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Multiple beneficial cardiovascular effects of HDL depend on sphingosine-1-phosphate (S1P). S1P associates with HDL by binding to apolipoprotein M (ApoM). Insulin resistance is a major driver of dyslipidemia and cardiovascular risk. However, the mechanisms linking alterations in insulin signaling with plasma lipoprotein metabolism are incompletely understood. The insulin-repressible FoxO transcription factors mediate key effects of hepatic insulin action on glucose and lipoprotein metabolism. This work tested whether hepatic insulin signaling regulates HDL-S1P and aimed to identify the underlying molecular mechanisms. We report that insulin-resistant, nondiabetic individuals had decreased HDL-S1P levels, but no change in total plasma S1P. This also occurred in insulin-resistant db/db mice, which had low ApoM and a specific reduction of S1P in the HDL fraction, with no change in total plasma S1P levels. Using mice lacking hepatic FoxOs (L-FoxO1,3,4), we found that hepatic FoxOs were required for ApoM expression. Total plasma S1P levels were similar to those in controls, but S1P was nearly absent from HDL and was instead increased in the lipoprotein-depleted plasma fraction. This phenotype was restored to normal by rescuing ApoM in L-FoxO1,3,4 mice. Our findings show that insulin resistance in humans and mice is associated with decreased HDL-associated S1P. Our study shows that hepatic FoxO transcription factors are regulators of the ApoM/S1P pathway., Introduction Individuals with insulin resistance and type 2 diabetes have low levels of HDL cholesterol, and low HDL-cholesterol levels are inversely correlated with cardiovascular disease (1). However, clinical trials have [...]

Genetic variants at the SORT1 locus in humans, which cause increased SORT1 expression in the liver, are significantly associated with reduced plasma levels of LDL cholesterol and apolipoprotein B (apoB). However, the role of hepatic sortilin remains controversial, as genetic deletion of sortilin in mice has resulted in variable and conflicting effects on apoB secretion. Here, we found that Sort7-KO mice on a chow diet and several Sort7-deficient hepatocyte lines displayed no difference in apoB secretion. When these models were challenged with high-fat diet or ER stress, the loss of Sort1 expression resulted in a significant increase in apoB-100 secretion. Sort1-overexpression studies yielded reciprocal results. Importantly, carriers of SORT1 variant with diabetes had larger decreases in plasma apoB, TG, and VLDL and LDL particle number as compared with people without diabetes with the same variants. We conclude that, under basal nonstressed conditions, loss of sortilin has little effect on hepatocyte apoB secretion, whereas, in the setting of lipid loading or ER stress, sortilin deficiency leads to increased apoB secretion. These results are consistent with the directionality of effect in human genetics studies and suggest that, under stress conditions, hepatic sortilin directs apoB toward lysosomal degradation rather than secretion, potentially serving as a quality control step in the apoB secretion pathway in hepatocytes., Introduction The protein sortilin, encoded by the gene SORT1, has been of great interest to the genetics and cardiometabolic disease communities over the last decade. Sortilin is a type I [...]

GDP-mannose-pyrophosphorylase-B (GMPPB) facilitates the generation of GDP-mannose, a sugar donor required for glycosylation. GMPPB defects cause muscle disease due to hypoglycosylation of [alpha]-dystroglycan ([alpha]-DG). AlphaDG is part of a protein complex, which links the extracellular matrix with the cytoskeleton, thus stabilizing myofibers. Mutations of the catalytically inactive homolog GMPPA cause alacrima, achalasia, and mental retardation syndrome (AAMR syndrome), which also involves muscle weakness. Here, we showed that Gmppa-KO mice recapitulated cognitive and motor deficits. As structural correlates, we found cortical layering defects, progressive neuron loss, and myopathic alterations. Increased GDP-mannose levels in skeletal muscle and in vitro assays identified GMPPA as an allosteric feedback inhibitor of GMPPB. Thus, its disruption enhanced mannose incorporation into glycoproteins, including [alpha]-DG in mice and humans. This increased [alpha]-DG turnover and thereby lowered [alpha]-DG abundance. In mice, dietary mannose restriction beginning after weaning corrected [alpha]-DG hyperglycosylation and abundance, normalized skeletal muscle morphology, and prevented neuron degeneration and the development of motor deficits. Cortical layering and cognitive performance, however, were not improved. We thus identified GMPPA defects as the first congenital disorder of glycosylation characterized by [alpha]-DG hyperglycosylation, to our knowledge, and we have unraveled underlying disease mechanisms and identified potential dietary treatment options., Introduction Cell surface proteins are important for the attachment of a cell to the extracellular matrix (ECM). They are modified by post-translational covalent attachment of branched carbohydrate oligomers. N-linked oligosaccharides [...]

Enhanced fibroblast growth factor 21 (FGF21) production and circulation has been linked to the metabolic adaptation to starvation. Here, we demonstrated that hepatic FGF21 expression is induced by dietary protein restriction, but not energy restriction. Circulating FGF21 was increased 10-fold in mice and rats fed a low-protein (LP) diet. In these animals, liver Fgf21 expression was increased within 24 hours of reduced protein intake. In humans, circulating FGF21 levels increased dramatically following 28 days on a LP diet. LP-induced increases in FGF21 were associated with increased phosphorylation of eukaryotic initiation factor 2a (eIF2α) in the liver, and both baseline and LP- induced serum FGF21 levels were reduced in mice lacking the eIF2α kinase general control nonderepressible 2 (GCN2). Finally, while protein restriction altered food intake, energy expenditure, and body weight gain in WT mice, FGF21-deficient animals did not exhibit these changes in response to a LP diet. These and other data demonstrate that reduced protein intake underlies the increase in circulating FGF21 in response to starvation and a ketogenic diet and that FGF21 is required for behavioral and metabolic responses to protein restriction. FGF21 therefore represents an endocrine signal of protein restriction, which acts to coordinate metabolism and growth during periods of reduced protein intake., Introduction Nutrient restriction engages a variety of metabolic and behavioral adaptations, and in the case of energy restriction, these adaptations are well described as including alterations in feeding behavior, energy [...]

