Milk protein composition and stability changes affected by iron in water sources.

Water makes up more than 80% of the total weight of milk. However, the influence of water chemistry on the milk proteome has not been extensively studied. The objective was to evaluate interaction of water-sourced iron (low, medium, and high levels) on milk proteome and implications on milk oxidative state and mineral content. Protein composition, oxidative stability, and mineral composition of milk were investigated under conditions of iron ingestion through bovine drinking water (infused) as well as direct iron addition to commercial milk in 2 studies. Four ruminally cannulated cows each received aqueous infusions (based on water consumption of 100L) of 0, 2, 5, and 12.5mg/L Fe(2+) as ferrous lactate, resulting in doses of 0, 200, 500 or 1,250mg of Fe/d, in a 4×4Latin square design for a 14-d period. For comparison, ferrous sulfate solution was directly added into commercial retail milk at the same concentrations: control (0mg of Fe/L), low (2mg of Fe/L), medium (5mg of Fe/L), and high (12.5mg of Fe/L). Two-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization-tandem time-of-flight (MALDI-TOF/TOF) high-resolution tandem mass spectrometry analysis was applied to characterize milk protein composition. Oxidative stability of milk was evaluated by the thiobarbituric acid reactive substances (TBARS) assay for malondialdehyde, and mineral content was measured by inductively coupled plasma mass spectrometry. For milk from both abomasal infusion of ferrous lactate and direct addition of ferrous sulfate, an iron concentration as low as 2mg of Fe/L was able to cause oxidative stress in dairy cattle and infused milk, respectively. Abomasal infusion affected both caseins and whey proteins in the milk, whereas direct addition mainly influenced caseins. Although abomasal iron infusion did not significantly affect oxidation state and mineral balance (except iron), it induced oxidized off-flavor and partial degradation of whey proteins. Direct iron addition to milk led to lipid oxidation during storage at 4°C. Oxidation level was positively associated with the concentration of added iron. Minerals (Mg, P, Na, K, Ca, Zn) in milk were not affected by the added iron in milk. This study indicated that a small amount of iron contamination in bovine drinking water at the farm or incidental iron addition from potable water sources causes oxidation, affects milk protein composition and stability, and affects final milk quality.

Proteomic evaluation of chicken brush-border membrane during the early posthatch period.

The chicken small intestine undergoes structural and functional changes during the early posthatch period to accommodate the transition from a lipid-rich diet inside the egg to a carbohydrate- and protein-based diet. Many of the enterocyte brush-border membrane-associated proteins responsible for mediating changes in nutrient utilization are unknown. The objective of this study was to conduct a proteomic analysis of chicken small intestine during the early posthatch period. We isolated brush-border membrane at day of hatch and days 1, 3, 7, and 14 posthatch from the small intestine of 2 genetic lines of broilers that differ in growth performance, and performed 2D gel-electrophoresis. A total of 1693 spots were analyzed by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry (MALDI-TOF/TOF). In total, 132 different proteins were identified and grouped according to biological function. Of these, there were 10 nutrient transporters, 9 digestive enzymes, and 17 proteins associated with cytoskeletal structure and microvilli organization. The remaining proteins were classified as basolateral membrane (3), endosomal/membrane trafficking (8), signaling (14), metabolic (33), degradative (5), stress-related (5), protein synthesis machinery/mitochondria/nucleus (19), immunologic (1), or unknown (8). Of the spots in which proteins were identified, there were 10 that showed an effect of broiler genetic line on protein spot density (P<0.001) and 19 spots showing a correlation of broiler genetic line x age (P<0.001). Identification of brush-border membrane-associated proteins is an important step in furthering our understanding of digestion and absorption in the chicken.

Helix Cracking Regulates the Critical Interaction between RetS and GacS in Pseudomonas aeruginosa.

Recent paradigm shifting discoveries have demonstrated that bacterial signaling kinases engage in unexpected regulatory crosstalk, yet the underlying molecular mechanisms remain largely uncharacterized. The Pseudomonas aeruginosa RetS/GacS system constitutes an ideal model for studying these mechanisms. The in-depth analysis of the kinase region of RetS and RetS/GacS interactions presented here refutes a longstanding model, which posited the formation of a catalytically inactive RetS/GacS heterodimer. Crystallographic studies uncovered structurally dynamic features within the RetS kinase region, suggesting that RetS uses the reversible unfolding of a helix, or helix cracking, to control interactions with GacS. The pivotal importance of this helical region for regulating GacS and, by extension, Pseudomonas aeruginosa virulence, was corroborated via in vivo assays. The implications of this work extend beyond the RetS/GacS system because the helix cracking occurs right next to a highly conserved catalytic residue histidine-424, suggesting this model could represent an emergent archetype for histidine kinase regulation.

Effect of lactoferrin on taste and smell abnormalities induced by chemotherapy: a proteome analysis.

Cancer patients receiving chemotherapy often experience taste and smell abnormalities (TSA). To date, the underlying molecular mechanisms of this frequent side-effect have not been determined and effective treatments are not available. This study assessed the feasibility of lactoferrin (LF) supplementation as a treatment for TSA and investigate the related mechanisms through salivary proteome analysis. Nineteen cancer patients with established TSA following chemotherapy administration were enrolled in this study. Cancer patients and additional 12 healthy subjects took LF supplements, 3 tablets per day (250 mg per tablet), for 30 days. Saliva was collected at three timepoints: baseline, 30-day LF supplementation, and 30-day post-LF supplementation. Patient's TSA level, salivary proteome, and salivary minerals at each LF treatment stage were analyzed. High TSA level was associated with high concentration of salivary Fe and loss of critical salivary immune proteins. LF supplementation significantly decreased the concentration of salivary Fe (P = 0.025), increased the abundance (P < 0.05) of salivary α-amylase and Zn-α-2-GP, and led to an overall increase of expression (≥2-fold changes) of immune proteins including immunoglobulin heavy chain, annexin A1, and proteinase inhibitor. Abundance of α-amylase and SPLUNC2 were further increased (P < 0.05) at 30-day post-LF supplementation in cancer patients. At the same time, total TSA score was significantly reduced (P < 0.001) in chemotherapy patients. This study demonstrated the feasibility of developing lactoferrin supplementation as a treatment to reduce TSA caused by chemotherapy and improve cancer patient's oral immunity.

Activation of SsoPK4, an Archaeal eIF2α Kinase Homolog, by Oxidized CoA.

The eukaryotic protein kinase (ePK) paradigm provides integral components for signal transduction cascades throughout nature. However, while so-called typical ePKs permeate the Eucarya and Bacteria, atypical ePKs dominate the kinomes of the Archaea. Intriguingly, the catalytic domains of the handful of deduced typical ePKs from the archaeon Sulfolobus solfataricus P2 exhibit significant resemblance to the protein kinases that phosphorylate translation initiation factor 2α (eIF2α) in response to cellular stresses. We cloned and expressed one of these archaeal eIF2α protein kinases, SsoPK4. SsoPK4 exhibited protein-serine/threonine kinase activity toward several proteins, including the S. solfataricus homolog of eIF2α, aIF2α. The activity of SsoPK4 was inhibited in vitro by 3',5'-cyclic AMP (Ki of ~23 µM) and was activated by oxidized Coenzyme A, an indicator of oxidative stress in the Archaea. Activation enhanced the apparent affinity for protein substrates, Km, but had little effect on Vmax. Autophosphorylation activated SsoPK4 and rendered it insensitive to oxidized Coenzyme A.