Frost survival and gene expression in timothy (Phleum pratense L.) cultivars as affected by age and selection in diverse field environments.

The sustainable production of perennial grasses in Northern Norway is at risk due to the ongoing climate change. The predicted increase in temperatures and variable weather patterns are further expected to create challenges for winter survival of timothy (Phleum pratense L.). Knowledge about the molecular mechanisms underlying freezing tolerance is crucial for developing robust cultivars. The current study is aimed at identifying genes involved in freezing stress response of timothy and studying gene expression differentiation due to field selection in contrasting environments using RNAseq. Four timothy cultivars were field tested for three years in Tromsø and Vesterålen, in Northern Norway. The surviving material from the field tests, along with plants raised from the original seed lots, were subjected to freezing tests. LT50 values varied across cultivars and materials. Many genes coding for transcription factors and proteins known to play an important role in freezing tolerance, like dehydrins, c-repeat binding factors, and late embryogenesis abundant proteins were upregulated with decreasing temperatures. Moreover, genes associated with glycolysis/gluconeogenesis, TCA cycle, glutathione metabolism, proteasome pathways and genes encoding autophagy-related proteins, plasma membrane-associated proteins, sugar and amino acid transporters had elevated expression in field survivors compared to plants raised from the original material. The lower freezing stress tolerance of field survivors despite the elevated expression of several stress-responsive genes might be due to a combination of selection in the field and the age effect. Furthermore, differences in freezing stress response between northern and southern adapted cultivars and surviving material from two field trial locations are discussed.

Oligomerization and Nitration of the Grass Pollen Allergen Phl p 5 by Ozone, Nitrogen Dioxide, and Peroxynitrite: Reaction Products, Kinetics, and Health Effects.

The allergenic and inflammatory potential of proteins can be enhanced by chemical modification upon exposure to atmospheric or physiological oxidants. The molecular mechanisms and kinetics of such modifications, however, have not yet been fully resolved. We investigated the oligomerization and nitration of the grass pollen allergen Phl p 5 by ozone (O3), nitrogen dioxide (NO2), and peroxynitrite (ONOO-). Within several hours of exposure to atmospherically relevant concentration levels of O3 and NO2, up to 50% of Phl p 5 were converted into protein oligomers, likely by formation of dityrosine cross-links. Assuming that tyrosine residues are the preferential site of nitration, up to 10% of the 12 tyrosine residues per protein monomer were nitrated. For the reaction with peroxynitrite, the largest oligomer mass fractions (up to 50%) were found for equimolar concentrations of peroxynitrite over tyrosine residues. With excess peroxynitrite, the nitration degrees increased up to 40% whereas the oligomer mass fractions decreased to 20%. Our results suggest that protein oligomerization and nitration are competing processes, which is consistent with a two-step mechanism involving a reactive oxygen intermediate (ROI), as observed for other proteins. The modified proteins can promote pro-inflammatory cellular signaling that may contribute to chronic inflammation and allergies in response to air pollution.

Tall fescue as an alternative to timothy fed with or without alfalfa to dairy cows.

