Voltage-clamp fluorometry in the local environment of the C255-C511 disulfide bridge of the Na+/glucose cotransporter

We recently identified a functionally important disulfide bridge between C255 and C511 of the human Na+/glucose cotransporter SGLT1. In this study, voltage-clamp fluorometry was used to characterize the fluorescence of four different dyes attached to C255 and C511 under various ionic and substrate/inhibitor conditions. State-dependent fluorescence changes (DeltaF) were observed when TMR5M or TMR6M dyes were attached to C255 and C511 or when Alexa488 was bound to C511. TMR5M-C511 was extremely sensitive to membrane potential (Vm) and to external Na+ and alphaMG (a nonmetabolizable glucose analog) concentrations. A progressive increase in alphaMG concentration drastically changed the maximal voltage-dependent DeltaF and produced a positive shift in the midpoint of the DeltaF-Vm curve. By determining specific fluorescence intensity for each state of the cotransporter, our steady-state fluorescence data could be reproduced using the rate constants previously proposed for a five-state kinetic model exclusively derived from electrophysiological measurements. Our results bring an independent support to the proposed kinetic model and show that the binding of alphaMG substrate significantly modifies the environment of C255 and C511.     

Mutational analysis of conserved aromatic residues in the A-loop of the ABC transporter ABCB1A (mouse Mdr3)

The A-loop is a recently described conserved region in the NBDs of ABC transporters [Ambudkar, S.V., Kim, I.-W., Xia, D. and Sauna, Z.E. (2006) The A-loop, a novel conserved aromatic acid subdomain upstream of the Walker A motif in ABC transporters, is critical for ATP binding. FEBS Lett. 580, 1049-1055; Kim, I.W., Peng, X.H., Sauna, Z.E., FitzGerald, P.C., Xia, D., Muller, M., Nandigama, K. and Ambudkar, S.V. (2006) The conserved tyrosine residues 401 and 1044 in ATP sites of human P-glycoprotein are critical for ATP binding and hydrolysis: evidence for a conserved subdomain, the A-loop in the ATP-binding cassette. Biochemistry 45, 7605-7616]. In mouse P-glycoprotein (Abcb1a), the aromatic residue of the A-loop in both NBDs is a tyrosine: Y397 in NBD1 and Y1040 in NBD2. Another tyrosine residue (618 in NBD1 and 1263 in NBD2) also appears to lie in proximity to the ATP molecule. We have mutated residues Y397, Y618, Y1040, and Y1263 to tryptophan and analyzed the effect of these substitutions on transport properties, ATP binding, and ATP hydrolysis by Abcb1a (mouse Mdr3). Y618W and Y1263W enzymes had catalytic characteristics similar to WT Abcb1a. On the other hand, Y397W and Y1040W showed impaired transport and greatly reduced ATPase activity, including a approximately 10-fold increase in Km for MgATP. Thus, Y397 and Y1040 play an important role in Abcb1a catalysis.     

Analysis of internal motions of interleukin-13 variant associated with severe bronchial asthma using (15)N NMR relaxation measurements

The single nucleotide polymorphism interleukin-13 (IL-13) R110Q is associated with severe bronchial asthma because its lower affinity leads to the augmentation of local IL-13 concentration, resulting in an increase in the signal transduction via IL-13R. Since the mutation site does not directly bind to IL-13Ralpha2, we carried out NMR relaxation analyses of the wild-type IL-13 and IL-13-R110Q in order to examine whether the R110Q mutation affects the internal motions in IL-13 molecules. The results showed that the internal motion in the micro- to millisecond time scale on helix D, which is suggested to be important for the interaction between IL-13 and IL-13Ralpha2, is increased in IL-13-R110Q compared with that in the wild-type IL-13. It therefore appears that the difference in the internal motions on helix D between the wild-type IL-13 and IL-13-R110Q may be involved in their affinity differences with IL-13Ralpha2.     

Ground,dry-rolled and steam-processed barley grain for midlactation Holstein cows

Fine grinding of barley grain has traditionally been considered to be a potential risk to rumen function, feed intake and milk yield. These concerns are thought to be reduced by steam-rolling or coarse dry rolling. We hypothesized that finely ground barley grain is as effective in stimulating feed intake and milk production as are dry- and steam-rolled barley grain, and so the objective was to determine effects of feeding either (1) finely ground, (2) steam-rolled, (3) finely dry-rolled, or (4) coarsely dry-rolled barley grain on rumen fermentation, digestibility and milk yield and composition. Eight multiparous midlactation Holstein cows were used in a replicated 4×4 Latin square design experiment with four periods of 21 d. Diets contained 256 g barley grain/kg on a dry matter (DM) basis. Processing method did not affect milk yield and composition, DM intake, rumen pH and volatile fatty acids, fecal and urine pH, and apparent total tract nutrient digestibility. Results suggest that finely ground barley grain is no different than dry-rolled and steam-rolled barley grains in stimulating feed intake and productivity of midlactation cows, when 256 g of dietary DM/kg is barley grain.     

