Splanchnic metabolism of nitrogenous compounds and urinary nitrogen excretion in steers fed alfalfa under conditions of increased absorption of ammonia and L-arginine supply across the portal-drained viscera

Effects of increased ammonia and/or arginine absorption across the portal-drained viscera (PDV) on net splanchnic (PDV and liver) metabolism of nitrogenous compounds and urinary N excretion were investigated in six cathetenzed Hereford x Angus steers (501 +/- 1 kg BW) fed a 75% alfalfa:25% (as-fed basis) corn-soybean meal diet (0.523 MJ of ME/[kg BW0.15.d]) every 2 h without (27.0 g of N/kg of dietary DM) and with 20 g of urea/kg of dietary DM (35.7 g of N/kg of dietary DM) in a split-plot design. Net splanchnic flux measurements were obtained immediately before beginning and ending a 72-h mesenteric vein infusion of L-arginine (15 mmol/h). For 3 d before and during arginine infusion, daily urine voided was measured and analyzed for N composition. Feeding urea increased PDV absorption (P < 0.01) and hepatic removal (P < 0.01) of ammonia N, accounting for 80% of increased hepatic urea N output (P < 0.01). Numerical increases in net hepatic removal of AA N could account for the remaining portion of increased hepatic urea N output. Arginine infusion increased hepatic arginine removal (P < 0.01) and hepatic urea N output (P < 0.03) and switched hepatic ornithine flux from net uptake to net output (P < 0.01), but numerical changes in net hepatic removal of ammonia and AA N could not account fully for the increase in hepatic urea N output. Increases in urine N excretion equaled quantities of N fed as urea or infused as arginine. Estimated salivary urea N excretion was not changed by either treatment. Urea cycle regulation occurs via a complex interaction of mechanisms and requires N sources other than ammonia, but the effect of increased ammonia absorption on hepatic catabolism of individual AA in the present study was not significant.

The effect of dietary nitrate on salivary, plasma, and urinary nitrate metabolism in humans

Dietary nitrate is metabolized to nitrite by bacterial flora on the posterior surface of the tongue leading to increased salivary nitrite concentrations. In the acidic environment of the stomach, nitrite forms nitrous acid, a potent nitrating/nitrosating agent. The aim of this study was to examine the pharmacokinetics of dietary nitrate in relation to the formation of salivary, plasma, and urinary nitrite and nitrate in healthy subjects. A secondary aim was to determine whether dietary nitrate increases the formation of protein-bound 3-nitrotyrosine in plasma, and if dietary nitrate improves platelet function. The pharmacokinetic profile of urinary nitrate excretion indicates total clearance of consumed nitrate in a 24 h period. While urinary, salivary, and plasma nitrate concentrations increased between 4- and 7-fold, a significant increase in nitrite was only detected in saliva (7-fold). High dietary nitrate consumption does not cause a significant acute change in plasma concentrations of 3-nitrotyrosine or in platelet function.

Evaluation of milk urea nitrogen as a diagnostic of protein feeding

An evaluation of milk urea nitrogen (MUN) as a diagnostic of protein feeding in dairy cows was performed using mean treatment data (n = 306) from 50 production trials conducted in Finland (n = 48) and Sweden (n = 2). Data were used to assess the effects of diet composition and certain animal characteristics on MUN and to derive relationships between MUN and the efficiency of N utilization for milk production and urinary N excretion. Relationships were developed using regression analysis based on either models of fixed factors or using mixed models that account for between-experiment variations. Dietary crude protein (CP) content was the best single predictor of MUN and accounted for proportionately 0.778 of total variance [ MUN (mg/dL) = -14.2 + 0.17 x dietary CP content (g/kg dry matter)]. The proportion of variation explained by this relationship increased to 0.952 when a mixed model including the random effects of study was used, but both the intercept and slope remained unchanged. Use of rumen degradable CP concentration in excess of predicted requirements, or the ratio of dietary CP to metabolizable energy as single predictors, did not explain more of the variation in MUN (R-2 = 0.767 or 0.778, respectively) than dietary CP content. Inclusion of other dietary factors with dietary CP content in bivariate models resulted in only marginally better predictions of MUN (R-2 = 0.785 to 0.804). Closer relationships existed between MUN and dietary factors when nutrients (CP to metabolizable energy) were expressed as concentrations in the diet, rather than absolute intakes. Furthermore, both MUN and MUN secretion (g/d) provided more accurate predictions of urinary N excretion (R-2 = 0.787 and 0.835, respectively) than measurements of the efficiency of N utilization for milk production (R-2 = 0.769). It is concluded that dietary CP content is the most important nutritional factor influencing MUN, and that measurements of MUN can be utilized as a diagnostic of protein feeding in the dairy cow and used to predict urinary N excretion.

