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Liquid feeding strategies have been devised with the aim of enhancing grain nutrient availability for livestock. It is characterized by a steeping/soaking period that softens the grains and initiates mobilization of seed storage reserves. The present study uses 2D gel-based proteomics to investigate the role of proteolysis and reduction by thioredoxins over a 48 h steeping period by monitoring protein abundance dynamics in barley-based liquid feed samples supplemented with either protease inhibitors or NADPH-dependent thioredoxin reductase/thioredoxin (NTR/Trx). Several full-length storage proteins were only identified in the water-extractable fraction of feed containing protease inhibitors, illustrating significant inhibition of proteolytic activities arising during the steeping period. Application of functional NTR/Trx to liquid feed reductively increased the solubility of known and potentially new Trx-target proteins, e.g., outer membrane protein X, and their susceptibility to proteolysis. Thus, the NTR/Trx system exhibits important potential as a feed additive to enhance nutrient digestibility in monogastric animals.

Hypokalaemic periodic paralysis is a rare genetic neuromuscular disease characterized by episodes of skeletal muscle paralysis associated with low serum potassium. Muscle fibre inexcitability during attacks of paralysis is due to an aberrant depolarizing leak current through mutant voltage sensing domains of either the sarcolemmal voltage-gated calcium or sodium channel. We report a child with hypokalaemic periodic paralysis and CNS involvement, including seizures, but without mutations in the known periodic paralysis genes. We identified a novel heterozygous de novo missense mutation in the ATP1A2 gene encoding the α2 subunit of the Na+/K+-ATPase that is abundantly expressed in skeletal muscle and in brain astrocytes. Pump activity is crucial for Na+ and K+ homeostasis following sustained muscle or neuronal activity and its dysfunction is linked to the CNS disorders hemiplegic migraine and alternating hemiplegia of childhood, but muscle dysfunction has not been reported. Electrophysiological measurements of mutant pump activity in Xenopus oocytes revealed lower turnover rates in physiological extracellular K+ and an anomalous inward leak current in hypokalaemic conditions, predicted to lead to muscle depolarization. Our data provide important evidence supporting a leak current as the major pathomechanism underlying hypokalaemic periodic paralysis and indicate ATP1A2 as a new hypokalaemic periodic paralysis gene.

Zinc is an essential trace element, highly important for a well functioning immune system. In case of zinc deficiency, proper immune functions are not ensured thus leading to various diseases. Weaning of pigs from the sow causes stress, increasing susceptibility to infections. Moreover, low feed intake during the first two weeks post-weaning, accompanied by low zinc intake, results in temporary zinc deficiency. Therefore, supporting the immune system by zinc supplementation might improve its function and thereby the pigs' health and well-being. In this study, the immune status of weaned pigs was analyzed under different conditions of zinc supplementation. More precisely, the daily porcine diet was either left unsupplemented (0 ppm), or was supplemented with low (100 ppm), or high (2500 ppm) amounts of additional zinc in the form of zinc oxide (ZnO) (Zn0, Zn100, and Zn2500, respectively). Porcine innate and adaptive immune cells of the different dietary groups were analyzed. Results revealed an improved innate immune capacity, represented by increased phagocytosis and slightly increased oxidative burst in cells from the Zn2500 pigs and Zn100 pigs, respectively. Apart from that, zinc supplementation improved adaptive immunity, as seen by increased numbers of CD3 + T cells as well as increased numbers of CD3 + CD4 + Foxp3 + regulatory T cells, elevated interleukin (IL)-2 production and decreased IL-10 production. Although not significant, supplementing 2500 ppm zinc slightly decreased killing activity of natural killer (NK) cells. Thus, the optimal concentration for zinc supplementation of weaned pigs two weeks post-weaning needs to be further studied, presumably establishing an optimal concentration between 100 ppm and 2500 ppm zinc. Genome comparisons indicate that the porcine genome is more closely related to the human genome than the murine genome is related to the human genome. Therefore, the pig seems to be a suitable organism to study human immunity and diseases. Results obtained in the current study might therefore be transferable to the human immune system., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

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The sodium and potassium gradients across the plasma membrane are used by animal cells for numerous processes, and the range of demands requires that the responsible ion pump, the Na,K-ATPase, can be fine-tuned to the different cellular needs. Therefore, several isoforms are expressed of each of the three subunits that make a Na,K-ATPase, the alpha, beta and FXYD subunits. This review summarizes the various roles and expression patterns of the Na,K-ATPase subunit isoforms and maps the sequence variations to compare the differences structurally. Mutations in the Na,K-ATPase genes encoding alpha subunit isoforms have severe physiological consequences, causing very distinct, often neurological diseases. The differences in the pathophysiological effects of mutations further underline how the kinetic parameters, regulation and proteomic interactions of the Na,K-ATPase isoforms are optimized for the individual cellular needs.

