Chronic subclinical levels of disease occur frequently in intensive swine production and compromise nutrient utilization efficiency. Sulfur amino acids (methionine plus cysteine; M+C) have been implicated in improving the animal’s response to immune system stimulation (ISS). Research objectives were to determine the effects of ISS on the optimal dietary methionine to methionine plus cysteine ratio (M:M+C) and on the fractional synthesis rate (FSR) of albumin, fibrinogen and total protein in plasma, liver, and small intestine (SI) of growing pigs. A nitrogen balance study showed that the optimal M:M+C was increased during ISS and greater than 0.62. In a flooding dose infusion study it was determined that total plasma protein FSR was increased during ISS and tended to decrease with reduced M+C intake. Plasma albumin FSR decreased with reduced M+C intake. The data implicates M+C as important nutrients involved in the immune response and careful dietary supplementation during ISS is necessary. / Funding sponsored by Evonik Degussa, Ontario Pork, the Ontario Ministry of Agriculture, Food and Rural Affairs and the Natural Sciences and Engineering Research Council of Canada.

No description available.

Standards of Amadori rearrangement products (ARP) were synthesized for the purpose of developing analytical techniques and performing mechanistic studies related to their thermal decomposition. Several synthetic strategies were explored. An HPLC analytical system with a diode array detector was coupled to a fluorometer and an electrochemical detector, in order to detect simultaneously and on-line, a wide variety of degradation products of ARPs and to follow their kinetics. The potential of such a system to analyze complex Maillard mixtures was demonstrated. The kinetics of the reaction of glucose with morpholine (a Strecker inactive analogue of proline was used in order to simplify the kinetics) to produce Amadori morpholine was studied under experimental conditions that minimize side reactions and maximize Amadori product formation. At specific time intervals, the samples were analyzed for the presence of reactants and Amadori product by the multidetector HPLC system. Color and fluorescence were also measured. The data obtained were used to calculate the rate constants for the formation and degradation of Amadori product. A mechanistic model that statistically fitted the kinetic data was proposed. To further understand the details of the decomposition mechanism of Amadori proline, different mass spectrometric experiment were performed. High resolution, linked-field scan and neutral loss experiments have indicated that 1-((2$ sp prime$-carboxyl)pyrrolidinyl)-1-deoxy- scD-fructose (Proline Amadori product) followed two main pathways of fragmentation under electron impact conditions; one initiated by the ring oxygen and the other by the amino acid nitrogen, producing two well stabilized fragment ions; oxonium and imminium ions. In addition, ortho-elimination reactions initiated by O or N-centered radical sites were shown to produce the most intense peaks and diagnostically important ions for the identification of Amadori products. However this approach can only pro

Since glucose is an essential nutrient for normal fetal growth and development, the impact of reduced maternal dietary glucose supply, on amniotic fluid (amf) amino acid composition was investigated. Furthermore, this study investigated whether any resulting changes in the concentrations of amf amino acids could be predictive of fetal growth and metabolic status. Pregnant rat dams were fed isocaloric diets containing graded levels of dietary glucose (0, 12, 24 and 60%) and the amf amino acid content was analysed on gestational days (gd) 18.5 to 21.5. Carbohydrate restriction produced significant increases in the concentrations of amf isoleucine (on gd 21.5), tryptophan (on gd 18.5 and 21.5) and 3-methylhistidine (on gd 20.5 and 21.5). An interaction between diet and day of gestation modified amf taurine levels such that dams fed low carbohydrate diets showed significant increases in amf taurine as pregnancy progressed. Specific amf amino acids correlated with fetal growth parameters and fetal tissue glycogen reserves indicating the ability of amf composition to reflect fetal distress under conditions of compromised maternal nutritional status. A greater statistical predictability of amf constituents was obtained with fetal growth parameters than with fetal tissue glycogen reserves. These results suggest that amf amino acids are better predictors of fetal growth status than of fetal metabolic status.

No description available.

