Obesity is an increasing health problem which is causally associated with insulin resistance and type 2 diabetes. Oxidative stress, i.e. overproduction of reactive oxygen species, is associated with insulin resistance and obesity and may be a major risk factor in the onset and progression of diabetes. Bernlohr Lab at University of Minnesota have study oxidative stress in adipocytes by silencing the enzyme glutathione S-transferase A-4 (GSTA4), an enzyme detoxifying 4-hydroxynonenal formed during oxidative stress. Their results indicate that lysine acetylation, an important post-translational modification, may be involved during oxidative stress. In this study lysine acetylation has been investigated in condition of oxidative stress in 3T3-L1 adipocytes and subcutaneous adipose tissue from mice using SDS-PAGE gel electrophoresis and western blot. Lysine acetylation was analyzed in different compartments of the cell such as in cytoplasm, mitochondria as well as in whole cell extracts. Silencing of GSTA4 and stimulation by TNF-α in 3T3-L1 adipocytes resulted in an increase of lysine acetylation in cytoplasm. Furthermore, stimulation by IL-6 did not have any effect on lysine acetylation. Surprisingly, subcutaneous adipose tissue from mice fed on a high-fat diet showed a decrease of lysine acetylation in cytoplasm compare to mice fed on a chow diet. In conclusion, lysine acetylation seems to change during oxidative stress and may be an important factor during insulin resistance, type 2 diabetes and obesity. Therefore, studying lysine acetylation and enzymes modulating acetylation may potentially increase our understanding of insulin resistance, type 2 diabetes and obesity and could lead to new therapies.

Two feeding trials were conducted to study the requirements of juvenile cobia for dietary methionine and lysine. Experiment I compared both fishmeal-based (fish meal and soybean meal as protein sources) and casein-based (casein and gelatin as protein sources) diets. All diets were isonitrogenous and isoenergetic. Crystalline amino acids was pre-coated with carboxymethylcellulose (CMC) and supplemented to simulate the amino acid pattern of juvenile cobia muscle protein except methionine. Five methionine levels of 0.45, 0.70, 0.95, 1.20 and 1.45% (1.07, 1.66, 2.26, 2.86 and 3.45% of protein) were studied. The test diets contained 0.16% cystine. Juvenile cobia (initial weight 35g) were fed test diets for 8 weeks. The results showed that the cobia used fish meal more effectively than casein. Final weight, percent weight gain (PWG), feed conversion ratio(FCR), hepatosomatic index (HSI), condition factor (CF), protein efficiency ratio (PER), protein retention (PR), carcass proximate composition and free methionine concentration in the serum were significantly enhanced (p<0.05) by increasing dietary methionine level. But profile of essential amino acids in the muscle and serum superoxidase and lysozyme activity were not significantly affected. Weight gain of the cobia fed the fishmeal-based diet containing 1.2% methionine was significantly higher than the other dietary groups. Methionine content of 0.45% resulted in reduced growth. No significant difference in weight gain was found in the casein-containing diets when methionine was 0.7% and over. The cobia fed diet containing 0.45% methionine started show apparent skin lesions in their heads on the sixth week of the growth trial and significant mortalities were observed since the seventh week (p<0.05). The optimum dietary level of methionine for cobia, estimated by the broken-line regression analysis on weight gain, was 1.10% (2.62% of protein) based on the fishmeal-based diet results and was 0.70% (1.67% of protein) based on the casein-based diet results. Experiment II used fish meal and wheat gluten as the protein sources. Pre-coated crystalline amino acids were supplemented to simulate the amino acid pattern of juvenile cobia muscle protein except lysine. Lysine levels including 1.49, 1.75, 2.00, 2.25, 2.50 and 2.75% (3.54, 4.16, 4.76, 5.35, 5.95 and 6.54%) were studied. Juvenile cobia (initially 12g) were fed test diets for 8 weeks. The results showed that the final weight, PWG, FCR, CF, PER, PR, carcass proximate composition and free lysine concentration in the serum were significantly enhanced (p<0.05) by increasing lysine levels. But HSI and essential amino acid profile of fish in the muscle were not significantly affected. Final weight and percent weight gain of the cobia fed diets containing 1.75% lysine or over were significantly higher than fish fed diet contained 1.49% lysine. FCR was lower for cobia fed diets containing 1.75% lysine or over than the 1.49% lysine group. The optimum dietary level of lysine, estimated by the broken-line regression analysis on weight gain was 1.76% (4.19% of protein), and was 1.95% (4.64% of protein) on serum free lysine concentration. There was no overt deficiency symptom, such as caudal fin rot in rainbow trout, in the cobia during the growth trial.

