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The high energetic demands and metabolism of amino acids (AA) within the lactating mammary gland have been ascribed to the requirements for milk component synthesis and tissue maintenance. Our objective in this work was to assess rates of protein synthesis from several AA so that the energetic costs of tissue maintenance could be better reflected. Lactating goats (n = 4) were given staggered infusions of 5 labeled forms of phenylalanine (Phe) initiated at 30, 12, 9, 6, and 3 h before goats were killed. [5-[sup.13]C[H.sub.3]] Methionine (Met), [1-[sup.13]C] leucine, and [1-[sup.13]C] valine were also infused for 30 h, during which time, the glands were milked hourly and arteriovenous flux measurements were performed the last 6 h. A dynamic, compartmental model capable of simulating fluxes of AA through extracellular and intracellular free, slow and fast turnover tissue-bound, and milk protein pools was developed and fitted to the observed data. The udder removed 81% of the Phe present in plasma using 31% for milk protein synthesis and releasing 66% back into plasma. Transamination accounted for 40% of Phe flux in the mammary and transmethylation accounted for a portion of mammary Met flux. Mammary tissue protein synthesis was >300% the value of milk protein synthesis with fractional protein synthesis rates >130%/d. Assuming 4 tool of ATP/mol of peptide bond formed, we estimate that ~50% of ATP generated by the lactating mammary glands is used for synthesis of tissue (nonmilk) protein.

This study examined the impact of leucine (Leu) derived from complete meals on stimulation of skeletal muscle protein synthesis (MPS). Expt. 1 examined time course changes in translation initiation and MPS after a meal. Male rats (~300 g) were trained for 5 d to eat 3 meals/d providing 20, 50, and 30% of energy from whey protein, carbohydrates, and fats, respectively. Plasma and skeletal muscle were collected at time 0 (baseline) after 12 h of food ,deprivation and then at 45, 90, 135, 180, and 300 min after a 4-g meal. Plasma Leu increased at 45 rain and remained elevated through 180 min. MPS peaked at 45-90 min and returned to baseline by 180 min. Plasma Leu correlated with phosphorylation of ribosomal protein p70 S6 kinase (r = 0.723; P< 0.05), eukaryotic initiation factor 4E binding protein-1 (r = 0.773; P< 0.05), and MPS (r = 0.608; P < 0.05) over time. Expt. 2 examined 3 levels of protein intake (10, 20, and 30% of energy) from 2 sources (wheat and whey) with different Leu contents (~6.8 and ~10.9%, respectively) on stimulation of initiation and IV!PS. Rats were trained to eat 3 meals/d providing 14, 56, and 30% of energy from protein, carbohydrates, and fats. On d 6, MPS was evaluated at 90 min after rats consumed 1 of the 6 test meals. Whey protein stimulated initiation and MPS more than wheat and the differential response related to greater plasma Leu responses in the whey groups. These studies demonstrate that peak activation but not duration of M PS is proportional to the Leu content of a meal.

At some stage in the origin of life, an informational polymer must have arisen by purely chemical means. According to one version of the 'RNA world' hypothesis (1-3) this polymer was RNA, but attempts to provide experimental support for this have fade (4,5). In particular, although there has been some success demonstrating that 'activated' ribonucleotides can polymerize to form RNA (6,7), it is far from obvious how such ribonucleotides could have formed from their constituent parts (ribose and nucleobases). Ribose is difficult to form selectively (8,9) and the addition of nucleobases to ribose is inefficient in the case of purines (10) and does not occur at all in the case of the canonical pyrimidines (11). Here we show that activated pyrimidine ribonucleotides can be formed in a short sequence that bypasses free ribose and the nucleobases, and instead proceeds through arabinose amino-oxazoline and anhydronucleoside intermediates. The starting materials for the synthesis--cyanamide, cyanoacetylene, glycolaldehyde, glyceraldehyde and inorganic phosphate--are plausible prebiotic feedstock molecules (12-15), and the conditions of the synthesis are consistent with potential early-Earth geochemical models. Although inorganic phosphate is only incorporated into the nucleotides at a late stage of the sequence, its presence from the start is essential as it controls three reactions in the earlier stages by acting as a general acid/base catalyst, a nucleophilic catalyst, a pH buffer and a chemical buffer. For prebiotic reaction sequences, our results highlight the importance of working with mixed chemical systems in which reactants for a particular reaction step can also control other steps., Because they comprise phosphate, ribose and nucleobases, it is tempting to assume that ribonucleotides must have prebiotically assembled from such building blocks. Thus, for example, it has previously been supposed [...]

Stress granules (SGs) arise as a consequence of cellular stress, contain stalled translation preinitiation complexes, and are associated with cell survival during environmental insults. SGs are dynamic entities with proteins relocating into and out of them during stress. Among the repertoire of proteins present in SGs is eukaryotic initiation factor 4E (eIF4E), a translation factor required for cap-dependent translation and that regulates a rate-limiting step for protein synthesis. Herein, we demonstrate that localization of eIF4E to SGs is dependent on the presence of a family of repressor proteins, eIF4E-binding proteins (4E-BPs). Our results demonstrate that 4E-BPs regulate the SG localization of eIF4E. eukaryotic initiation factor 4E; stress granules

The aim of this study was to assess the metabolic fate of AA (endogenous or export protein synthesis, gluconeogenesis, or oxidation) after an imbalanced supply of energy and N in the diet of growing lambs. Eighteen INRA 401 lambs (3 mo old, 29.7 [+ or -] 0.45 kg of BW) were fed 3 experimental diets, one providing a N and energy supply according to recommended allowances (control), one with 23% less N supply relative to energy (LN), and one with 19% less ME supply relative to N (LE). Animals were assigned to 6 blocks of 3, with each animal receiving 1 of the 3 diets, and the animals from each block were slaughtered on the same day. Liver slices from these lambs were incubated in a minimum salt medium (Krebs-Henseleit) containing physiological concentrations of propionate and AA as energy and N sources, similarly across all 3 treatments. Protein synthesis (endogenous and export) using [U-[sup.14]C]valine and [[sup.35]S]methionine, gluconeogenesis from [1-[sup.14]C]propionate and [U-[sup.14]C]alanine, and oxidation were measured. A relative sparing of AA at the liver level was observed with the LN diet because of reduced urinary N (-42%, LN vs. control, P < 0.001). The AA were also directed toward anabolic purposes in the LN diet via an increased endogenous and total export protein synthesis ([+ or -]51%, LN vs. control, P = 0.01; also observed for fibrinogen synthesis, but not for albumin or transferrin) associated with a tendency for increased gluconeogenesis from alanine ([+ or -]58%, LN vs. LE, P = 0.08). On the other hand, the LE diet resulted in a marked loss of N in urine ([+ or -]24%, LE vs. control, P < 0.05), but no notable effect of the LE diet was demonstrated for protein synthesis or gluconeogenesis ex vivo. These data demonstrate a more efficient utilization of AA for anabolic purposes in the lambs fed LN, probably via an activation of some AA transport systems, to address the shortage of nitrogenous nutrients in the LN diet. By contrast, no such adaptation occurred in the LE lambs, probably because the regulatory mechanisms that prevailed in this case were the nutrient supply or hormones, which were not altered in our ex vivo experimental model. Key words: amino acid, ex vivo, gluconeogenesis, lamb, liver, protein synthesis

