2D-HPLC and MALDI-TOF/TOF analysis of barley proteins glycated during brewing

The barley proteins have been the subject of interests of many research groups dealing with barley grains, malt and beer. The proteins which remain intact after harsh malting conditions influence the quality and flavor of beer. The characteristic feature of the proteins present in malt and beer is their extensive modification with carbohydrates, mainly glucose that comes from the starch degradation during technological processes. The degree of the protein glycation has an effect on the quality of malt and beer and on the properties of the beer foam. A combination of two-dimensional high performance liquid chromatography (2D-HPLC) and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF MS) was used for the analysis of the protein extracts that were reduced, alkylated, and degraded enzymatically without prior protein separation. This so-called "shot-gun" approach enabled us to determine glycation sites in one third of the proteins identified in the study and to propose potential glycation markers for fast and efficient monitoring during malting.     

Validation and application of an LC–MS/MS method for quantitation of three fatty acid ethanolamides as biomarkers for fatty acid hydrolase inhibition in human plasma

Endogenous ethanolamides (fatty acid amides), including arachidonyl ethanolamide (anandamide, AEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA), are substrates of fatty acid amide hydrolase (FAAH). FAAH may play an important role for pain, anxiety/depression, and metabolic disorders. Ethanolamides are considered to be potential pharmacodynamic biomarkers to determine target engagement for FAAH inhibition by novel pharmaceutical agents. A highly selective, sensitive, and high-throughput liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous quantitation of AEA, OEA, and PEA in human plasma. The method employed D₄-AEA, D₄-OEA, and ¹³C₂-PEA as “surrogate analytes” to establish the concentration–mass response relationship, i.e. a regression equation. The concentrations of AEA, OEA, and PEA were calculated based on the regression equations derived from the surrogate analytes. This approach made it possible to prepare calibration standard and quality control (QC) samples in plasma devoid of interferences from the endogenous analytes. The analytical methodology required 150 μL of human plasma that was processed via liquid–liquid extraction (LLE) using a 96-well plate format. Chromatographic separation was achieved with a reversed-phase high performance liquid chromatography (HPLC) column using gradient elution, and the run time was 3 min. The method was fully validated and it demonstrated acceptable accuracy, precision, linearity, and specificity. The lower limit of quantitation (LLOQ) was 0.1/0.5/0.5 ng/mL for AEA/OEA/PEA, which was sensitive enough to capture the basal plasma levels in healthy subjects. Bench-top stability in plasma, freeze–thaw stability in plasma, frozen long-term stability in plasma, autosampler stability, and stock solution stability all met acceptance criteria (%Bias within ±12.0%). Characterization of stability in purchased/aged blood indicated that ethanolamides are subject to degradation mediated by intracellular membrane-bound FAAH, which has been shown to be inhibited by phenylmethylsulfonyl fluoride (PMSF). In the presence of PMSF, ethanolamide levels increased slightly over time, suggesting that blood cells release ethanolamides into plasma. Whole blood stability conducted in fresh blood immediately following collection revealed that there was significant elevation of ethanolamide concentrations (∼1.3–2.0-fold on ice and ∼1.5–3.0-fold at room temperature by 2 h), indicating that de novo synthesis and release from blood cells were the predominant factors affecting ethanolamide concentrations ex vivo. Accordingly, conditions that ensured rapid separation of plasma from blood cells and consistency in the blood harvesting procedures were established and implemented for clinical studies to minimize the ex vivo elevation of plasma ethanolamide concentrations. The variability (intra-subject and inter-subject) of plasma ethanolamide levels was evaluated in healthy subjects during a Phase 0 study (no drug administration) that simulated the design of single-ascending dose and multiple-ascending dose clinical trials in terms of sample collection time points, population, food, and activity. The data indicated there was relatively large inter- and intra-subject variation in plasma ethanolamide concentrations. In addition, apparent variations due to time of day and/or food effects were also revealed. Understanding the variability of ethanolamide levels in humans is very important for study design and data interpretation when changes in ethanolamide levels are used as target engagement biomarkers in clinical trials.     

Determination of human serum 8-hydroxy-2′-deoxyguanosine (8-OHdG) by HPLC-ECD combined with solid phase extraction (SPE)

Human serum 8-hydroxy-2′-deoxyguanosine (8-OHdG) was measured by HPLC-ECD method combined with solid phase extraction (SPE) developed by our group: (our proprietary kit, named 8-OHdG Pre-treatment Kit (TANITA Corporation)). The major interfering substances and proteins in serum were removed by 8-OHdG Pre-treatment Kit. This measurement method was highly reproducible (CV = 2.2–7.1%) and demonstrated the lower detection limit for control serum sample of less than 10 pg/ml without the sample evaporation. The other hand 8-OHdG concentration in serum for healthy people was in the range of 0–70 pg/ml (25.5 ± 13.8 pg/ml, n = 37). Secondary a relationship between the HPLC-ECD and ELISA methods was investigated. ELISA method could not detect 8-OHdG concentration in serum for healthy people, because the detection limit of 130 pg/ml was higher than the normal range for healthy people. These results show our SPE method has high sensitivity and quantitative accuracy for 8-OHdG analysis.     

