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The compatible solute glycine betaine accumulates in many plants including spinach (Spinacea oleracea) under conditions of water deficit stress. The precursor to glycine betaine is choline, a ubiquitous metabolite in plants as a component of phosphotidylcholine. In spinach choline is synthesized from phosphocholine, a product of three sequential N-methylations of phosphoethanolamine catalysed by the cytosolic enzyme S-adenosyl-L-methionine: phosphoethanolamine-N-methyltransferase (PEAMT). PEAMT activity shows diurnal changes with peak activity at the end of the photoperiod and a decrease overnight. The activity of this enzyme is up-regulated 2 to 3-fold in salt-stressed plants relative to unstressed plants. The objective of this thesis is to determine how PEAMT activity is regulated in vivo. Thus, PEAMT activity, protein and transcript levels were quantified in spinach leaves from plants subjected to different light and salinity conditions. A spinach PEAMT eDNA sequence was used to over-express recombinant PEAMT in the protein expression vector pET30a (+). The presence of a polyhistidine-tag on the overexpressed protein allowed for purification by a cobalt metal affinity column. The affinity purified protein was used to produce polyclonal antibodies for immunoblot hybridization analysis. For these studies, PEAMT protein was first immunoaffinity purified from soluble extracts prepared from leaves and then the protein subjected to electrophoresis by SDS-p AGE. Enzyme assays and immunoblot analysis show PEAMT activity and protein levels increase and become relatively constant in leaves of plants exposed to continuous light. In continuous darkness, PEAMT activity and protein levels decrease and remain low and constant. Thus the pattern of changes in PEAMT activity levels are associated with changes in PEAMT protein levels. In contrast, Northern blot hybridizations show that under conditions of constant light, peamt transcript levels undergo cyclical changes with peak levels at 20 and 40 h and troughs at 28 and 52 h after the continuous light treatment was imposed. These peaks coincide with the dark and light cycles of the normal photoperiod. The same cyclical changes in peamt transcript levels was seen for plants transferred from a normal photoperiod to continuous darkness. Since these changes persist in the absence of a day/night cue we conclude that peamt transcript levels are circadian-regulated. The peamt transcript levels of control unstressed and salt-stressed plants also show circadian rhythms, however the levels found in salt-stressed plants were 0.5 to 2-fold higher than the controls. Therefore, while salinization of plants increases peamt transcript abundance, it does not alter the circadian rhythm that transcripts of this gene display. Changes in PEAMT activity and protein levels are likely controlled by other as yet unknown post-translational mechanisms, processes that override and obscure operation of a circadian rhythm in regulating the level of peamt transcripts. / Thesis / Master of Science (MS)

Two conditions of alkaline hydrolysis of proteins, (1) 2M NaOH, 18 hours, 100°C and (2) 3M NaOH, 16 hours 110°C, prior to ion-exchange chromatography were tested on free amino acids and model protein systems to determine the better set of conditions for measurement of methionine sulfoxide in food proteins. Recoveries of methionine, methionine sulfoxide, and methionine sulfone from base-hydrolyzed amino acid mixtures were, respectively, 89, 100, and 105% with the 2M NaOH conditions and 83, 90, and 98% with the 3M NaOH conditions. The percentages of total methionine, determined by performic acid oxidation, recovered as methionine, methionine sulfoxide, and methionine sulfone after hydrolysis with 2M NaOH were, respectively 101, 0, and 0% in lysozyme, 68, 25, and 0% in oxidized lysozyme, 74, 3, and 0% in casein and 0, 74, and 0% in oxidized casein. The presence of glucose in the hydrolysis mixture with the model proteins caused as much as 8% oxidation of methionine to methionine sulfoxide. The presence of copper (II) and iron (II) ions along with sugars did not increase the amount of methionine generated in casein and a soy isolate. Methionine sulfone was never generated in any of the model systems. These results suggested that determination of methionine sulfoxide after basic hydrolysis with 2M NaOH in foods low in carbohydrates is valid but in foods high in carbohydrates the procedure may slightly overestimate the methionine sulfoxide content. Acid hydrolysis of free methionine sulfoxide reduced 15% of the methionine sulfoxide to methionine while acid hydrolysis of oxidized lysozyme and oxidized casein led to reduction of all the methionine sulfoxide in these proteins. Eight food products were analyzed for methionine, methionine sulfoxide, and methionine sulfone. Total methionine was measured by the performic acid oxidation method, methionine sulfone by ion-exchange chromatography after acid hydrolysis, methionine sulfoxide by ion-exchange chromatography after hydrolysis with 2M NaOH for 18 hours at 100°C, and methionine by the difference between total methionine and the sum of methionine sulfoxide and sulfone. Only a trace of methionine sulfone and less than 6% of total methionine was present as methionine sulfoxide in a soy flour and a concentrate. Two soy isolates contained 74 and 8% of total methionine as sulfoxide and 6 and 4%, respectively, as sulfone. Two soy-based infant formulas contained 17 and 12% of total methionine as the sulfoxide and 12 and 8%, respectively, as sulfone. Two milk-based formulas contained 18 and 9% as sulfoxide and 8 and 13%, respectively, as sulfone. The feasibility of using ATP:L-methionine S-adenosyltransferase to determine the number of unaltered methionine residues in food proteins was also explored. Di- and tripeptides composed of methionine appeared to be able to function as well as L-methionine as substrates. Spectrophotometric studies suggested that the enzyme could act on methionine residues in two soy isolates; however, these results could not be confirmed by amino acid analyses of the isolates after incubation with ATP and the enzyme. / Ph. D.

