Temperature-Caused Changes in Raman Pattern and Protein Profiles of Winter Triticale (x Triticosecale, Wittm.) Field-Grown Seedlings.

Climate change, which causes periods with relatively high temperatures in winter in Poland, can lead to a shortening or interruption of the cold hardening of crops. Previous research indicates that cold acclimation is of key importance in the process of acquiring cereal tolerance to stress factors. The objective of this work was to verify the hypothesis that both natural temperature fluctuations and the plant genotype influence the content of metabolites as well as proteins, including antioxidant enzymes and photosystem proteins. The research material involved four winter triticale genotypes, differing in their tolerance to stress under controlled conditions. The values of chlorophyll a fluorescence parameters and antioxidant activity were measured in their seedlings. Subsequently, the contribution of selected proteins was verified using specific antibodies. In parallel, the profiling of the contents of chlorophylls, carotenoids, phenolic compounds, and proteins was carried out by Raman spectroscopy. The obtained results indicate that a better PSII performance along with a higher photosystem II proteins content and thioredoxin reductase abundance were accompanied by a higher antioxidant activity in the field-grown triticale seedlings. The Raman studies showed that the cold hardening led to a variation in photosynthetic dyes and an increase in the phenolic to carotenoids ratio in all DH lines.

The Spectral Compositions of Light Changes Physiological Response of Chinese Cabbage to Elevated Ozone Concentration.

The effects of ozone combined with other environmental factors remain an important topic of the research, both in connection with climate change and the possibility of using modern solutions in horticulture. In our experiment, we compared the influence of ozone (100 ppb) on photosynthesis and changes in the pigment composition of Chinese cabbage (Brassica rapa subsp. pekinensis) leaves depending on the spectral composition of light. We used white LED light (WL), a combination of red + green + blue (RGBL) with a dominant red component and white +blue (WBL) with a dominant blue component in comparison with the classic sodium lamp lighting (yellow light-YL). The values of the parameters describing the light-dependent phase of photosynthesis and the parameters of the gas exchange, as well as non-photosynthesis pigment contents, show that the spectral composition strongly differentiates the response of Chinese cabbage leaves to ozone. In general, the efficiency of photochemical reactions was the highest in YL, but after O3 fumigation, it decreased. In plants growing in WL and WBL, the increase of O3 concentration stimulated light photosynthesis reactions and led to the enhancement of transpiration, stomatal conductance and intracellular CO2 concentration. Changes in photosynthetic activity were accompanied by an increase in the content of anthocyanins and flavonols.

Does Nitrogen Fertilization Affect the Secondary Structures of Gliadin Proteins in Hypoallergenic Wheat?

One of the macronutrients indispensable for plant growth and development is nitrogen (N). It is responsible for starch and storage protein (gliadins and glutenins) biosynthesis and, in consequence, influences kernels' quality and yields. However, applying N-fertilizers increases gluten content in wheat, and it may intensify the risk of developing allergy symptoms in gluten-sensitive individuals. The purpose of our research was to analyse whether and how the elimination of N-fertilizers during the cultivation of wasko.gl- wheat (modified genotype lacking ω-gliadins) changes the secondary structures of gliadin proteins. To this aim, using the FT-Raman technique, we examined flour and gliadin protein extracts obtained from kernels of two winter wheat lines: wasko.gl+ (with a full set of gliadin proteins) and wasko.gl- (without ω-gliadin fraction) cultivated on two different N-fertilization levels-0 and 120 kg N·ha-1. On the basis of the obtained results, we proved that nitrogen fertilization does not have a major impact on the stability of the secondary structures of gliadin proteins for wasko.gl- wheat line with reduced allergenic properties. Furthermore, the results presented herein suggest the possibility of increasing the stability of glutenin structures as a result of the N-fertilization of wasko.gl- wheat line, which gives hope for its use in the production of wheat articles devoted to people suffering from diseases related to gluten sensitivity.

FT-Raman Spectroscopy as a Tool to Study the Secondary Structures of Wheat Gliadin Proteins.

Raman spectroscopy is a useful method in biological, biomedical, food, and agricultural studies, allowing the simultaneous examination of various chemical compounds and evaluation of molecular changes occurring in tested objects. The purpose of our research was to explain how the elimination of ω-fractions from the wheat gliadin complex influences the secondary structures of the remaining αßγ-gliadins. To this aim, we analyzed the endosperm of wheat kernels as well as gliadin proteins extracted from two winter wheat genotypes: wasko.gl+ (control genotype containing the full set of gliadins) and wasko.gl- (modified genotype lacking all ω-gliadins). Based on the decomposition of the amide I band, we observed a moderate increase in ß-forms (sheets and turns) at the expense of α-helical and random coil structures for gliadins isolated from the flour of the wasko.gl- line. Since ω-gliadins contain no cysteine residues, they do not participate in the formation of the disulfide bridges that stabilize the protein structure. However, they can interact with other proteins via weak, low-energetic hydrogen bonds. We conclude that the elimination of ω-fractions from the gliadin complex causes minor modifications in secondary structures of the remaining gliadin proteins. In our opinion, these small, structural changes of proteins may lead to alterations in gliadin allergenicity.

