An LC-MS method for analyzing total resveratrol in grape juice, cranberry juice, and in wine.

Resveratrol is an antioxidant found in grapes, grape products, and some other botanical sources with antiinflammatory and anticancer properties. In grapes and wine, it occurs both as free resveratrol and piceid, the 3beta-glucoside of resveratrol. Here we report a liquid chromatography-mass spectrometry method to analyze total resveratrol (including free resveratrol and resveratrol from piceid) in fruit products and wine. Samples were extracted using methanol, enzymatically hydrolyzed, and analyzed using reversed phase HPLC with positive ion atmospheric pressure chemical ionization (APCI) mass spectrometric detection. Following APCI, the abundance of protonated molecules was recorded using selected ion monitoring (SIM) of m/z 229. An external standard curve was used for quantitation, which showed a linear range of 0.52-2260 pmol of trans-resveratrol injected on-column with a correlation coefficient 0.9999. The coefficient of variance of the response factor over the same concentration range was determined to be 5.8%, and the intra-assay coefficient of variance was determined to be 4.2% (n = 7). The limit of quantitation, defined as signal-to-noise 10:1, was determined to be 0.31 pmol injected on-column. The extraction efficiency of the method was determined to be 92%. The stability of resveratrol under different conditions was also examined. For example, resveratrol was stable for up to 5 days at 4 degrees C in the dark but was not stable at room temperature without protection from light. Resveratrol was detected in grape, cranberry, and wine samples. Concentrations ranged from 1.56 to 1042 nmol/g in Concord grape products, and from 8.63 to 24.84 micromol/L in Italian red wine. The concentrations of resveratrol were silmilar in cranberry and grape juice at 1.07 and 1.56 nmol/g, respectively.     

Purification and characterization of a thermostable alpha-galactosidase from Thermoanaerobacterium polysaccharolyticum

Food ingredients containing alpha-1,6-galactoside bonds elicit gastrointestinal disturbances in monogastric animals, including humans. Pretreatment of such ingredients with alpha-galactosidase (EC 3.2.1.22) has the potential to alleviate this condition. For this purpose, a thermostable alpha-galactosidase from Thermoanaerobacterium polysaccharolyticum was purified by a combination of anion exchange and size exclusion chromatographies. The enzyme has a monomeric molecular weight of approximately 80 kDa; however, it is active as a dimer. The optimum temperature for enzyme activity is 77.5 degrees C. Approximately 84 and 88% of enzyme activity remained after 36.5 h of incubation at 70 and 65 degrees C, respectively. Optimum activity was observed at pH 8.0, with a broad range of activity from pH 5.0 to 9.0. Different transition metals had weak to strong inhibitory effects on enzyme activity. The K(m) and V(max) of the enzyme are 0.29-0.345 mM and 200-232 micromol/min/mg of protein, respectively. Importantly, enzyme activity was only slightly inhibited by 75-100 mM galactose, an end product of hydrolysis. Enzyme activity was specific for the alpha-1,6-galactosyl bond, and activity was demonstrated on melibiose and soy molasses.     

Suppression of methane oxidation in aerobic soil by nitrogen fertilizers,nitrification inhibitors,and urease inhibitors

Concentrations of CH₄, a potent greenhouse gas, have been increasing in the atmosphere at the rate of 1% per year. The objective of these laboratory studies was to measure the effect of different forms of inorganic N and various N-transformation inhibitors on CH₄ oxidation in soil. NH ₄ ⁺ oxidation was also measured in the presence of the inhibitors to determine whether they had differential activity with respect to CH₄ and NH ₄ ⁺ oxidation. The addition of NH₄Cl at 25 g N g^-1 soil strongly inhibited (78–89%) CH₄ oxidation in the surface layer (0–15 cm) of a fine sandy loam and a sandy clay loam (native shortgrass prairie soils). The nitrification inhibitor nitrapyrin (5 g g^-1 soil) inhibited CH₄ oxidation as effectively as did NH₄Cl in the fine sandy loam (82–89%), but less effectively in the sandy clay loam (52–66%). Acetylene (5 mol mol^-1 in soil headspace) had a strong (76–100%) inhibitory effect on CH₄ consumption in both soils. The phosphoroamide (urease inhibitor) N-(n-butyl) thiophosphoric triamide (NBPT) showed strong inhibition of CH₄ consumption at 25 g g^-1 soil in the fine sandy loam (83%) in the sandy clay loam (60%), but NH ₄ ⁺ oxidation inhibition was weak in both soils (13–17%). The discovery that the urease inhibitor NBPT inhibits CH₄ oxidation was unexpected, and the mechanism involved is unknown.     

Crystal structure of the human K2P TRAAK, a lipid- and mechano-sensitive K+ ion channel

TRAAK channels, members of the two-pore domain K(+) (potassium ion) channel family K2P, are expressed almost exclusively in the nervous system and control the resting membrane potential. Their gating is sensitive to polyunsaturated fatty acids, mechanical deformation of the membrane, and temperature changes. Physiologically, these channels appear to control the noxious input threshold for temperature and pressure sensitivity in dorsal root ganglia neurons. We present the crystal structure of human TRAAK at a resolution of 3.8 angstroms. The channel comprises two protomers, each containing two distinct pore domains, which create a two-fold symmetric K(+) channel. The extracellular surface features a helical cap, 35 angstroms tall, that creates a bifurcated pore entryway and accounts for the insensitivity of two-pore domain K(+) channels to inhibitory toxins. Two diagonally opposed gate-forming inner helices form membrane-interacting structures that may underlie this channel's sensitivity to chemical and mechanical properties of the cell membrane.     