Tall fescue might be an alternative to timothy in northeastern North America because of its tolerance of recurring drought periods and its good summer regrowth, but is not always considered as an option in dairy rations because of its possible lack of palatability. The objective of this study was to evaluate the effects on the performance of lactating dairy cows of (1) replacing timothy silage by tall fescue silage, offered as sole forage in the diet or in combination with alfalfa silage, and (2) feeding tall fescue as silage (35% dry matter, DM) or haylage (55% DM). Experimental diets with a forage-to-concentrate ratio of 70:30 were (1) 100% timothy silage (TS); (2) 100% tall fescue silage (TFS); (3) 55:45 timothy:alfalfa silages (TS + AS); (4) 55:45 tall fescue:alfalfa silages (TFS + AS); and (5) 100% tall fescue haylage (TFH). Fifteen Holstein cows in mid-lactation (5 fitted with a rumen fistula) were randomly assigned to treatments in a triple 5 × 5 Latin square design with treatment periods of 21 d. Preplanned contrasts were timothy versus tall fescue silages, sole grass species versus grass-alfalfa, interaction between sole grass species and grass-alfalfa, and TFS versus TFH. Grass species did not affect dry matter intake (DMI) or milk yield and fat concentration. Milk protein concentration was not affected by grass species when offered in combination with alfalfa, but it was higher with the TS diet than the TFS diet when offered as sole forages. Adding alfalfa to either tall fescue or timothy silage resulted in greater DMI and milk yield, but lower milk fat concentration, than when the grass silages were the sole forage in the diet. The molar proportion of propionate in the rumen was greater when cows were fed diets with tall fescue silage compared with timothy silage, which resulted in a lower acetate-to-propionate ratio. Milk fat concentrations of fatty acids from microbial origin, namely branched-chain fatty acids, were greater when grass silage, and especially timothy silage, were fed as sole forages rather than with alfalfa silage. Feeding TFH rather than TFS caused a decrease in DMI and tended to lower milk protein concentration, but did not affect milk yield. A more fibrolytic fermentation profile was observed in rumen of cows fed TFH compared with TFS, as indicated by the increase in the molar proportion of acetate and the higher acetate-to-propionate ratio in rumen fluid, and a concomitant increase in branched-chain fatty acid concentration in milk fat. Tall fescue as silage or haylage is a valuable alternative to timothy silage for lactating dairy cows.

Cold acclimation in warmer extended autumns impairs freezing tolerance of perennial ryegrass (Lolium perenne) and timothy (Phleum pratense).

The effect of variable autumn temperatures in combination with decreasing irradiance and daylength on photosynthesis, growth cessation and freezing tolerance was investigated in northern- and southern-adapted populations of perennial ryegrass (Lolium perenne) and timothy (Phleum pratense) intended for use in regions at northern high latitudes. Plants were subjected to three different acclimation temperatures; 12, 6 and 9/3°C (day/night) for 4 weeks, followed by 1 week of cold acclimation at 2°C under natural light conditions. This experimental setup was repeated at three different periods during autumn with decreasing sums of irradiance and daylengths. Photoacclimation, leaf elongation and freezing tolerance were studied. The results showed that plants cold acclimated during the period with lowest irradiance and shortest day had lowest freezing tolerance, lowest photosynthetic activity, longest leaves and least biomass production. Higher acclimation temperature (12°C) resulted in lower freezing tolerance, lower photosynthetic activity, faster leaf elongation rate and higher biomass compared with the other temperatures. Photochemical mechanisms were predominant in photoacclimation. The northern-adapted populations had a better freezing tolerance than the southern-adapted except when grown during the late autumn period and at the highest temperature; then there were no differences between the populations. Our results indicate that the projected climate change in the north may reduce freezing tolerance in grasses as acclimation will take place at higher temperatures and shorter daylengths with lower irradiance.

A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal Bacteroides thetaiotaomicron Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass.

Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a ß-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B. thetaiotaomicron, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the levansucrase Lsc3 of Pseudomonas syringae pv. tomato, its mutant Asp300Asn, levansucrases of Zymomonas mobilis, Erwinia herbicola, Halomonas smyrnensis as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (<60 kDa) levans, including timothy grass levan and levan synthesized from sucrose by the Lsc3Asp300Asn, were degraded most rapidly whilst levan produced by Lsc3 from raffinose least rapidly. BT1760 catalyzed finely at human body temperature (37°C) and in moderately acidic environment (pH 5-6) that is typical for the gut lumen. According to differential scanning fluorimetry, the Tm of the endo-levanase was 51.5°C. All tested levans were sufficiently stable in acidic conditions (pH 2.0) simulating the gastric environment. Therefore, levans of both bacterial and plant origin may serve as a prebiotic fiber for B. thetaiotaomicron and contribute to short-chain fatty acids synthesis by gut microbiota. In the genome of Bacteroides xylanisolvens of human origin a putative levan degradation locus was disclosed.

Performance and nitrogen use efficiency in mid-lactation dairy cows fed timothy cut in the afternoon or morning.

