The nutritional status, especially circulating amino acid (AA) levels, can drastically change during a non-infectious (i.e., LPS) or infectious (e.g., Salmonella) challenge. Thus, study 1 examined the effect of LPS treatment (N = 9, 26.9 ± 1.07 kg BW) on plasma AA and metabolite levels in pigs. Data were used to generate prediction equations establishing mathematical relationships between plasma AA levels and numerous blood metabolites (e.g., total lipid, LDL, HDL, blood urea nitrogen, etc). These equations have the potential to improve the nutritional treatment and recovery of acute and chronically ill patients. Study 2 (19.1 ± 0.37 kg) was a continuation of study 1 except the sampling time was increased from 12 to 24 h. One-half of the pigs in study 2 were treated with LPS (N=15) and the other one-half were saline treated control animals (N = 16). This design allows for monitoring changes in plasma AA and their catabolism in response to endotoxemia. Area under the curve (AUC) was calculated for a selected AA to report AA balances. During the induction phase of an acute challenge (t = -2 to 12 h), analyzed AA were in a negative balance indicating heavy AA catabolism. However, during the recovery phase (t = 12 to 24 h) half of the AA were in a positive balance while the other half were still negative. The ability of equations to accurately predict AA concentrations was tested. Results indicate poor performance possibly due to heavy term biases. Thus, it was concluded that equations need to be revisited and non-linear terms need to be evaluated. Nonetheless, routine clinical blood metabolites can be used to estimate plasma AA levels during immune activation. We successfully established a porcine Salmonella enterica serovar Typhimurium model. Pigs infected with Salmonella had a febrile response for 4 d and exhibited marked changes in their fecal bacterial populations Finally, we investigated plasma changes in N-τ-methyl histidine (NτMH) in healthy and LPS-treated pigs. NτMH— is a post-translationally modified AA that has historically been used as an indirect marker of muscle protein breakdown in rodents and humans. However, the major form (i.e., free or acetylated) of NτMH in pig plasma was unknown. Results indicate that only 15% of plasma NτMH is in the free form and the remainder is acetylated. Furthermore, LPS treated pigs had increased acetylated and total NτMH fractions while free NτMH did not change. Therefore, to accurately monitor plasma changes in NτMH as an indicator of muscle proteolysis, plasma samples must be subjected to acid hydrolysis. / Ph. D.

The esterification of amino acids to the tRNA-like structures of viral genomic RNA was studied as catalyzed by amino acyl tRNA synthetases from E. coli, yeast, bean and rat. Peanut stunt virus RNA was esterified with tyrosine in the presence of bean tyrosyl-tRNA synthetase to a maximum of 22 mole percent (average MW = 1 x 10⁶ ). The rate of TYMV-RNA aminoacylation was extensively studied. In the presence of enzymes from E. coli, yeast and bean, the rate of aminoacylation was inhibited 4 fold by 47.5 mM KCl. In the presence of rat enzyme a 1.5 fold increase in rate was observed. In identical studies using tRNA, added KCl generally favored aminoacylation of tRNA by the homologous enzyme but disfavored heterologous reactions. The kinetic parameters (K_m and V_max of TYMV-RNA aminoacylation in the presence of valyl-tRNA synthetases from the four sources were determined and compared to results obtained for yeast and E. coli tRNA controls. TYMV-RNA was found to be a uniquely competent and versatile substrate as compared to tRNA. The K_m of yeast valyl-tRNA synthetase for TYMV-RNA (7 nM) is the second lowest K_m reported for any aminoacyl tRNA synthetase. Kinetic studies using TYMV-RNA with or without the 3' terminal AMP indicate that this AMP is involved in the binding of TYMV-RNA to yeast valyl-tRNA synthetase. / Master of Science

