Horse heart myoglobin catalyzes the H2O2-dependent oxidative dehalogenation of chlorophenols to DNA-binding radicals and quinones

The heme-containing respiratory protein, myoglobin (Mb), best known for oxygen storage, can exhibit peroxidase-like activity under conditions of oxidative stress. Under such circumstances, the initially formed ferric state can react with H2O2 (or other peroxides) to generate a long-lived ferryl [Fe(IV)=O] Compound II (Cpd II) heme intermediate that is capable of oxidizing a variety of biomolecules. In this study, the ability of Mb Cpd II to catalyze the oxidation of carcinogenic halophenols is demonstrated. Specifically, 2,4,6-trichlorophenol (TCP) is converted to 2,6-dichloro-1,4-benzoquinone in a H2O2-dependent process. The fact that Mb Cpd II is an active oxidant in halophenol dehalogenation is consistent with a traditional peroxidase order of addition of H2O2 followed by TCP. With 4-chlorophenol, a dimerized product is formed, consistent with a mechanism involving generation of a reactive phenoxy radical intermediate by an electron transfer process. The radical nature of this process may be physiologically relevant since recent studies have revealed that phenoxy radicals and electrophilic quinones, specifically of the type described herein, covalently bind to DNA [Dai, J., Sloat, A. L., Wright, M. W., and Manderville, R. A. (2005) Chem. Res. Toxicol. 18, 771-779]. Thus, the stability of Mb Cpd II and its ability to oxidize TCP may explain why such compounds are carcinogenic. Furthermore, the initial rate of dehalogenation catalyzed by Mb Cpd II is nearly comparable to that of the same reaction carried out by turnover of the ferric state, demonstrating the potential physiological danger of this long-lived, high-valent intermediate.     

Glutamate 107 in subunit I of the cytochrome bd quinol oxidase from Escherichia coli is protonated and near the heme d/heme b595 binuclear center

Cytochrome bd is a quinol oxidase from Escherichia coli, which is optimally expressed under microaerophilic growth conditions. The enzyme catalyzes the two-electron oxidation of either ubiquinol or menaquinol in the membrane and scavenges O2 at low concentrations, reducing it to water. Previous work has shown that, although cytochrome bd does not pump protons, turnover is coupled to the generation of a proton motive force. The generation of a proton electrochemical gradient results from the release of protons from the oxidation of quinol to the periplasm and the uptake of protons used to form H2O from the cytoplasm. Because the active site has been shown to be located near the periplasmic side of the membrane, a proton channel must facilitate the delivery of protons from the cytoplasm to the site of water formation. Two conserved glutamic acid residues, E107 and E99, are located in transmembrane helix III in subunit I and have been proposed to form part of this putative proton channel. In the current work, it is shown that mutations in either of these residues results in the loss of quinol oxidase activity and can result in the loss of the two hemes at the active site, hemes d and b595. One mutant, E107Q, while being totally inactive, retains the hemes. Fourier transform infrared (FTIR) redox difference spectroscopy has identified absorption bands from the COOH group of E107. The data show that E107 is protonated at pH 7.6 and that it is perturbed by the reduction of the heme d/heme b595 binuclear center at the active site. In contrast, mutation of an acidic residue known to be at or near the quinol-binding site (E257A) also inactivates the enzyme but has no substantial influence on the FTIR redox difference spectrum. Mutagenesis shows that there are several acidic residues, including E99 and E107 as well as D29 (in CydB), which are important for the assembly or stability of the heme d/heme b595 active site.     

Non-additive genetic effects for fertility traits in Canadian Holstein cattle (Open Access publication )

The effects of additive, dominance, additive by dominance, additive by additive and dominance by dominance genetic effects on age at first service, non-return rates and interval from calving to first service were estimated. Practical considerations of computing additive and dominance relationships using the genomic relationship matrix are discussed. The final strategy utilized several groups of 1000 animals (heifers or cows) in which all animals had a non-zero dominance relationship with at least one other animal in the group. Direct inversion of relationship matrices was possible within the 1000 animal subsets. Estimates of variances were obtained using Bayesian methodology via Gibbs sampling. Estimated non-additive genetic variances were generally as large as or larger than the additive genetic variance in most cases, except for non-return rates and interval from calving to first service for cows. Non-additive genetic effects appear to be of sizeable magnitude for fertility traits and should be included in models intended for estimating additive genetic merit. However, computing additive and dominance relationships for all possible pairs of individuals is very time consuming in populations of more than 200 000 animals.     