Shifting cutting from morning to afternoon has been shown to increase the concentration of nonstructural carbohydrates in forages. We hypothesized that, compared with a total mixed ration containing timothy baleage and silage cut in the morning (a.m.-cut TIM), a total mixed ration containing timothy baleage and silage cut in the afternoon (p.m.-cut TIM) would improve animal performance and N use efficiency in mid-lactation Holstein cows due to enhanced supply of ruminal fermentable energy. The objective of this study was to compare the effects of p.m.- versus a.m.-cut TIM on milk yield, concentrations and yields of milk components, ruminal metabolism, and plasma concentrations of AA in mid-lactation Holstein cows. Ten (6 ruminally cannulated) primiparous cows averaging 139±13 d in milk and 550±56 kg of body weight, and 6 (2 ruminally cannulated) multiparous cows averaging 128±11 d in milk and 632±57 kg of body weight at the beginning of the experiment, were used in a crossover design. Each period lasted 21 d with 14 d for diet adaptation and 7 d for data and sample collection. The concentration of nonstructural carbohydrates (water-soluble carbohydrates plus starch) was numerically greater in the p.m.- versus the a.m.-cut TIM and averaged 13.2±1.06% and 12.2±1.13%, respectively. Treatment × parity effects were observed for milk urea N, feed efficiency, and milk N efficiency, whereas parity effects were observed for nutrient intake, milk yield, and plasma concentration of several essential and nonessential AA. Intakes of dry matter (19.3 versus 18.6 kg/d) and nonstructural carbohydrates (2.56 versus 2.31 kg/d), and yields of 4% fat-corrected milk (23.1 versus 22.2 kg/d), energy-corrected milk (25.0 versus 24.1 kg/d), milk fat (0.91 versus 0.88 kg/d), and milk protein (0.77 versus 0.73 kg/d) were all greatest with feeding p.m.-cut TIM. Milk yield (23.5 versus 22.7 kg/d) tended to increase in cows fed p.m.-cut TIM. The ruminal fermentation profiles and plasma concentrations of AA were mostly unaffected by treatments. However, ruminal valerate (1.01 versus 1.17 mol/100 mol) and plasma Gly (172 versus 188 µM) were lowest with feeding p.m.-cut TIM. Overall, feeding mid-lactation dairy cows a total mixed ration that consisted of p.m.-cut timothy baleage and silage significantly increased dry matter intake and yields of milk, milk fat, and milk protein.

Allergen Microarray Indicates Pooideae Sensitization in Brazilian Grass Pollen Allergic Patients.

BACKGROUND: Grass pollen, in particular from Lolium multiflorum is a major allergen source in temperate climate zones of Southern Brazil. The IgE sensitization profile of Brazilian grass pollen allergic patients to individual allergen molecules has not been analyzed yet. OBJECTIVE: To analyze the IgE sensitization profile of a Brazilian grass pollen allergic population using individual allergen molecules. METHODS: We analyzed sera from 78 grass pollen allergic patients for the presence of IgE antibodies specific for 103 purified micro-arrayed natural and recombinant allergens by chip technology. IgE-ELISA inhibition experiments with Lolium multiflorum, Phleum pratense extracts and a recombinant fusion protein consisting of Phl p 1, Phl p 2, Phl p 5 and Phl p 6 were performed to investigate cross-reactivities. RESULTS: Within the Brazilian grass pollen allergic patients, the most frequently recognized allergens were Phl p 1 (95%), Phl p 5 (82%), Phl p 2 (76%) followed by Phl p 4 (64%), Phl p 6 (45%), Phl p 11 (18%) and Phl p 12 (18%). Most patients were sensitized only to grass pollen allergens but not to allergens from other sources. A high degree of IgE cross-reactivity between Phleum pratense, Lolium multiflorum and the recombinant timothy grass fusion protein was found. CONCLUSIONS: Component-resolved analysis of sera from Brazilian grass pollen allergic patients reveals an IgE recognition profile compatible with a typical Pooideae sensitization. The high degree of cross-reactivity between Phleum pratense and Lolium multiflorum allergens suggests that diagnosis and immunotherapy can be achieved with timothy grass pollen allergens in the studied population.

Does vegetation affect the methane oxidation efficiency of passive biosystems?