Pregnane X receptor (PXR) is an important component of the body's adaptive defense system responsible for the elimination of various toxic xenobiotics. PXR activation by endogenous and exogenous chemicals, including steroids, antibiotics, bile acids, and herbal compounds, results in induction of drug metabolism. We investigated the ability of the isoflavones genistein, daidzein, and the daidzein metabolite equol to activate human and mouse PXR in vitro using cell-based transient transfection studies and primary hepatocytes and in vivo in a mouse model. In transient transfection assays, the isoflavones genistein and daidzein activate full-length, wild-type mouse PXR, but not a mutant form, with genistein being the most potent. In contrast, equol was a more potent activator of human PXR than genistein or daidzein. In a mammalian 2-hybrid assay, isoflavones induced recruitment of the coactivator steroid receptor coactivator 1 to PXR. When tested against the native human Cytochrome P450 3A4 (CYP3A4) promoter, equol was the more potent activator and treatment of human hepatocytes with equol increased CYP3A4 mRNA and immunoreactive protein expression. Treatment of wild-type, but not [PXR.sup.-/-], mouse hepatocytes showed that genistein and daidzein induced the expression of Cytochrome P450 3A11 (Cyp3A11) mRNA, whereas equol had no effect. Cyp3A11 mRNA was also induced in vivo in mice fed a soy protein-containing diet. The results presented herein demonstrate that there is a species-specific difference in the activation of PXR by isoflavones and equol.

Food deprivation induces a repression of protein synthesis in skeletal muscle in part due to reduced signaling through the mammalian target of rapamycin complex 1 (mTORC1). Previous studies have identified upregulated expression of the protein Regulated in DNA Damage and Development (REDD1) as an important mechanism in the regulation of mTORC1 activity in response to a variety of stresses. Our goal in this investigation was to determine whether modulation of REDD1 expression occurs in response to food deprivation and refeeding, and, if it does, to ascertain if changes in REDD1 expression correlate with altered mTORC1 signaling. As expected, mTORC1 signaling was repressed after 18 h of food deprivation compared with freely-fed control rats and quickly recovered after refeeding for 45 min. Food deprivation caused a dramatic rise in REDD1 mRNA and protein expression; refeeding resulted in a reduction to baseline. Food deprivation is characterized by low-serum insulin and elevated glucocorticoid concentrations. Therefore, initially, alloxan-induced type I diabetes was used to minimize the food deprivation- and refeeding-induced changes in insulin. Although diabetic rats exhibited upregulated REDD1 expression compared with nondiabetic controls, there was no direct correlation between REDD1 mRNA expression and serum insulin levels, and insulin treatment of diabetic rats did not affect REDD1 expression. In contrast, serum corticosterone levels correlated directly with REDD1 mRNA expression (r = 0.68; P = 0.01). Moreover, inhibiting corticosterone-mediated signaling via administration of the glucocorticoid receptor antagonist RU486 blocked both the food deprivation- and diabetes-induced increase in REDD1 mRNA expression. Overall, the results demonstrate that changes in REDD1 expression likely contribute to the regulation of mTORC1 signaling during food deprivation and refeeding.

Stress-induced phosphorylation of elF2[alpha] inhibits global protein synthesis to conserve energy for repair of stress-induced damage. Stress-induced translational arrest is observed in cells expressing a non-phosphorylatable elF2[alpha] mutant (S51A), which indicates the existence of an alternative pathway of translational control. In this paper, we show that arsenite, heat shock, or ultraviolet irradiation promotes transfer RNA (tRNA) cleavage and accumulation of tRNA-derived, stress-induced small RNAs (tiRNAs). We show that angiogenin, a secreted ribonuclease, is required for stress-induced production of tiRNAs. Knockdown of angiogenin, but not related ribonucleases, inhibits arsenite-induced tiRNA production and translational arrest. In contrast, knockdown of the angiogenin inhibitor RNH1 enhances tiRNA production and promotes arsenite-induced translational arrest. Moreover, recombinant angiogenin, but not RNase 4 or RNase A, induces tiRNA production and inhibits protein synthesis in the absence of exogenous stress. Finally, transfection of angiogenin-induced tiRNAs promotes phospho-elF2[alpha]-independent translational arrest. Our results introduce angiogenin and tiRNAs as components of a phospho-elF2[alpha]-independent stress response program.

Previous studies identified G791 in Escherichia coli 16S rRNA as an invariant residue for ribosome function. In order to establish the functional role of this residue in protein synthesis, we searched for multicopy suppressors of the mutant ribosomes that bear a G-to-U substitution at position 791. We identified relA, a gene whose product has been known to interact with ribosomes and trigger a stringent response. Overexpression of RelA resulted in the synthesis of approximately 1.5 times more chloramphenicol acetyltransferase (CAT) protein than could be synthesized by the mutant ribosomes in the absence of RelA overexpression. The ratio of mutant rRNA to the total ribosome pool was not changed, and the steady-state level of CAT mRNA was decreased by RelA overexpression. These data confirmed that the phenotype of RelA as a multicopy suppressor of the mutant ribosome did not result from the enhanced synthesis of mutant rRNA or CAT mRNA from the plasmid. To test whether the phenotype of RelA was related to the stringent response induced by the increased cellular level of (p)ppGpp, we screened for mutant RelA proteins whose overexpression enhances CAT protein synthesis by the mutant ribosomes as effectively as wild-type RelA overexpression and then screened for those whose overexpression does not produce sufficiently high levels of (p)ppGpp to trigger the stringent response under the condition of amino acid starvation. Overexpression of the isolated mutant RelA proteins resulted in the accumulation of (p)ppGpp in cells, which was amounted to approximately 18.2 to 38.9% of the level of (p)ppGpp found in cells that overexpress the wild-type RelA. These findings suggest that the function of RelA as a multicopy suppressor of the mutant ribosome does not result from its (p)ppGpp synthetic activity. We conclude that RelA has a previously unrecognized role in ribosome function.

The micronutrient selenium is found in proteins as selenocysteine (Sec), the 21st amino acid cotranslationally inserted in response to a UGA codon. In vitro studies in archaea and mouse showed that Sec-[tRNA.sup.sec] formation is a 3-step process starting with serylation of [tRNA.sup.sec] by seryl-tRNA synthetase (SerRS), phosphorylation of serine to form phosphoserine (Sep)-[tRNA.sup.sec] by phosphoseryl[tRNA.sup.sec] kinase (PSTK), and conversion to Sec-[tRNA.sup.sec] by Sep-tRNA:Sec-tRNA synthase (SepSecS). However, a complete study of eukaryotic selenoprotein synthesis has been lacking. Here, we present an analysis of Sec-tRNAsec formation in the parasitic protozoon Trypanosoma bruceiin vivo. Null mutants of either PSTK or SepSecS abolished selenoprotein synthesis, demonstrating the essentiality of both enzymes for Sec-[tRNA.sup.sec] formation. Growth of the 2 knockout strains was not impaired; thus, unlike mammals, trypanosomes do not require selenoproteins for viability. Analysis of conditional RNAi strains showed that SerRS, selenophosphate synthase, and the Sec-specific elongation factor, EFSec, are also essential for selenoprotein synthesis. These results with T. brucei imply that eukaryotes have a single pathway of Sec-tRNAsec synthesis that requires Sep-tRNAsec as an intermediate. phosphoseryl-tRNASec kinase | selenocysteine tRNA | Sep-tRNA:Sec-tRNA synthase | Trypanosoma brucei | selenoprotein

