Long-range interactions within a nonnative protein

Protein folding and unfolding are coupled to a range of biological phenomena, from the regulation of cellular activity to the onset of neurodegenerative diseases. Defining the nature of the conformations sampled in nonnative proteins is crucial for understanding the origins of such phenomena. We have used a combination of nuclear magnetic resonance (NMR) spectroscopy and site-directed mutagenesis to study unfolded states of the protein lysozyme. Extensive clusters of hydrophobic structure exist within the wild-type protein even under strongly denaturing conditions. These clusters involve distinct regions of the sequence but are all disrupted by a single point mutation that replaced residue Trp62 with Gly located at the interface of the two major structural domains in the native state. Thus, nativelike structure in the denatured protein is stabilized by the involvement of Trp62 in nonnative and long-range interactions.     

Effects of soybean meal supplementation on ruminal kinetics of feed particle size reduction and passage in dairy cows fed rice straw

Six ruminal-cannulated nonlactating Holstein Friesian cows (mean body weight:660 ± 42.9 kg) were used to investigate the effect of soybean meal (SBM) supplementation on voluntary rice straw (RS) intake, feed particle size reduction, and passage kinetics in the rumen. They were allocated to two dietary treatments: RS alone or RS supplemented with SBM. Voluntary dry matter intake of RS and total tract fiber digestibility was increased by SBM supplementation (p < 0.05). Supplementation with SBM decreased rumination time per dietary dry matter (DM) and neutral detergent fiber (NDFom) intake (p < 0.01). Particle size distribution in the rumen and total ruminal NDFom digesta weights were not affected by SBM supplementation. However, the disappearance rates of total digesta and large and small particles from the rumen were increased by SBM supplementation (p < 0.01). Moreover, SBM supplementation increased the rate of size reduction in ruminal particles (p < 0.05). In situ disappearance of DM and NDFom of RS in the rumen was greater in SBM-supplemented cows than in nonsupplemented cows (p < 0.05). This study clearly showed that increased ruminal RS particle size reduction, passage, and fermentation due to SBM supplementation accelerated the RS particle clearance from the rumen and resulted in increased voluntary RS intake of dairy cows.     

Nitrate Loss for Denitrification during High Frequency Research in Floodplain Groundwater of the Tama River

This study aims to identify the possible roles of a floodplain inbiogeochemical nitrogen cycles, based on analysis of groundwaterdynamics of inorganic nitrogen species. Groundwater samples collected from boreholes made of poly vinyl chloride located onthe floodplain in the middle reach of the Tama River, Nagata district were analyzed for dissolved organic carbon, dissolved oxygen, inorganic nitrogen compounds, major anions and the stableisotope ratio of nitrate-nitrogen.Dissolved oxygen in groundwater samples collected from marked borehole (G-5) was low (50 ± 16 μM) and nitrate decreasedfrom 63 μM to < 10 μM during our research period. Concentrations of nitrate, nitrite and nitrous oxide decreased one after the other. These decreases in concentration, combinedwith ¹⁵N-enrichment of groundwater nitrate suggest denitrification as a significant nitrate sink, yielding an isotopic fractionation of nitrate-nitrogen with an enrichment factor (ε= –17.9‰) that is comparable tothose in various soil and groundwater systems. This study provesthat floodplains can perform as nitrogen sinks for groundwaterin river catchments.     

Model Intercomparison Study of Long Range Transport and Sulfur Deposition in East Asia (MICS-ASIA)

To help improve the use of models in science & policy analysis in Asia it is necessary to have a better understanding of model performance and uncertainties. Towards this goal an intercomparison exercise has been initiated as a collaborative study of scientists interested in long-range transport in East Asia. An overview of this study is presented in this paper. The study consists of a set of prescribed test calculations with carefully controlled experiments. Models used the same domain, emission inventory, model parameters, meteorological conditions, etc. Two periods (January and May 1993) were selected to reflect long-range transport conditions under two distinct seasons. During these periods measurements of sulfur concentrations and deposition were made throughout the study region using identical sampling and analysis protocols. The intercomparison activity consists of four tasks (Blind Test, Fixed Parameter Test, Source Receptor test, and Tuning Test). All participants were asked to do Task A, and as many of the other tasks as possible. To date seven different models have participated in this study. Results and key findings are presented.     