Genetic variation in pea (Pisum sativum L.) demonstrates the importance of root but not shoot C/N ratios in the control of plant morphology and reveals a unique relationship between shoot length and nodulation intensity

Nodule numbers are regulated through systemic auto‐regulatory signals produced by shoots and roots. The relative effects of shoot and root genotype on nodule numbers together with relationships to organ biomass, carbon (C) and nitrogen (N) status, and related parameters were measured in pea (Pisum sativum) exploiting natural genetic variation in maturity and apparent nodulation intensity. Reciprocal grafting experiments between the early (Athos), intermediate (Phönix) and late (S00182) maturity phenotypes were performed and Pearson's correlation coefficients for the parameters were calculated. No significant correlations were found between shoot C/N ratios and plant morphology parameters, but the root C/N ratio showed a strong correlation with root fresh and dry weights as well as with shoot fresh weight with less significant interactions with leaf number. Hence, the root C/N ratio rather than shoot C/N had a predominant influence on plant morphology when pea plants are grown under conditions of symbiotic nitrogen supply. The only phenotypic characteristic that showed a statistically significant correlation with nodulation intensity was shoot length, which accounted for 68.5% of the variation. A strong linear relationship was demonstrated between shoot length and nodule numbers. Hence, pea nodule numbers are controlled by factors related to shoot extension, but not by shoot or root biomass accumulation, total C or total N. The relationship between shoot length and nodule numbers persisted under field conditions. These results suggest that stem height could be used as a breeding marker for the selection of pea cultivars with high nodule numbers and high seed N contents.     

Newer and select maize,wheat, and rice varieties can help mitigate N footprint while producing more grain

Sustainably feeding the growing population amid a changing climate and dwindling resources is a grand challenge facing mankind. Decades‐long advancement in crop breeding has progressively elevated yield potential, markedly enhancing global food production capacity. However, relevant impact on reactive N (Nr) emissions associated with crop variety improvement has not been explicitly described. Here, we report multitiered evidence that newer and select maize, wheat, and rice varieties developed in China have the capacity to substantially lower Nr losses while producing more grain. First, we pooled studies featuring side‐by‐side comparison of different varieties, totaling 269 paired observations, to demonstrate that collectively, relatively newer varieties of maize, wheat, and rice had less Nr emissions (9.6%–23.5%) while yielding more grains (7.3%–11.2%) compared to older varieties under wide‐ranging conditions. Next, we built an extended database (142 field studies with 833 observations) and comprehensively evaluated the Nr‐loss reduction potential of newer varieties (2000 and after) versus older ones (1985–1999). We found that newer varieties had Nr emission factors (N loss as a percentage of N applied after correcting for background emissions) 18.2%–75.7% less for N₂O, 18.3%–75.7% less for , and ?8.5% to 22.8% less for NH₃, while producing more grains (16.0%–24.4%). Individual varieties differed markedly in yield‐emission scores. A nationwide farmer survey (2.47 million responses) indicated tremendous opportunities for a new way of management intervention. Coupling variety selection with sound N and other agronomic management can help lower N footprint while producing more grain.     

Comparative analyses of albumin/globulin grain proteome fraction in differentially salt‐tolerant Tunisian barley landraces reveals genotype‐specific and defined abundant proteins

• Salinity is one of the major abiotic stresses threatening crop production and yield worldwide. Breeding programmes are therefore needed to improve yield under cultivation in soil. Traits from locally adopted landraces provide a resource to assist breeding of novel elite genotypes. Here, we examine differentially expressed proteins by performing comparative proteomic profiling of the albumin/globulin grain fraction of Tunisian barley genotype landraces with contrasting salinity tolerance.
• Tunisian barley Boulifa (B, tolerant) and Testour (T, sensitive) mature grains were assessed in 2‐DE profiles. Differentially expressed spots, with an abundance enhanced 1.5‐fold in the grain, were subjected to MALDI TOF/TOF MS for identification.
• Distinctiveness between tolerant and sensitive genotypes was proved in the albumin/globulin fraction using PCA; 64 spots showed significant differential abundance. Increased accumulation of 40 spots was confirmed in Boulifa with, interestingly, four genotype‐specific spots. Two of these four spots were sHSP. Proteins with highest abundance were serpin Z7, 16.9 KDa Class I HSP and phosphogluconolactonase 2. Proteins such as expansin, kiwellin, kinesin and succinyl‐CoA ligase were identified for the first time in barley grain. Moreover, ß‐amylase, LEA family and others were identified as abundant in Boulifa. On the other hand, proteins more accumulated in Testour are implicated mainly in ROS scavenging and protease inhibition.
• Our results clearly indicate proteomic contrast between the two selected genotypes. With identification of specific HSP, high abundant stress‐protective and other defined proteins, we provide biochemical traits that will support breeding programmes to address the threat of salinity in agricultural production.