Integrating ancient patterns and current dynamics of insect–plant interactions: Taxonomic and geographic variation in herbivore specialization

Abstract The search for pattern in the ecology and evolutionary biology of insect–plant associations has fascinated biologists for centuries. High levels of tropical (low-latitude) plant and insect diversity relative to poleward latitudes and the disproportionate abundance of host-specialized insect herbivores have been noted. This review addresses several aspects of local insect specialization, host use abilities (and loss of these abilities with specialization), host-associated evolutionary divergence, and ecological (including “hybrid”) speciation, with special reference to the generation of biodiversity and the geographic and taxonomic identification of “species borders” for swallowtail butterflies (Papilionidae). From ancient phytochemically defined angiosperm affiliations that trace back millions of years to recent and very local specialized populations, the Papilionidae (swallowtail butterflies) have provided a model for enhanced understanding of localized ecological patterns and genetically based evolutionary processes. They have served as a useful group for evaluating the feeding specialization/physiological efficiency hypothesis. They have shown how the abiotic (thermal) environment interacts with host nutrirional suitability to generate “voltinism/suitability” gradients in specialization or preference latitudinally, and geographical mosaics locally. Several studies reviewed here suggest strongly that the oscillation hypothesis for speciation does have considerable merit, but at the same time, some species-level host specializations may lead to evolutionary dead-ends, especially with rapid environmental/habitat changes involving their host plants. Latitudinal gradients in species richness and degree of herbivore feeding specialization have been impacted by recent developments in ecological genetics and evolutionary ecology. Localized insect–plant associations that span the biospectrum from polyphenisms, polymorphisms, biotypes, demes, host races, to cryptic species, remain academically contentious, with simple definitions still debated. However, molecular analyses combined with ecological, ethological and physiological studies, have already begun to unveil some answers for many important ecological/evolutionary questions.     

Economic consequences of reproductive performance in dairy cattle

The net economic value of reproductive efficiency in dairy cattle was estimated using a stochastic dynamic simulation model. The objective was to compare the economic consequences of reproductive performance scenarios (“average” and “poor”) of a cow having a good reproductive performance and to explore which reproductive factors have an important impact on economic efficiency. A “good” reproductive performance scenario was defined with 1 ovulation rate (POVU_i), 0.7 estrus detection rate (PEst), 0.7 conception rate (PCon), 0.03 incidence rate of postpartum disorders prolonging the ovarian cyclicity (CO), 0.2 incidence rate of postpartum disorders reducing conception (ME), 0.05 embryonic death rate (ED), and voluntary waiting period (VWP) of 9 wks pp (post partum). In the current situation of dairy cows in the Netherlands, an “average” reproductive scenario (0.95 POVU_i, 0.5 PEst, 0.5 Pcon, 0.07 CO, 0.27 ME, 0.07 ED and VWP of 12 wks pp) and a “poor” reproductive scenario (0.90 POVU_i, 0.3 PEst, 0.3 Pcon, 0.11 CO, 0.33 ME, 0.09 ED and VWP of 15 wks pp) were identified. A sensitivity analysis was performed by comparing changes of single effect of factors in a good and poor scenario with the average scenario. The mean net economic loss (NEL_i) compared with the good scenario was €34 and €231 per cow per year for the average and poor reproductive performance scenario, respectively. Increasing the calving interval resulted in greater economic loss. The important factors on the cost of reproductive efficiency were the involuntary culling cost and the return of milk production. Variation in PCon, PEst, ME, ED, and VWP had large impacts on economic benefits.     

Silent ovulation, based on walking activity and milk progesterone concentrations, in Holstein cows housed in a free-stall barn

The objectives of the current study were to determine the incidence of silent ovulation (based on walking activity and milk progesterone profiles), identify risk factors for silent ovulation, and investigate its impact on reproductive performance in high-yielding dairy cows in free-stall housing. Overall, 277 lactations in 161 Holstein Friesian cows from a commercial dairy herd in northern Japan were studied. Walking activity (measured with pedometers) >80% above the mean for the preceding 2 d was defined as estrus, whereas day of ovulation was estimated using milk progesterone concentrations. Ovulation not preceded by increased walking activity was considered silent ovulation; the incidence was 55.2%, 23.8%, 21.3%, and 10.5% at the first, second, third, and fourth ovulations postpartum, respectively. Moderate and high milk yield significantly increased the risk of silent ovulation at second (odds ratio [OR] = 2.7 and 1.2; P = 0.04) and third and/or fourth ovulations (OR = 6.7 and 12.9; P = 0.03). Based on survival analysis, silent ovulations at the first, second, third, and/or fourth ovulations were associated with 28% (hazard ratio [HR] = 0.72), 55% (HR = 0.45), and 47% (HR = 0.53) reductions in pregnancy rate, respectively, and 41% (HR = 0.59), 66% (HR = 0.34), and 65% (HR = 0.35) reductions in artificial insemination (AI) submission rate. Cows with at least one silent ovulation (with the exception of the first ovulation) had a longer interval from calving to first AI (72 vs. 54 d, P < 0.001) and to achievement of pregnancy (133 vs. 80 d, P < 0.001). In conclusion, approximately one third of the ovulations (based on milk progesterone concentrations) in Holstein cows within 90 d postpartum were silent. Silent ovulations at the second to fourth ovulations were associated with high milk yields and at all ovulations were associated with impaired reproductive performance.     