It is often reported in the technical literature that the presence of vegetation improves the methane oxidation efficiency of biosystems; however, the phenomena involved and biosystem performance results are still poorly documented, particularly in the field. This triggered a study to assess the importance of vegetation in methane oxidation efficiency (MOE). In this study, 4 large scale columns, each filled with sand, topsoil and a mixture of compost and topsoil were tested under controlled conditions in the laboratory and partially controlled conditions in the field. Four series of laboratory tests and two series of field tests were performed. 4 different plant covers were tested for each series: Trifolium repens L. (White clover), Phleum pratense L. (Timothy grass), a mixture of both, and bare soil as the control biosystem. The study results indicated that up to a loading equal to 100 g CH4/m(2)/d, the type of plant cover did not influence the oxidation rates, and the MOE was quite high (⩾ 95%) in all columns. Beyond this point, the oxidation rate continued to increase, reaching 253 and 179 g CH4/m(2)/d in laboratory and field tests respectively. In the end, the bare soil achieved as high or higher MOEs than vegetated biosystems. Despite the fact that the findings of this study cannot be generalized to other types of biosystems and plants and that the vegetation types tested were not fully grown, it was shown that for the short-term tests performed and the types of substrates and plants used herein, vegetation does not seem to be a key factor for enhancing biosystem performance. This key conclusion does not corroborate the conclusion of the relatively few studies published in the technical literature assessing the importance of vegetation in MOE.

Kinetic of NO2 uptake by Phleum pratense pollen: chemical and allergenic implications.

Phleum pratense pollen was exposed to NO(2) in a reactor allowing a continuous analysis of NO(2) concentration by FTIR. The uptake coefficient of NO(2) on pollen was calculated postulating a first order kinetic reaction and a value of (1.1 ± 0.1) x 10(-7) was determined. NO(2) uptake was faster when the pollen water content was increased and when the pollen was pre-treated with ozone. The effect of NO(2) exposure on pollen allergic properties was investigated by quantifying Th2- and Th1-associated chemokines in a model of human dendritic cells. Cellular analysis clearly showed that cells exposed to fumigated pollen favored the production of chemokines known to promote Th2-cell responses. Altogether these data demonstrate that NO(2) uptake by pollen directly correlates with increased Th2 response in human cells,and are in favor of the involvement of NO(2) pollution in the increase of allergic diseases.

Gastrointestinal metabolism of phytoestrogens in lactating dairy cows fed silages with different botanical composition.

Dietary phytoestrogens are metabolized or converted in the gastrointestinal tract of ruminants, only limited knowledge exists on the extent and location of this conversion in vivo. The objective of this study was to quantify the gastro-intestinal metabolism of phytoestrogens in lactating dairy cows fed silages with different botanical composition. Four lactating rumen cannulated Norwegian Red cattle were assigned to a 4 × 4 Latin square with 1 cow per treatment period of 3 wk. The 4 treatment silages were prepared from grasslands with different botanical compositions: organically managed short-term timothy (Phleum pratense L.) and red clover (Trifolium pratense L.) ley (2 yr old: ORG-SG); organically managed long-term grassland with a high proportion of unsown species (6 yr old; ORG-LG); conventionally managed perennial ryegrass (Lolium perenne L.) ley (CON-PR); and conventionally managed timothy ley (CON-TI). The herbages were cut, wilted, and preserved with additive in round bales, fed as a mix of the first and third cut at 90% of ad libitum intake, and contributed to 70% of the total dry matter intake. Milk, feed, omasal digesta, urine, and feces were collected at the end of each period and analyzed for the concentrations of phytoestrogens by using a liquid chromatography-tandem mass spectrometry technique. Concentration of total isoflavones was highest in ORG-SG and lowest in CON-TI silage, whereas the content of total lignans was highest in the grass silages. The isoflavones were extensively metabolized in the rumen on all diets, and the recovery of formononetin and daidzein in omasum, mainly as equol, averaged 0.11 mg/mg. The apparent intestinal metabolism was less severe as, on average, 0.29 mg/mg of the omasal flow was recovered in feces. The plant lignans were also strongly degraded in the rumen. However, the flow of lignans to omasum and excretion in feces were, on average, 7.2- and 5.2-fold higher, respectively, than the intake of the plant lignans matairesinol and secoisolariciresinol, known as precursors of mammalian lignans. Thus, excretion to milk could not be directly related to intake, implying that plant lignans other than matairesinol and secoisolariciresinol in forage are precursors for enterolactone production in the rumen and for its content in milk. Equol followed mainly the flow of large particles out of the rumen, whereas the mammalian lignans were distributed between phases proportional to dry matter flow. The main metabolism of phytoestrogens occurred in the rumen and the main route of excretion was through feces and urine, with only a small part being excreted in milk. The concentration of phytoestrogens in milk can be manipulated through intake but the intermediate transfer capacity to milk appears to be limited by saturation.