Early life stages of the coral Seriatopora caliendrum were used to test the hypothesis that the depression of dark respiration in coral recruits by high pC[O.sub.2] is caused by perturbed protein metabolism. First, the contribution of protein anabolism to respiratory costs under high pC[O.sub.2] was evaluated by measuring the aerobic respiration of S. caliendrum recruits with and without the protein synthesis inhibitor emetine following 1 to 4 days at 45 Pa versus 77 Pa pC[O.sub.2]. Second, protein catabolism under high pC[O.sub.2] was evaluated by measuring the flux of ammonium (N[H.sub.4.sup.+]) from juvenile colonies of S. caliendrum incubated in darkness at 47 Pa and 90 Pa pC[O.sub.2]. Two days after settlement, respiration of recruits was affected by an interaction between emetine and pC[O.sub.2], with emetine reducing respiration 63% at 45 Pa pC[O.sub.2] and 27% at 77 Pa pC[O.sub.2]. The interaction disappeared 5 days after settlement, when respiration was reduced 27% by emetine under both pC[O.sub.2] conditions. These findings suggest that protein anabolism accounted for a large proportion of metabolic costs in coral recruits and was affected by high pC[O.sub.2], with consequences detected in aerobic respiration. Juvenile S. caliendrum showed net uptake of N[H.sub.4.sup.+] at 45 Pa pC[O.sub.2] but net release of N[H.sub.4.sup.+] at 90 Pa pC[O.sub.2], indicating that protein catabolism, N[H.sub.4.sup.+] recycling, or both were affected by high pC[O.sub.2]. Together, these results are consistent with the hypothesis that high pC[O.sub.2] affects protein metabolism in corals., Introduction Ocean acidification (OA) caused by the dissolution of atmos[pH.sub.e]ric pC[O.sub.2] in seawater is a serious threat to marine life (Hoegh-Guldberg et al., 2007). Of the many taxa likely to [...]

Ocean acidification (OA) is expected to have a major impact on marine species, particularly during early life-history stages. These effects appear to be species-specific and may include reduced survival, altered morphology, and depressed metabolism. However, less information is available regarding the bioenergetics of development under elevated [CO.sub.2] conditions. We examined the biochemical and morphological responses of Strongylocentrotus purpuratus during early development under ecologically relevant levels of p[CO.sub.2] (365, 1030, and 1450 µatm) that may occur during intense upwelling events. The principal findings of this study were (1) lipid utilization rates and protein content in S. purpuratus did not vary with p[CO.sub.2]; (2) larval growth was reduced at elevated p[CO.sub.2] despite similar rates of energy utilization; and (3) relationships between egg phospholipid content and larval length were found under control but not high p[CO.sub.2] conditions. These results suggest that this species may either prioritize endogenous energy toward development and physiological function at the expense of growth, or that reduced larval length may be strictly due to higher costs of growth under OA conditions. This study highlights the need to further expand our knowledge of the physiological mechanisms involved in OA response in order to better understand how present populations may respond to global environmental change., Introduction Ocean acidification--the decrease of ocean pH via absorption of anthropogenic [CO.sub.2] by surface waters--has emerged as a major priority in the marine science research community (e.g., National Research Council, [...]

Evidence has been presented for a metabolic multienzyme complex, the purinosome, that participates in de novo purine biosynthesis to form clusters in the cytoplasm of living cells under purine-depleted conditions. Here we identified, using fluorescent live cell imaging, that a microtubule network appears to physically control the spatial distribution of purinosomes in the cytoplasm. Application of a cell-based assay measuring the rate of de novo purine biosynthesis confirmed that the metabolic activity of purinosomes was significantly suppressed in the absence of microtubules. Collectively, we propose a microtubule-assisted mechanism for functional purinosome formation in HeLa cells. metabolism | protein complex | purine biosynthesis doi/ 10.1073/pnas.1008451107

To compare the change in lipoprotein metabolism with aging, we analyzed the lipid and protein compositions of individual lipoprotein fractions. Healthy and nonobese elderly participants (elderly group, n = 26) had a serum lipid profile within the normal range, although slightly higher than in young participants (control group, n = 18). However. the elderly group had a twofold higher serum uric acid level and triglyceride (TG):high-density lipoprotein cholesterol ratio. The elderly group had less antioxidant ability and elevated TG content in high-density lipoprotein (HDL) with enhanced cholesteryl ester transfer activity. An elevated level of advanced glycated end products in lipoproteins and fragmentation of apoA-I were present in the elderly group, with detected lower apoA-I level and more multimerized apoA-I in HDL. The protein levels of apoA-I, apoC-IIl, and serum amyloid A in lipoprotein-deficient serum were increased in the elderly group. Key Words: Aging--Apolipoprotein--Lipoprotein--Cholesteryl ester transfer protein--Advanced glycated end products. doi: 10.1093/gerona/glq034

CspA, a small protein that is highly induced by cold shock, is encoded by a monocistronic mRNA of 428 nucleotides (nt) whose half-life and abundance are greatly increased following cold shock We show here that in vitro cspA mRNA can bind multiple copies of Hfq, a hexameric Sm-like protein which promotes a variety of RNA-RNA interactions. Binding of the first Hfq hexamer occurs with an apparent [K.sub.d] (dissociation constant) of doi: 10.1128/JB.01619-09