Development of a novel stall design for dairy cattle: Part II. The effect of minimal stall partitioning on lying behavior,rumination, stall cleanliness,and preference

Free-stalls for dairy cows promote cleanliness, provide cows with a defined space to lie down, and decrease labor and bedding required for maintenance. However, current stall features can restrict behavior and reduce stall use. The objective of this study was to assess the short-term effect of a novel free-stall design (flexible single-bar partition, no neck rail, increased slope) on stall cleanliness and the lying behavior, rumination, milk production, and preferences of dairy cows in comparison to standard free-stalls (metal loop partitions, neck rail). In the first experiment, 60 Holstein cows were randomly divided into two groups and switched between standard and novel stalls in a replicated crossover design with four 7-day periods. Lying behavior and rumination were recorded continuously. Milk yields were recorded 2x/day, and stall cleanliness scores were collected on the last four days of weeks 3 and 4. The second experiment was a 6-day preference test where 14 cows from experiment 1 were given free access to 16 standard and 16 novel stalls. Lying behavior was recorded continuously. On day 6, standing, perching, and intention, lying, and rising movements were recorded. In experiment 1, cows spent 12 min less time lying down (14.1 vs 14.3 ± 0.48 h/d), had one fewer lying bouts (8.8 vs 9.8 ± 0.23 bouts/d), and had longer lying bouts by 6 min (1.4 vs 1.5 ± 0.05 h/bout) in novel stalls. Rumination time (547.3 vs 548.9 ± 4.66 min/d) and milk yield (35.0 vs 35.2 ± 0.51 kg/d) did not differ between stall types. However, novel stalls were more than twice as soiled as standard stalls (32.8 vs 14.2 ± 1.38% of rear half soiled). In experiment 2, the cows most recently housed in the novel stalls showed a clear preference for lying down in those stalls, whereas cows most recently housed in standard stalls showed no preference. Intention and lying down movements were longer in standard stalls when other cows were present in diagonally opposite stalls, but were similar between stall types when not occupied. All cows preferred standing in novel stalls. Novel stalls may have improved stall use compared to standard free-stalls, and the design requires further development to address cleanliness concerns. Future work is required to provide comfortable stalls without sacrificing cleanliness.     

Yield responses of wild C3 and C4 crop progenitors to subambient CO2: a test for the role of CO2 limitation in the origin of agriculture

Limitation of plant productivity by the low partial pressure of atmospheric CO₂ (C_a) experienced during the last glacial period is hypothesized to have been an important constraint on the origins of agriculture. In support of this hypothesis, previous work has shown that glacial C_a limits vegetative growth in the wild progenitors of both C₃ and C₄ founder crops. Here, we present data showing that glacial C_a also reduces grain yield in both crop types. We grew four wild progenitors of C₃ (einkorn wheat and barley) and C₄ crops (foxtail and broomcorn millets) at glacial and postglacial C_a, measuring grain yield and the morphological and physiological components contributing to these yield changes. The C₃ species showed a significant increase in unthreshed grain yield of ~50% with the glacial to postglacial increase in C_a, which matched the stimulation of photosynthesis, suggesting that increases in photosynthesis are directly translated into yield at subambient levels of C_a. Increased yield was controlled by a higher rate of tillering, leading to a larger number of tillers bearing fertile spikes, and increases in seed number and size. The C₄ species showed smaller, but significant, increases in grain yield of 10–15%, arising from larger seed numbers and sizes. Photosynthesis was enhanced by C_a in only one C₄ species and the effect diminished during development, suggesting that an indirect mechanism mediated by plant water relations could also be playing a role in the yield increase. Interestingly, the C₄ species at glacial C_a showed some evidence that photosynthetic capacity was upregulated to enhance carbon capture. Development under glacial C_a also impacted negatively on the subsequent germination and viability of seeds. These results suggest that the grain production of both C₃ and C₄ crop progenitors was limited by the atmospheric conditions of the last glacial period, with important implications for the origins of agriculture.     

Delayed coral recovery in a warming ocean

Climate change threatens coral reefs across the world. Intense bleaching has caused dramatic coral mortality in many tropical regions in recent decades, but less obvious chronic effects of temperature and other stressors can be equally threatening to the long‐term persistence of diverse coral‐dominated reef systems. Coral reefs persist if coral recovery rates equal or exceed average rates of mortality. While mortality from acute destructive events is often obvious and easy to measure, estimating recovery rates and investigating the factors that influence them requires long‐term commitment. Coastal development is increasing in many regions, and sea surface temperatures are also rising. The resulting chronic stresses have predictable, adverse effects on coral recovery, but the lack of consistent long‐term data sets has prevented measurement of how much coral recovery rates are actually changing. Using long‐term monitoring data from 47 reefs spread over 10 degrees of latitude on Australia's Great Barrier Reef (GBR), we used a modified Gompertz equation to estimate coral recovery rates following disturbance. We compared coral recovery rates in two periods: 7 years before and 7 years after an acute and widespread heat stress event on the GBR in 2002. From 2003 to 2009, there were few acute disturbances in the region, allowing us to attribute the observed shortfall in coral recovery rates to residual effects of acute heat stress plus other chronic stressors. Compared with the period before 2002, the recovery of fast‐growing Acroporidae and of “Other” slower growing hard corals slowed after 2002, doubling the time taken for modest levels of recovery. If this persists, recovery times will be increasing at a time when acute disturbances are predicted to become more frequent and intense. Our study supports the need for management actions to protect reefs from locally generated stresses, as well as urgent global action to mitigate climate change.     

