Analysis of expressed sequence tags from a single wheat cultivar facilitates interpretation of tandem mass spectrometry data and discrimination of gamma gliadin proteins that may play different functional roles in flour

<p>Abstract</p> <p>Background</p> <p>The gamma gliadins are a complex group of proteins that together with other gluten proteins determine the functional properties of wheat flour. The proteins have unusually high levels of glutamine and proline and contain large regions of repetitive sequences. While most gamma gliadins are monomeric proteins containing eight conserved cysteine residues, some contain an additional cysteine residue that enables them to be linked with other gluten proteins into large polymers that are critical for flour quality. The ability to differentiate among the gamma gliadins is important for studies of wheat flour quality because proteins with similar sequences can have different effects on functional properties.</p> <p>Results</p> <p>The complement of gamma gliadin genes expressed in the wheat cultivar Butte 86 was evaluated by analyzing publicly available expressed sequence tag (EST) data. Eleven contigs were assembled from 153 Butte 86 ESTs. Nine of the contigs encoded full-length proteins and four of the proteins contained nine cysteine residues. Only one of the encoded proteins was a perfect match with a sequence reported in NCBI. Contigs from four different publicly available EST assemblies encoded proteins that were perfect matches with some, but not all, of the Butte 86 gamma gliadins and the complement of identical proteins was different for each assembly. A specialized database that included the sequences of Butte 86 gamma gliadins was constructed for identification of flour proteins by tandem mass spectrometry (MS/MS). In a pilot experiment, proteins corresponding to six Butte 86 gamma gliadin contigs were distinguished by MS/MS, including one containing the extra cysteine residue. Two other proteins were identified as one of two closely related Butte 86 proteins but could not be distinguished unequivocally. Unique peptide tags specific for Butte 86 gamma gliadins are reported.</p> <p>Conclusions</p> <p>Inclusion of cultivar-specific gamma gliadin sequences in databases maximizes the number and quality of peptide identifications and increases sequence coverage of these gamma gliadins by MS/MS. This approach makes it possible to distinguish closely related proteins, to associate individual proteins with sequences of specific genes, and to evaluate proteomic data in a biological context to better address questions about wheat flour quality.</p

Deciphering the complexities of the wheat flour proteome using quantitative two-dimensional electrophoresis, three proteases and tandem mass spectrometry

BACKGROUND: Wheat flour is one of the world's major food ingredients, in part because of the unique end-use qualities conferred by the abundant glutamine- and proline-rich gluten proteins. Many wheat flour proteins also present dietary problems for consumers with celiac disease or wheat allergies. Despite the importance of these proteins it has been particularly challenging to use MS/MS to distinguish the many proteins in a flour sample and relate them to gene sequences. RESULTS: Grain from the extensively characterized spring wheat cultivar Triticum aestivum 'Butte 86' was milled to white flour from which proteins were extracted, then separated and quantified by 2-DE. Protein spots were identified by separate digestions with three proteases, followed by tandem mass spectrometry analysis of the peptides. The spectra were used to interrogate an improved protein sequence database and results were integrated using the Scaffold program. Inclusion of cultivar specific sequences in the database greatly improved the results, and 233 spots were identified, accounting for 93.1% of normalized spot volume. Identified proteins were assigned to 157 wheat sequences, many for proteins unique to wheat and nearly 40% from Butte 86. Alpha-gliadins accounted for 20.4% of flour protein, low molecular weight glutenin subunits 18.0%, high molecular weight glutenin subunits 17.1%, gamma-gliadins 12.2%, omega-gliadins 10.5%, amylase/protease inhibitors 4.1%, triticins 1.6%, serpins 1.6%, purinins 0.9%, farinins 0.8%, beta-amylase 0.5%, globulins 0.4%, other enzymes and factors 1.9%, and all other 3%. CONCLUSIONS: This is the first successful effort to identify the majority of abundant flour proteins for a single wheat cultivar, relate them to individual gene sequences and estimate their relative levels. Many genes for wheat flour proteins are not expressed, so this study represents further progress in describing the expressed wheat genome. Use of cultivar-specific contigs helped to overcome the difficulties of matching peptides to gene sequences for members of highly similar, rapidly evolving storage protein families. Prospects for simplifying this process for routine analyses are discussed. The ability to measure expression levels for individual flour protein genes complements information gained from efforts to sequence the wheat genome and is essential for studies of effects of environment on gene expression

Catch-effort estimation of white-tailed deer population size

Estimation of population size is important for most research in population biology and in the management of game species. Using a stochastic, catch-effort, competing risks model (Dupont 1983), we estimated the population size of the Savannah River Site white-tailed deer (Odocotleus virginianus) herd for 1965-86. Population size varied markedly in response to changes in both hunting method and pressure. Still hunters preferentially harvested older animals compared to dog hunters. Deer were 2.37 times more susceptible to harvest from dog hunting than from still hunting. Hunter-induced mortality was estimated as 1.73 and 4.10 times as large as nonhunting mortality for still and dog hunting, respectively. The temporal pattern of estimated prehunt population sizes was significantly correlated with the temporal pattern of car-deer accidents recorded on the site during the same time period, suggesting that the temporal pattern of the population estimates is accurate. If the number of cohorts is large and an accurate estimate of hunter effort can be obtained, this technique may provide more reliable population estimates than previously available techniques because it imposes fewer and less stringent biological assumptions