Effects of essential oils on ruminal microorganisms and their protein metabolism

A commercial blend of essential oil (EO) compounds was added to a grass, maize silage, and concentrate diet fed to dairy cattle in order to determine their influence on protein metabolism by ruminal microorganisms. EO inhibited (P < 0.05) the rate of deamination of amino acids. Pure-culture studies indicated that the species most sensitive to EO were ammonia-hyperproducing bacteria and anaerobic fungi.

Role of Nrf2 in the regulation of CD36 and stress protein expression in murine macrophages - Activation by oxidatively modified LDL and 4-hydroxynonenal

CD36 is an important scavenger receptor mediating uptake of oxidized low- density lipoproteins ( oxLDLs) and plays a key role in foam cell formation and the pathogenesis of atherosclerosis. We report the first evidence that the transcription factor Nrf2 is expressed in vascular smooth muscle cells, and demonstrate that oxLDLs cause nuclear accumulation of Nrf2 in murine macrophages, resulting in the activation of genes encoding CD36 and the stress proteins A170, heme oxygenase- 1 ( HO- 1), and peroxiredoxin I ( Prx I). 4- Hydroxy- 2- nonenal ( HNE), derived from lipid peroxidation, was one of the most effective activators of Nrf2. Using Nrf2- deficient macrophages, we established that Nrf2 partially regulates CD36 expression in response to oxLDLs, HNE, or the electrophilic agent diethylmaleate. In murine aortic smooth muscle cells, expressing negligible levels of CD36, both moderately and highly oxidized LDL caused only limited Nrf2 translocation and negligible increases in A170, HO- 1, and Prx I expression. However, treatment of smooth muscle cells with HNE significantly enhanced nuclear accumulation of Nrf2 and increased A170, HO- 1, and Prx I protein levels. Because PPAR-gamma can be activated by oxLDLs and controls expression of CD36 in macrophages, our results implicate Nrf2 as a second important transcription factor involved in the induction of the scavenger receptor CD36 and antioxidant stress genes in atherosclerosis.

Proteomic profiling of neuromas reveals alterations in protein composition and local protein synthesis in hyper-excitable nerves

Neuropathic pain may arise following peripheral nerve injury though the molecular mechanisms associated with this are unclear. We used proteomic profiling to examine changes in protein expression associated with the formation of hyper-excitable neuromas derived from rodent saphenous nerves. A two-dimensional difference gel electrophoresis ( 2D-DIGE) profiling strategy was employed to examine protein expression changes between developing neuromas and normal nerves in whole tissue lysates. We found around 200 proteins which displayed a > 1.75-fold change in expression between neuroma and normal nerve and identified 55 of these proteins using mass spectrometry. We also used immunoblotting to examine the expression of low-abundance ion channels Nav1.3, Nav1.8 and calcium channel alpha 2 delta-1 subunit in this model, since they have previously been implicated in neuronal hyperexcitability associated with neuropathic pain. Finally, S(35)methionine in vitro labelling of neuroma and control samples was used to demonstrate local protein synthesis of neuron-specific genes. A number of cytoskeletal proteins, enzymes and proteins associated with oxidative stress were up-regulated in neuromas, whilst overall levels of voltage-gated ion channel proteins were unaffected. We conclude that altered mRNA levels reported in the somata of damaged DRG neurons do not necessarily reflect levels of altered proteins in hyper-excitable damaged nerve endings. An altered repertoire of protein expression, local protein synthesis and topological re-arrangements of ion channels may all play important roles in neuroma hyper-excitability.