Plant maturity and nitrogen fertilization affected fructan metabolism in harvestable tissues of timothy (Phleum pratense L.).

Timothy (Phleum pratense L.) is an important grass forage used for pasture, hay, and silage in regions with cool and humid growth seasons. One of the factors affecting the nutritive value of this grass is the concentration of non-structural carbohydrates (NSC), mainly represented by fructans. NSC concentration depends on multiple factors, making it hardly predictable. To provide a better understanding of NSC metabolism in timothy, the effects of maturity stage and nitrogen (N) fertilization level on biomass, NSC and N-compound concentrations were investigated in the tissues used for forage (leaf blades and stems surrounded by leaf sheaths) of hydroponically grown plants. Moreover, activities and relative expression level of enzymes involved in fructan metabolism were measured in the same tissues. Forage biomass was not altered by the fertilization level but was strongly modified by the stage of development. It increased from vegetative to heading stages while leaf-to-stem biomass ratio decreased. Total NSC concentration, which was not altered by N fertilization level, increased between heading and anthesis due to an accumulation of fructans in leaf blades. Fructan metabolizing enzyme activities (fructosyltransferase-FT and fructan exohydrolase-FEH) were not or only slightly altered by both maturity stage and N fertilization level. Conversely, the relative transcript levels of genes coding for enzymes involved in fructan metabolism were modified by N supply (PpFT1 and Pp6-FEH1) or maturity stage (PpFT2). The relative transcript level of PpFT1 was the highest in low N plants while that of Pp6-FEH1 was the highest in high N plants. Morevoer, transcript level of PpFT1 was negatively correlated with nitrate concentration while that of PpFT2 was positively correlated with sucrose concentration. This distinct regulation of the two genes coding for 6-sucrose:fructan fructosyltransferase (6-SFT) may allow a fine adequation of C allocation towards fructan synthesis in response to carbon and N availability. Contrary to fructans, starch content increased in low N plants, suggesting different regulatory mechanisms and/or sensitivity of starch and fructan metabolism in relation to the N status.

Fructan metabolism and expression of genes coding fructan metabolic enzymes during cold acclimation and overwintering in timothy (Phleum pratense).

Metabolism of fructans in temperate grasses dynamically fluctuates before and during winter and is involved in the overwintering activity of plants. We monitored three candidate factors that may be involved in seasonal fructan metabolism in timothy (Phleum pratense): transcription levels of two fructosyltransferase (PpFT1 and PpFT2) genes and one fructan exohydrolase (Pp6-FEH1) gene during fall and winter and under artificially cold conditions. Functional analysis using a recombinant enzyme for PpFT2, a novel fructosyltransferase cDNA, revealed that it encoded sucrose:fructan 6-fructosyltransferase, with enzymatic properties different from previously characterized PpFT1. PpFT1 transcripts decreased from September to December as the amount of fructans increased, whereas PpFT2 transcripts increased in timothy crowns. PpFT2 was transcriptionally more induced than PpFT1 in response to cold and sucrose in timothy seedlings. A rapid increase in Pp6-FEH1 transcripts and increased monosaccharide content were observed in timothy crowns when air temperature was continuously below 0°C and plants were not covered by snow. Transcriptional induction of Pp6-FEH1 by exposure to -3°C was also observed in seedlings. These findings suggest Pp6-FEH1 involvement in the second phase of hardening. PpFT1 and PpFT2 transcription levels decreased under snow cover, whereas Pp6-FEH1 transcription levels were constant, which corresponded with the fluctuation of fructosyltransferase and fructan exohydrolase activities. Inoculation with snow mold fungi (Typhula ishikariensis) increased Pp6-FEH1 transcription levels and accelerated hydrolysis of fructans. These results suggest that transcriptional regulation of genes coding fructan metabolizing enzymes is partially involved in the fluctuation of fructan metabolism during cold acclimation and overwintering.

