O-glycosylation of protein subpopulations in alcohol-extracted rice proteins

Mucin-type O-glycosylation has been well characterized in mammalian systems but not in plants. In this study, the purified alcohol-soluble, non-reduced protein (prolamin) fraction from rice seed was investigated for the occurrence of O-linked oligosaccharides. As storage prolamins are unlikely to be O-glycosylated, any O-glycosylation found was likely to belong to co-extracted proteins, whether because of association with the protein body or solubility. SDS-PAGE and MS analyses revealed 14 and 16kDa protein families in fractions that bound to the lectins peanut agglutinin (PNA), Vicia villosa lectin (VVL) and Jacalin, indicative of the presence of O-linked saccharides. Enzymatic cleavage, fluorescent labeling and high-performance liquid chromatography (HPLC) analysis demonstrated a peak consistent with Gal-beta-(1-->3)-GalNAc, with similar MS/MS fragmentation. Additionally, upon chemical analysis, a GlcNAc-containing O-linked carbohydrate moiety was discovered. Protein blotting with anti-O-GlcNAc antibody (clone CTD110.6) was positive in a subpopulation of the 14kDa alcohol-soluble protein fraction, but a hot capping experiment was negative. Therefore, the GlcNAc residue in this case is unlikely to be terminal. Additionally, a positive reaction with CTD110.6mAb cannot be taken as absolute proof of O-GlcNAc modification and further confirmatory experiments should be employed. We hypothesize that O-glycosylation may contribute to protein functionality or regulation. Further investigation is required to identify the specific proteins with these modifications. This 'reverse' approach could lead to the identification of proteins involved in mRNA targeting, signaling, translation, anchoring or maintenance of translational quiescence and may be applied to germinating rice seed extracts for further elucidation of protein function and regulation.     

State-Dependency Effects on TMS: A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.^1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.^3,4,5In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.⁶Download video file.^{(78M, mov)}     

Identification of jellyfish from Continuous Plankton Recorder samples

Previous analysis of the Continuous Plankton Recorder (CPR) long-term data set for the presence of ‘coelenterate’ tissue revealed changes in the frequency of jellyfish occurrence in the North Atlantic Ocean and North Sea; however, the identities of the jellyfish were unknown, causing uncertainty in interpreting these findings. To improve the utility of the ‘coelenterate’ data from the CPR, 62 CPR samples were selected for re-analysis from an area where the widespread occurrence of the holoplanktonic scyphomedusan, Pelagia noctiluca (Forsskål, 1775), was previously documented from net tows to test if the CPR sampled P. noctiluca. Examination of the samples revealed smears (<1 mm) of golden gelatinous tissue on the sampling mesh. Subsequent microscopic examination and measurement of nematocysts identified most of the smears as P. noctiluca. Indeed, P. noctiluca was identified on 53% of the CPR samples from the area that best coincided with the documented bloom area. Although hydrozoans were also identified in the samples, the characteristic golden colour of P. noctiluca and its distinct suite of nematocysts allowed identification to species level. The identification of other jellyfish may be more complicated due to the labour-intensive examination of samples and unclear delineation of nematocyst types when multiple similar species are present. Nevertheless, re-analysis of CPR samples holds great promise for identifying the underlying reason for increases in jellyfish occurrence in the CPR records.     

The Coevolution of Secrecy and Stigmatization

We propose a coevolutionary model of secrecy and stigmatization. According to this model, secrecy functions to conceal potential fitness costs detected in oneself or one’s genetic kin. In three studies, we found that the content of participants’ distressing secrets overlapped significantly with three domains of social information that were important for inclusive fitness and served as cues for discriminating between rewarding and unrewarding interaction partners: health, mating, and social-exchange behavior. These findings support the notion that secrecy functions primarily as a defense against stigmatization by suppressing information about oneself or one’s kin that evolutionarily has been devalued in mating and social exchange.     

Comparison of kinetic variables and muscle activity during a squat vs. a box squat