Denitrification, N2O and CO2 fluxes in rice-wheat cropping system as affected by crop residues, fertilizer N and legume green manure

Use of renewable N and C sources such as green manure (GM) and crop residues in rice-wheat cropping systems of South Asia may lead to higher crop productivity and C sequestration. However, information on measurements of gaseous N losses (N₂O+N₂) via denitrification and environmental problems such as N₂O and CO₂ production in rice-wheat cropping systems is not available. An acetylene inhibition-intact soil core technique was employed for direct measurement of denitrification losses, N₂O and CO₂ production, in an irrigated field planted to rice (Oryza sativa L.) and wheat (Triticum aestivum L.) in an annual rotation. The soil was a coarse-textured Tolewal sandy loam soil (Typic Ustochrept) and the site a semi-arid subtropical Punjab region of India. Wheat residue (WR, C:N=94) was incorporated at 6 t ha^-1 and sesbania (Sesbania aculeata L.) was grown as GM crop for 60 days during the pre-rice fallow period. Fresh biomass of GM (C:N.=18) at 20 or 40 t ha^-1 was incorporated into the soil 2 days before transplanting rice. Results of this study reveal that (1) denitrification is a significant N loss process under wetland rice amounting to 33% of the prescribed dose of 120 kg N ha^-1 applied as fertilizer urea-N (FN); (2) integrated management of 6 t WR ha^-1 and 20 t GM ha^-1 supplying 88 kg N ha^-1 and 32 kg FN ha^-1 significantly reduced cumulative gaseous N losses to 51.6 kg N ha^-1 as compared with 58.2 kg N ha^-1 for 120 kg FN ha^-1 alone; (3) application of excessive N and C through applying 40 t GM ha^-1 (176 kg N ha^-1) resulted in the highest gaseous losses of 70 kg N ha^-1; (4) the gaseous N losses under wheat were 0.6% to 2% of the applied 120 kg FN ha^-1 and were eight- to tenfold lower (5-8 kg N ha^-1) than those preceding rice; (5) an interplay between the availability of NO₃^- and organic C largely controlled denitrification and N₂O flux during summer-grown flooded rice whereas temperature and soil aeration status were the primary regulators of the nitrification-denitrification processes and gaseous N losses during winter-grown upland wheat; (6) the irrigated rice-wheat system is a significant source of N₂O as it emits around 15 kg N₂O-N ha^-1 year^-1; (7) incorporation of WR in rice and rice residue (C:N=63) in wheat increased soil respiration, and increased CO₂ production in WR- and GM-amended soils under anaerobic wetland rice coincided with enhanced rates of denitrification; and (8) with adequate soil moisture, most of the decomposable C fraction of added residues was mineralized within one crop-growing season and application of FN and GM further accelerated this process.     

Social rank in house mice: differentiation revealed by ultraviolet visualization of urinary marking patterns

Ultraviolet light has been used to examine urine marks deposited by adult male house mice on filter paper on the floors of their cages during overnight tests. Both the urination frequency and the pattern in which urine was deposited on the filter paper depended upon social rank. Dominant males vigorously marked their entire cage floor, whereas subordinate males typically voided urine in only two to four pools in the corners of their cages.     

Metagenomic discovery of biomass-degrading genes and genomes from cow rumen

The paucity of enzymes that efficiently deconstruct plant polysaccharides represents a major bottleneck for industrial-scale conversion of cellulosic biomass into biofuels. Cow rumen microbes specialize in degradation of cellulosic plant material, but most members of this complex community resist cultivation. To characterize biomass-degrading genes and genomes, we sequenced and analyzed 268 gigabases of metagenomic DNA from microbes adherent to plant fiber incubated in cow rumen. From these data, we identified 27,755 putative carbohydrate-active genes and expressed 90 candidate proteins, of which 57% were enzymatically active against cellulosic substrates. We also assembled 15 uncultured microbial genomes, which were validated by complementary methods including single-cell genome sequencing. These data sets provide a substantially expanded catalog of genes and genomes participating in the deconstruction of cellulosic biomass.     

Quantitative variability inPisum seed globulins: its assessment and significance

Five different techniques have been used with sevenPisum (pea) genotypes to demonstrate the existence of extensive genotypic variability in the proportions of the major pea seed globulins, legumin, vicilin and convicilin. The accuracy and reproducibility of each method have been assessed and the significance of the observed variability discussed both in relation to studies of the control of globulin synthesis and to breeding for pea seed protein with higher nutritional quality.     

Effects of plant protection chemicals on vesicular-arbuscular mycorrhizas

The effects of eight plant protection chemicals on the development of vesiculararbuscular mycorrhizas were influenced by the identity of the host plant and fungal endophyte, the soil type, and the composition and concentration of the biocide. These effects ranged from stimulatory (as with the fungicide chloroneb, applied to inocula of Glomus microcarpus used with pot-grown lettuce), through negligible (aldicarb on G. mosseae in pot-grown onions and field grown barley with indigenous mycorrhizal fungi) to short-term inhibition (drazoxolon on G. mosseae in onion) and longer term inhibition (the fungicides benomyl and triadimefon on G. mosseae in onion).