Effect of lignin content and subunit composition on digestibility in clones of timothy (Phleum pratense L.).

Lignin amount and subunit composition were analyzed from stems and leaf sheaths of timothy (Phleum pratense L.) clones of different in vitro digestibility. Lignin concentration in stems and leaf sheaths was higher in clones of low digestibility than those of high digestibility. No change in lignin concentration occurred in stems as digestibility decreased. Intriguingly, the lignin concentration was lower and the syringyl/guaiacyl (S/G) ratio was higher in stems compared to leaf sheaths at all developmental stages studied. The developmental-associated decrease in digestibility correlated with the increase in S units in lignin in stems and leaf sheaths and in the amounts of p-coumaric acid and ferulic acid residues in the cell wall of stems. Yields of copper oxidation products increased in stems during maturation indicating qualitative changes in the lignin structure. This correlated strongly with the developmentally linked decrease in digestibility. The information obtained is valuable for breeding and for DNA marker development.

Degradation of microcrystalline cellulose and non-pretreated plant biomass by a cell-free extracellular cellulase/hemicellulase system from the extreme thermophilic bacterium Caldicellulosiruptor bescii.

Caldicellulosiruptor bescii is a cellulolytic/hemicellulolytic anaerobe, which extracellularly secretes various proteins, including multidomain cellulases with two-catalytic domains, for plant biomass degradation. Degradation by C. bescii cells has been well characterized, but degradation by the cell-free extracellular cellulase/hemicellulase system (CEC) of C. bescii has not been as well studied. In the present study, C. bescii CEC was prepared from cell-free culture supernatant, and the degradation properties for defined substrates and non-pretreated plant biomass were characterized. Four multidomain cellulases (Cbes_1857, Cbes_1859, Cbes_1865, and Cbes_1867), composed of the glycoside hydrolase families 5, 9, 10, 44, and 48, were the major enzymes identified in the CEC by mass spectrometry. The CEC degraded xylan, mannose-based substrates, ß-1,4-linked glucans, including microcrystalline cellulose (Avicel), and non-pretreated timothy grass and rice straw. However, degradation of chitin, pectin, dextran, and wheat starch was not observed. The optimum temperatures for degradation activities were 75°C for timothy grass and Avicel, 85°C for carboxylmethyl cellulose, and >85°C for xylan. The optimum pH for these substrates was 5-6. The degradation activities were compared with a CEC derived from the fungus Trichoderma reesei, the most common enzyme used for plant biomass saccharification. The amounts of degraded Avicel, timothy grass, and rice straw by C. bescii CEC were 2.2-2.4-fold larger than those of T. reesei CEC. The high hydrolytic activity of C. bescii CEC might be attributed to the two-catalytic domain architecture of the cellulases.

A contaminant trypsin-like activity from the timothy grass pollen is responsible for the conflicting enzymatic behavior of the major allergen Phl p 1.

We intend to solve whether or not Phl p 1 can be regarded as a protease. A group reported that Phl p 1 has papain-like properties and later on, that this allergen resembles cathepsin B, while another one demonstrated that Phl p 1 lacks proteinase activity and suggested that the measured activity may rise either from a recombinant Phl p 1 contaminant or as a result of an incompletely purified natural allergen. A third group reported Phl p 1 to act by a non-proteolytic activity mechanism. We report the purification of the natural Phl p 1 by means of hydrophobic interaction, gel filtration and STI-Sepharose affinity chromatographies. The Phl p 1 purity was assessed by silver-stained SDS-PAGE and by 'in-gel' and 'gel-free' approaches associated to mass spectrometry analyses. The proteolytic activity was measured using Boc-Gln-Ala-Arg-AMC and Z-Phe-Arg-AMC as substrates. While amidolytic activity could be measured with Phl p 1 after rechromatography on gel filtration, it however completely disappeared after chromatography on STI-Sepharose. The contaminant activity co-eluting with Phl p 1 was not affected by cysteine proteases inhibitors and other thiol-blocking agents, by metalloproteases inhibitors and by aspartic proteases inhibitors. However, it was completely inhibited by low molecular weight and proteinaceous serine proteases inhibitors. TLCK, but not TPCK, inhibited the contaminant activity, showing a trypsin-like behavior. The pH and temperature optimum were 8.0 and 37°C, respectively. These data indicated that Phl p 1 is not a protease. The contaminant trypsin-like activity should be considered when Phl p 1 allergenicity is emphasized.