Metabolism of phenolic compounds during loquat fruit development.

Phenolic compounds in loquat fruit were identified as 5-caffeoylquinic acid (chlorogenic acid), neochlorogenic acid, hydroxybenzoic acid, 5-p-feruloylquinic acid, protocatechuic acid, 4-caffeoylquinic acid, epicatechin, o-coumaric acid, ferulic acid, and p-coumaric acid. Neochlorogenic acid was found to be dominant in the early stages of loquat fruit development. Both the concentrations and types of phenolic compounds were high in young fruit but then decreased steadily during growth. However, the concentration of chlorogenic acid increased during ripening and became predominant in ripe fruit. The large rise in chlorogenic acid concentration appears to be a characteristic of loquat fruit ripening. In all of the cultivars tested, the types of phenolic compounds were similar but the total phenolic content varied from 81.8 to 173.8 mg/100 g of fresh pulp. In the biosynthetic pathway of chlorogenic acid, the enzyme activities of phenylalanine ammonia-lyase (PAL), 4-coumarate:CoA ligase (CL), and hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase (CQT) were high at the early stage of growth, diminished to low levels approximately 3 weeks prior to harvest, but then rose to a peak at 1 week before harvest. The changes of these enzyme activities seemed to be associated with variations in chlorogenic acid concentration during development, maturation, and ripening of loquat fruit.     

Hydrogen bonding in alcoholic beverages (distilled spirits) and water-ethanol mixtures

The hydrogen-bonding properties of water-ethanol of alcoholic beverages and water-ethanol mixtures of the corresponding ethanol contents were examined on the basis of OH proton NMR chemical shifts and the Raman OH stretching spectra of water and ethanol. Japanese shochu, an unaged distilled spirit of 25% (v/v) alcoholic content made from various grains, was provided for the samples; it is a high-purity spirit as it contains only small amounts of dissolved components, like typical vodka, gin, and white rum. The hydrogen-bonding structure in shochu containing some acids was found to be different from that of the water-ethanol mixture with corresponding ethanol content. It was concluded that, by the presence of small amounts of organic acids, the water-ethanol hydrogen-bonding structure was strengthened, at the same time, the proton exchange between water and ethanol molecules was promoted in shochu, compared with the water-ethanol mixture. The NMR chemical shifts of fruit cocktail drinks suggested that the hydrogen bonding of water-ethanol in the solution was developed by organic acids and (poly)phenols from fruit juices.     

Identification and validation of a quantitative trait locus associated with wheat yellow mosaic virus pathotype I resistance in a Japanese wheat variety

Yellow mosaic disease, caused by wheat yellow mosaic virus (WYMV), is one of the most serious diseases of winter wheat (Triticum aestivum L.) in Japan. The three pathotypes of WYMV are distributed in different geographical areas: pathotype I is found mainly in western and central Japan (Kanto), pathotype II in northern Japan (Tohoku and Hokkaido) and pathotype III on the southern island of Japan (Kyushu). A total of 246 doubled‐haploid (DH) lines, derived from a cross between ‘Yumechikara’ (resistant) and ‘Kitahonami’ (susceptible), were evaluated for 2 years for their resistance to WYMV pathotype I. A single major quantitative trait locus, Q.Ymym, mapping to chromosome 2D was associated with resistance to pathotype I in ‘Yumechikara’. This is the first time a QTL responsible for pathotype I resistance has been identified. Fine mapping of Q.Ymym indicated that it was on a tight linkage block originating from ‘Yumechikara’, and the markers associated with this block will accelerate the development of varieties resistant to WYMV pathotype I.     