Dairy cows are extremely inefficient at converting dietary nitrogen (N) to productive N. Approximately 25-30% of dietary N is used for milk protein while the remaining N is lost to the environment. According to National Research Council (NRC, 2001) recommendations, dairy cow rations are formulated in terms of metabolizable protein (MP) which often causes many amino acids (AA) to be fed in excess. A better understanding of protein and AA requirements could help to improve the nitrogen efficiency of dairy cows. The objective of this work was to examine the effects of feeding a low protein diet supplemented with rumen protected (RP) AA on production and N efficiency of dairy cows. Twenty-four Holstein and 24 Holstein x Jersey crossbred cows were used in a Youden square design consisting of 3 periods. Cows were randomly assigned to one of 8 treatments: 1) a standard diet containing 17% crude protein (+Con), 2) a 15% crude protein diet (-Con), 3) â Con plus RP methionine (+M, 16g/d), 4) â Con plus RP lysine (+K, 47g/d), 5) â Con plus RP leucine (+L, 181g/d), 6) â Con plus RP methionine and lysine (+MK), 7) â Con plus RP methionine and leucine (+ML), and 8) â Con plus RP methionine, lysine, and leucine (+MKL). Cows fed the â Con as well as the +MKL diet experienced a reduction in milk production and milk protein yield (P < 0.05). Dry matter intake decreased only for those animals on the +ML diet (P < 0.05). Milk urea N (MUN) decreased for all diets when compared to the +Con treatment (P < 0.05). In accordance with the decrease in MUN, N efficiency was numerically increased in the diets supplemented with RP AA, but this improvement was not significant. Phosphorylation of signaling proteins important for protein synthesis were also examined. Animals fed the +MK treatment increased phosphorylated and total forms of eukaryotic elongation factor 2 (eEF2) when compared to the +Con and â Con (P < 0.05), but this increase in abundance did not affect the ratio of phosphorylated to total abundance. Feeding dairy cows a low protein diet supplemented with RP AA has the ability to alleviate the loss in milk production associated with feeding a low protein diet as well as to increase nitrogen efficiency. / Master of Science

Currently accepted optimum protein levels for growth in the horse may be reduced with amino acid supplementation. This study investigated the effects on growth and protein status of Thoroughbred foals offered a supplement with a typical CP level to a supplement that had a lower CP level fortified with limiting amino acids. The control supplement (CS) contained 14% CP, 3.0 Mcal/kg DM, 10% corn oil, 22% soybean meal, 1.4% calcium, and three sources of fiber. The experimental supplement (LTS) contained 9% CP, 3.0 Mcal/kg DM, 10% corn oil, 3% soybean meal, 1.4% calcium, three sources of fiber, and was fortified with .6% lysine and .4% threonine. Lysine and threonine are the first two limiting amino acids in common diets of the horse. Mares and foals were fed twice daily (0700 and 1400) and kept on 30-acre pastures (mixed grass/white clover) until weaning (7 months). Weanlings continued on specified supplements and pastures for seven additional months. Physical measurements and blood samples were taken monthly. Measurements included weight, average daily gain (ADG), body condition (BC), wither height, hip height, length, girth, forearm length, front and hind cannon length, physis and fetlock circumference. Blood analysis included total protein (TP), albumin (ALB), creatinine (CREA), and plasma urea nitrogen (PUN). Effect of diet and time were evaluated by analysis of variance with repeated measures. No differences were found in physical measurements between the CS and LTS groups (ADG 0.8(0.4 kg/day, BC 4.9(0.05) for the observational period. Blood data also showed no difference for the period (ALB 2.9 ( 0.03 g/dl, TP 5.7 ( 0.10 g/dl, CREA 1.1 ( 0.02 mg/dl). These results suggest that the foals offered the LTS faired as well or better than foals on the CS. Lower levels of crude protein (CP) in the diet will result in less nitrogen pollution of pasture lands due to better utilization in the horse. By fortifying a low protein diet with the first two limiting amino acids, the protein is used more efficiently for growth and development, thereby benefiting both the horse and the land. / Master of Science