N-glycosylation, a common cotranslational modification, is thought to be critical for plasma membrane expression of glycoproteins by enhancing protein folding, trafficking, and stability through targeting them to the ER folding cycles via lectin-like chaperones. In this study, we show that N-glycans, specifically core glycans, enhance the productive folding and conformational stability of a polytopic membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), independently of lectin-like chaperones. Defective N-glycosylation reduces cell surface expression by impairing both early secretory and endocytic traffic of CFTR. Conformational destabilization of the glycan-deficient CFTR induces ubiquitination, leading to rapid elimination from the cell surface. Ubiquitinated CFTR is directed to lysosomal degradation instead of endocytic recycling in early endosomes mediated by ubiquitin-binding endosomal sorting complex required for transport (ESCRT) adaptors Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and TSG101. These results suggest that cotranslational N-glycosylation can exert a chaperone-independent profolding change in the energetic of CFTR in vivo as well as outline a paradigm for the peripheral trafficking defect of membrane proteins with impaired glycosylation.

Knocking out myostatin activity during development increases the rate of muscle protein synthesis. The present study was done to determine whether postdevelopmental loss of myostatin activity stimulates myofibrillar protein synthesis and the phosphorylation of some of the proteins involved in regulation of protein synthesis rate. Myostatin activity was inhibited for 4 days, in 4- to 5-mo-old male mice, with injections of an anti-myostatin antibody (JA16). The mean myofibrillar synthesis rate increased 19% (P < 0.01) relative to the mean rate in saline-treated mice, as determined by incorporation of deuterium-labeled phenylalanine. JA16 increased phosphorylation of p70 S6 kinase (S6K) and ribosomal protein S6 (rpS6) 1.9-fold (P < 0.05). It did not affect phosphorylation of eukaryotic initiation factor 4E-binding protein-1 or Akt. Microarrays and real-time PCR analyses indicated that JA16 administration did not selectively enrich levels of mRNAs encoding myofibrillar proteins, ribosomal proteins, or translation initiation and elongation factors. Rapamycin treatment did not affect the rate of myofibrillar protein synthesis whether or not the mice received JA16 injections, although it eliminated the phosphorylation of S6K and rpS6. We conclude that the normal level of myostatin activity in mature muscle is sufficient to inhibit myofibrillar synthesis rate and phosphorylation of S6K and rpS6. Reversal of the inhibition of myofibrillar synthesis with an anti-myostatin antibody is not dependent on mTOR activation. rapamycin; mammalian target of rapamycin; Akt; eukaryotic initiation factor 4E-binding protein- 1; translation; JA 16 anti-myostatin antibody

This experiment evaluated the effect of 2 levels of diet concentrate (20 and 40% of DM) and 2 levels of ruminally undegraded protein (RUP: 25 and 40% of CP) on nutrient intake, total and partial apparent nutrient digestibility, microbial protein synthesis, and ruminal and physiological variables. Eight Nellore heifers (233 [+ or -] 14 kg of BW) fitted with ruminal, abomasal, and ileal cannulas were used. The animals were held in individual sheltered pens of approximately 15 [m.sup.2] and fed twice daily at 0800 and 1600 h for ad libitum intake. Heifers were allocated in two 4 x 4 Latin square designs, containing 8 heifers, 4 experimental periods, and 4 treatments in a 2 x 2 factorial arrangement. All statistical analyses were performed using PROC MIXED of SAS. Titanimn dioxide (Ti[O.sub.2]) and chromic oxide ([Cr.sub.2][O.sub.3]) were used to estimate digesta fluxes and fecal excretion. Purine derivative (PD) excretion and abomasal purine bases were used to estimate the microbial N (MN) synthesis. No significant interaction (P > 0.10) between dietary levels of RUP and concentrate was observed. There was no effect of treatment (P = 0.24) on DMI. Both markers led to the same estimates of fecal, abomasal, and ileal DM fluxes, and digestibilities of DM and individual nutrients. Ruminal pH was affected by sampling time (P < 0.001), but no interaction between treatment and sampling time was observed (P = 0.71). There was an interaction between treatment and sampling time (P < 0.001) for ruminal [NH.sub.3]-N concentration. A linear decrease (P = 0.04) over sampling time was observed for the higher level of RUP, whereas a quadratic effect (P < 0.001) of sampling time was observed for the lower level of RUP. The higher level of dietary concentrate led to greater MN yield regardless of the level of RUP. The MN yield and the efficiency of microbial yield estimated from urinary PD excretion produced greater (P < 0.01) values than those estimated by either Ti[O.sub.2] or [Cr.sub.2][O.sub.3], which did not differ (P = 0.63) from each other. However, all methods yielded values that were within the range reported in the literature. In conclusion, no interactions between dietary levels of RUP and concentrate were observed for ruminal and digestive parameters. Neither RUP nor concentrate level affected DMI. Titanium dioxide showed to be similar to [Cr.sub.2][O.sub.3] as an external marker to measure digestibility and nutrient fluxes in cattle. Key words: energy, microbial synthesis, Nellore, protein

The thiazolylpeptides are a family of >50 bactericidal antibiotics that block the initial steps of bacterial protein synthesis. Here, we report a biosynthetic gene cluster for thiocillin and establish that it, and by extension the whole class, is ribosomally synthesized. Remarkably, the C-terminal 14 residues of a 52-residue peptide precursor undergo 13 posttranslational modifications to give rise to thiocillin, making this antibiotic the most heavily posttranslationally-modified peptide known to date. biosynthesis | natural products | thiazolylpeptides

Regulation of RNA polymerase I (Pol I) transcription is critical for controlling ribosome synthesis. Most previous investigations into Pol I transcription regulation have focused on transcription initiation. To date, the factors involved in the control of Pol I transcription elongation are poorly understood. The Paf1 complex (Paf1C) is a well-defined factor that influences polymerase II (Pol II) transcription elongation. We found that Paf1C associates with rDNA. Deletion of genes for Paf1C subunits (CDC73, CTR9, or PAF1) reduces the rRNA synthesis rate; however, there is no significant alteration of rDNA copy number or Pol I occupancy of the rDNA. Furthermore, EM analysis revealed a substantial increase in the frequency of large gaps between transcribing polymerases in ctr9[DELTA] mutant cells compared with WT. Together, these data indicate that Paf1C promotes Pol I transcription through the rDNA by increasing the net rate of elongation. Thus, the multifunctional, conserved transcription factor Paf1C plays an important role in transcription elongation by Pol I in vivo. gene expression | ribosome