Objectives: To generate tryptophan-overproducing Bacillus subtilis strains for in situ use in pigs, to reduce the feed cost for farmers and nitrogen pollution., Results: A novel concept has been investigated-to generate B. subtilis strains able to produce tryptophan (Trp) in situ in pigs. Mutagenesis by UV was combined with selection on Trp and purine analogues in an iterative process. Two mutants from different wild types were obtained, mutant 1 (M1) produced 1 mg Trp/l and mutant 2 (M2) 14 mg Trp/l. Genome sequence analysis revealed that M1 had three single nuclear polymorphisms (SNPs) and M2 had two SNPs compared to the wild type strains. In both mutants SNPs were found in genes regulating tryptophan synthesis. Reverse transcription PCR confirmed up-regulation of the tryptophan synthesis genes in both mutants, the expression was up to 3 times higher in M2 than in M1., Conclusions: Tryptophan-excreting B. subtilis strains were obtained with UV-mutagenesis and analogue selection and can be used in animal feed applications., Competing Interests: The authors declare that they have no conflict of interest.

The angiopoietin-like 4 (ANGPLT4) protein is involved in lipid metabolism and is known to inhibit lipoprotein lipase in the bloodstream. We investigated the effect of milk on intestinal ANGPTL4 and the metabolic profile of growing pigs and the effect of free fatty acids (FFAs) on ANGPTL4 in ex vivo and in vitro assays. Feeding pigs whole milk increased intestinal ANGPTL4 mRNA and increased fecal excretion of long-chain FFA compared to the control group fed soybean oil (n = 9). Furthermore, FFAs (C4-C8) induced ANGPTL4 gene expression in porcine intestinal tissue mounted in Ussing chambers and ANGPTL4 protein secretion to both the apical and basolateral sides of intestinal Caco-2 cells on permeable membranes. Altogether, these results support an ANGPTL4-induced secretion of fecal FFAs. Urinary levels of FFAs (C4-C12), 3-hydroxyadipic acid, and suberic acid were also increased by milk consumption, indicating higher energy expenditure compared to the control group.

Background: To increase self-supply of protein and phosphorus (P) in European pig and poultry diets and reduce nitrogen (N) and P excretion, attention is directed to approaches increasing protein and P digestibility of rapeseed, sunflower and faba beans. Wheat bran is rich in enzymes degrading and solubilizing protein and phytate. Herein, solubilization of protein, N and P was investigated when increasing ratios of wheat bran were fermented with rapeseed meal (RSM), sunflower meal (SFM), faba beans (FB) or a combination of these (RSM/SFM/FB)., Results: Protein, N and P solubility was greater, for all mixtures, the more wheat bran was included and the longer the mixtures were fermented. The increase in N (FB > RSM/SFM/FB > SFM > RSM) and protein solubility (RSM/SFM/FB > RSM > SFM > FB) was greatest from day 0 to day 3 and thereafter limited, whereas P solubility increased during the whole period (5 days; FB > RSM/SFM/FB > SFM > RSM). In general, FB showed the highest solubility and highest increase in N and P solubility, while RSM showed the highest protein solubility and RSM/SFM/FB the highest increase in protein solubility., Conclusion: Fermentation of RSM, SFM, FB and RSM/SFM/FB without or with wheat bran uncovers a potential for increased protein and P digestibility and thereby reduced N and P excretion from pigs and poultry. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