The Effects of a Korean Ginseng, GINST15, on Hypo-Pituitary-Adrenal and Oxidative Activity Induced by Intense Work Stress

The effect of GINST15, an enzyme fermented ginseng supplement, on hormonal and inflammatory responses to physical stress in humans is unknown. The purpose of this investigation was to examine the constitutive and stress-induced effects of GINST15 supplement on hypo-pituitary-adrenal (HPA) and antioxidant activity in addition to muscle damage. Ten women (age: 38.7 ± 7.8 years; height: 163.81 ± 4.4 cm; body mass 76.0 ± 11.6 kg) and nine men (age: 41.2. ± 9.7 years; height: 177.4 ± 5.3 cm; body mass: 88.5 ± 5.0 kg) participated in a double-blinded, placebo-controlled, counterbalanced within-group study. Participants completed three 14-day treatment cycles with different doses (high: 960 mg; low: 160 mg; placebo: 0 mg) separated by a 1-week washout period. At the end of treatment, physical stress was imposed with intense resistance exercise work stress. Participants provided blood at rest and various time points after exercise (immediately [IP], 30 min [30], 60 min [60], 24 h [+24HR]). Cortisol (CORT), superoxide dismutase (SOD), total glutathione, nonspecific antioxidant activity, total antioxidant power (TAP), and creatine kinase were measured. GINST15 supplementation produced stress-inducible dose-dependent reductions in circulating cortisol and increased enzymatic and nonspecific antioxidant activity. Twenty-four hours after intense exercise, a high dose GINST15, a bioactive ginsenoside metabolite, significantly reduces muscle damage and HPA responses to physical stress in humans; these effects may result from increased antioxidant expression

The Effects of a Korean Ginseng, GINST15, on Perceptual Effort, Psychomotor Performance, and Physical Performance in Men and Women

The purpose of this double-blind, placebo-controlled investigation was to examine the effects of a Korean Ginseng (GINST15) on measures of perception and physical performance following an acute bout of resistance exercise. Ten women (age: 38.7 ± 7.8 years; height: 1.64 ± 0.05 m; body mass: 76.0 ± 11.6 kg) and nine men (age: 41.2. ± 9.7 years; height: 1.77 ± 0.05 m; body mass: 88.5 ± 5.0 kg) completed the investigation. Participants were randomized to a three-cycle testing scheme consisting of high dose ginseng (HIGH: 960 mg/day), low dose ginseng (LOW: 160 mg/day) and placebo (PBO: 0 mg/day). After 14 days of supplementation participants returned to the laboratory for an acute resistance exercise trial (5 sets of 12 repetitions of the leg press at 70% of one-repetition-maximum [1RM]). Ratings of perceived exertion (RPE) were assessed after each set. Muscle pain/soreness was assessed before exercise and 24 hours post exercise. Psychomotor performance and peak power were measured before exercise, immediately post exercise and 24 hours after exercise. Each treatment cycle was separated by a minimum one-week washout period. HIGH significantly reduced perceived exertion during exercise. HIGH and LOW significantly reduced change in muscle soreness at 24 hours post exercise. Analysis of peak power demonstrated the presence of responders (n = 13) and non-responders (n = 6). Responders showed a significant effect of HIGH GINST15 on maintenance of neuromuscular function. The appearance of responders and non-responders, could explain the mixed literature base on the ergogenic properties of ginseng

Bioactive growth hormone in older men and women: Its relationship to immune markers and healthspan

Objective: The consequences of age-related decline in the somatotropic axis of humans are complex and remain largely unresolved. We tested the hypothesis that hGH measurements of plasma by bioassay vs immunoassay from samples obtained from free-living, elderly individuals would reveal a dichotomy in GH activities that are correlated with the functional status of the donors, i.e. their healthspan. Design: Forty-one men and women of advanced age (men: N=16, age, 80.5±6.5years; height, 173.1±6.9cm; body mass, 81.8±13.0kg) and (women: N=25, age, 80.7±7.2years; height, 157.7±6.0cm; body mass, 68.8±17kg), were recruited for a cross-sectional study. Participants filled out PROMIS (Patient-Reported Outcomes Measurement Information System, U. S. Department of Health and Human Services) scales, undertook physical performance tests and had fasted blood samples obtained at rest for measurement of hormonal and immunology biomarkers. Results: When measured by the well-established rat tibial line GH bioassay, one half of the plasma samples (n=20) contained bioassayable GH (bGH), but the other half (n=21) failed to mount increases in tibial plate width above saline injected controls. This difference did not correlate with the age, sex or physical functionality of the plasma donor. It also did not correlate with hGH concentrations measured by immunoassay. In those cases in which bGH was detected, various hierarchical regression models predicted that GHRH, c-peptide, VEGF, NPY, IL-4 and T-regulatory lymphocytes were associated with the difference and predicted bGH. Conclusion: Results from this study suggest that the actions of bGH at the cellular level may be modified by other factors and that this may explain the lack of correlations observed in this study