     

Potassium‐induced decrease in cytosolic Na+ alleviates deleterious effects of salt stress on wheat (Triticum aestivum L.)

• Accumulation of NaCl in soil causes osmotic stress in plants, and sodium (Na⁺) and chloride (Cl^?) cause ion toxicity, but also reduce the potassium (K⁺) uptake by plant roots and stimulate the K⁺ efflux through the cell membrane. Thus, decreased K⁺/Na⁺ ratio in plant tissue lead us to hypothesise that elevated levels of K⁺ in nutrient medium enhance this ratio in plant tissue and cytosol to improve enzyme activation, osmoregulation and charge balance.
• In this study, wheat was cultivated at different concentrations of K⁺ (2.2, 4.4 or 8.8 mm ) with or without salinity (1, 60 or 120 mm NaCl) and the effects on growth, root and shoot Na⁺ and K⁺ distribution and grain yield were determined. Also, the cytosolic Na⁺ concentration was investigated, as well as photosynthesis rate and water potential.
• Salinity reduced fresh weight of both shoots and roots and dry weight of roots. The grain yield was significantly reduced under Na⁺ stress and improved with elevated K⁺ fertilisation. Elevated K⁺ level during cultivation prevented the accumulation of Na⁺ into the cytosol of both shoot and root protoplasts. Wheat growth at vegetative stage was transiently reduced at the highest K⁺ concentration, perhaps due to plants' efforts to overcome a high solute concentration in the plant tissue, nevertheless grain yield was increased at both K⁺ levels.
• In conclusion, a moderately elevated K⁺ application to wheat seedlings reduces tissue as well as cytosolic Na⁺ concentration and enhances wheat growth and grain yield by mitigating the deleterious effects of Na⁺ toxicity.

     

Individual diet specialisation in sparrows is driven by phenotypic plasticity in traits related to trade‐offs in animal performance

Individual diet specialisation (IS) is frequent in many animal taxa and affects population and community dynamics. The niche variation hypothesis (NVH) predicts that broader population niches should exhibit greater IS than populations with narrower niches, and most studies that examine the ecological factors driving IS focus on intraspecific competition. We show that phenotypic plasticity of traits associated with functional trade‐offs is an important, but unrecognised mechanism that promotes and maintains IS. We measured nitrogen isotope (δ¹⁵N) and digestive enzyme plasticity in four populations of sparrows (Zonotrichia capensis) to explore the relationship between IS and digestive plasticity. Our results show that phenotypic plasticity associated with functional trade‐offs is related in a nonlinear fashion with the degree of IS and positively with population niche width. These findings are opposite to the NVH and suggest that among individual differences in diet can be maintained via acclimatisation and not necessarily require a genetic component.     

Sn‐Alloy Foil Electrode with Mechanical Prelithiation: Full‐Cell Performance up to 200 Cycles

Self‐supporting Sn foil is a promising high‐volumetric‐capacity anode for lithium ion batteries (LIBs), but it suffers from low initial Coulombic efficiency (ICE). Here, mechanical prelithiation is adopted to improve ICE, and it is found that Sn foils with coarser grains are prone to cause electrode damage. To mitigate damage and prepare thinner lithiated electrodes, 3Ag0.5Cu96.5Sn foil is used that has more refined grains (5–10 µm) instead of Sn (50–100 µm), where the abundant grain boundaries (GBs) offer more sliding systems to release stress and reduce deep fractures. Thus, the thickness of Li_x3Ag0.5Cu96.5Sn can be reduced to 50 µm, compared to 100 µm Li_xSn. When the foils contact open air, the Sn‐Li‐O(H) products are more stable than Li‐O(H), thus Li_x3Ag0.5Cu96.5Sn shows outstanding air stability. The as‐prepared 50 µm foil anode achieves stable 200 cycles in LiFePO₄//Li_x3Ag0.5Cu96.5Sn full cell (≈2.65 mAh cm^?2) and the capacity retention is 95%. Even at 5C, the capacity of Li_x3Ag0.5Cu96.5Sn is still up to ≈1.8 mAh cm^?2. The cycle life of NCM523//Li_x3Ag0.5Cu96.5Sn full cell exceeds that of NCM523//Li. Furthermore, 70 µm Li_x3Ag0.5Cu96.5Sn is used as double‐sided anode for a 3 cm × 2.8 cm pouch cell and its actual volumetric capacity density is 674 mAh cm^?3 after 50 cycles.