The global DNA methylation pattern is erased and re-established during oogenesis and again in preimplantation (PI) embryo development. Understanding where these methyl groups come from and how the process of methylation is regulated is important, as disruptions could result in detrimental effects. The methionine cycle that produces the cellular methyl pool is linked to the folate cycle. The key enzyme linking theses cycles is Methylenetetrahydrofolate Reductase (MTHFR) which converts 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. Mthfr RNA and protein are present throughout mouse oocyte and PI embryo development, including the germinal vesicle, MII egg, 1-cell embryo, 2-cell embryo, morula and blastocysts. In MII eggs the protein appears to be heavier than in any other stage. This was reversed by treatment with Lambda Protein Phosphatase (LPP), indicating that MTHFR is phosphorylated in MII eggs. MTHFR was progressively phosphorylated beginning shortly after initiation of meiotic maturation, reaching maximal levels in MII eggs before decreasing after egg activation using strontium chloride. Potential kinases responsible for the phosphorylation of MTHFR have been identified however not in oocytes or PI embryos. DYRK1A/1 and GSK3A/B have both been suggested to mediate the phosphorylation, however when inhibited showed no effect on the oocyte sample. An LC-MS/MS assay was attempted to measure the activity of MTHFR in wildtype and knockout mouse liver samples, however unsuccessful in the amounts needed to be used for comparison to oocytes. Overall, MTHFR is present in the developing stages of interest and is mediated in some capacity by phosphorylation modifications around the MII stage of development.

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Call number: LD2668 .T4 1984 O53 / Master of Science

Sarcopenia is a condition that is most common in aged animals, and is characterized by the loss of skeletal muscle mass and integrity, and can lead to physical disability and poor quality of life. Since skeletal muscle protein synthesis can be limited by the availability of amino acids, supplementation of limiting amino acids to ameliorate the progression of sarcopenia has become a topic of interest in companion animal research. Although there is some data to support the idea that amino acid supplementation improves maintenance of muscle mass in aged horses, the cellular mechanisms behind that improvement have yet to be elucidated. Therefore, the objective of this study was to examine the effect of amino acid supplementation in aged horses on markers of whole body and muscle protein metabolism. In a cross-over design, six old horses were studied while receiving each of three treatments in a replicated Latin square design. For all three treatments, horses received 1.8% BW/d of timothy hay cubes and 0.5% BW/d of experimental concentrate. The three treatments included a control (CON) treatment concentrate that was designed to meet all requirements of mature horses when fed in combination with the timothy hay cubes, and two supplemented concentrates, LYS/THR with additional lysine and threonine (40 mg/kg BW/d and 31 mg/kg BW/d, respectively), and LYS/THR/MET with additional lysine, threonine, and methionine (40 mg/kg BW/d, 31 mg/kg BW/d and 11mg/kg BW/d respectively). In each 15 d period, following a 9-day adaptation, horses were fitted with a collection harness, and total urine and feces were collected for 72 hours for assessment of nitrogen balance and creatinine output. Blood samples were taken directly before feeding and 30, 60, 90, 120, 150, 180, 210, and 240 minutes post-feeding for analysis of plasma urea nitrogen (PUN), glucose, insulin, and plasma amino acid concentrations. Muscle biopsy samples were taken for analysis of proteins in the mTOR pathway. Additionally, horses underwent stable isotope infusion procedures, and comparisons of phenylalanine kinetics were used to determine whole-body rates of protein synthesis and degradation. There was no significant effect of treatment on creatinine output (P=0.58), relative abundance of proteins in the mTOR pathway (P>0.05), nitrogen retention (P=0.70), or phenylalanine kinetics (P>0.05). PUN concentrations were significantly (P=0.0058) higher for LYS/THR and LYS/THR/MET than for CON. Atrogin-1 activation was significantly higher for the pre-feeding CON sample compared to the post-feeding CON sample. Lack of significant difference in creatinine output suggests that there were not significant differences in muscle mass between treatments. Lack of significant differences in mTOR protein activation suggests that amino acid supplementation did not result in improvements in protein synthesis. Lack of significant differences in nitrogen retention and phenylalanine kinetics suggests that whole-body protein metabolism was not improved. Additionally, higher PUN concentrations in the supplemented diets suggests that the supplemented amino acids being provided were catabolized. However, increased activation of Atrogin-1 in the pre-feeding CON samples, but not the pre-feeding samples of supplemented treatments, suggests amino acid supplementation may have reduced protein degradation in the post-absorptive state. Data from the present study suggests that amino acid availability may not have been limiting protein synthesis in the sedentary aged horses in the present study.

published_or_final_version / Medicine / Master / Master of Philosophy

Nonalcoholic steatohepatitis (NASH) has been identified as a source of significant inter individual variation in drug metabolism. A previous ex vivo study demonstrated significant changes in hepatic Cytochrome P450 (CYP) activity in human NASH. This study evaluated the in vivo activities of multiple CYP isoforms simultaneously in prominent diabetic NASH mouse models. The pharmacokinetics of CYP selective substrates: caffeine, losartan, and omeprazole changed significantly in a diabetic NASH mouse model, indicating attenuation of the activity of Cyp1a2 and Cyp2c29, respectively. Decreased mRNA expression of Cyp1a2 and Cyp2c29, as well as an overall decrease in CYP protein expression, was found in the diabetic NASH mice. Overall, these data suggest that the diabetic NASH model only partially recapitulates the human ex vivo CYP alteration pattern. Therefore, in vivo determination of the effects of NASH on CYP activity should be conducted in human, and more appropriate models are required for future drug metabolism studies in NASH.