Urea-nitrogen recycling to the gastrointestinal tract (GIT), N metabolism, and urea transporter-B (UT-B) mRNA abundance in ruminal epithelium were evaluated in partially defaunated (PDFAUN) and faunated (FAUN) growing lambs fed 2 levels (10%, low, or 15%, high) of dietary CP (DM basis). Four Suffolk ram lambs (43.9 [+ or -] 1.4 kg initial BW) were used in a 4 x 4 Latin square design with 27-d periods. Sunflower oil was fed (6%; DM basis) as an anti-protozoal agent. Nitrogen balance was measured from d 22 to 26, with concurrent measurement of urea-N kinetics using continuous intrajugular infusions of [[sup.15]N[sup.15]N]-urea. Feeding sunflower oil decreased (P < 0.01) total ruminal protozoa by 88%, and this was associated with a decrease (P < 0.01) in ruminal ammonia-N concentrations. Endogenous production of urea-N (UER; 26.1 vs. 34.6 g/d) and urea-N loss in urine (UUE; 10.1 vs. 15.7 g/d) were less (P < 0.01), and urea-N entering the GIT (GER; 16.0 vs. 18.9 g/d) tended to be less (P = 0.06) in PDFAUN as compared with FAUN lambs. However, as a proportion of UER, GER was greater (P < 0.01) and the proportion of recycled urea-N that was utilized for anabolism (i.e., UUA) tended to be greater (P = 0.09) in PDFAUN lambs. Partial defaunation increased (P < 0.01) microbial N supply. The UER, GER, and UUE were greater (P < 0.01) in lambs fed the high diet. However, as a proportion of UER, GER and its anabolic use were greater (P < 0.01) in lambs fed the low diet. The expression of UT-B mRNA in PDFAUN lambs was numerically greater (by 20%; P = 0.15) compared with FAUN lambs. In summary, results indicate that part of the mechanism for improved N utilization in defaunated ruminants is an increase in the proportion of endogenous urea-N output that is recycled to the GIT, thus potentially providing additional N for microbial growth. Key words: defaunation, dietary crude protein, microbial protein synthesis, nitrogen metabolism, sheep, urea-nitrogen recycling doi:10.2527/jas.2009-2218

Fish consumption is associated with a reduced colorectal cancer risk. A possible mechanism by which fish consumption could decrease colorectal cancer risk is by reducing inflammation. However, thus far, intervention studies investigating both systemic and local gut inflammation markers are lacking. Our objective in this study was to investigate the effects of fatty and lean fish consumption on inflammation markers in serum, feces, and gut. In an intervention study, participants were randomly allocated to receive dietary advice (DA) plus either 300 g of fatty fish (salmon) or 300 g of lean fish (cod) per week for 6 mo, or only DA. Serum C-reactive protein (CRP) concentrations were measured pre- and postintervention (n = 161). In a subgroup (n = 52), we explored the effects of the fish intervention on fecal calprotectin and a wide range of cytokines and chemokines in fecal water and in colonic biopsies. Serum CRP concentrations were lower in the salmon (-0.5 mg/L; 95% CI -0.9, -0.2) and cod (-0.4 mg/L; 95% CI -0.7, 0.0) groups compared with the DA group. None of the inflammation markers in fecal water and colonic biopsies differed between the DA group and the groups that consumed extra fish. In conclusion, increasing salmon or cod consumption for 6 mo resulted in lower concentrations of the systemic inflammation marker CRP. However, exploratory analysis of local markers of inflammation in the colon or feces did not reveal an effect of fish consumption. J. Nutr. 140: 371-376, 2010. doi: 10.3945/jn.109.113472.

Four ruminally and duodenally cannulated Suffolk wether lambs (34.5 [+ or -] 2.0 kg initial BW) were used in a 4 x 4 Latin square-designed experiment to examine the effects of ruminal protein degradability and supplementation frequency on site and extent of digestion in lambs consuming a low-quality forage diet. Wethers were fed a basal diet of mature crested wheatgrass hay (4.2% CP) for ad libitum consumption plus 1 of 4 supplements: 1) a high RDP supplement provided daily (RDP-D), 2) the high RDP supplement provided on alternate days (RDP-A), 3) a high RUP supplement provided on alternate days (RUP-A), or 4) a 50:50 mixture of the RDP and RUP supplements, provided on alternate days (MIX-A). Forage OM, N, NDF, or ADF intakes were not affected by treatment. True ruminal OM digestibility was greater (P < 0.001) for MIX-A lambs compared with other treatments. True ruminal N digestibility was less (P < 0.01) in RUP-A lambs compared with other treatments. Ruminal digestibilities of NDF and ADF were greater (P [less than or equal to] 0.01) for MIX-A lambs compared with other treatments. There was a treatment x hour interaction (P < 0.001) with a delay in peak concentrations within the RDP-A lambs. However, lambs supplemented with RUP had less (P < 0.001) ruminal ammonia concentrations compared with RDP-D lambs, with RUP-A lambs exhibiting the least concentrations and least variation over time. Ruminal urease activity was not affected by treatment. Microbial N flow was not affected by treatment; however, there was an increase (P = 0.004) in microbial efficiency for RDP-D lambs. Alternate day protein supplementation with a mixture of RDP and RUP may improve digestibility in lambs consuming low-quality forage, which may be related to decreased fluctuation in ruminal ammonia concentrations as a result of greater endogenous N recycling. Key words: growing lamb, low-quality forage, nitrogen metabolism, ruminal protein degradability, supplementation frequency doi: 10.2527/jas.2009-2245