The plant glutamate receptors (GLRs) are homologs of mammalian ionotropic glutamate receptors (iGluRs) and are hypothesized to be potential amino acid sensors in plants. Since their first discovery in 1998, the members of plant GLRs have been implicated in diverse processes such as C/N ratio sensing, root formation, pollen germination and plant-pathogen interaction. However, the exact properties of these channels, such as the spectrum of ligands, ion specificities, and subunit compositions are still not well understood. It is well established that animal iGluRs form homo- or hetero-tetramers in order to form ligand-gated cation channels. The first aspect of this research was to determine if plant GLRs likewise require different subunits to form functional channels. A modified yeast-2-hybrid system approach was initially taken and applied to 14 of the 20 AtGLRs to identify a number of candidate interactors in yeast. Forster resonance energy transfer (FRET), which measures the transfer of energy between interacting molecules, was performed in mammalian cells to confirm interaction between a few of those candidates. Interestingly, despite an abundance of overlapping co-localization between heteromeric combinations, only homomeric interactions were identified between GLRs 1.1 and 3.4 in HEK293 cells. Further, amino acids have been implicated in signaling between plants and microbes, but the mechanisms for amino acid perception in defense responses are far from being understood. Recently it was demonstrated that calcium responses initiated by bacterial and fungal microbe-associated molecular patterns (MAMPs) were diminished in seedlings treated with known agonists and antagonists of mammalian iGluRs, suggesting potential roles of GLRs in pathogen responses. Analysis of publicly available microarray data shows altered gene expression of a sub-fraction of GLRs in response to pathogen infection and bacterial elicitors. Thus, the second goal of my PhD research was aimed at determining whether GLRs are involved in the interaction between plants and pathogens. Gene expression changes of a number of candidate GLRs as well as pathogen growth was examined in response to the plant pathogen Pseudomonas syringae pv. tomato DC3000. Interestingly, single gene and multi-gene deficient plants responded differently with regards to pathogen susceptibility, likely as a result of functional compensation between GLRs. / Ph. D.

The amino acid requirement for milk protein synthesis was investigated in the rat mammary cells in tissue culture. Studies involved: 1) the amino acid reduction from a positive control (3X) back to a negative control (1X) for each of the essential amino acids that were present in the 1X-MEM (Omission Study) [X refers to the amino acid concentration of Eagle's minimal essential medium (MEM)], 2) the addition experiments which resulted in an inverse relation to those of the Omission studies, and 3) a three-dimensional central composite experiment with three variables (X₁ = lysine; X₂ = methionine, valine and arginine; X₃ = isoleucine, tryptophan, threonine, phenylalanine and histidine), embracing 15 treatment combinations. In addition, supplemental studies were conducted which determined: 1) the effect of varied labeled amino acid precursors (¹⁴C-U-lysine, phenylalanine and leucine) on the measured synthesis of milk protein, 2) the effect of varying cell density (6 x 10⁶ to 6 x 10⁷) on protein synthesis, and 3) the response to graded amino acid complement (1X through 5X) on milk protein synthesis. Supplemental studies indicated that: 1) synthesis of β-lactoglobulin and β-casein in response to the labeled amino acids were significant (P<.05), 2) cell numbers between 6 x 10⁶ to 6 x 10⁷ did not significantly (P>.05) alter the rates of the milk protein synthesis, and 3) the response of amino acid complement addition to the 1X-MEM was linear (P<.01) for β-lactoglobulin and β-casein synthesis. Data analysis from Omission and Addition experiments revealed that: 1) the first limiting amino acid was lysine, 2) the second limiting group of amino acids were methionine, valine and arginine, and 3) the least limiting amino acids were tyrosine, leucine and glutamine. The response surface study determined that the optimal combination of essential amino acids for the maximum milk protein synthesis were: 1) lysine = 15X, 2) methionine, valine and arginine = 4.5X, and 3) isoleucine, tryptophan, threonine, phenylalanine and histidine = 1. 5X elevation over that present in the 1X-MEM, and 4) leucine, tyrosine, cystine and glutamine = 1X. On the basis of these findings, it was possible to construct the optimal essential amino acid complement unique to the rat mammary cells in tissue culture for the maximum milk protein synthesis. / Ph. D.