The Effects Combining Cryocompression Therapy following an Acute Bout of Resistance Exercise on Performance and Recovery

Compression and cold therapy used separately have shown to reduce negative effects of tissue damage. The combining compression and cold therapy (cryocompression) as a single recovery modality has yet to be fully examined. To examine the effects of cryocompression on recovery following a bout of heavy resistance exercise, recreationally resistance trained men (n =16) were recruited, matched, and randomly assigned to either a cryocompression group (CRC) or control group (CON). Testing was performed before and then immediately after exercise, 60 minutes, 24 hours, and 48 hours after a heavy resistance exercise workout (barbell back squats for 4 sets of 6 reps at 80% 1RM, 90 sec rest between sets, stiff legged deadlifts for 4 sets of 8 reps at 1.0 X body mass with 60 sec rest between sets, 4 sets of 10 eccentric Nordic hamstring curls, 45 sec rest between sets). The CRC group used the CRC system for 20-mins of cryocompression treatment immediately after exercise, 24 hours, and 48 hours after exercise. CON sat quietly for 20-mins at the same time points. Muscle damage [creatine kinase], soreness (visual analog scale, 0-100), pain (McGill Pain Q, 0-5), fatigue, sleep quality, and jump power were significantly (p \u3c 0.05) improved for CRC compared to CON at 24 and 48 hours after exercise. Pain was also significantly lower for CRC compared to CON at 60-mins post exercise. These findings show that cryocompression can enhance recovery and performance following a heavy resistance exercise workout

Changes of Hydration Measures in Elite National Collegiate Athletic Association Division I Wrestlers

Purpose: To evaluate the changes in the state of hydration in elite National Collegiate Athletic Association (NCAA) Division I college wrestlers during and after a season. Methods: Ohio State University wrestling team members (N = 6; mean [SD] age = 19.6 [1.1] y; height = 171.6 [2.9] cm; body mass = 69.5 [8.1] kg) gave informed consent to participate in the investigation with measurements (ie, body mass, urine-specific gravity [USG; 2 methods], Visual Analog Scale thirst scale, plasma osmolality) obtained during and after the season. Results: Measurements for USG, regardless of methods, were not significantly different between visits, but plasma osmolality was significantly (P = .001) higher at the beginning of the season—295.5 (4.9) mOsm·kg−1 compared with 279.6 (6.1) mOsm·kg−1 after the season. No changes in thirst ratings were observed, and the 2 measures of USG were highly correlated (r \u3e .9, P = .000) at each time point, but USG and plasma osmolality were not related. Conclusions: A paradox in the clinical interpretation of euhydration in the beginning of the season was observed with the USG, indicating that the wrestlers were properly hydrated, while the plasma osmolality showed they were not. Thus, the tracking of hydration status during the season is a concern when using only NCAA policies and procedures. The wrestlers did return to normal euhydration levels after the season on both biomarkers, which is remarkable, as previous studies have indicated that this may not happen because of the reregulation of the osmol-regulatory center in the brain

The modern pollen-vegetation relationship of a tropical forest-savannah mosaic landscape, Ghana, West Africa

Transitions between forest and savannah vegetation types in fossil pollen records are often poorly understood due to over-production by taxa such as Poaceae and a lack of modern pollen-vegetation studies. Here, modern pollen assemblages from within a forest-savannah transition in West Africa are presented and compared, their characteristic taxa discussed, and implications for the fossil record considered. Fifteen artificial pollen traps were deployed for 1 year, to collect pollen rain from three vegetation plots within the forest-savannah transition in Ghana. High percentages of Poaceae and Melastomataceae/Combretaceae were recorded in all three plots. Erythrophleum suaveolens characterised the forest plot, Manilkara obovata the transition plot and Terminalia the savannah plot. The results indicate that Poaceae pollen influx rates provide the best representation of the forest-savannah gradient, and that a Poaceae abundance of >40% should be considered as indicative of savannah-type vegetation in the fossil record

Density dependent regulation of inflammatory responses in macrophages

Macrophage distribution density is tightly regulated within the body, yet the importance of macrophage crowding during in vitro culture is largely unstudied. Using a human induced pluripotent stem cell (iPSC)-derived macrophage model of tissue resident macrophages, we characterize how increasing macrophage culture density changes their morphology and phenotype before and after inflammatory stimulation. In particular, density drives changes in macrophage inflammatory cytokine and chemokine secretion in both resting and activated states. This density regulated inflammatory state is also evident in blood monocyte derived-macrophages, the human monocytic THP-1 immortalized cell line, and iPSC-derived microglia. Density-dependent changes appear to be driven by a transferable soluble factor, yet the precise mechanism remains unknown. Our findings highlight cell plating density as an important but frequently overlooked consideration of in vitro macrophage research relevant to a variety of fields ranging from basic macrophage cell biology to disease studies