Two experiments were conducted to determine the effect of ruminal protein degradability and supplementation frequency on intake, apparent digestibility, N retention, and nutrient flux across visceral tissues of lambs fed a low-quality forage diet. In both experiments, wethers were fed a basal diet of mature crested wheatgrass hay (4.2% CP) for ad libitum consumption plus 1 of 4 supplements: 1) a high RDP supplement provided daily (RDP-D), 2) the high RDP supplement provided on alternate days (RDP-A), 3) a high RUP provided on alternate days (RUP-A), or 4) a 50:50 mixture of the RDP and RUP supplements provided on alternate days. In Exp. 1, 12 lambs (29.9 [+ or -] 2.7 kg initial BW) were used. Forage OM, NDF, and ADF intake were not affected by treatment. Total tract digestibilities (OM, NDF, ADF, and N) were unaffected (P [greater than or equal to] 0.15) by treatment. Neither protein degradability nor supplementation frequency had an effect (P [greater than or equal to] 0.52) on N retention. In Exp. 2, 15 lambs (34 [+ or -] 4 kg initial BW) fitted with indwelling catheters in a hepatic vein, the hepatic portal vein, a mesenteric vein, and a mesenteric artery were used. Release of ammonia N by the portal-drained viscera (PDV) was reduced (P = 0.004) in alternate-day-supplemented lambs compared with RDP-D. Consequently, hepatic uptake of ammonia N was least (P = 0.003) in all alternate-day lambs. Alpha-amino nitrogen (AAN) release by the PDV and hepatic uptake of AAN were not affected by treatment or supplementation frequency. Additionally, hepatic output and PDV uptake of urea N were not affected by treatment. Hepatic N uptake (ammonia N + AAN) accounted for urea synthesized by the liver in all treatments; however, hepatic urea synthesis was approximately 4.5-fold less for RUP-A lambs. This suggests that the provision of AA as RUP may provide a delay in ureagenesis, thus altering the timing of N recycling. Key words: lamb, low-quality forage, nitrogen balance, nitrogen recycling, nutrient flux, protein degradability doi: 10.2527/jas.2009-2246

Fouillet H, Juillet B, Gaudichon C, Mariotti F, Tome D, Bos C. Absorption kinetics are a key factor regulating postprandial protein metabolism in response to qualitative and quantitative variations in protein intake. Am J Physiol Regul lntegr Comp Physiol 297: R1691-R1705, 2009. First published October 7, 2009; doi: 10.1152/ajpregu.00281.2009.--We have previously demonstrated that increasing the habitual protein intake widened the gap in nutritional quality between proteins through mechanisms that arc not yet fully understood. We hypothesized that the differences in gastrointestinal kinetics between dietary proteins were an important factor affecting their differential response to an increased protein intake. To test this hypothesis, we built a 13-compartment model providing integrative insight into the sequential dynamics of meal nitrogen (Nm) absorption, splanchnic uptake, and metabolism, and subsequent peripheral transfer and deposition. The model was developed from data on postprandial Nm kinetics in certain accessible pools, obtained from subjects having ingested a [sup.5]N-labeled milk or soy protein meal, after adaptation to normal (NP) or high (HP) protein diets. The faster absorption of Nm after soy vs. milk caused its earlier and stronger splanchnic delivery, which favored its local catabolic utilization (up to +30%) and limited its peripheral accretion (down to -20%). Nm absorption was also accelerated after HP vs. NP adaptation, and this kinetic effect accounted for most of the HP-induced increase (up to +20%) in splanchnic Nm catabolic use, and the decrease (down to -25%) in peripheral Nm anabolic utilization. The HP-induced acceleration in Nm absorption was more pronounced with soy than with milk, as were the HP effects on Nm regional metabolism. Our integrative approach identified Nm absorption kinetics, which exert a direct and lasting impact on Nm splanchnic catabolic use and peripheral delivery, as being critical in adaptation to both qualitative and quantitative changes in protein intake. interorgan amino acid metabolism; milk protein; soy protein; highprotein diets; compartmental model doi: 10.1152/ajpregu.00281.2009

While Eukarya, Bacteria, and Archaea are all capable of protein N glycosylation, the archaeal version of this posttranslational modification is the least understood. To redress this imbalance, recent studies of the halophilic archaeon Haloferax volcanii have identified a gene cluster encoding the Agl proteins involved in the assembly and attachment of a pentasaccharide to select Asn residues of the surface layer glycoprotein in this species. However, because the automated tools used for rapid annotation of genome sequences, including that of H. volcanii, are not always accurate, a reannotation of the agl cluster was undertaken in order to discover genes not previously recognized. In the present report, reanalysis of the gene cluster that includes aglB, aglE, aglF, aglG, aglI, and aglJ, which are known components of the H. volcanii protein N-glycosylation machinery, was undertaken. Using computer-based tools or visual inspection, together with transcriptional analysis and protein expression approaches, genes encoding AglP, AglQ, and AglR are now described.

Copper uptake proteins (CTRs), mediate cellular acquisition of the essential metal copper in all eukaryotes. Here, we report the structure of the human CTR1 protein solved by electron crystallography to an in plane resolution of 7 [Angstrom]. Reminiscent of the design of traditional ion channels, trimeric hCTR1 creates a pore that stretches across the membrane bilayer at the interface between the subunits. Assignment of the helices identifies the second transmembrane helix as the key element lining the pore, and reveals how functionally important residues on this helix could participate in Cu(I)-coordination during transport. Aligned with and sealing both ends of the pore, extracellular and intracellular domains of hCTR1 appear to provide additional metal binding sites. Consistent with the existence of distinct metal binding sites, we demonstrate that hCTR1 stably binds 2 Cu(I)-ions through 3-coordinate Cu-S bonds, and that mutations in one of these putative binding sites results in a change of coordination chemistry. copper homeostasis | electron crystallography | EXAFS | membrane protein