Biochemical theories postulate that deficient serotonergic functioning may be etiologically related to affective illness and aggressive behavior. In Study I mood and aggressivity were measured in thirty-six normal male subjects before and after ingestion of a Tryptophan Depleted, Tryptophan Loaded or Balanced amino acid mixture. While no differences in aggressivity were found, the Tryptophan Depleted group scored significantly higher at posttest on the MAACL Depression Scale than the control groups and demonstrated selective attention for dysphoric themes. In Study II a Balanced or Tryptophan Depleted amino acid mixture was administered to eighty normal male subjects prior to placing them in either a positive or negative environment, with or without instructions concerning the potential amino acid effects. The tryptophan depleted group became significantly more depressed than the control group regardless of environmental condition or instructional set. These findings suggest that lowered tryptophan may result in a central serotonergic dysfunction which is causally related to depressive affect and possibly to the pathogenesis of clinical forms of depression.

Biotin is a water soluble vitamin that is mainly used as a cofactor in carboxylation reactions by a class of enzyme known as biotin-dependent carboxylases. In order to act as a cofactor, the biotin molecule has to be covalently attached to a lysine residue by an enzyme called holocarboxylase synthetase (HCS). Inherited deficiency of the biotin-dependent propionyl-CoA carboxylase (PCC) results in the inborn error of metabolism propionic acidemia. Mutations in either the alpha (PCCA gene) or beta (PCCB gene) subunit of the enzyme have been shown to cause propionic acidemia. Mutation analysis of the PCCB gene have revealed several mutations. However, few PCCalpha mutations have been described. The first goal of this thesis was to determine the molecular etiology of alpha subunit deficiency at the mRNA as well as at the protein level. I found that most mutations destabilized either the mRNA or the protein. Two other mutations were found to affect the biotinylation of PCCalpha, defining residues important for the folding of the domain or for interaction with HCS. The second part of my thesis was to study in more details the interactions between HCS and the biotinylation domain of PCCalpha, represented by the last 67 amino acids of the subunit (p-67). I expressed and purified p-67 from Pichia pastoris. I compared p-67 with the E. coli biotinylation domain (BCCP87) as substrates for the E. coli orthologous enzyme BirA, using steady-state as well as stopped-flow kinetics. I noticed some differences between these two substrates and how it might relate to the biotinylation reaction. I generated N-terminal and C-terminal deletions of HCS and I tested their activity in vivo and in vitro using purified susbtrates. I was able to map the minimal sequence requirement for HCS activity to the last 348 amino acids of the enzyme. I also found that some longer HCS were either almost or totally inactive or some that were active showed a differential activity towards the different susb

Submitted in partial fulfillment of the academic requirements for the degree of Master of Technology: Biotechnology, 2009. / Production of numerous biotechnologically-important products such as threonine is based on cultivation of bacterial cultures. Infection of these bacterial cultures by bacteriophages has a detrimental effect in the production of these bioproducts. Despite this, most people controlling these bioprocesses do not recognize the early signs of bacteriophage infection. SA Bioproducts (Ply) Ltd was no exception and has suffered tremendous loss of production time after bacteriophages infected threonine producing E. coli strain B. This study was aimed at developing assays to control and prevent bacteriophage infection at this company. These included determining the source of phages by monitoring the process plant environment, optimising the detection and enumeration methods so as to monitor the levels of bacteriophages in the environment, identification of bacteriophages in order to determine the number of bacteriophages capable of infection threonine producing E. coli strain B, treatment and of phages, and possible prevention of phage infection. Adam's DAL method was very efficient at detecting phages in the samples collected at various areas (sumps, odour scrubber, process water, and soil) around the plant for 16 weeks. High levels of phages were found in the sumps and this was identified as the source of infection. Samples collected were grouped together according to their source. The samples were enriched and purified in order to characterise them. The prevalent phage in all samples was identified as a T1-like phage. Bacterial strains that grew on the plate in the presence of phages were assumed to be resistant to phages or contained lysogenic phages which would explain the new lytic cycles that were observed whenever these resistant strains were used for production. UV light, green v indicator plates, and a mutagen (Mitomycin C) were used to detect Iysogens. Mitomycin C at 1 IJg/ml was found to be most effective in detecting lysogenic phages. This was shown by new plaque forming units that were visible on the DAL plates. Temperature (heat), chemicals, and inhibitors (vitamins) were investigated as strategies for prevention and treatment of bacteriophage infection. Bacteriophage samples were exposed to 70, 80, 100, and 120°C. At these temperatures pfu counts in the samples were reduced significantly. At 120°C there was a complete inactivation of bacteriophages within 30 minutes. Chemicals investigated such as sodium hydroxide and Albrom 100T were capable of complete deactivation of bacteriophages at a very low concentration (0.1%). Therefore, these chemicals can be used to clean the plant area and sumps. Vitamins C, K and E solutions were investigated to determine their inhibitory effect on bacteriophages. Vitamin C, K and E reduced pfu counts by 3, 2, and 4 logs, respectively. Therefore vitamin C and E solutions were mixed and to determine if mixing them would enhance their inactivation capabilities. This resulted in a reduction greater than 9 logs of phage in the sample (from 7.7 x 109 to 3 pfu/ml). The host bacterium was also exposed to this mixture to determine effect of the vitamin mixture on its growth. It was found that there was no effect exerted by this mixture on the host bacteria. This proved to be an ideal mixture for combating phages during fermentation. However, vitamin E is not cost effective for co-feeding in 200 m' fermenters, and therefore vitamin C solution was a cost-effective alternative. It was concluded that bacteriophage contaminated bioprocessing plant should be properly cleaned using a combination of heat and chemicals. Bacteriophage infection should be prevented by employing inhibitors.