Glutathione and related thiol compounds II: the importance of protein bound glutathione and related protein-bound compounds in gluten proteins

Protein-bound glutathione (PSSG) and protein-bound related thiol compounds, i.e. cysteine (PSSCys), glutamyl-cysteine (PSSGlu-Cys) and cysteinyl-glycine (PSSCys-Gly), were analysed in proteins of Osborne fractions, i.e. gliadin, glutenin and gliadin-, glutenin-subfractions separated by gel filtration chromatography, gel protein and the total gluten proteins separated from wheat varieties with varying breadmaking performances. The results showed that PSSG and some protein-bound related thiol compounds were found in monomeric gliadins, indicating that glutathione and some related thiol compounds are able to form disulphide bonds (SS) with sulphydryl group (SH) of those proteins and the formation of those disulphide bonds may prevent those monomeric proteins from binding to other proteins. It was also observed that a larger amount of PSSG in glutenin proteins was negatively correlated with the molecular weight (M-w) distribution of glutenin polymers, suggesting that PSSG and protein-bound related thiol compounds may play an important role in controlling polymerisation of glutenin. Furthermore, it was found that the level of PSSG in gel protein from flours with poor breadmaking performances was constantly higher and significantly different (p < 0.05) from that of flours with good breadmaking performance. The same trend was observed with gluten samples from breadmaking and biscuitmaking flours. (C) 2003 Elsevier Ltd. All rights reserved.

Stress relaxation behavior of wheat dough, gluten, and gluten protein fractions

Relaxation behavior was measured for dough, gluten and gluten protein fractions obtained from the U.K. biscuitmaking flour, Riband, and the U.K. breadmaking flour, Hereward. The relaxation spectrum, in which relaxation times (tau) are related to polymer molecular size, for dough showed a broad molecular size distribution, with two relaxation processes: a major peak at short times and a second peak at times longer than 10 sec, which is thought to correspond to network structure, and which may be attributed to entanglements and physical cross-links of polymers. Relaxation spectra of glutens were similar to those for the corresponding doughs from both flours. Hereward gluten clearly showed a much more pronounced second peak in relaxation spectrum and higher relaxation modulus than Riband gluten at the same water content. In the gluten protein fractions, gliadin and acetic acid soluble glutenin only showed the first relaxation process, but gel protein clearly showed both the first and second relaxation processes. The results show that the relaxation properties of dough depend on its gluten protein and that gel protein is responsible for the network structure for dough and gluten.

Localization to the nucleolus is a common feature of coronavirus nucleoproteins and the protein may disrupt host cell division

The subcellular localization of transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV) (group I and group II coronaviruses, respectively) nucleoproteins (N proteins) were examined by confocal microscopy. The proteins were shown to localize either to the cytoplasm alone or to the cytoplasm and a structure in the nucleus. This feature was confirmed to be the nucleolus by using specific antibodies to nucleolin, a major component of the nucleolus, and by confocal microscopy to image sections through a cell expressing N protein. These findings are consistent with our previous report for infectious bronchitis virus (group III coronavirus) (J. A. Hiscox et al., J. Virol. 75:506-512, 2001), indicating that nucleolar localization of the N protein is a common feature of the coronavirus family and is possibly of functional significance. Nucleolar localization signals were identified in the domain III region of the N protein from all three coronavirus groups, and this suggested that transport of N protein to the nucleus might be an active process. In addition, our results suggest that the N protein might function to disrupt cell division. Thus, we observed that approximately 30% of cells transfected with the N protein appeared to be undergoing cell division. The most likely explanation for this is that the N protein induced a cell cycle delay or arrest, most likely in the G2/M phase. In a fraction of transfected cells expressing coronavirus N proteins, we observed multinucleate cells and dividing cells with nucleoli (which are only present during interphase). These findings are consistent with the possible inhibition of cytokinesis in these cells.

Effects of ammonia treatment and undegradable protein supplementation on nutrient digestion of sheep fed on wheat straw based diets.

A study was conducted to investigate the effects of wheat straw ammonisation and supplementation with a rumen undegradable protein (UDP) source on nutrient digestion and nitrogen balance by lambs while diets were supplemented with kibbled carob pods as energy source. Ammonisation increased the crude protein content of wheat straw by nearly 100% and decreased the contents of neutral detergent fibre and acid detergent fibre by 7% and 1.7% respectively. Treating the straw with ammonia resulted in significant (P<0.01) increase in nitrogen (N) intake and intakes of organic matter (OM) and dry matter (DM) tended toward significance (P<0.1). The UDP source had no effect (P>0.05) on DM and OM intakes but resulted in an increase (P<0.05) of N intakes. Both, ammonization and UDP supplementation increased (P<0.01) the DM, OM and N digestibility. In conclusion, the results of this study suggest that ammonisation and UDP supplementation is a practical dietary manipulation option to improve the nutritional status of ruminants fed on roughage-based diets.