Mono-ADP-ribosylation is a reversible posttranslational modification that modulates the function of target proteins. The enzymes that catalyze this reaction in mammalian cells are either bacterial pathogenic toxins or endogenous cellular ADP-ribosyltransferases. For the latter, both the enzymes and their targets have largely remained elusive, mainly due to the lack of specific techniques to study this reaction. The recent discovery of the macro domain, a protein module that interacts selectively with ADP-ribose, prompted us to investigate whether this interaction can be extended to the identification of ADP-ribosylated proteins. Here, we report that macro domains can indeed be used as selective baits for high-affinity purification of mono-ADP-ribosylated proteins, which can then be identified by mass spectrometry. Using this approach, we have identified a series of cellular targets of ADP-ribosylation reactions catalyzed by cellular ADP-ribosyltransferases and toxins. These proteins include most of the known targets of ADP-ribosylation, indicating the validity of this method, and a large number of other proteins, which now need to be individually validated. This represents an important step toward the discovery of new ADP-ribosyltransferase targets and an understanding of the physiological role and the pharmacological potential of this protein modification. ADP-ribosylation | ADP-ribosyltransferase | NAD | toxin | posttranslational modification

Since maximum anabolism occurs postprandially, we developed a simulated fed state with clamped hyperinsulinemia, physiological hyperglycemia, and hyperaminoacidemia (Hyper-3) and explored muscle cellular mechanisms. Whole body [1-[sup.13]C]leucine and [3-[sup.3]H]glucose kinetics in healthy men were compared between hyperinsulinemic, euglycemic, isoaminoacidemic (Hyper-1, n = 10) and Hyper-3 (n = 9) clamps. In Hyper-3 vs. Hyper-1, nonoxidative leucine [R.sub.d] [rate of disappearance (synthesis)] was stimulated more (45 [+ or -] 4 vs. 24 [+ or -] 4 [micro]mol/min, P < 0.01) and endogenous [R.sub.a] [rate of appearance (breakdown)] was inhibited similarly; hence net balance increased more (86 [+ or -] 6 vs. 49 [+ or -] 2 [micro]mol/min, P < 0.001). Glucose [R.sub.d] was similar; thus Hyper-3 metabolic clearance rate (331 [+ or -] 23 vs. 557 [+ or -] 41 ml/min, P < 0.0005) and [R.sub.d]/insulin (M, 0.65 [+ or -] 0.10 vs. 1.25 [+ or -] 0.10 mg x [min.sup.-1] x [pmol.sup.-1] x 1, P < 0.001) were less, despite higher insulin (798 [+ or -] 74 vs. 450 [+ or -] 24 pmol/l, P < 0.005). In vastus lateralis muscle biopsies, phosphorylation of Akt (P = 0.025), mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p[70.sup.S6K1]; P = 0.008), S6 (P = 0.049), and 4E-binding protein 1 (4E-BP1; P = 0.001) increased. With decreased eukaryotic initiation factor-4E (eIF4E) x 4E-BP1 complex (P = 0.01), these are consistent with increased roTOR complex 1 (mTORC1) signaling and translation initiation of protein synthesis. Although mRNA expression of ubiquitin, MAFbx 1, and MuRF-1 was unchanged, total ubiquitinated proteins decreased 20% (P < 0.01), consistent with proteolysis suppression. The Hyper-3 clamp increases whole body protein synthesis, net anabolism, and muscle protein translation initiation pathways and decreases protein ubiquitination. The main contribution of hyperaminoacidemia is stimulation of synthesis rather than inhibition of proteolysis, and it attenuates the expected increment of glucose disposal. translation initiation; ubiquitin pathway; leucine kinetics; glucose turnover; insulin resistance

Amino acids are known to be anabolic factors that affect protein metabolism, but the response of animals to daily amino acid changes is little understood. We aimed to test the effects of feeding birds with alternations of diets varying in lysine content on the expression of genes related to proteolysis in chicken muscle. Cyclic feeding programs with 2 diets, each given for 24 h during 48-h cycles, were carried out from 10 d of age. Three programs were used: 1) control treatment with continuous distribution of a complete diet containing standard medium lysine level (ML; 11.9 g/kg); 2) alternation of diets with high (HL) and low (LL) lysine levels; 3) alternation of ML and LL diets, where LL = 70%, ML = 100%, HL = 130% of standard lysine level. The Pectoralis major muscles were sampled after 2 wk of cyclic feeding. Measurements included the expression patterns of 6 genes involved in proteolysis, and mammalian target of rapamycin and Forkhead box-O transcription factor (FoxO) signaling. Cathepsin B, m-calpain, and E3 ubiquitin ligases Muscle Ring Finger-1 and Muscle Atrophy F box were significantly overexpressed in chickens transiently fed the LL diet, whereas the mRNA levels of 20S proteasome C2 subunit and ubiquitin remained unchanged. Modifications of E3 ubiquitin ligase expression can be partly explained by significant changes in FoxO phosphorylation with cyclic dietary treatments. Our results suggest timing-sensitive regulation of proteolysis in chicken muscle according to dietary treatment and a high metabolism capacity to compensate for changes in amino acid supply, which might be used for nutritional purposes.

We have combined nonresonant Raman microspectroscopy and spectral imaging with stable isotope labeling by amino acids in cell culture (SILAC) to selectively detect the incorporation of deuterium-labeled phenylalanine, tyrosine, and methionine into proteins in intact, single HeLa cells. The C-D stretching vibrational bands in these amino acids are observed in the 2100-2300 [cm.sup.-1] spectral region that is devoid of vibrational contributions from other, nondeuterated intracellular constituents. We found that incubation with deuterated amino acids for 8 h in cell culture already led to clearly detectable isotope-related signals in Raman spectra of HeLa cells. As expected, the level of isotope incorporation into proteins increased with incubation time, reaching 55% for deuterated phenylalanine after 28 h. Raman spectral imaging of HeLa cells incubated with deuterium-labeled amino acids showed similar spatial distributions for both isotope-labeled and unlabeled proteins, as evidenced by Raman ratio imaging. The SILAC-Raman methodology presented here combines the strengths of stable isotopic labeling of cells with the nondestructive and quantitative nature of Raman chemical imaging and is likely to become a powerful tool in both cell biology applications and research on tissues or whole organisms.