Diverse cellular stress responses are linked to phosphorylation of serine 51 on the alpha subunit of translation initiation factor 2. The resultant attenuation of protein synthesis and activation of gene expression figure heavily in the adaptive response to stress, but dephosphorylation of elF2([alpha]P), which terminates signaling in this pathway, is less well understood. GADD34 and CReP, the products of the related mammalian genes Ppp1r15a and Ppp1r15b, can recruit phosphatase catalytic subunits of the PPP1 class to elF2([alpha]P), but the significance of their contribution to its dephosphorylation has not been explored systematically. Here we report that unlike Ppp1r15a mutant mice, which are superficially indistinguishable from wild type, [Ppp1r15b.sup.-/-] mouse embryos survive gestation but exhibit severe growth retardation and impaired erythropoiesis, and loss of both Ppp1r15 genes leads to early embryonic lethality. These loss-of-function phenotypes are rescued by a mutation, [Eif2a.sup.s51A], that prevents regulated phosphorylation of elF2[alpha]. These findings reveal that the essential process of elF2([alpha]P) dephosphorylation is the predominant role of PPP1R15 proteins in mammalian development. mouse genetics | phosphatase regulation | protein synthesis | unfolded protein response

Staphylococcus aureus synthesizes polyglycerol-phosphate lipoteichoic acid (LTA) from phosphatidylglycerol. LtaS, a predicted membrane protein with 5 N-terminal transmembrane helices followed by a large extracellular part (eLtaS), is required for staphylococcal growth and LTA synthesis. Here, we report the first crystal structure of the eLtaS domain at 1.2-A resolution and show that it assumes a sulfatase-like fold with an [alpha]/[beta] core and a C-terminal part composed of 4 anti-parallel [beta]-strands and a long [alpha]-helix. Overlaying eLtaS with sulfatase structures identified active site residues, which were confirmed by alanine substitution mutagenesis and in vivo enzyme function assays. The cocrystal structure with glycerolphosphate and the coordination of a [Mn.sup.2+] cation allowed us to propose a reaction mechanism, whereby the active site threonine of LtaS functions as nucleophile for phosphatidylglycerol hydrolysis and formation of a covalent threonine-glycerolphosphate intermediate. These results will aid in the development of LtaS-specific inhibitors for S. aureus and many other Gram-positive pathogens. Gram-positive | cell wall | enzyme

The objectives of this study were to determine the relative effect of feeding corn silage (CS), fermented whole-crop wheat (FWCW), and urea-treated processed whole-crop wheat (UPWCW) compared with grass silage (GS), each supplemented with concentrates, on forage intake, ruminal fermentation, microbial protein synthesis, some plasma metabolites, and ruminal and total tract digestibility in cattle. Four ruminally fistulated steers with a mean BW of 509 kg (SD 6.3) were used in a 4 x 4 Latin square-designed experiment with each period lasting 21 d. The omasal sampling technique in combination with a triple marker method was used to measure nutrient flows to the omasum with Co-EDTA, Yb acetate, and indigestible NDF as liquid, small particle, and large particle phase markers, respectively. Microbial N flow was assessed from purine base concentrations. Steers fed CS, FWCW, and UPWCW consumed 2.7, 2.4, and 2.6 kg/d more (P < 0.05) forage and total DMI, respectively, than those fed GS-based diets. Rumen pH (P = 0.07) and lactic acid (P = 0.11) concentration did not differ between the forages. Rumen concentration of N[H.sub.3]-N was greatest for UPWCW and least for CS (P < 0.001). Total VFA concentrations were greater (P < 0.05) for CS than GS and UPWCW, with FWCW being intermediate. Acetate-to-propionate ratio (P < 0.05) was greater (P < 0.05) for UPWCW than the other forages, which did not differ. Apparent ruminal digestion of OM (P < 0.05) was less for CS, FWCW, and UPWCW than GS. Ruminal NDF digestibility was greater (P < 0.01) for GS than the other forages, which did not differ (P [greater than or equal to] 0.06). Total tract NDF digestibility was less (P < 0.05) for UPWCW than the other forages, with GS being greatest and CS and FWCW being intermediate. Starch intake was less (P < 0.001) for GS than the other forages, but there was no effect of forage on omasal starch flow (P = 0.23) or ruminal digestibility (P = 0.88). Flow of non-N[H.sub.3]-N and microbial N was greater (P < 0.05) for CS, FWCW, and UPWCW than GS. Efficiency of microbial N synthesis was greater (P < 0.05) for FWCW than GS and CS, with UPWCW being intermediate. Plasma [beta]-hydroxybutyrate concentrations were greatest with CS and least for GS (P < 0.001), whereas concentrations of plasma urea were least for CS and greatest for UPWCW (P < 0.001). In conclusion, feeding alternative forages to GS can significantly increase feed DMI and alter rumen fermentation and site of nutrient digestion when offered to cattle supplemented with 3 kg of concentrate daily. Key words: cattle, corn silage, grass silage, nutrient flow, whole-crop wheat

Protein synthesis in skeletal muscle is known to decrease during exercise, and it has been suggested that this may depend on the magnitude of the relative metabolic stress within the contracting muscle. To examine the mechanisms behind this, the effect of exercise intensity on skeletal muscle eukaryotic elongation factor 2 (eEF2) and eukaryotic initiation factor 4E binding protein 1 (4EBP1) phosphorylation, key components in the mRNA translation machinery, were examined together with AMP-activated protein kinase (AMPK) in healthy young men. Skeletal muscle eEF2 phosphorylation at [Thr.sup.56] increased during exercise but was not influenced by exercise intensity, and was lower than rest 30 min after exercise. On the other hand, 4EBP1 phosphorylation at [Thr.sup.37/46] decreased during exercise, and this decrease was greater at higher exercise intensities and was similar to rest 30 min after exercise. AMPK activity, as indexed by AMPK [alpha]-subunit phosphorylation at [Thr.sup.172] and phosphorylation of the AMPK substrate ACCI[beta] at [Ser.sup.221], was higher with higher exercise intensities, and these indices were higher than rest after high-intensity exercise only. Using immunohistochemistry, it was shown that the increase in skeletal muscle eEF2 [Thr.sup.56] phosphorylation was restricted to type I myofibers. Taken together, these data suggest that the depression of skeletal muscle protein synthesis with endurance-type exercise may be regulated at both initiation (i.e., 4EBP 1) and elongation (i.e., eEF2) steps, with eEF2 phosphorylation contributing at all exercise intensities but 4EBP1 dephosphorylation contributing to a greater extent at high vs. low exercise intensities. protein synthesis; translation; elongation; initiation; AMPK

One of biology's critical ironies is the need to adapt to periods of energy limitation by using the energy-intensive process of protein synthesis. Although previous work has identified the individual energy-requiring steps in protein synthesis, we still lack an understanding of the dependence of protein biosynthesis rates on [ATP] and [GTP]. Here, we used an integrated Escherichia coli cell-free platform that mimics the intracellular, energy-limited environment to show that protein synthesis rates are governed by simple Michaelis-Menten dependence on [ATP] and [GTP] ([K.sub.m.sup.ATP], 27 [+ or -] 4 [micro]M; [K.sub.m.sup.GTP], 14 [+ or -] 2 [micro]M). Although the system-level GTP affinity agrees well with the individual affinities of the GTP-dependent translation factors, the system-level [K.sub.m.sup.ATP] is unexpectedly low. Especially under starvation conditions, when energy sources are limited, cells need to replace catalysts that become inactive and to produce new catalysts in order to effectively adapt. Our results show how this crucial survival priority for synthesizing new proteins can be enforced after rapidly growing cells encounter energy limitation. A diminished energy supply can be rationed based on the relative ATP and GTP affinities, and, since these affinities for protein synthesis are high, the cells can adapt with substantial changes in protein composition. Furthermore, our work suggests that characterization of individual enzymes may not always predict the performance of multicomponent systems with complex interdependencies. We anticipate that cell-free studies in which complex metabolic systems are activated will be valuable tools for elucidating the behavior of such systems.