The purpose of this investigation was to determine if there was a difference in kinetic variables and muscle activity when comparing a squat to a box squat. A box squat removes the stretch-shortening cycle component from the squat, and thus, the possible influence of the box squat on concentric phase performance is of interest. Eight resistance trained men (Height: 179.61 ± 13.43 cm; Body Mass: 107.65 ± 29.79 kg; Age: 24.77 ± 3.22 years; 1 repetition maximum [1RM]: 200.11 ± 58.91 kg) performed 1 repetition of squats and box squats using 60, 70, and 80% of their 1RM in a randomized fashion. Subjects completed the movement while standing on a force plate and with 2 linear position transducers attached to the bar. Force and velocity were used to calculate power. Peak force and peak power were determined from the force-time and power-time curves during the concentric phase of the lift. Muscle activity (electromyography) was recorded from the vastus lateralis, vastus medialis, biceps femoris, and longissimus. Results indicate that peak force and peak power are similar between the squat and box squat. However, during the 70% of 1RM trials, the squat resulted in a significantly lower peak force in comparison to the box squat (squat = 3,269 ± 573 N, box squat = 3,364 ± 575 N). In addition, during the 80% of 1RM trials, the squat resulted in significantly lower peak power in comparison to the box squat (squat = 2,050 ± 486 W, box squat = 2,197 ± 544 W). Muscle activity was generally higher during the squat in comparison to the box squat. In conclusion, minimal differences were observed in kinetic variables and muscle activity between the squat and box squat. Removing the stretch-shortening cycle during the squat (using a box) appears to have limited negative consequences on performance.     

Derivation of Man-1 and Man-2 research grade human embryonic stem cell lines

We report here the derivation of two new human embryonic stem cell lines, Man-1 and Man-2, and their full characterization as novel pluripotent stem cell lines. Man-1 was derived from an embryo surplus to requirement from routine IVF, while Man-2 was obtained from an oocyte classified as failed to fertilise and subsequently chemically activated. We report the characterisation of pluripotency and the differentiation potential of these lines. Work is in progress to establish novel methods of stem cell derivation and culture, which will avoid the use of xenobiotics and be relevant to clinical production of human embryonic stem cell lines. Both newly derived human embryonic stem cell lines will be available for the research community from the UK Stem Cell Bank (http://www.ukstemcellbank.org.uk).     

Behavioural Response of Bats to Perceived Predation Risk While Foraging

The ability to detect and respond to predation risk while foraging may have important fitness consequences for prey organisms. Anti‐predator behaviours may reduce the probability of mortality because of predation, but they may also be associated with reduced foraging efficiency. Several behaviours of bats have been suggested to serve as anti‐predator responses, and there is evidence that predation, particularly by avian predators such as owls, may be an important cause of bat mortality. Previous studies have attempted to determine whether predator presence affects bat behaviour when emerging from roost sites, but few have examined effects of predator presence on bat behaviour while foraging. In this study, we investigated whether foraging bats respond to predator cues by presenting bats with an acoustic cue simulating the presence of an owl. Within matched trials, which were conducted at different locations each of 18 nights, significantly fewer bat detections were recorded at owl playback stations than at control stations (no auditory cue), suggesting an avoidance response by bats. An acoustic control (i.e. station playing woodpecker calls), however, did not have significantly more detections than the stations playing the owl calls, suggesting that bats may simply be avoiding noise and more detailed investigation is warranted. Although evidence for owl predation on bats is minimal in North America, results of this study may indicate that the perceived presence of owls may represent a factor influencing the behaviour of bats while foraging.     

A novel yeast two-hybrid approach to identify CDPK substrates: characterization of the interaction between AtCPK11 and AtDi19, a nuclear zinc finger protein

Calcium-dependent protein kinases (CDPKs) are sensor-transducer proteins capable of decoding calcium signals in diverse phosphorylation-dependent calcium signaling networks in plants and some protists. Using a novel yeast two-hybrid (YTH) approach with constitutively active and/or catalytically inactive forms of AtCPK11 as bait, we identified AtDi19 as an AtCPK11-interacting protein. AtDi19 is a member of a small family of stress-induced genes. The interaction was confirmed using pull-down assays with in vitro translated AtCPK11 and GST-AtDi19 and localization studies in Arabidopsis protoplasts cotransfected with AtCPK11:GFP and AtDi19:DsRed2 protein fusions. We further showed that the interaction of AtDi19 is specific to both AtCPK4 and AtCPK11, whereas other closely related CPKs from Arabidopsis interacted weakly (e.g., AtCPK12) or did not interact (e.g., AtCPK26, AtCPK5 and AtCPK1) with AtDi19. Deletion analyses showed that a region containing two predicted nuclear localization signals (NLS) and a nuclear export signal (NES) of AtDi19 is essential for interaction with AtCPK11. We further demonstrated that AtDi19 is phosphorylated by AtCPK11 in a Ca(2+)-dependent manner at Thr105 and Ser107 within the AtDi19 bipartite NLS using in vitro kinase assays. Our data suggest that disruption of the autoinhibitor domain leading to the formation of a constitutively active CDPK may stabilize kinase-substrate interactions without affecting specificity.