The objective of this study was to investigate the effect of CP intake from 2 grass silage-only diets, differing in CP concentration, fed at similar DMI on the equine colon ecosystem after an abrupt feed change between the diets. Four adult right ventral colon-fistulated geldings were fed one silage-only diet high in CP (HP, 873 g of CP/d) and one diet providing recommended intakes (RP, 615 g of CP/d). An adaptation period of 15 d on either the HP or the RP diet was followed by 2 experimental periods when the diets were fed for 22 d each in a crossover design. Colon samples were taken before and at 4, 12, and 24 h, and at 7, 14, and 22 d after the feed change. During the first 24 h after the abrupt feed change, the concentrations of total anaerobic bacteria and lactobacilli were greater on the HP than the RP diet (7.1 vs. 6.7 [log.sub.10] cfu/mL, P = 0.021, 6.0 vs. 5.5 [log.sub.10] cfu/mL, P = 0.021, respectively). During the first 24 h post feed change, VFA concentrations did not differ between the diets. From 7 to 22 d, total VFA concentrations were greater on the HP diet than on the RP diet (51.8 vs. 45.1 mmol/L, P = 0.034), and colon pH was lower on the HP diet than on the RP diet (6.9 vs. 7.2, P = 0.035). After an adaptation period of 22 d, N, ammonia, and urea concentrations and osmolality of the colon fluid did not differ between diets. Fecal pH and colon and fecal DM were unchanged throughout the experiment. The results suggest that, in horses fed at the maintenance level of energy intake, a feed change between silages with different CP content may alter the colon bacterial counts within the first 24 h. Moreover, during the subsequent 3 wk, pH decreased slightly and VFA concentrations increased, but no other major alterations occurred in the composition and activities of the colon ecosystem or fecal DM. Key words: colon microflora, equine, nitrogen, pH, protein intake, silage

Aging is characterized by a progressive loss of muscle mass that could be partly explained by a defect in the anabolic effect of food intake. We previously reported that this defect resulted from a decrease in the protein synthesis response to leucine in muscles from old rats. Because aging is associated with changes in oxidative status, we hypothesized that reactive oxygen species--induced oxidative damage may be involved in the impairment of the anabolic effect of leucine with age. The present study assessed the effect of antioxidant supplementation on leucine-regulated protein metabolism in muscles from adult and old rats. Four groups of 8- and 20-mo-old male rats were supplemented or not for 7 wk with an antioxidant mixture containing rutin, vitamin E, vitamin A, zinc, and selenium. At the end of supplementation, muscle protein metabolism was examined in vitro using epitrochlearis muscles incubated with increasing leucine concentrations. In old rats, the ability of leucine to stimulate muscle protein synthesis was significantly decreased compared with adults. This defect was reversed when old rats were supplemented with antioxidants. It was not related to increased oxidative damage to 70-kDa ribosomal protein S6 kinase that is involved in amino acid signaling. These effects could be mediated through a reduction in the inflammatory state, which decreased with antioxidant supplementation. Antioxidant supplementation could benefit muscle protein metabolism during aging, but further studies are needed to determine the mechanism involved and to establish if it could be a useful nutritional tool to slow down sarcopenia with longer supplementation.

Ruminally cannulated Rambouillet wether lambs were used in three 6 x 6 Latin square experiments (n = 6/experiment) to determine which essential AA limit N retention. Lambs (BW = 36.9 [+ or -] 1.9 kg for Exp. 1, 35.1 [+ or -] 1.4 kg for Exp. 2, and 46.0 [+ or -] 1.3 kg for Exp. 3) were housed in metabolism crates and limit-fed (DMI = approx. 1.8% of BW daily) twice daily a soybean hull-based diet low in ruminally undegradable protein. Treatments for Exp. 1 were continuous abomasal infusions of a solution (500 mL/d) containing 1) no AA (CON), 2) a mixture of 10 essential AA and 2 nonessential AA (10EAA), 3) 10EAA with Met removed, 4) 10EAA with Lys removed, 5) 10EAA with His removed, and 6) 10EAA with Thr removed. Treatments for Exp. 2 were abomasal infusions of 1) CON, 2) 10EAA, 3) 10EAA with Leu, Ile, and Val removed (-BCAA), 4) 10EAA with Arg removed, 5) 10EAA with Phe removed, and 6) 10EAA with Trp removed. Treatments for Exp. 3 were abomasal infusions of 1) CON, 2) 10EAA, 3) -BCAA, 4) 10EAA with Leu removed, 5) 10EAA with Ile removed, and 6) 10EAA with Val removed. All lambs received continuous infusions of acetate and propionate into the rumen and dextrose into the abomasum to supply additional energy. Periods were 7 d: 3 d for adaptation to abomasally infused treatments and 4 d for fecal and urinary collections. Blood samples were collected 3 h after feeding on d 7. In all 3 experiments, N retention was greater (P < 0.10) for lambs receiving 10EAA vs. CON, demonstrating that the basal AA supply from CON was limiting. Removal of each of the essential AA from 10EAA decreased (P < 0.10) their concentrations in plasma (except for Trp), indicating that 10EAA supplied these AA in excess of the animal's requirement. In Exp. 1, N retention (g/d) decreased (P < 0.10) in response to the removal of Met and Thr, but was not affected by removal of Lys and His from 10EAA. In Exp. 2, N retention decreased (P < 0.10) in response to removal of all 3 branched-chain AA, Arg, and Trp, whereas the removal of Phe from 10EAA did not affect N retention. In Exp. 3, N retention decreased (P < 0.10) in response to removal of branched-chain AA and Val, but was not affected by the omission of Leu and Ile from 10EAA. The results of this research demonstrated that Met, Thr, Arg, Trp, and Val limited N retention of lambs fed a diet low in ruminally undegradable protein. Key words: lamb, limiting amino acid, nitrogen retention