Effects of ear corn silage supplementation on milk production and milk fatty acid profiles in grazing dairy farms

This study investigated the effects of substituting ear corn silage (ECS) for commercial formula feed on milk production and milk fatty acid profiles in grazing dairy farms during the summer season. A field survey was conducted on five grazing dairy farms in every summer month of 2017, 2018, and 2019. Three of the five farms substituted fresh ECS for the commercial formula feed at a ratio of 2:1 from July of each year (ECS farms). Other farms maintained the same feeding management as before (non‐ECS farms). An interview survey was conducted on each farm to calculate feed intake and milk yield per cow. Feed and milk samples were collected in each survey. Milk compositions and milk fatty acid profiles were determined. The substitution of ECS for the commercial formula feed did not affect milk yield or milk composition, but ECS farms maintained low levels of milk urea compared with non‐ECS farms (p < .01). The ECS substitution also influenced some of the milk fatty acid proportions; C16:0 and C16:1 increased, and trans‐11 C18:1, cis‐9,trans‐11 C18:2, and the sum of polyunsaturated fatty acids decreased, while these fatty acid proportions were maintained in non‐ECS farms throughout the summer season (p < .05).     

Alanine racemase activity in the microalga <Emphasis Type="Italic">thalassiosira</Emphasis> sp.

ABSTRACT:   In this paper, the authors report the detection of alanine racemase activity in the marine diatom Thalassiosira sp. Since the Thalassiosira sp. was cultured under germ-free conditions, it appeared that D-alanine was not derived from bacteria but was produced through catalysis by algal alanine racemase. The rate of conversion of L-alanine to D-alanine was approximately the same as that for the reverse reaction, and the enzyme catalyzed the equilibration of the D- and L-forms. The crude enzyme preparation obtained from the cells at the stationary phase of the growth cycle had an optimal pH of approximately 9.5. The Lineweaver–Burk analysis showed that the K _m for D- and L-alanine was 16.5 mM and 29.4 mM, respectively. It appears that the enzyme is highly specific for D- or L-alanine because it does not catalyze the racemization of other amino acids. In addition, after gel filtration, the enzyme did not require exogenous pyridoxal 5'-phosphate (PLP) for its activity, however, the effects of several chemicals suggest that the enzyme may be PLP-dependent. The enzyme is more similar to that found in invertebrates when compared with that found in bacteria. This is the first report on the occurrence of alanine racemase activity in the microalga Thalassiosira sp.     

Midgut juice of Plutellaxylostella highly resistant to Bacillusthuringiensis Cry1Ac contains a three times larger amount of glucosinolate sulfatase which binds to Cry1Ac compared to that of susceptible strain

Midgut juice of Plutellaxylostella strain PXR which is resistant to Cry1Ac was biochemically characterized relative to the susceptible PXS strain. The midgut juice of PXR (PXR-Juice) was shown to process Cry1Ac protoxin to 60 kDa active toxin with the same processing pattern as that of juice from PXS (PXS-Juice) in SDS–PAGE. PXS larvae which were given the Cry1Ac toxin pre-processed with PXR-Juice were killed with the same rate as that with Cry1Ac pre-activated by trypsin. PXR-Juice was found to contain three times larger amount of 66 kDa protein (P66) than PXS-Juice and the N-terminal amino acid sequence of P66 was matched to that of glucosinolate sulfatase in data base search. The protein band of P66 was coincided with the band of p-nitro phenyl sulfatase activity in zymogram. P66 purified to homogeneity in SDS-PAGE bound to Cry1Ac and soybean agglutinin, and K_D for Cry1Ac was estimated to be 718 nM with surface plasmon resonance analysis. Using purified sulfatase, K_m and V_max were estimated and involvement of the enzyme in the PXR resistance was discussed.