Short/branched chain acyl-CoA dehydrogenase (SBCAD), a member of the acyl-CoA dehydrogenase (ACD) family of enzymes, catalyzes the oxidation of branched chain fatty acids and the branched chain amino acids isoleucine and valine. This research project focuses on expression studies of the SBCAD gene. Northern blot analysis detected two SBCAD mRNA species of 2.7 and 6.5 kb in various human tissues and cell types. A single 4.1 and 2.0 kb SBCAD message was detected in rat and pig tissues, respectively, revealing a species difference in SBCAD mRNA size. Studies of human and rat SBCAD tissue-specificity and relative abundance, at both the RNA and protein levels, identified liver and kidney as the tissues with the highest levels of SBCAD expression, establishing a unique tissue-specific expression pattern that is not seen among the other members of the ACD family. Furthermore, a fetal and adult difference in SBCAD expression was observed in human kidney, suggesting that the SBCAD gene may be developmentally regulated in some tissues. Finally, an attempt was made to isolate and characterize the SBCAD promoter region in order to provide valuable data for future SBCAD promoter studies.

The objective of this thesis was to determine the molecular basis of neonatal multiple carboxylase deficiency (MCD) produced by an impairment in holocarboxylase synthetase (HCS) activity and the origin of the biotin-responsiveness that characterizes this disease. To determine HCS activity, I developed a peptide substrate and used the biotinylation system of E: coli to determine its properties. C-terminal fragments of the $ alpha$ subunit of human propionyl-CoA carboxylase (PCC-$ alpha$) were expressed in E. coli and site-directed mutagenesis was used to define the residues required for biotinylation by the bacterial biotin ligase, BirA. These experiments showed that the biotin region of PCC-$ alpha$ can act as an autonomous domain for biotinylation and suggested its use as substrate for human HCS. For the molecular characterization of MCD, I isolated several cDNA clones encoding human HCS by functional complementation of an E. coli mutant with a temperature-sensitive BirA. Comparison of the predicted amino acid sequence of HCS with bacterial biotin ligases allowed the identification of the putative biotin-binding domain of this protein. Mutation analysis of DNA from HCS deficient patients showed that most of the changes in the HCS sequence are clustered in the biotin-binding domain. All the patients tested in this study showed deficiency of HCS activity as determined using the PCC-$ alpha$ peptide as substrate for biotinylation. The biotin-responsiveness was demonstrated by obtaining a stimulation of HCS activity of MCD cells at high biotin concentrations while remaining unstimulated in extracts of normal cells. Together with the mutation studies, these results showed that neonatal MCD is caused by mutations in the biotin binding domain of HCS which reduce the affinity of the enzyme towards biotin. This change in the kinetic properties of HCS results in the inefficient biotinylation of carboxylases at physiological concentrations of biotin. The defect can be over