Air pollution in the places of Betula pendula growth and development changes the physicochemical properties and the main allergen content of its pollen.

Pollen allergy becomes an increasing problem for humans, especially in the regions, where the air pollution level increases due to the traffic and urbanization. These factors may also affect the physiological activity of plants, causing changes in pollen allergenicity. The aim of the study was to estimate the influence of air pollutants on the chemical composition of birch pollen and the secondary structures of the Bet v1 protein. The research was conducted in seven locations in Malopolska region, South of Poland of a different pollution level. We have found slight fluctuations in the values of parameters describing the photosynthetic light reactions, similar spectra of leaf reflectance and the negligible differences in the discrimination values of the δ13C carbon isotope were found. The obtained results show a minor effect of a degree of pollution on the physiological condition B. pendula specimen. On the other hand, mean Bet v1 concentration measured in pollen samples collected in Kraków was significantly higher than in less polluted places (p = .03886), while FT-Raman spectra showed the most distinct variations in the wavenumbers characteristic of proteins. Pollen collected at sites of the increased NOx and PM concentration, show the highest percentage values of potential aggregated forms and antiparallel ß-sheets in the expense of α-helix, presenting a substantial impact on chemical compounds of pollen, Bet v1 concentration and on formation of the secondary structure of proteins, what can influence their functions.

Cold-modulated leaf compounds in winter triticale DH lines tolerant to freezing and Microdochium nivale infection: LC-MS and Raman study.

Tolerance to freezing and seedling diseases caused by Microdochium spp. is an essential trait for the wintering of triticale (×Triticosecale Wittmack) and other cereals. Preceding multi-year studies indicate that after long-term exposure to the low temperature, cereal seedlings acquire a genotype-dependent cross-tolerance to other subsequent stresses. This paper presents the first non-gel protein profiling performed via high performance liquid chromatography coupled with Mass Spectrometry as well as Fourier Transform-Raman spectroscopy measurements performed directly on leaves of triticale seedlings growing under different conditions. The research used doubled haploid lines selected from the mapping population, with extreme tolerance/susceptibility to freezing and M. nivale infection. These non-targeted methods led to the detection of twenty two proteins cold-accumulated in the most tolerant seedlings in relation to susceptible ones, classified as involved in protein biosynthesis, response to different stimuli, energy balancing, oxidative stress response, protein modification, membrane structure and anthocyanin synthesis. Additionally, in seedlings of the most freezing- and M. nivale -tolerant line, cold-hardening caused decrease of the carotenoid and chlorophyll content. Moreover, a decrease in the band intensity typical for carbohydrates as well as an increase in the band intensity characteristic for protein compounds were detected. Both studied lines revealed a different answer to stress in the characteristics of phenolic components.

Application of high pressure laser flash photolysis in studies on selected hemoprotein reactions.

This article focuses on the application of high pressure laser flash photolysis for studies on selected hemoprotein reactions with the objective to establish details of the underlying reaction mechanisms. In this context, particular attention is given to the reactions of small molecules such as dioxygen, carbon monoxide, and nitric oxide with selected hemoproteins (hemoglobin, myoglobin, neuroglobin and cytochrome P450(cam)), as well as to photo-induced electron transfer reactions occurring in hemoproteins (particularly in various types of cytochromes). Mechanistic conclusions based on the interpretation of the obtained activation volumes are discussed in this account.

Interaction of selected divalent metal ions with human ataxin-3 Q36.

The mode of interaction of ataxin-3 Q36 (AT-3 Q36) with selected endogenous and exogenous metal ions, namely, Zn(2+), Cu(2+), Ni(2+), and Cd(2+), was examined. Metal-ion-induced structural changes of the protein were monitored by fluorescence as well as Fourier transform Raman spectroscopy. We found that the cations tested lead to a decrease in alpha-helical content and a concurrent increase in beta-sheet as well as undefined (beta-turn and random-coil) structures. The most evident effect was observed for copper and nickel cations. After titration with these cations, the AT3 Q36 secondary structure content (27% alpha-helices in the presence of either ion, 31 and 27% beta-sheets for Cu(2+) and Ni(2+), respectively) was similar to that observed for the aggregated form of the protein (27% alpha-helices, 36% beta-sheets). Using the 1-anilinonaphthalene-8-sulfonate hydrophobic fluorescence probe, we showed that the presence of the metal ions tested led to the formation of solvent-exposed hydrophobic patches of AT-3 Q36, and that such an effect decreased with increasing ionic radius.