Tumor necrosis factor (TNF) synthesis is known to play a major part in numerous inflammatory disorders, and multiple transcriptional and post-transcriptional regulatory mechanisms have therefore evolved to dampen the production of this key proinflammatory cytokine (1,2). The high expression of nicotinamide phosphoribosyltransferase (Nampt), an enzyme involved in the nicotinamide-dependent NAD biosynthetic pathway, in cells of the immune system (3) has led us to examine the potential relationship between NAD metabolism and inflammation. We show here that intracellular NAD concentration promotes TNF synthesis by activated immune cells. Using a positive screen, we have identified Sirt6, a member of the sirtuin family (4), as the NAD-dependent enzyme able to regulate TNF production by acting at a post-transcriptional step. These studies reveal a previously undescribed relationship between metabolism and the inflammatory response and identify Sirt6 and the nicotinamide-dependent NAD biosynthetic pathway as novel candidates for immunointervention in an inflammatory setting., NAD is an essential cofactor regulating numerous cellular metabolic pathways that has been recently recognized as a substrate for a growing number of NAD-dependent enzymes with multiple roles in cellular [...]

Previous work showed that sleep is associated with increased brain protein synthesis and that arrest of protein synthesis facilitates sleep. Arrest of protein synthesis is induced during the endoplasmic reticulum (ER) stress response, through phosphorylation of eukaryotic initiation factor 2[alpha] (p-eIF2[alpha]). We tested a hypothesis that elevation of p-eIF2[alpha] would facilitate sleep. We studied the effects of intracerebroventricular infusion of salubrinal (Salub), which increases p-eIF2[alpha] by inhibiting its dephosphorylation. Salub increased deep slow wave sleep by 255%, while reducing active waking by 49%. Delta power within non-rapid eye movement (NREM) sleep was increased, while power in the sigma, beta, and gamma bands during NREM was reduced. We found that Salub increased expression of p-eIF2[alpha] in the basal forebrain (BF) area, a sleep-wake regulatory brain region. Therefore, we quantified the p-eIF2[alpha]-immunolabeled neurons in the BF area: Salub administration increased the number of p-eIF2[alpha]-expressing noncholinergic neurons in the caudal BF. In addition, Salub also increased the intensity of p-eIF2[alpha] expression in both cholinergic and noncholinergic neurons, but this was more widespread among the noncholinergic neurons. Our findings support a hypothesis that sleep is facilitated by signals associated with the ER stress response. protein synthesis inhibition; endoplasmic reticulum stress; phosphorylated eukaryotic initiation factor 2[alpha]; basal forebrain

When gene regulatory networks operate in regimes where the number of protein molecules is so small that the molecular species are on the verge of extinction, the death and resurrection of the species greatly modifies the attractor landscape. Deterministic models and the diffusion approximation to the master equation break down at the limits of protein populations in a way very analogous to the breakdown of geometrical optics that occurs at distances cooperativity | regulation | stochasticity

[beta]-Hydroxy-[beta]-methylbutyrate (HMB; 50 [micro]M) has been shown to attenuate the depression in protein synthesis in murine myotubes in response to lipopolysaccharide (LPS), tumor necrosis factor-[alpha] (TNF-[alpha] with or without interferon-[gamma] (IFN-[gamma]), and angiotensin II (ANG II). The mechanism for the depression of protein synthesis by all three agents was the same and was attributed to activation of double-stranded RNA-dependent protein kinase (PKR) with the subsequent phosphorylation of eukaryotic initiation factor 2 (eIF2) on the [alpha]-subunit as well as increased phosphorylation of the elongation factor (eEF2). Myotubes expressing a catalytically inactive PKR variant, PKR[DELTA]6, showed no depression of protein synthesis in response to either LPS or TNF-[alpha], confirming the importance of PKR in this process. There was no effect of any of the agents on phosphorylation of mammalian target of rapamycin (mTOR) or initiation factor 4E-binding protein (4E-BP1), and thus no change in the amount of eIF4E bound to 4E-BP1 or the concentration of the active eIF4E x eIF4G complex. HMB attenuated phosphorylation of eEF2, possibly by increasing phosphorylation of mTOR, and also attenuated phosphorylation of eIF2[alpha] by preventing activation of PKR. These results suggest that HMB may be effective in attenuating muscle atrophy in a range of catabolic conditions. eukaryotic initiation factor 2[alpha]

This experiment was conducted to investigate the effect of lemongrass [Cymbopogon citratus (DC.) Stapf.] powder (LGP) on rumen ecology, rumen microorganisms, and digestibility of nutrients. Four ruminally fistulated crossbred (Brahman native) beef cattle were randomly assigned according to a 4 x 4 Latin square design. The dietary treatments were LGP supplementation at 0, 100, 200, and 300 g/d with ureatreated rice straw (5%) fed to allow ad libitum intake. Digestibilities of DM, ether extract, and NDF were significantly different among treatments and were greatest at 100 g/d of supplementation. However, digestibility of CP was decreased with LGP supplementation (P < 0.05), whereas ruminal N[H.sub.3]-N and plasma urea N were decreased with incremental additions of LGP (P < 0.05). Ruminal VFA concentrations were similar among supplementation concentrations (P > 0.05). Total viable bacteria, amylolytic bacteria, and cellulolytic bacteria were significantly different among treatments and were greatest at 100 g/d of supplementation (4.7 x [10.sup.9], 1.7 x [10.sup.7], and 2.0 x [10.sup.9] cfu/mL, respectively). Protozoal populations were significantly decreased by LGP supplementation. In addition, efficiency of rumen microbial N synthesis based on OM truly digested in the rumen was enriched by LGP supplementation, especially at 100 g/d (34.2 g of N/kg of OM truly digested in the rumen). Based on this study, it could be concluded that supplementation of LGP at 100 g/d improved digestibilities of nutrients, rumen microbial population, and microbial protein synthesis efficiency, thus improving rumen ecology in beef cattle. Key words: lemongrass powder, manipulation, microbial protein synthesis, rumen ecology, rumen fermentation, supplementation

Translocation during the elongation phase of protein synthesis involves the relative movement of the 30S and 50S ribosomal subunits. This movement is the target of tuberactinomycin antibiotics. Here, we describe the isolation and characterization of mutants of Thermus thermophilus selected for resistance to the tuberactinomycin antibiotic capreomycin. Two base substitutions, A1913U and mU1915G, and a single base deletion, [DELTA]mU1915, were identified in helix 69 of 23S rRNA, a structural element that forms part of an interribosomal subunit bridge with the decoding center of 16S rRNA, the site of previously reported capreomycin resistance base substitutions. Capreomycin resistance in other bacteria has been shown to result from inactivation of the TlyA methyltransferase which 2'-O methylates C1920 of 23S rRNA. Inactivation of the tlyA gene in T. thermophilus does not affect its sensitivity to capreomycin. Finally, none of the mutations in helix 69 interferes with methylation at C1920 or with pseudouridylation at positions 1911 and 1917. We conclude that the resistance phenotype is a consequence of structural changes introduced by the mutations.