The existing data on the safe upper limits of amino acid intake in humans is essentially observational; how much do individuals ingest and what side effects do they have? There are numerous studies in humans comparing the effects of high doses of amino acids given as protein bound vs. as free amino acids. These studies have shown that protein-bound amino acids have much less effect on plasma levels of the test amino acid, because protein intake stimulates protein synthesis as another sink for the increased amino acid intake. In practice, the highest amino acid intakes occur with free amino acid supplements that may be ingested by athletes who believe that the amino acids will benefit them in training and/or performance. Previously, in a piglet study, we were able to define the point at which maximal phenylalanine oxidation occurred, above which plasma phenylalanine concentration and body balance rose exponentially. We regard this value of maximal disposal (oxidation) of an amino acid as one metabolic marker of the upper limit of intake. Recently, others have demonstrated a similar maximal oxidation rate for leucine in rats. Based on these experimental data and the paucity of published human data in controlled experiments, we think that a systematic approach needs to be undertaken to define the maximal oxidation rate for all dietary indispensable amino acids and other amino acids that may be ingested in excess by humans. We believe that this will provide a rational basis to begin to define the upper limits of tolerance for dietary amino acids. However, some amino acids, such as threonine and methionine, will be more difficult to study, because they have more than 1 route of disposal or very complex metabolic regulation, in which case defining their upper limits will be more multifaceted.

A study was conducted to evaluate the extent and dynamics of whole body protein deposition (Pd) and changes in chemical and physical body composition after a period of AA intake restriction in entire male pigs with high lean-tissue growth potentials. Fifty-eight entire male pigs (initial BW 15.8 [+ or -] 0.9 kg) were allotted to 1 of 3 dietary AA levels between 15 and 38 kg of BW: control (15% above requirements), AA-15% (15% below requirements), and AA-30% (30% below requirements). Thereafter, pigs were fed diets not limiting in AA content. Throughout the experiment, pigs were scale-fed at 90% of estimated voluntary daily DE intake. Representative pigs were slaughtered at 15, 38, 53, 68, or 110 kg of BW to monitor changes in body composition. Between 15 and 38 kg of BW, restriction of AA intake reduced BW gain (P < 0.01; 794, 666, and 648 g/d for control, AA-15%, and AA-30%, respectively). At 38 kg of BW, AA intake restriction increased whole body lipid (LB) content (P < 0.01; 11.3, 14.3, 17.5% of empty BW), and the LB-to-whole body protein (PB) ratio (LB/PB; P < 0.02; 0.68, 0.88, 1.10 for control, AA-15%, and AA-30%, respectively). Relationships between PB versus whole body water and PB versus whole body ash were not affected by dietary treatments (P > 0.10). At 110 kg of BW and based on BW, PB, and LB/PB, complete compensatory growth (CG) was achieved. Body weight gain between 38 and 110 kg of BW was inversely related to previous dietary AA levels (P < 0.01; 1,089, 1,171, and 1,185 g/d for control, AA-15%, and AA-30%, respectively). For pigs on the control diet, and based on N-balance data, Pd increased with BW, from 172 g/d at 40 kg of BW to 226 g/d at 82 kg of BW. At 40 kg of BW, Pd was greater (P < 0.05) for pigs on the AA-15% (205 g/d) and AA-30% (191 g/d) diets than pigs on the control diet (172 g/d). These findings indicate that pigs with high lean-tissue growth potentials are more likely to express compensatory Pd and their genetically determined upper limit to Pd (PdMax) after a period of AA intake restriction. This study confirms previous findings that BW effects on PdMax are small in growing pigs between 40 and 80 kg of BW. It is suggested that CG and compensatory Pd after a period of AA intake restriction is constrained by the pig's PdMax and is driven by a target LB/PB. Combined with previous observations in our laboratory, these results suggest that CG after a period of AA intake restriction tends to occur only when pigs are within the energy-dependent phase of lean-tissue growth and not when the genetically determined upper limit to lean-tissue growth, or PdMax, determines growth performance. Key words: amino acid intake, body composition, compensatory growth, pig

A novel fluorescence polarization assay based on the natural fluorophore epicocconone has been developed. This assay allows the rapid and accurate determination of enzyme kinetic parameters as well as inhibition constants through the measurement of fluorescence anisotropy on the actual substrate of the protease. It takes advantage of epicocconone's ability to reversibly react with proteins to form an internal charge-transfer complex that is highly fluorescent. The protein-substrate is labeled in situ without the need for prior incubation and/or derivatization steps, which saves time and effort compared to methods employing specifically labeled protein-substrates. The assay can be carried out in 96- or 384-well plates, making it suitable for high-throughput applications in drug development and biotechnology.

An on-chip microreactor was proposed toward the acceleration of protein digestion through the construction of a nanozeolite-assembled network. The nanozeolite microstructure was assembled using a layer-by-layer technique based on poly(diallyldimethylammonium chloride) and zeolite nanocrystals. The adsorption of trypsin in the nanozeolite network was theoretically studied based on the Langmuir adsorption isotherm model. It was found that the controlled trypsin-containing nanozeolite networks assembled within a microchannel could act as a stationary phase with a large surface-to-volume ratio for the highly efficient proteolysis of both proteins at low levels and with complex extracts. The maximum proteolytic rate of the adsorbed trypsin was measured to be 350 mM [min.sup.-1] [micro][g.sup.-1], much faster than that in solution. Moreover, due the large surface-to-volume ratio and biocompatible microenvironment provided by the nanozeolite-assembled films as well as the microfluidic confinement effect, the low-level proteins down to 16 fmol per analysis were confidently identified using the as-prepared microreactor within a very short residence time coupled to matrix-assisted laser desorption-time-of-flight mass spectrometry. The on-chip approach was further demonstrated in the identification of the complex extracts from mouse macrophages integrated with two-dimensional liquid chromatography-electrospray ionization-tandem mass spectrometry. This microchip reactor is promising for the development of a facile means for protein identification.