In their journey from the male to the female reproductive tract, spermatozoa are confronted with a constantly changing environment. To cope with the associated challenges, spermatozoa express a distinct set of transporters, channels and pumps. One of the membrane proteins unique to spermatozoa is the α4 isoform of the Na(+) ,K(+) -ATPase. In addition to α4, spermatozoa express the ubiquous α1 variant. To get a detailed understanding of how α1 and α4 differ, and why spermatozoa need an additional Na(+) ,K(+) -ATPase, we have conducted an electrophysiological comparison of the rodent isoforms (rat α4 versus mouse α1-3) using the Xenopus oocyte expression system. We demonstrate isoform-specific differences in the voltage sensitivity of steady-state turnover, with α2 being the more sensitive, and α1 and α2 having faster Na(+) release kinetics than α3 and α4. Our data further show that the α1 and α2 turnover rates are fast compared with those of α3 and α4. Finally, α4 is less influenced by changes in extracellular Na(+) and temperature than α1. Based on these findings, we discuss the possibility that evolution has selected robust activity rather than rapid turnover for α4., (© 2015 FEBS.)

The transport of three Na(+) per two K(+) means that the Na,K-ATPase is electrogenic, and though the currents generated by the ion pump are small compared to ion channel currents, they can be measured using electrophysiology, both steady-state pumping and individual steps in the transport cycle. Various electrophysiological techniques have been used to study the endogenous pumps of the squid giant axon and of cardiac myocytes from for example rabbits. Here, we describe the characterization of heterologously expressed Na,K-ATPases using two-electrode voltage clamping (TEVC) and oocytes from the Xenopus laevis frog as the model cell. With this system, the effects of particular mutations can be studied, including the numerous mutations that in later years have been found to cause human diseases.

The Na(+), K(+)-adenosine triphosphatase (ATPase) maintains the electrochemical gradients of Na(+) and K(+) across the plasma membrane--a prerequisite for electrical excitability and secondary transport. Hitherto, structural information has been limited to K(+)-bound or ouabain-blocked forms. We present the crystal structure of a Na(+)-bound Na(+), K(+)-ATPase as determined at 4.3 Å resolution. Compared with the K(+)-bound form, large conformational changes are observed in the α subunit whereas the β and γ subunit structures are maintained. The locations of the three Na(+) sites are indicated with the unique site III at the recently suggested IIIb, as further supported by electrophysiological studies on leak currents. Extracellular release of the third Na(+) from IIIb through IIIa, followed by exchange of Na(+) for K(+) at sites I and II, is suggested.

All animals are characterized by steep gradients of Na(+) and K(+) across the plasma membrane, and in spite of their highly similar chemical properties, the ions can be distinguished by numerous channels and transporters. The gradients are generated by the Na(+),K(+)-ATPase, or sodium pump, which pumps out Na(+) and takes up K(+) at the expense of the chemical energy from ATP. Because the membrane is more permeable to K(+) than to Na(+), the uneven ion distribution causes a transmembrane voltage difference, and this membrane potential forms the basis for the action potential and for much of the neuronal signaling in general. The potential energy stored in the concentration gradients is also used to drive a large number of the secondary transporters responsible for transmembrane carriage of solutes ranging from sugars, amino acids, and neurotransmitters to inorganic ions such as chloride, inorganic phosphate, and bicarbonate. Furthermore, Na(+) and K(+) themselves are important enzymatic cofactors that typically lower the energy barrier of substrate binding.In this chapter, we describe the roles of Na(+) and K(+) in the animal cell with emphasis on the creation and usage of the steep gradients across the membrane. More than 50 years of Na(+),K(+)-ATPase research has revealed many details of the molecular machinery and offered insights into how the pump is regulated by post-translational modifications and specific drugs.

The Na+,K+-ATPase transforms the energy of ATP to the maintenance of steep electrochemical gradients for sodium and potassium across the plasma membrane. This activity is tissue specific, in particular due to variations in the expressions of the alpha subunit isoforms one through four. Several mutations in alpha2 and 3 have been identified that link the specific function of the Na+,K+-ATPase to the pathophysiology of neurological diseases such as rapid-onset dystonia parkinsonism and familial hemiplegic migraine type 2. We show a mapping of the isoform differences and the disease-related mutations on the recently determined crystal structure of the pig renal Na+,K+-ATPase and a structural comparison to Ca2+-ATPase. Furthermore, we present new experimental data that address the role of a stretch of three conserved arginines near the C-terminus of the alpha subunit (Arg1003-Arg1005).