OBJECTIVE: To investigate the clinical importance of cytokines, surfactant protein D (SFTPD, formerly SP-D), and Kerbs von Lungren 6 antigen (KL-6) in bronchoalveolar lavage fluid (BALF) and blood in patients [...]

We have studied the role of the pga1 gene of Penicillium chrysogenum, encoding the alpha subunit of a heterotrimeric G protein, in secondary metabolite production. The dominant activating [pga1.sup.G42R] mutation caused an increase in the production of the three secondary metabolites penicillin, the yellow pigment chrysogenin and the mycotoxin roquefortine, whereas the dominant inactivating [pga1.sup.G203R] allele and the deletion of the pga1 gene resulted in a decrease of the amount of produced penicillin and roquefortine. Chrysogenin is produced in solid medium as a yellow pigment, and its biosynthesis is clearly enhanced by the presence of the dominant activating [pga1.sup.G42R] allele. Roquefortine is produced associated with mycelium during the first 3 days in submerged cultures, and is released to the medium afterwards; dominant activating and inactivating pga1 mutations result in upregulation and downregulation of roquefortine biosynthesis recpectively. Pga1 regulates penicillin biosynthesis by controlling expression of the penicillin biosynthetic genes; the three genes pcbAB, pcbC and penDE showed elevated transcript levels in transformants expressing the [pga1.sup.G42R] allele, whereas in transformants with the inactivating [pga1.sup.G203R] allele and in the pga1-deleted mutant their transcript levels were lower than those in the parental strains. Increase of intracellular cAMP levels had no effect on penicillin production. In summary, the dominant activating [pga1.sup.G42R] allele upregulates the biosynthesis of three secondary metabolites in Penicilfium chrysogenum to a different extent.

Elsinochromes are nonhost-selective, light-activated, polyketide-derived toxins produced by many phytopathogenic Elsinoe species. We recently showed that the polyketide synthase-encoding gene EfPKS1 is essential for elsinochrome biosynthesis in the citrus scab fungus Elsinoe fawcettii. Sequence analysis beyond the EfPKS1 gene identified nine putative ORFs: four genes, designated RDT1, TSF1, PRF1 and ECT1, all encode polypeptides likely to have biosynthetic or efflux functions; five additional genes, OXR1 and EfHP1 to EfHP4, encode hypothetical proteins of unknown function. Northern-blot analysis revealed that expression of these genes in E. fawcettii was not completely correlated with accumulation of elsinochromes under nitrogen limitation, alkaline pH or high concentrations of glucose. Targeted disruption of the TSF1 gene, encoding a putative transcriptional activator, yielded fungal mutants unable to produce elsinochromes, and defective in both conidiation and expression of RDT1, EfPKS1, PRF1 and EfHP1, whereas expression of RDT1, TSF1, PRF1 and ECT1 was nearly abolished in EfPKS1-disrupted mutants. By contrast, expression of QXR1, EfHP2 and EfHP3 was not affected by disrupting either EfPKS1 or TSF1. Taken together, the results indicate that in addition to polyketide synthase, the products of TSF1 and other adjacent genes may also play a crucial role in elsinochrome production.

Transcription-induced hypernegative supercoiling is a hallmark of Escherichia coli topoisomerase I (topA) mutants. However, its physiological significance has remained unclear. Temperature downshift of a mutant yielded transient growth arrest and a parallel increase in hypernegative supercoiling that was more severe with lower temperature. Both properties were alleviated by overexpression of RNase HI. While ribosomes in extracts showed normal activity when obtained during growth arrest, mRNA on ribosomes was reduced for fis and shorter for crp, polysomes were much less abundant relative to monosomes, and protein synthesis rate dropped, as did the ratio of large to small proteins. Altered processing and degradation of lacA and fis mRNA was also observed. These data are consistent with truncation of mRNA during growth arrest. These effects were not affected by a mutation in the gene encoding RNase E, indicating that this endonuclease is not involved in the abnormal mRNA processing. They were also unaffected by spectinomycin, an inhibitor of protein synthesis, which argued against induction of RNase activity. In vitro transcription revealed that R-loop formation is more extensive on hypernegatively supercoiled templates. These results allow us, for the first time, to present a model by which hypernegative supercoiling inhibits growth. In this model, the introduction of hypernegative supercoiling by gyrase facilitates degradation of nascent RNA; overproduction of RNase HI limits the accumulation of hypernegative supercoiling, thereby preventing extensive RNA degradation.

We tested the relative ability of rapidly digested whey and slowly digested casein to stimulate net whole-body protein synthesis during prolonged physical inactivity. We studied 8 young male volunteers after they consumed isonitrogenous casein or whey mixed meals on d 12 or d 14 of experimental bed rest. Rates of phenylalanine hydroxylation were measured by primed-constant oral administration of L[2-[sup.2][H.sub.2]]tyrosine and L[ring-[sup.2][H.sub.5]]phenylalanine for 3 h in the postabsorptive state and 6 h after an isonitrogenous bolus meal containing sucrose (0.27 g/kg) and casein or whey (0.40 g/kg). Net protein synthesis in the fed state was calculated during the first 6 h postmeal as the difference between phenylalanine hydroxylation and phenylalanine content in the ingested casein or whey. In the fed state, the integrated changes in phenylalanine hydroxylation were lower (P < 0.05) after whey (-2 [+ or -] 8 [micro]mol x [kg.sup.-1] x 6 [h.sup.-1]) than after casein ingestion (34 [+ or -] 7 [micro]mol x [kg.sup.-1] x 6 [h.sup.-1]), During bed rest, net postprandial protein synthesis was greater (P < 0.05) after whey (96 [+ or -] 8 [micro]mol phenylalanine x [kg.sup.-1] x 6 [h.sup.-1]) than after casein ingestion (82 [+ or -] 7 [micro]mol phenylalanine x [kg.sup.-1] x 6 [h.sup.-1]). The rapidly digested whey protein was more efficient than the slowly digested casein in increasing postprandial net protein synthesis during short-term bed rest.

We investigated the effect of carbohydrate and protein hydrolysate ingestion on whole-body and muscle protein synthesis during a combined endurance and resistance exercise session and subsequent overnight recovery. Twenty healthy men were studied in the evening after consuming a standardized diet throughout the day. Subjects participated in a 2-h exercise session during which beverages containing both carbohydrate (0.15 g x [kg.sup.-1] x [h.sup.-1]) and a protein hydrolysate (0.15 g x [kg.sup.-1] x [h.sup.-1])) (C+ P, n = 10) or water only (W, n = 10) were ingested. Participants consumed 2 additional beverages during early recovery and remained overnight at the hospital. Continuous i.v. infusions with L-[ring-[sup.13][C.sub.6]]-phenylalanine and L-[ring-[sup.2][H.sub.2]]-tyrosine were applied and blood and muscle samples were collected to assess whole-body and muscle protein synthesis rates. During exercise, whole-body and muscle protein synthesis rates increased by 29 and 48% with protein and carbohydrate coingestion (P < 0.05). Fractional synthetic rates during exercise were 0.083 [+ or -] 0.011%/h in the C+P group and 0.056 [+ or -] 0.003%/h in the W group, (P < 0.05). During subsequent overnight recovery, whole-body protein synthesis was 19% greater in the C+P group than in the W group (P < 0.05). However, mean muscle protein synthesis rates during 9 h of overnight recovery did not differ between groups and were 0.056 [+ or -] 0.004%/h in the C+P group and 0.057 [+ or -] 0.004%/h in the W group (P = 0.89). We conclude that, even in a fed state, protein and carbohydrate supplementation stimulates muscle protein synthesis during exercise. Ingestion of protein with carbohydrate during and immediately after exercise improves whole-body protein synthesis but does not further augment muscle protein synthesis rates during 9 h of subsequent overnight recovery.