Soils and waste water purification from oil products using combined methods under the North conditions.

Oil and gas production and transportation in Russia is increasingly moving to the north regions. Such regions are characterized by relatively low self-purification capacity of the natural environments from the contaminants due to slow character of the energy exchange and mass transfer processes. Off-shore field development in the Barents Sea and oil product transportation can result in contamination, as confirmed by the national and international practice of the developed oil and gas regions. The research aims at development of the soil bioremediation methods and industrial waste water purification contaminated by oil products in the north-western region of Russia. The dynamics of oil products carry-over have been investigated under the field model experiments in podzolic soils: gas condensate, diesel fuel and mazut from oil and the plants were selected for phyto-remediation of contaminated soils under high north latitudes. It is shown that soil purification from light hydrocarbons takes place during one vegetation period. In three months of the vegetation period the gas condensate was completely removed from the soil, diesel fuel - almost completely (more than 90%). Residual amounts of heavy hydrocarbons were traced, even 1.5 later. The following plants that were highly resistant to the oil product contamination were recommended for bioremediation: Phalaroides arundinacea, Festuca pratensis, Phleum pratense, Leymus arenarius. There has been developed and patented the combined method of treatment of waste water contaminated with hydrocarbons based on inorganic coagulants and local oil-oxidizing bacteria.

Optimisation of grass pollen nasal allergen challenge for assessment of clinical and immunological outcomes.

Nasal allergen challenge can be used to assess the clinical and immunological aspects of rhinitis due to inhalant allergens. We aimed to develop a reproducible technique for grass pollen nasal allergen challenge and to study biomarkers within nasal secretions. 20 Grass pollen allergic individuals underwent nasal challenges with purified Timothy grass allergen. An initial dose-titration challenge was used to determine dose-response characteristics. Subsequently, volunteers underwent 3 further challenges using individualised threshold doses. Symptom scores, visual analogue scores, and peak nasal inspiratory flow (PNIF) were recorded at baseline and up to 6h after challenge. Nasal secretions were collected at each time point using synthetic filter papers or absorptive polyurethane sponges and analysed for IL-4, -5, -10, -13, IFN-γ, Tryptase and Eosinophil Cationic Protein (ECP). Challenges gave reproducible symptom scores and decreased PNIF. Tryptase levels in nasal fluid peaked at 5 min after challenge and returned to baseline levels at 1h. ECP, IL-5, IL-13 and IL-4 levels were increased from 2-3 h and showed progressive increases to 5-6 h. Sponges proved the superior nasal fluid sampling technique. We have developed a reproducible nasal allergen challenge technique. This may be used as a surrogate clinical endpoint in trials assessing the efficacy of treatments for allergic rhinitis. Tryptase in local nasal secretions is a potential biomarker of the early phase response; ECP and the Th2 cytokines IL-5, -13 and -4 markers of late phase allergic responses. Our model allows correlation between clinical responses and local biomarkers following nasal allergen challenge.

Surfactant Protein D modulates allergen particle uptake and inflammatory response in a human epithelial airway model.