Inhibition of synaptic transmission and G protein modulation by synthetic CaV2.2 Ca2+ channel peptides

Abstract: Modulation of presynaptic voltage-dependent Ca+ channels is a major means of controlling neurotransmitter release. The CaV 2.2 Ca2+ channel subunit contains several inhibitory interaction sites for Gβγ subunits, including the amino terminal (NT) and I–II loop. The NT and I–II loop have also been proposed to undergo a G protein-gated inhibitory interaction, whilst the NT itself has also been proposed to suppress CaV 2 channel activity. Here, we investigate the effects of an amino terminal (CaV 2.2[45–55]) ‘NT peptide’ and a I–II loop alpha interaction domain (CaV 2.2[377–393]) ‘AID peptide’ on synaptic transmission, Ca2+ channel activity and G protein modulation in superior cervical ganglion neurones (SCGNs). Presynaptic injection of NT or AID peptide into SCGN synapses inhibited synaptic transmission and also attenuated noradrenaline-induced G protein modulation. In isolated SCGNs, NT and AID peptides reduced whole-cell Ca2+ current amplitude, modified voltage dependence of Ca2+ channel activation and attenuated noradrenaline-induced G protein modulation. Co-application of NT and AID peptide negated inhibitory actions. Together, these data favour direct peptide interaction with presynaptic Ca2+ channels, with effects on current amplitude and gating representing likely mechanisms responsible for inhibition of synaptic transmission. Mutations to residues reported as determinants of Ca2+ channel function within the NT peptide negated inhibitory effects on synaptic transmission, Ca2+ current amplitude and gating and G protein modulation. A mutation within the proposed QXXER motif for G protein modulation did not abolish inhibitory effects of the AID peptide. This study suggests that the CaV 2.2 amino terminal and I–II loop contribute molecular determinants for Ca2+ channel function; the data favour a direct interaction of peptides with Ca2+ channels to inhibit synaptic transmission and attenuate G protein modulation. Non-technical summary: Nerve cells (neurones) in the body communicate with each other by releasing chemicals (neurotransmitters) which act on proteins called receptors. An important group of receptors (called G protein coupled receptors, GPCRs) regulate the release of neurotransmitters by an action on the ion channels that let calcium into the cell. Here, we show for the first time that small peptides based on specific regions of calcium ion channels involved in GPCR signalling can themselves inhibit nerve cell communication. We show that these peptides act directly on calcium channels to make them more difficult to open and thus reduce calcium influx into native neurones. These peptides also reduce GPCR-mediated signalling. This work is important in increasing our knowledge about modulation of the calcium ion channel protein; such knowledge may help in the development of drugs to prevent signalling in pathways such as those involved in pain perception.

Phenolic acid intake, delivered via moderate champagne wine consumption, improves spatial working memory via the modulation of hippocampal and cortical protein expression/activation.

Aims: While much data exist for the effects of flavonoid-rich foods on spatial memory in rodents, there are no such data for foods/beverages predominantly containing hydroxycinnamates and phenolic acids. To address this, we investigated the effects of moderate Champagne wine intake, which is rich in these components, on spatial memory and related mechanisms relative to the alcohol- and energy-matched controls. Results: In contrast to the isocaloric and alcohol-matched controls, supplementation with Champagne wine (1.78 ml/kg BW, alcohol 12.5% vol.) for 6 weeks led to an improvement in spatial working memory in aged rodents. Targeted protein arrays indicated that these behavioral effects were paralleled by the differential expression of a number of hippocampal and cortical proteins (relative to the isocaloric control group), including those involved in signal transduction, neuroplasticity, apoptosis, and cell cycle regulation. Western immunoblotting confirmed the differential modulation of brain-derived neurotrophic factor, cAMP response-element-binding protein (CREB), p38, dystrophin, 2',3'-cyclic-nucleotide 3'-phosphodiesterase, mammalian target of rapamycin (mTOR), and Bcl-xL in response to Champagne supplementation compared to the control drink, and the modulation of mTOR, Bcl-xL, and CREB in response to alcohol supplementation. Innovation: Our data suggest that smaller phenolics such as gallic acid, protocatechuic acid, tyrosol, caftaric acid, and caffeic acid, in addition to flavonoids, are capable of exerting improvements in spatial memory via the modulation in hippocampal signaling and protein expression. Conclusion: Changes in spatial working memory induced by the Champagne supplementation are linked to the effects of absorbed phenolics on cytoskeletal proteins, neurotrophin expression, and the effects of alcohol on the regulation of apoptotic events in the hippocampus and cortex. Antioxid. Redox Signal. 00, 000-000.