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.

Here, we present two bifunctional protein building blocks that coassemble to form a bioelectrocatalytic hydrogel that catalyzes the reduction of dioxygen to water. One building block, a metalIopolypeptide based on a previously designed triblock polypeptide, is electron-conducting. A second building block is a chimera of artificial [alpha]-helical leucine zipper and random coil domains fused to a polyphenol oxidase, small laccase (SLAC). The metal-lopolypeptide has a helix-random-helix secondary structure and forms a hydrogel via tetrameric coiled coils. The helical and random domains are identical to those fused to the polyphenol oxidase. Electron-conducting functionality is derived from the divalent attachment of an osmium bis-bipyrdine complex to histidine residues within the peptide. Attachment of the osmium moiety is demonstrated by mass spectroscopy (MS-MALDI-TOF) and cyclic voltammetry. The structure and function of the [alpha]-helical domains are confirmed by circular dichroism spectroscopy and by theological measurements. The metallopolypeptide shows the ability to make electrical contact to a solid-state electrode and to the redox centers of modified SLAC. Neat samples of the modified SLAC form hydrogels, indicating that the fused a-helical domain functions as a physical cross-linker. The fusion does not disrupt dimer formation, a necessity for catalytic activity. Mixtures of the two building blocks coassemble to form a continuous supramolecular hydrogel that, when polarized, generates a catalytic current in the presence of oxygen. The specific application of the system is a biofuel cell cathode, but this protein-engineering approach to advanced functional hydrogel design is general and broadly applicable to biocatalytic, biosensing, and tissue-engineering applications. biocatalysis | biofuel cell | biomaterial | laccase | protein

Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were maintained at fasting levels. This study aimed to determine whether the pST-induced increase in insulin promotes skeletal muscle protein synthesis when amino acids are provided at fed levels and whether the response is associated with enhanced translation initiation factor activation. Growing pigs were treated with pST (0 or 180 [micro]g x [kg.sup.-1] x [day.sup.-1]) for 7 days, and then pancreatic-glucose-amino acid clamps were performed. Amino acids were raised to fed levels in the presence of either fasted or fed insulin concentrations; glucose was maintained at fasting throughout. Muscle protein synthesis was increased by pST treatment and by amino acids (with or without insulin) (P < 0.001). In pST-treated pigs, fed, but not fasting, amino acid concentrations further increased muscle protein synthesis rates irrespective of insulin level (P < 0.02). Fed amino acids, with or without raised insulin concentrations, increased the phosphorylation of S6 kinase (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1), decreased inactive 4EBP1 x eIF4E complex association, and increased active eIF4E x eIF4G complex formation (P < 0.02). pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of muscle protein synthesis requires fed amino acid levels, but not fed insulin levels. However, under the current conditions, the response to amino acids is not mediated by the activation of translation initiation factors that regulate mRNA binding to the ribosomal complex. translation initiation; growth hormone; mammalian target of rapamycin; eukaryotic initiation factor 4G

Biochemically, one-third of the collagen molecule is composed of glycine. The next largest amino acid component is formed by proline (PRO) and hydroxyproline, which together comprise ~23% of the collagen molecule. The best method to support wound collagen biosynthesis is to provide adequate host nutrition, assuring adequate provision of calories and protein. However, despite adequate nutrition, clinically, there is a need to enhance collagen synthesis and research has focused on methods to enhance collagen precursor availability. PRO biosynthesis is related to both the citric acid cycle and the urea cycle. During the early phases of wound healing, wound fluid PRO levels are at least 50% higher than plasma levels, suggesting active import of PRO into the wound. Providing additional PRO in the diet to enhance PRO bioavailability for collagen biosynthesis does not result in increased collagen accumulation. Provision of other citric cycle precursors such as glutamine also does not enhance wound collagen synthesis. In looking at other PRO biosynthetic pathways, the arginine (ARG) [right arrow] ornithine (ORN) [right arrow] glutamic semialdehyde [right arrow] PRO pathway looks the most promising. ARG administration in quantities above those required for growth and reproduction results in a marked enhancement in wound collagen deposition. This effect is also shared by ORN, which cannot replace ARG for growth requirement but shares many of its biological and pharmacological activities. Several mechanisms have been postulated to explain the positive effect of ARG on wound healing, although none have been firmly proven. In conclusion, ARG and ORN supplementation are most effective in increasing collagen deposition, but whether this is accomplished by conversion to PRO is uncertain.

Fractional protein synthesis rates (FSR) are widely measured by the flooding dose technique via either an i.g. or an i.v. route. This study was conducted to compare differences in tracer incorporation and FSR in organs and tissues of fed piglets. The piglets were surgically implanted with catheters and randomly assigned to receive a flooding dose of Phe (1.5 mmol/kg body weight, 40 percent molar enrichment with [[sup.2][H.sub.5]]Phe) in saline administered via an i.p. or an i.v. route. [[sup.2][H.sub.5]]Phe free-pool enrichment in plasma increased logarithmically (P < 0.05) from 0 to 25% in the i.p. group, whereas it rose to a peak level within 3 min of the tracer injection and then decreased linearly (P < 0.05) in the i.v. group. Intracellular free-pool tracer enrichments in organs and tissues were within the range of the values measured for the plasma-free pool (25-27%), reaching the flooding status. Administration of the tracer via the i.p. and i.v. routes induced a logarithmical pattern (P < 0.05) of a surge in plasma cortisol concentrations within 30 min. Measurements of FSR in plasma, cardiac muscle, and skeletal muscles were lower (P < 0.05) in the i.p. than in the i.v. group due to the adverse effect of cortisol surge being more dramatic (P < 0.05) in the i.p. than in the i.v. group at 30 min of the post-tracer administration. We conclude that FSR may be measured by the flooding dose through an i.p. or an i.v. route and the i.p. route may underestimate FSR by the flooding dose for plasma, cardiac muscle, and skeletal muscles. This concern may be addressed by a fast regimen of sampling to be completed within 12-20 min after an i.p. route of tracer injection.