Overexpression of γ-tocopherol methyl transferase gene in transgenic Brassica juncea plants alleviates abiotic stress: Physiological and chlorophyll a fluorescence measurements

Tocopherols (vitamin E) are lipid soluble antioxidants synthesized by plants and some cyanobacteria. We have earlier reported that overexpression of the γ-tocopherol methyl transferase (γ-TMT) gene from Arabidopsis thaliana in transgenic Brassica juncea plants resulted in an over six-fold increase in the level of α-tocopherol, the most active form of all the tocopherols. Tocopherol levels have been shown to increase in response to a variety of abiotic stresses. In the present study on Brassica juncea, we found that salt, heavy metal and osmotic stress induced an increase in the total tocopherol levels. Measurements of seed germination, shoot growth and leaf disc senescence showed that transgenic Brassica juncea plants overexpressing the γ-TMT gene had enhanced tolerance to the induced stresses. Analysis of the chlorophyll a fluorescence rise kinetics, from the initial “O” level to the “P” (the peak) level, showed that there were differential effects of the applied stresses on different sites of the photosynthetic machinery; further, these effects were alleviated in the transgenic (line 16.1) Brassica juncea plants. We show that α-tocopherol plays an important role in the alleviation of stress induced by salt, heavy metal and osmoticum in Brassica juncea.     

Genetic variation in competition traits at different ages and time periods and correlations with traits at field tests of 4-year-old Swedish Warmblood horses

For many years, the breeding value estimation for Swedish riding horses has been based on results from Riding Horse Quality Tests (RHQTs) of 4-year-olds only. Traits tested are conformation, gaits and jumping ability. An integrated index including competition results is under development to both get as reliable proofs as possible and increases the credibility of the indexes among breeders, trainers and riders. The objectives of this study were to investigate the suitability of competition data for use in genetic evaluations of horses and to examine how well young horse performance agrees with performance later in life. Competition results in dressage and show jumping for almost 40 000 horses from the beginning of the 1960s until 2006 were available. For RHQT data of 14 000 horses judged between 1988 and 2007 were used. Genetic parameters were estimated for accumulated competition results defined for different age groups (4 to 6 years of age, 4 to 9 years of age and lifetime), and for different birth year groups. Genetic correlations were estimated between results at RHQT and competitions with a multi-trait animal model. Heritabilities were higher for show jumping than dressage and increased with increasing age of the horse and amount of information. For dressage, heritabilities increased from 0.11 for the youngest group to 0.16 for lifetime results. For show jumping corresponding values increased from 0.24 to 0.28. Genetic correlations between competition results for the different age groups were highly positive (0.84 to 1.00), as were those between jumping traits at RHQT and competition results in show jumping (0.87 to 0.89). For dressage-related traits as 4-year-old and dressage competition results the estimated genetic correlations were between 0.47 and 0.77. We suggest that lifetime results from competitions should be integrated into the genetic evaluation system. However, genetic parameters showed that traits had changed during the over 35-year period covered due to the development of the sport, which needs to be considered in future genetic evaluations.     

The effect of dietary laminarin and fucoidan in the diet of the weanling piglet on performance, selected faecal microbial populations and volatile fatty acid concentrations

A 2 × 2 factorial experiment (n = 12 replicates per treatment, 4 pigs per replicate) was performed to investigate the effects of seaweed extracts, laminarin (derived ß-glucans) and fucoidan (sulphated polysaccharides), independently or in combination on post-weaning piglet performance and selected microbial populations. At weaning, the piglets (24 days of age, 6.4 kg live weight) were assigned to one of the four dietary treatments: (T1) basal diet, (T2) basal diet with 300 p.p.m. laminarin, (T3) basal diet with 240 p.p.m. fucoidan, (T4) basal diet with 300 p.p.m. laminarin and 240 p.p.m. fucoidan. Pigs offered diets supplemented with laminarin had an increased daily gain (P < 0.01), and gain-to-feed ratio (P < 0.05) compared to pigs offered diets without laminarin supplementation during the experimental period (days 0 to 21). Pigs offered laminarin-supplemented diets had an increased faecal dry matter and reduced diarrhoea (P < 0.05) during the critical 7 to 14 day period. Pigs offered diets containing laminarin had reduced faecal Escherichia coli populations. There was a significant interaction (P < 0.01) on faecal Lactobacilli populations between laminarin and fucoidan. Pigs offered the fucoidan diet had an increased Lactobacilli population compared to pigs offered the basal diet. However, there was no effect of fucoidan on faecal Lactobacilli populations when laminarin was added. Overall, the reduction in E. coli population and the increase in daily gain suggest that laminarin may provide a dietary means to improve gut health after weaning.