Improving our understanding of milk protein production regulation and AA transport is important for successfully formulating diets for AA and improving N efficiency. The objectives were to study protein synthesis regulation and AA transport using in vitro and in vivo models. In the first experiment, the objective was to evaluate the ability of five distinct AA profiles and balancing Lys to Met ratio to 3:1 to stimulate protein translation. No single AA profile uniquely stimulated phosphorylation of translational machinery related proteins suggesting identification of a single optimal AA profile as unlikely. In the second experiment, an in vitro method using three different AA isotopes was developed to trace AA movement. The method assesses bi-directional transport of multiple AA simultaneously enabling evaluation of unidirectional uptake kinetics. This method was used to evaluate AA concentrations representing 16, 100, 186, and 271% of cow plasma AA concentrations. Amino acid uptake was not saturable within the in vivo range for eleven AA. Arginine, Val, and Pro exhibited saturation with the Michaelis-Menten km being 95, 49, and 65% of in vivo concentrations. Results suggest that AA transport is generally non-saturable and that high bi-directional transport exists which enables a mechanism for mitigating AA shortages. In experiment 3, the objective was to evaluate milk protein production and regulation from infusing Met, Lys, and His (MKH) or Ile and Leu (IL). The two EAA groups independently and additively increased milk protein yield. This finding contradicts the single limiting AA theory that a single nutrient will limit milk protein yield. Changes in udder AA extraction and blood flow from supplemental EAA reveal flexible delivery mechanisms. The phosphorylation state of proteins associated with the mTOR pathway was impacted by both EAA treatments. Changes in the udder proteome suggest negative feedback on mTOR pathway activation when milk protein yield was increased by the EAA groups separately but when supplemented together, negative feedback was lessened. Results indicate that multiple EAA can stimulate milk protein production, the ability of AA transport to match intracellular needs, and that the single limiting AA theory or existence of a unique optimal AA profile is likely irrelevant in dairy cows. / Doctor of Philosophy / Post absorptive metabolism of dietary protein and conversion to milk protein is not well described in dairy nutrition models resulting in poor predictions of response to changing protein supply. This partially constrains diet formulation with respect to successfully balancing diets for protein and amino acids or for improving N efficiency. The efficiency of absorbed AA into milk protein varies and udder AA uptake may contribute to this varying efficiency through transport regulation in an attempt to maintain intracellular AA homeostasis. Amino acid transport was assessed when AA supplies were varied below and above in vivo supplies. High bi-directional AA transport was saturating uptake for Arg, Pro, and Val within the normal in vivo range for lactating dairy cows. The high AA exchange suggest strong ability to manage changes in AA supply to meet needs for milk protein translation. When intracellular AA supply declines, efflux of the limiting AA out of the cell declines which results in greater uptake of the limiting AA by the cell. The theory that milk protein yield is limited by a single most limiting amino acid (e.g., the barrel and stave analogy) and that a single optimal EAA profile exists predominates in the field of animal nutrition, but implementation of this theory has not greatly improved N efficiency or been adopted widely. We observed that various AA profiles can equally stimulate milk protein translational machinery in mammary epithelial cells and that balancing Lys to Met ratio to 3 to 1 only had a minor effect. Multiple EAA can regulate milk protein production through signaling to synthesis machinery and delivery of AA to the udder. Supplementation of two iv groups of EAA, 1) methionine, lysine, and histidine and 2) isoleucine and leucine, independently and additively increased milk protein yield in dairy cows. These increases were achieved by changes in blood flow in the udder, AA uptake, and nutrient signaling related to protein translation regulation. Hence, results of this dissertation tend to not support the idea of a single limiting amino acid or a unique optimal profile of AA for milk protein production in dairy cattle.