Introgression and repeated co‐option facilitated the recurrent emergence of C4 photosynthesis among close relatives

The origins of novel traits are often studied using species trees and modeling phenotypes as different states of the same character, an approach that cannot always distinguish multiple origins from fewer origins followed by reversals. We address this issue by studying the origins of C₄ photosynthesis, an adaptation to warm and dry conditions, in the grass Alloteropsis. We dissect the C₄ trait into its components, and show two independent origins of the C₄ phenotype via different anatomical modifications, and the use of distinct sets of genes. Further, inference of enzyme adaptation suggests that one of the two groups encompasses two transitions to a full C₄ state from a common ancestor with an intermediate phenotype that had some C₄ anatomical and biochemical components. Molecular dating of C₄ genes confirms the introgression of two key C₄ components between species, while the inheritance of all others matches the species tree. The number of origins consequently varies among C₄ components, a scenario that could not have been inferred from analyses of the species tree alone. Our results highlight the power of studying individual components of complex traits to reconstruct trajectories toward novel adaptations.     

Cardiorespiratory dynamics: sensitivity of the on-transition to endurance-training status

This project investigated the sensitivity of oxygen uptake (VO(2)) dynamics to training-induced physiological changes, across a broad spectrum of endurance-training histories. Forty subjects participated: sedentary (n = 10), active healthy (n = 10), regular runners (n = 10), and competitive distance runners (n = 10). Subjects completed a cycle step-function protocol, to elicit a steady state at 60% maximal work rate. Breath-by-breath data were collected for VO(2) and cardiac frequency (f(c)), and modelled mathematically, and used to determine the average response times to attain 20%, 40%, 60%, 80% and 100% of the respective steady states. The between-group comparisons for both VO(2) and f(c) revealed significantly faster response times to 40%, 60%, 80% and 100% of the induced response, for the better trained subjects (P < 0.05). In general, this technique permitted differentiation between the VO(2) and f(c) response dynamics of non-elite subjects from a broad range of endurance-training histories, with differences becoming more pronounced as subjects approached the steady state.     

Performance of open-fronted microbiological safety cabinets: the value of operator protection tests during routine servicing

The performance of class I and II microbiological safety cabinets over 7 years, employed in a force-ventilated containment level 3 (CL-3) laboratory, is described. Operator Protection (OP) provided by the cabinets, assessed by still and latterly limited 'in-use' KI-Discus tests, showed no overall deterioration during the review period. Comparisons show that a selected class II unit, but not a second, and a new class II MSC in a recently commissioned, similar CL-3 facility, provide the same order of OP as a class I cabinet. From the experiences described, it is strongly recommended that OP tests (OPTs) should be part of the routine servicing regime to ensure that cabinets meet required performance levels, and additionally to allow detection and rectification of poor containment, particularly where induced by environmental factors. The value of OPTs is discussed with reference to certain national standards.     

Sulfation of chondroitin sulfate in human articular cartilage. The effect of age, topographical position, and zone of cartilage on tissue composition.

The chondroitin ABC lyase digestion products of normal human femoral condyle articular cartilage and of purified aggrecan were analyzed for their mono- and nonsulfated disaccharide composition. Changes in the total tissue chemistry were most pronounced during the period from birth to 20 years of age, when the -[GlcAbeta,3GalNAc6]- disaccharide content increased from approximately 50% to 85% of the total disaccharide content and there was a concomitant decrease in the content of the 4-sulfated disaccharide. In general, the disaccharide content of the deeper layers of immature cartilage were richer in the 4-sulfated residue than the upper regions of the tissue. As the tissue aged and decreased in thickness, the disaccharide composition became more evenly 6-sulfated. The newly synthesized chondroitin sulfate chains had a similar composition to the endogenous chains and also underwent the same age and zonal changes. The monoclonal antisera 3B3(+) and 2B6(+) were used to immunolocalize the unsaturated 6- and 4-sulfated residues generated at the reducing termini of the chondroitin sulfate chains by digestion with chondroitin ABC lyase, and these analyses indicated that the sulfation pattern at this position did not necessarily reflect the internal disaccharide composition of the chains. In summary, the sulfation pattern of chondroitin sulfate disaccharides from human normal articular cartilage varies with the age of the specimen, the position (topography) on the joint surface, and the zone of cartilage analyzed. Furthermore, these changes in composition are a consequence of both extracellular, post-translational processing of the core protein of aggrecan and changes in the sulfotransferase activity of the chondrocyte.