Antibody reactivity in patients with IgE-mediated wheat allergy to various subunits and fractions of gluten and non-gluten proteins from ω-gliadin-free wheat genotypes.

[b]Abstract Introduction and objective[/b]. Gluten proteins (gliadins and glutenins) are polymorphic wheat storage proteins of allergenic properties. Significant differences in chemical composition between both protein groups allow to expect highly specific immunological response of individual subunits and fractions in reactions with IgE sera of people allergic to wheat. The aim of these studies was to identify and characterize the most allergenic gluten proteins (GP) and nongluten proteins (NGP) occurred in two closely related wheat hybrid genotypes. [b]Materials and method.[/b] 3xC and 3xN wheat hybrids, which differ strongly in regard of gliadin composition, were analyzed. Seven people manifesting different symptoms of wheat allergy donated sera for the experiment. The technique of immunoblotting after SDS-PAGE was used for identification of allergenic subunits and fractions among GP and NGP. Immunologically active protein bands were visualized by chemiluminescence. [b]Results[/b]. Great variation of immunodetection spectra was observed. Results of immunoblotting showed LMW glutenins to be of highest, gliadins of medium, while NGP of lowest allergenicity for selected patients. The 43-kDa and 47-kDa LMW glutenin subunits, 40-kDa and 43-kDa γ-gliadin fractions and 49-kDa NGP can be considered as the most immunoreactive among all protein bands [b]separated by SDS-PAGE. CONCLUSION: [/b] The observed differentiation of immunodetection spectra allows to model highly specific IgE-binding profiles of allergenic wheat proteins attributed to individual patients with symptoms of gluten intolerance. Highly immunoreactive subunits and fractions among GP and NGP were identified. The observed immunoreactivity of 49 kDa NGP is worth to emphasize, as it has never been reported as wheat allergenic protein before.

The reduction of (ImH)[trans-RuIIICl4(dmso)(Im)] under physiological conditions: preferential reaction of the reduced complex with human serum albumin.

A systematic study of the reduction of (ImH)[trans-RuCl(4)(dmso)(Im)] (NAMI-A; dmso is dimethyl sulfoxide, Im is imidazole), a promising antimetastasing agent, by L-ascorbic acid under physiological conditions is reported. Under blood plasma conditions (pH 7.4, 0.1-0.15 M NaCl , 37 degrees C) the rapid reduction of trans-[Ru(III)Cl(4)(dmso)(Im)](-) results in the formation of trans-[Ru(II)Cl(4)(dmso)(Im)](2-) within seconds, and is followed by successive dissociation of the chloride ligands, whereas neither dmso nor imidazole ligands are released during the reaction. Under our experimental conditions, the formation of the ascorbate dianion is the rate-determining step, and once it has formed it reacts rapidly with NAMI-A. Moreover, the NAMI-A complex is very unstable at physiological pH (7.4); therefore, the hydrolysis of NAMI-A cannot be excluded as a competing reaction. During hydrolysis, aquated derivatives via stepwise dissociation of chloride and dmso ligands are formed, and most of these species have a higher redox potential and are expected to be even more easily reduced by ascorbic acid. Thus, it is very likely that the reduced form of NAMI-A or the reduction products of its hydrolytic derivatives react with albumin. The reaction of reduced NAMI-A with human serum albumin leads to the formation of stable adducts, with a binding efficiency very similar to that of the parent complex, viz., 3.2+/-0.3 and 4.0+/-0.4 mol of Ru(II) and Ru(III) per mole of albumin, respectively, however with a significantly higher reactivity.

Central metal determines pharmacokinetics of chlorophyll-derived xenobiotics.

Chlorophyll derivatives are potentially dangerous xenobiotics of dietary origin. The interactions of water-soluble derivatives of chlorophyll a with the animal organism were investigated using chlorophyllide a and its Zn-substituted analogue as model xenobiotics. The chlorophyllides were administered to tumor-bearing mice and their uptake, distribution, and clearance were compared. The centrally bound metal determines important aspects of the in vivo behavior of metallochlorophyllides as xenobiotics. The uptake and clearance of chlorophyllide a were significantly faster than those of [Zn]-chlorophyllide a. Chlorophyllide a showed some tissue selectivity, while [Zn]-chlorophyllide a was uniformly distributed among tissues. Interestingly, the tissue levels of the latter compound were ten times higher than those of the Mg-derivative. These differences indicate that [Zn]-chlorophyllide a, in contrast to chlorophyllide a, is only weakly recognized by the system of active transport of xenobiotics and by enzymes involved in chlorophyll metabolism. The dependence of chlorophyllide pharmacokinetics on the central metal is of great relevance to chlorophyll-based phototherapy.