll and in carotenoids as well as in a change in the lipid : chlorophyll : protein ratio. Upon treatment a decrease in the unsaturation of PSII membrane fragments was also monitored together with a degradation towards more saturated molecular species of monogalactosyldiacylglycerol.

Activated oxygen generation from thylakoids: a novel spin trap.

Using a novel phosphorylated spin trap, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), an analogue of the commonly used spin trap 5,5'-dimethyl-1-pyrroline N-oxide (DMPO), we have investigated the production of oxygen radical species under illumination of thylakoids from wheat (Triticum durum Desf cv. Ofanto). DEPMPO reacted with superoxide (O2*-) and hydroxyl radical (HO*) forming distinctive spin trap adducts. Spectra of (O2*-) and HO* adducts of DEPMPO were recorded in the presence of xanthine/xanthine oxidase and FeSO4/H2O2, respectively, and computer simulation of spectra was performed. During illumination of thylakoids both O2*- and HO* were detected as well. Transition metals catalysed transformation of O2*- into HO*. The conversion was enhanced by H2O2 and prevented by exogenous superoxide dismutase and catalase. The presence of a thylakoid-bound superoxide dismutase, whose activity was inhibited by H2O2 and diethyldithiocarbamic acid, was responsible for H2O2 production from O2*- and thus for HO* generation.

Elements of light-cured epoxy-based dental polymer systems.

The greatest problem with current dental composite systems is their polymerization shrinkage. Extensive work is being done by many investigators to alleviate this problem. Our approach has been to examine epoxy- and spiro-orthocarbonate (SOC)-based resins. The hypothesis to be tested in this study was that the cure characteristics of experimental visible-light-cured epoxy resin systems are governed by the types and concentrations of co-reactants and activators. Resin samples containing onium salt initiators and a thiozanthone sensitizer were successfully cured by means of either an experimental visible-light irradiation system or a commercially available dental lamp. Test resins consisted of di-epoxies alone or in combination, epoxy mixtures in combination with an SOC, or an epoxy in combination with a caprolactone-derived polyol. Significant findings were as follows: (a) Resins containing the SOC had longer cure times than their counterparts; (b) the optimum ratios of epoxy to polyol for most rapid cure were 50:50 or 60:40 under conditions tested; (c) resins containing TONE 305 polyol generally were faster to cure than those containing no polyol, or TONES 201 or 310; and (d) a resin mixture was found that had a cure time of 1 to 3 min when irradiated with a commercial dental lamp. Based on this exploratory study, it should be possible for clinically relevant cure times to be achieved for visible-light-cured epoxy-based resins by careful manipulation and optimization of key elements.

Design and development of isocyanatoacrylates as dental adhesives.

During the last 12 years, significant progress has been made in the development of dental adhesive systems. Some of the more promising systems are based on multifunctional structures that contain polymerizable vinyl double bonds and reactive isocyanate groups. The utility of compounds with such structures as adhesives arises in part because their isocyanate functionality is available for reaction independently, without compromising the reactivity of the vinyl groups. The hypotheses tested in this investigation were: (1) that the monomer reactivity ratios (r1, r2) for the free-radical-initiated copolymerization of ethyl alpha-isocyanatoacrylate (alpha-EIA) and 2-isocyanatoethyl methacrylate (IEM) with selected vinyl monomers can be determined; (2) that these reactivity ratios can be used to establish Q (reactivity) and e (polarity) values for alpha-EIA and IEM; and (3) that these reactivity parameters can be useful in designing copolymers with controlled compositions for dental adhesive applications. The free-radical copolymerization characteristics of alpha-EIA and IEM were studied. The isocyanate monomers were copolymerized at seven comonomer ratios with n-butyl acrylate (NBA), methyl methacrylate (MMA), and styrene (STY). Reactivity ratios, r1 and r2, were calculated for each of the copolymer systems, giving:IEM (r1) = 0.38 and STY (r2) = 0.44; IEM (r1) = 1.19 and MMA (r2) = 0.84; IEM (r1) = 2.50 and NBA (r2) = 0.40; alpha-EIA (r1) = 2.20 and STY (r2) = 0.06; alpha-EIA (r1) = 7.00 and MMA (r2) = 0.10; and alpha-EIA (r1) = 23.50 and NBA (r2) = 0.04. The Q (reactivity) and e (polarity) values for IEM and alpha-EIA were calculated from r1 and r2 with use of the Alfrey-Price equations, giving, for IEM, Q = 0.89 and e = 0.60, and, for alpha-EIA, Q = 7.64 and e = 0.74. These reactivity parameters are useful for tailoring copolymers with controlled compositions and properties. Based on these calculated reactivity parameters, several copolymers of IEM [for example, IEM/2-hydroxyethyl methacrylate (HEMA)] are currently being prepared and evaluated as adhesives.

Lipid Composition and Protein Dynamics in Thylakoids of Two Wheat Cultivars Differently Sensitive to Drought.

Two wheat (Triticum durum Desf.) cultivars with different sensitivities to drought were either grown under regular irrigation or subjected to water deficit by withholding water for 14 d. Water-stressed plants of both cultivars underwent similar decreases in leaf water potential, but the drought-tolerant cultivar showed higher relative water content and turgor. Neither osmotic nor elastic adjustment mechanisms appeared to be active under the conditions described here. Thylakoids isolated from the stressed, drought-tolerant wheat showed an increase in lipid-to-protein ratio, in comparison with the control, whereas this ratio remained unchanged in the sensitive wheat. In both cultivars, water deficit determined different rearrangements in the composition of the thylakoid individual polar lipids, but their unsaturation level remained unaffected with the exception of monogalactosyldiacylglycerol. In the drought-sensitive cultivar, an accumulation of free fatty acids together with a reduction in polar lipid amount was observed. Electron paramagnetic resonance measurements of spin-labeled proteins of stressed plants from the sensitive cv Adamello showed a higher spin label rotational correlation time together with lower sulphydryl group and mobile proteic portion levels, in comparison with the control. In the tolerant cv Ofanto, the first two parameters changed to a lesser extent following water depletion, and the mobile proteic portion was not altered.

Activity and conformational changes of alpha-chymotrypsin in reverse micelles studied by spin labeling.

alpha-Chymotrypsin (CT), spin-labeled at the active site by using an acylating label which constitutes a substrate for this protein, has been investigated in reverse micelles formed by AOT in isooctane. The electron spin resonance spectra provided information on conformation, dynamics and deacylation activity. The dynamics of the label bound to CT appears to be more hindered in reverse micelles than in aqueous solution, probably owing to the effect of the micellar environment on protein conformation. The deacylation rate in reverse micelles does not show the characteristic bell-shaped dependence on water content which is generally found for CT enzymatic activity.