Alfalfa (Medicago sativa L.) proteins ingested by dairy cows typically degrade at rapid rates and exhibit extensive ruminal degradability. Although the effects of conservation method (hay or silage) on these characteristics have been evaluated extensively, agronomic factors, such as harvest timing, have not. Our objective was to quantify rumen degradable protein (RDP) for 'Affinity' alfalfa harvested over a range of ages (0, 5, 10, 15, and 20 d following Stage 2) within each of four harvest periods (spring, early and late summer, and fall). For 2004, there were no interactions (P [greater than or equal to] 0.372) between harvest period and days within harvest period for any protein component. Crude protein (CP), neutral-detergent soluble CP (NDSCP; g [kg.sup.-1] dry matter [DM]), and RDP (g [kg.sup.-1] DM) declined in a quadratic (P [less than or equal to] 0.026) relationship with days following Stage 2. A quadratic (P = 0.002) pattern also was observed for rumen undegradable protein (RUP), but the overall range was small (60.4-66.5 g [kg.sup.-1] DM). On a CP basis, RDP declined linearly (P < 0.001) from 720 to 659 g [kg.sup.-1] CP during 2004. For 2005, there were interactions (P [less than or equal to] 0.020) of harvest period and days within period for all protein-related response variables, but trends over time within each harvest period generally were similar to those observed in 2004. Overall, RDP declined as alfalfa plants aged within harvest period, but these responses were due primarily to reduced concentrations of CP within the cell-soluble fraction.

The effects of the +TIP protein EB1 on microtubule dynamic instability are determined at a steady state in the absence and presence of the CAP-Gly domain of [p150.sup.glued]. It is found that EB1 by itself stabilizes plus ends by suppressing shortening dynamics without significantly affecting growth dynamics.

A study report on the accumulation of lipid derived oxidative protein modifications or isolevuglandin-modified proteins, as also elevated levels of inactive calpain-1, in glaucomatous trabecular meshwork (TM), is presented.

[Zn.sup.2+] ion plays a vital role in stabilising the structure and enhancing the chaperone function of alpha-crystallin. In the absence of protein turnover in the lens, this role of [Zn.sup.2+] is crucial for the long term transparency of the lens.

Surfactant protein A (SP-A) enhances phagocytosis of Pseudomonas aeruginosa. SP-A1 and SP-A2 encode human (h) SP-A; SP-A2 products enhance phagocytosis more than SP-A1. Oxidation can affect SP-A function. We hypothesized that in vivo and in vitro ozone-induced oxidation of SP-A (as assessed by its carbonylation level) negatively affects its function in phagocytosis (as assessed by bacteria cell association). To test this, we used P. aeruginosa, rat alveolar macrophages (AMs), hSP-As with varying levels of in vivo (natural) oxidation, and ozone-exposed SP-A2 (1A, [1A.sup.0]) and SP-A1 (6[A.sup.2], 6[A.sup.4]) variants. SP-A oxidation levels (carbonylation) were measured; AMs were incubated with bacteria in the presence of SP-A, and the phagocytic index was calculated. We found: 1) the phagocytic activity of hSP-A is reduced with increasing levels of in vivo SP-A carbonylation; 2) in vitro ozone exposure of hSP-A decreases its function in a dose-dependent manner as well as its ability to enhance phagocytosis of either gram-negative or gram-positive bacteria; 3) the activity of both SP-A1 and SP-A2 decreases in response to in vitro ozone exposure of proteins with SP-A2 being affected more than SP-A1. We conclude that both in vivo and in vitro oxidative modifications of SP-A by carbonylation reduce its ability to enhance phagocytosis of bacteria and that the activity of SP-A2 is affected more by in vitro ozone-induced oxidation. We speculate that functional differences between SP-A1 and SP-A2 exist in vivo and that the redox status of the lung microenvironment differentially affects function of SP-A1 and SP-A2. air pollution; host defense; carbonylation; macrophage

The characterization of CGFS-type monothiol Grxs of cyanobacterium Synechocystis as a new family of proteins containing a GSH-ligated Fe-S cluster is described. The findings provide insight into the role of CGFS-type monothiol Grxs in Fe-S cluster biogenesis and/or Fe regulation, in model organism as well as humans where deficiency of HsGrx5p generates anemia.

Although often supersaturated with mineral salts such as calcium phosphate and calcium oxalate, normal urine possesses an innate ability to keep them from forming harmful crystals. This inhibitory activity has been attributed to the presence of urinary macromolecules, although controversies abound regarding their role, or lack thereof, in preventing renal mineralization. Here, we show that 10% of the mice lacking osteopontin (OPN) and 14.3% of the mice lacking Tamm-Horsfall protein (THP) spontaneously form interstitial deposits of calcium phosphate within the renal papillae, events never seen in wild-type mice. Lack of both proteins causes renal crystallization in 39.3% of the double-null mice. Urinalysis revealed elevated concentrations of urine phosphorus and brushite (calcium phosphate) supersaturation in THP-null and OPN/ THP-double null mice, suggesting that impaired phosphorus handling may be linked to interstitial papillary calcinosis in THP- but not in OPN-null mice. In contrast, experimentally induced hyperoxaluria provokes widespread intratubular calcium oxalate crystallization and stone formation in OPN/THP-double null mice, while completely sparing the wild-type controls. Whole urine from OPN-, THP-, or double-null mice all possessed a dramatically reduced ability to inhibit the adhesion of calcium oxalate monohydrate crystals to renal epithelial cells. These data establish OPN and THP as powerful and functionally synergistic inhibitors of calcium phosphate and calcium oxalate crystallization in vivo and suggest that defects in either molecule may contribute to renal calcinosis and stone formation, an exceedingly common condition that afflicts up to 12% males and 5% females. kidney stone; urolithiasis; uromodulin