Several stress conditions are characterized by activation of 5'-AMP-activated protein kinase (AMPK) and the development of leucine resistance in skeletal muscle. In the present study, we determined whether direct activation of the AMPK by 5-aminoimidazole-4-carboxamide-1-[beta]-D-ribonucleoside (AICAR) prevents the characteristic leucine-induced increase in protein synthesis by altering mammalian target of rapamycin (mTOR) signal transduction. Rats were injected with AICAR or saline (Sal) and 1 h thereafter received an oral gavage of leucine (or Sal). Efficacy of AICAR was verified by increased AMPK phosphorylation. AICAR decreased basal in vivo muscle (gastrocnemius) protein synthesis and completely prevented the leucine-induced increase, independent of a change in muscle adenine nucleotide concentration. AICAR also prevented the hyperphosphorylation of eukaryotic initiation factor (elF) 4E binding protein (4E-BP1), ribosomal protein S6 kinase (S6K1), S6, and elF4G in response to leucine, suggesting a decrease in mTOR activity. Moreover, AICAR prevented the leucine-induced redistribution of elF4E from the inactive elF4E x 4E-BP1 to the active elF4E x elF4G complex. This ability of AICAR to produce muscle leucine resistance could not be attributed to a change in phosphorylation of tuberous sclerosis complex (TSC)2, the formation of a TSC1 x TSC2 complex, the binding of raptor with mTOR, or the phosphorylation of eukaryotic elongation factor-2. However, the inhibitory actions of AICAR were associated with reduced phosphorylation of proline- rich Akt substrate-40 and increased phosphorylation of raptor, which represent potential mechanisms by which AICAR might be expected to inhibit leucine-induced increases in mTOR activity and protein synthesis under in vivo conditions.

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.

Branched-chain amino acids are the most abundant amino acids in proteins. The Bacillus subtilis ilv-leu operon is involved in the biosynthesis of branched-chain amino acids. This operon exhibits a RelA-dependent positive stringent response to amino acid starvation. We investigated this positive stringent response upon lysine starvation as well as decoyinine treatment. Deletion analysis involving various lacZ fusions revealed two molecular mechanisms underlying the positive stringent response of ilv-leu, i.e., CodY-dependent and -independent mechanisms. The former is most likely triggered by the decrease in the in vivo concentration of GTP upon lysine starvation, GTP being a corepressor of the CodY protein. So, the GTP decrease derepressed ilv-leu expression through detachment of the CodY protein from its cis elements upstream of the ilv-leu promoter. By means of base substitution and in vitro transcription analyses, the latter (CodY-independent) mechanism was found to comprise the modulation of the transcription initiation frequency, which likely depends on fluctuation of the in vivo RNA polymerase substrate concentrations after stringent treatment, and to involve at least the base species of adenine at the 5' end of the ilv-leu transcript. As discussed, this mechanism is presumably distinct from that for B. subtilis rrn operons, which involves changes in the in vivo concentration of the initiating GTP.

BACKGROUND: Exposure to excessive amounts of fluoride ([F.sup.-]) cause dental fluorosis in susceptible individuals; however, the mechanism of ([F.sup.-]) induced toxicity is unclear. Previously, we have shown that high-dose F.sup.-]) [...]

Six rumen-fistulated Merino sheep were used in a crossover design experiment to evaluate the effects of an exogenous fibrolytic enzyme preparation (12 g/d; ENZ), delivered directly into the rumen, on diet digestibility, ruminal fermentation, and microbial protein synthesis. The enzyme contained endoglucanase and xylanase activities. Sheep were fed a mixed grass hay:concentrate (70:30; DM basis) diet at a daily rate of 46.1 g/kg of [BW.sup.0.75.] Samples of grass hay were incubated in situ in the rumen of each sheep to measure DM and NDF degradation. The supplementation with ENZ did not affect diet digestibility (P = 0.30 to 0.66), urinary excretion of purine derivatives (P = 0.34), ruminal pH (P = 0.46), or concentrations of N[H.sub.3]-N (P = 0.69) and total VFA (P = 0.97). In contrast, molar proportion of propionate were greater (P = 0.001) and acetate: propionate ratio was lower (P < 0.001) in ENZ-supplemented sheep. In addition, ENZ supplementation tended to increase (P = 0.06) numbers of cellulolytic bacteria at 4 h after feeding. Both the ruminally insoluble potentially degradable fraction of grass hay DM and its fractional rate of degradation were increased (P = 0.002 and 0.05, respectively) by ENZ treatment. Supplementation with ENZ also increased (P = 0.01 to 0.02) effective and potential degradability of grass hay DM and NDF. Ruminal fluid endoglucanase and xylanase activities were greater (P < 0.001 and 0.03, respectively) in ENZ-supplemented sheep than in control animals. It was found that ENZ supplementation did not affect either exoglucanase (P = 0.12) or amylase (P = 0.83) activity. The results indicate that supplementing ENZ directly into the rumen increased the fibrolytic activity and stimulated the growth of cellulolytic bacteria without a prefeeding feed-enzyme interaction. Key words: enzymatic activity, fibrolytic enzyme, microbial protein synthesis, ruminal fermentation

Two trials were conducted to evaluate the effect of rumen-degradable protein balance (OEB) deficit on voluntary intake (trial 1), microbial protein synthesis, and N metabolism (trial 2) in growing double-muscled Belgian Blue bulls. In trial 1, six bulls (339 [+ or -] 26 kg of initial BW) were used in a replicated 3 x 3 Latin square and received a diet of 60% corn silage and 40% concentrate with ad libitum intake (DM basis). Three concentrates were formulated by adding urea at the expense of barley to give similar dietary contents of intestinal digestible proteins, NE for fattening, and fermentable OM, but with different levels of OEB. Thus, 2 levels of OEB deficit (-23.7 and -9.2 g of OEB/kg of DM) were compared with a diet providing a slight OEB surplus (5.3 g of OEB/kg of DM). Voluntary DMI decreased linearly (P = 0.02) with decreasing rumen-degradable protein balance. This decrease in intake could explain the linear decrease in ADG observed when negative OEB diets were fed. In trial 2, six bulls (304 [+ or -] 12 kg of initial BW) with cannulas in the rumen and proximal duodenum were used in a replicated 3 x 3 Latin square and fed diets similar to those used in trial 1 at an intake level of 85 g of DM/kg of [BW.sup.0.75]. Diurnal variations of ruminal [NH.sub.3]-N and plasma urea-N concentrations were greatly influenced by the level of OEB in the diet. No differences in NDF and starch degradation in the rumen, microbial N flow at the duodenum, or efficiency of microbial protein synthesis in the rumen were noted among the levels of OEB in diets. The reductions of the OEB value from 5.3 g/kg of DM to -9.2 g/ kg of DM and -23.7 g/kg of DM were associated with reductions of 26.5 and 48.8% in urinary N output. Absolute amounts of N retained by the bulls increased significantly with the level of OEB in diets. Indeed, 51.4% of the incremental supply of N was excreted between -23.7 and -9.2 g of OEB/kg of DM diets, and 74.6% of the incremental supply of N was excreted between -9.2 and 5.3 g of OEB/kg of DM diets. Feeding diets characterized by an adequate intestinal digestible protein supply and a OEB close to -10 g of OEB/kg of DM could be a feeding strategy to reduce N losses from the farm with little effect on the animal performance and voluntary intake. Reduced OEB may reduce N excretion in the environment but may also result in decreased N retention. Key words: bull, nitrogen utilization, rumen-degradable protein balance, rumen fermentation, voluntary intake