Environmental changes threaten plant-pollinator mutualisms and their critical ecosystem service. Drivers such as land use, invasions and climate change can affect pollinator diversity or species encounter rates. However, nitrogen deposition, climate warming and CO(2) enrichment could interact to disrupt this crucial mutualism by altering plant chemistry in ways that alter floral attractiveness or even nutritional rewards for pollinators. Using a pumpkin model system, we show that these drivers non-additively affect flower morphology, phenology, flower sex ratios and nectar chemistry (sugar and amino acids), thereby altering the attractiveness of nectar to bumble bee pollinators and reducing worker longevity. Alarmingly, bees were attracted to, and consumed more, nectar from a treatment that reduced their survival by 22%. Thus, three of the five major drivers of global environmental change have previously unknown interactive effects on plant-pollinator mutualisms that could not be predicted from studies of individual drivers in isolation.

We have demonstrated two novel reactive species on radical-modified proteins which are relatively long-lived, one oxidizing and one reducing. The two species are reactive with critical biological components, and so may be of physiological and pathological importance. The oxidizing species, which have been identified as protein hydroperoxides, can consume key cellular reductants, such as ascorbate and glutathione. The reducing species can act on both free and metalloprotein forms of copper and iron ions, which participate in radical generation. These findings suggest that proteins may act as traps for the chemical energy released by free radicals, with the capacity to pass it on to other molecules. The long-lived nature of both the reactive moieties indicates that they may be able to diffuse and transfer damaging reactions to distant sites.

Proteins and aromatic amino acids previously exposed to hydroxyl radicals reduced cytochrome c, free iron, and copper ions. A major product of hydroxyl radical addition to tyrosine is 3,4-dihydroxyphenylalanine (DOPA), which has these reducing properties. The reduction of nitro blue tetrazolium by radical-damaged protein was consistent with the generation of quinones in the protein. By acid hydrolysis followed by high-performance C18 reversed-phase liquid chromatography we have shown that hydroxyl radical-damaged proteins contain significant amounts of protein-bound DOPA (PB-DOPA). The authenticity of the DOPA measured was confirmed by gas chromatography-mass spectrometry. PB-DOPA was also generated enzymatically using mushroom tyrosinase, which catalyzes the hydroxylation of tyrosine residues. By comparing the levels of DOPA in radical-damaged or enzyme-treated protein with that of cytochrome c reduction, we show that PB-DOPA is a major source of the observed reducing activity. PB-DOPA may have a role in the replenishment of reduced transition metal ions involved in free radical generating systems in vivo.

The oxidative resistance of low density lipoprotein (LDL) can be experimentally described by the length of time during which no significant lipid peroxidation is observed in a pro-oxidant environment. This period of inhibited oxidation, termed the 'lag phase', is partially due to the radical scavenging reactions of the anti-oxidants contained in the LDL particle. We have shown that the LDL lag time decreases with increasing copper concentration, leveling out at a relatively high copper-to-LDL ratio. This behaviour demonstrates the existence of a finite number of saturable pro-oxidant copper binding sites within the LDL particle. The relationship is described by the equation, lag time = [Cu]-' . K. tti + tti where the constant, K, is the negative reciprocal of the x-axis intercept of the graphed function, and tti, is given by the y-axis intercept. By this definition of the constant, K is the amount of copper that will produce a lag time of twice tti, while tmi,, is the minimum time a particular LDL will resist oxidation at a maximum copper concentration.

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Rugby union is a sport governed by the impacts of high force and high frequency. Analysis of physiological markers following a game can provide an understanding of the physiological response of an individual and the time course changes in response to recovery. Urine and saliva were collected from 11 elite amateur rugby players 24 h before, immediately after, and at 17, 25, 38, 62 and 86 h post-game. Myoglobin, salivary immunoglobulin A and cortisol were analysed by ELISA, whereas neopterin and total neopterin were analysed by high-performance liquid chromatography. There was a significant post-game increase of all four markers. The increases were cortisol 4-fold, myoglobin 2.85-fold, neopterin 1.75-fold and total neopterin 2.3-fold when corrected with specific gravity. All significant changes occurred post-game only, with markers returning to and remaining at baseline within 17 h. The intensity of the game caused significant changes in key physiological markers of stress. They provide an understanding of the stress experienced during a single game of rugby and the time course changes associated with player recovery. Neopterin provides a new marker of detecting an acute inflammatory response in physical exercise, while specific gravity should be considered for urine volume correction post-exercise.

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Rugby union is a physically intense intermittent sport coupled with high force collisions. Each position within a team has specific requirements which are typically based on speed, size and skill. The aim of this study was to investigate the contemporary demands of each position and whether they can explain changes in psychophysiological stress. Urine and saliva samples were collected before and after five selected Super 15 rugby games from 37 players. Total neopterin (NP), cortisol and immunoglobulin A were analysed by SCX-high performance liquid chromatography and enzyme linked immunosorbent assay. Global positioning system software provided distance data, while live video analysis provided impact data. All contemporary demands were analysed as events per minute of game time. Forwards were involved in more total impacts, tackles and rucks compared to backs (p < 0.001), while backs were involved in more ball carries and covered more total distance and distance at high speed per minute of game time (p < 0.01). Loose forwards, inside and outside backs covered significantly more distance at high speed (p < 0.01), while there was a negligible difference with number of impacts between the forward positions. There was also minimal difference between positions in the percentage change in NP, cortisol and sIgA. The results indicate distance covered and number of impacts per minute of game time is position-dependent whereas changes in psychophysiological stress are independent. This information can be used to adapt training and recovery interventions to better prepare each position based on the physical requirements of the game.