Peptide, plasma free, erythrocyte free and serum protein amino acid arteriovenous differences were quantified across the gastrointestinal tract (GIT) and liver of growing calves during a "steady state" and fasting metabolism. Six Holstein steer calves (136 kg) surgically equipped with cannulae in the aorta, portal vein and hepatic vein were maintained under constant light and fed a natural diet as 24 hourly meals per day. Blood was obtained simultaneously from all cannulae at 9 to 12 d postsurgery during the midpoint of a 1-hr feeding interval and following a 72-hr fast. Most amino acids were added to plasma by the GIT but fasting reduced the magnitude of this addition. GLN and GLU were the only plasma amino acids removed by the GIT. The liver removed a large portion of amino acids in portal plasma but a net splanchnic plasma free amino acid output occurred. Hepatic removal of amino acids, particularly VAL, ILE and LEU, increased after fasting. The GIT generally removed amino acids from the erythrocyte while the liver contributed amino acids to this pool. Peptide amino acids increased as blood crossed the GIT. Responses paralleled those of plasma free amino acids in direction but were threefold greater for peptide amino acids. A net output of peptide amino acids from the splanchnic bed was observed. Generally amino acids were added to the primarily globulin, albumin and mixed protein serum fractions while the pure albumin fraction contributed amino acids to the GIT. Fasting reversed the direction of response in the four fractions monitored. Arteriovenous differences for amino acids in two largely albumin fractions responded oppositely indicating a potential transport role of other proteins in these fractions. A more variable response was observed across the liver with direction and magnitude dependent upon individual amino acids. Fasting resulted in a release of amino acids by the liver into all four fractions. Several blood pools appear to be involved in the interorgan transport of amino acids. The extent and direction of transport varies with tissue, blood pool, nutritional status and amino acid. Quantitatively, peptides appear to be highly involved in interorgan movement of amino acids. / Ph. D.

No / D-amino acids are useful building blocks for de novo peptide design and they play a role in aging-related diseases associated with gradual protein racemization. For amino acids with achiral side chains, one should be able to presume that the conformational propensities of L- and D-amino acids are a reflection of one another due to the straightforward geometric inversion at the Cα atom. However, this presumption does not account for the directionality of the backbone dipole and the inverted propensities have never been definitively confirmed in this context. Furthermore, there is little known of how alternative side chain chirality affects the backbone conformations of isoleucine and threonine. Using a GGXGG host-guest pentapeptide system, we have completed exhaustive sampling of the conformational propensities of the D-amino acids, including D-allo-isoleucine and D-allo-threonine, using atomistic molecular dynamics simulations. Comparison of these simulations with the same systems hosting the cognate L-amino acids verifies that the intrinsic backbone conformational propensities of the D-amino acids are the inverse of their cognate L-enantiomers. Where amino acids have a chiral center in their side chain (Thr, Ile) the β-configuration affects the backbone sampling, which in turn can confer different biological properties. / NIH

Yes / The operation of many nanostructured biomolecular sensors and catalysts critically hinges on the manipulation of non-covalent adsorption of biomolecules on unfunctionalised noble-metal nanoparticles (NMNPs). Molecular-level structural details of the aqueous biomolecule/NMNP interface are pivotal to the successful realisation of these technologies, but such experimental data are currently scarce and challenging to obtain. Molecular simulations can generate these details, but are limited by the assumption of non-preferential adsorption to NMNP features. Here, via first principles calculations using a vdW-DF functional, and based on nanoscale sized NMNPs, we demonstrate that adsorption preferences to NP features vary with adsorbate chemistry. These results show a clear distinction between hydrocarbons, that prefer adsorption to facets over edges/vertices, over heteroatomic molecules that favour adsorption onto vertices over facets. Our data indicate the inability of widely used force-fields to correctly capture the adsorption of biomolecules onto NMNP surfaces under aqueous conditions. Our findings introduce a rational basis for the development of new force-fields that will reliably capture these phenomena.