BACKGROUND: Allergen-containing subpollen particles (SPP) are released from whole plant pollen upon contact with water or even high humidity. Because of their size SPP can preferentially reach the lower airways where they come into contact with surfactant protein (SP)-D. The aim of the present study was to investigate the influence of SP-D in a complex three-dimensional human epithelial airway model, which simulates the most important barrier functions of the epithelial airway. The uptake of SPP as well as the secretion of pro-inflammatory cytokines was investigated. METHODS: SPP were isolated from timothy grass and subsequently fluorescently labeled. A human epithelial airway model was built by using human Type II-pneumocyte like cells (A549 cells), human monocyte derived macrophages as well as human monocyte derived dendritic cells. The epithelial cell model was incubated with SPP in the presence and absence of surfactant protein D. Particle uptake was evaluated by confocal microscopy and advanced computer-controlled analysis. Finally, human primary CD4+ T-Cells were added to the epithelial airway model and soluble mediators were measured by enzyme linked immunosorbent assay or bead array. RESULTS: SPP were taken up by epithelial cells, macrophages, and dendritic cells. This uptake coincided with secretion of pro-inflammatory cytokines and chemokines. SP-D modulated the uptake of SPP in a cell type specific way (e.g. increased number of macrophages and epithelial cells, which participated in allergen particle uptake) and led to a decreased secretion of pro-inflammatory cytokines. CONCLUSION: These results display a possible mechanism of how SP-D can modulate the inflammatory response to inhaled allergen.

Quantifying instantaneous regeneration rates of plant leaf waxes using stable hydrogen isotope labeling.

Leaf waxes protect terrestrial plants from biotic and abiotic stresses and are important sedimentary biomarkers for terrestrial plants. Thus, understanding the production and ablation of leaf waxes is critical in plant physiology and for geochemical studies. However, there have been no accurate approaches to quantify leaf wax production at different time scales. In this study, we demonstrate a novel approach to study leaf wax regeneration by irrigating plants with a pulse of deuterium-enriched water, followed by measurements of leaf wax D/H ratios by gas chromatography/isotope-ratio mass spectrometry (GC/IRMS). We demonstrate the efficacy of this approach using the grass species Phleum pratense in a greenhouse environment. Using a binary isotope mass balance model, we are able to quantify the regeneration rates of the C(16), C(18) acids and leaf waxes (C(23)-C(31) n-alkanes; C(22)-C(30) n-acids) over a diurnal cycle. Our results show that within one day 33-47% of C(16) and C(18) acids are regenerated, and thus the recycling time for these compounds is 2-3 days. For C(22)-C(26) n-alkyl lipids, 7-21% are regenerated within one day and thus they require 5-16 days to recycle. In comparison, the recycling time for long-chain n-alkyl lipids (C(27)-C(31)) is as long as 71-128 days. Our approach can be applied to different plants at shorter or longer time scales by adjusting the degree of isotopic labeling, sampling intervals and the amount of irrigation water.

Pp6-FEH1 encodes an enzyme for degradation of highly polymerized levan and is transcriptionally induced by defoliation in timothy (Phleum pratense L.).

The ability of grasses to regrow after defoliation by cutting or grazing is a vital factor in their survival and an important trait when they are used as forage crops. In temperate grass species accumulating fructans, defoliation induces the activity of a fructan exohydrolase (FEH) that degrades fructans to serve as a carbon source for regrowth. Here, a cDNA from timothy was cloned, named Pp6-FEH1, that showed similarity to wheat fructan 6-exohydrolase (6-FEH). The recombinant enzyme expressed in Pichia pastoris completely degraded fructans that were composed mainly of ß(2,6)-linked and linear fructans (levan) with a high degree of polymerization (DP) in the crown tissues of timothy. The substrate specificity of Pp6-FEH1 differed from previously characterized enzymes with 6-FEH activity in fructan-accumulating plants: (i) Pp6-FEH1 showed 6-FEH activity against levan (mean DP 20) that was 4-fold higher than against 6-kestotriose (DP 3), indicating that Pp6-FEH1 has a preference for ß(2,6)-linked fructans with high DP; (ii) Pp6-FEH1 had significant activity against ß(2,1)-linked fructans, but considerably less than against ß(2,6)-linked fructans; (iii) Pp6-FEH1 had weak invertase activity, and its 6-FEH activity was inhibited slightly by sucrose. In the stubble of seedlings and in young haplocorms from adult timothy plants, transcripts of Pp6-FEH1 were significantly increased within 3 h of defoliation, followed by an increase in 6-FEH activity and in the degradation of fructans. These results suggest that Pp6-FEH1 plays a role in the degradation of fructans and the mobilization of carbon sources for regrowth after defoliation in timothy.