Influence of temperature during grain filling on gluten viscoelastic properties and gluten protein composition

BACKGROUND The aim of this study was to investigate the effects of low to moderate temperatures on gluten functionality and gluten protein composition. Four spring wheat cultivars were grown in climate chambers with three temperature regimes (day/night temperatures of 13/10, 18/15 and 23/20 °C) during grain filling. RESULTS The temperature strongly influenced grain weight and protein content. Gluten quality measured by maximum resistance to extension (Rmax) was highest in three cultivars grown at 13 °C. Rmax was positively correlated with the proportion of sodium dodecyl sulfate-unextractable polymeric proteins (%UPP). The proportions of ω-gliadins and D-type low-molecular-weight glutenin subunits (LMW-GS) increased and the proportions of α- and γ-gliadins and B-type LMW-GS decreased with higher temperature, while the proportion of high-molecular-weight glutenin subunits (HMW-GS) was constant between temperatures. The cultivar Berserk had strong and constant Rmax between the different temperatures. CONCLUSION Constant low temperature, even as low as 13 °C, had no negative effects on gluten quality. The observed variation in Rmax related to temperature could be explained more by %UPP than by changes in the proportions of HMW-GS or other gluten proteins. The four cultivars responded differently to temperature, as gluten from Berserk was stronger and more stable over a wide range of temperature

Effect of prolonged intravenous glucose and essential amino acid infusion on nitrogen balance, muscle protein degradation and ubiquitin-conjugating enzyme gene expression in calves

Background: Intravenous infusions of glucose and amino acids increase both nitrogen balance and muscle accretion. We hypothesised that co-infusion of glucose ( to stimulate insulin) and essential amino acids (EAA) would act additively to improve nitrogen balance by decreasing muscle protein degradation in association with alterations in muscle expression of components of the ubiquitin-proteasome proteolytic pathway. Methods: We examined the effect of a 5 day intravenous infusions of saline, glucose, EAA and glucose + EAA, on urinary nitrogen excretion and muscle protein degradation. We carried out the study in 6 restrained calves since ruminants offer the advantage that muscle protein degradation can be assessed by excretion of 3 methyl-histidine and multiple muscle biopsies can be taken from the same animal. On the final day of infusion blood samples were taken for hormone and metabolite measurement and muscle biopsies for expression of ubiquitin, the 14-kDa E2 ubiquitin conjugating enzyme, and proteasome sub-units C2 and C8. Results: On day 5 of glucose infusion, plasma glucose, insulin and IGF-1 concentrations were increased while urea nitrogen excretion and myofibrillar protein degradation was decreased. Co-infusion of glucose + EAA prevented the loss of urinary nitrogen observed with EAA infusions alone and enhanced the increase in plasma IGF-1 concentration but there was no synergistic effect of glucose + EAA on the decrease in myofibrillar protein degradation. Muscle mRNA expression of the ubiquitin conjugating enzyme, 14-kDa E2 and proteasome sub-unit C2 were significantly decreased, after glucose but not amino acid infusions, and there was no further response to the combined infusions of glucose + EAA. Conclusion: Prolonged glucose infusion decreases myofibrillar protein degradation, prevents the excretion of infused EAA, and acts additively with EAA to increase plasma IGF-1 and improve net nitrogen balance. There was no evidence of synergistic effects between glucose + EAA infusion on muscle protein degradation or expression of components of the ubiquitin-proteasome proteolytic pathway.