The human microbiome has important roles in maintaining health, but dysbiosis of the microbiota can lead to disease. Polymicrobial interactions can result in synergy, producing disease that is worse than the sum of the respective single species infections. Despite this significant impact, synergy is understudied due to the complexity of polymicrobial interactions. Periodontitis is a microbiome-associated disease, and is one of the most common infectious diseases worldwide. Therefore, we have used periodontal disease as a model to study polymicrobial synergy. I have used two complementary approaches to study polymicrobial infections. The opportunistic periodontal pathogen Aggregatibacter actinomycetemcomitans exhibits synergy with streptococci in model murine infections. Because polymicrobial interactions are dependent on organisms’ abilities to sense their environments, I have examined the genetic regulatory mechanisms used by A. actinomycetemcomitans to interact with its environment. Through Northern blot analyses and biochemical approaches, I show that A. actinomycetemcomitans uses non-coding RNAs to regulate amino acid transport. Taking a comparative genomics approach, I demonstrate that A. actinomycetemcomitans DNA uptake systems are evolutionarily linked to genome defense. To describe host-influenced changes in gene expression, I develop a new technique to transcriptionally profile A. actinomycetemcomitans in a murine abscess infection, thereby revealing the importance of specific fermentative and anaerobic respiratory genes for in vivo survival. The long-term goal is to use these studies as a basis to characterize genetic regulatory mechanisms mediating synergy in polymicrobial A. actinomycetemcomitans infections with streptococci and other oral microbes. As a second approach to study polymicrobial infections, I analyze gene expression of healthy and diseased human plaque communities from aggressive periodontitis patients. Profiling ribosome content of healthy and diseased communities, I show that disease communities adopt similar less diverse population structures distinct from healthy populations. In addition to changes in population composition, using community transcriptional profiling I show that a keystone species within diseased communities up-regulates expression of genes involved in making the oral inflammatory molecule butyrate. These studies demonstrate for the first time that microbiome based diseases are marked by gene expression changes in addition to compositional changes. / text

Multiple lines of evidence suggest that elevated plasma lipoprotein(a) (Lp(a)) concentrations are a significant risk factor for the development of a number of vascular diseases including coronary heart disease and stroke. Lp(a) consists of a low-density lipoprotein (LDL)-like moiety and an unique glycoprotein, apolipoprotein(a) (apo(a)), that is covalently attached to the apolipoproteinB-100 (apoB-100) component of LDL by a single disulfide bond. Many studies have suggested a role for Lp(a) in the process of endothelial dysfunction. Indeed, Lp(a) has been shown to increase both the expression of adhesion molecules on endothelial cells (EC), as well as monocyte and leukocyte chemotactic activity in these cells. We have previously demonstrated that Lp(a), through its apo(a) moiety, increases actomyosin-driven EC contraction which, as a consequence, increases EC permeability. In this thesis, we have demonstrated a role for the strong lysine-binding site in the kringle IV type 10 domain of apo(a) in increasing EC permeability, which occurs through a Rho/Rho kinase-dependent pathway. We have further validated these findings using mouse mesenteric arteries in a pressure myograph system. We also have dissected another major signaling pathway initiated by apo(a) that involves in a disruption of adherens junctions in EC. In this pathway, apo(a)/Lp(a) activates the PI3K/Akt/GSK3β-dependent pathway to facilitate nuclear translocation of beta-catenin. In the nucleus beta-catenin induced the expression of cyclooxygenase-2 (COX-2) and the secretion of prostaglandin E2 (PGE2) from the EC. Finally, we have presented data to suggest a novel inflammatory role for apo(a) in which it induces the activation of nuclear factor-kappaB through promotion of the dissociation of IkappaB from the inactive cytoplasmic complex; this allows the nuclear translocation of NFkappaB with attendant effects on the transcription of pro-inflammatory genes. Taken together, our findings may facilitate the development of new drug targets for mitigating the harmful effects of Lp(a) on vascular EC which corresponds to an early step in the process of atherogenesis. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2009-09-22 19:24:04.594

No description available.

This work investigates two distinct roles of lysine deacetylases (KDACs) in the budding yeast Saccharomyces cerevisiae. The first part focused on the classical, well characterized role of KDACs as transcriptional regulators and deciphering their role in G1 transcription. I show that two yeast KDACs, Rpd3 and Hos3 are recruited to G1 promoters through their interactions with the negative regulator Whi5 and that these KDACs are necessary for proper Whi5-mediated repression. The second part examines a newly discovered role for KDACs extending their role beyond the chromatin as modifiers of proteins other than the histones. I present here the first systematic approach that comprehensively examines these non-histone targets of KDACs in vivo. I identified 73 non-histone proteins acetylated in vivo involved in diverse cellular processes. Swi4, a component of the G1 transcription factor SBF, was identified in the Rpd3 screen and I show that the interaction between Swi4 and its heterodimeric partner Swi6 was regulated by acetylation. My findings significantly expand the scope of the yeast acetylome and demonstrate the utility of systematic functional genomic screens to explore enzymatic pathways.

This work investigates two distinct roles of lysine deacetylases (KDACs) in the budding yeast Saccharomyces cerevisiae. The first part focused on the classical, well characterized role of KDACs as transcriptional regulators and deciphering their role in G1 transcription. I show that two yeast KDACs, Rpd3 and Hos3 are recruited to G1 promoters through their interactions with the negative regulator Whi5 and that these KDACs are necessary for proper Whi5-mediated repression. The second part examines a newly discovered role for KDACs extending their role beyond the chromatin as modifiers of proteins other than the histones. I present here the first systematic approach that comprehensively examines these non-histone targets of KDACs in vivo. I identified 73 non-histone proteins acetylated in vivo involved in diverse cellular processes. Swi4, a component of the G1 transcription factor SBF, was identified in the Rpd3 screen and I show that the interaction between Swi4 and its heterodimeric partner Swi6 was regulated by acetylation. My findings significantly expand the scope of the yeast acetylome and demonstrate the utility of systematic functional genomic screens to explore enzymatic pathways.

Maize genetic diversity includes an array of kernel colors (red, blue, purple) with blue concentrated in the aleurone and red primarily in the pericarp. Quality protein maize (QPM) is improved over normal maize in regards to grain concentration of the essential amino acids lysine and tryptophan but has not been widely adapted in part due to lower than conventional yields. These are minimally-utilized specialty corns when compared to the yellows and whites commonly grown. Red, blue, and purple pigments are antioxidant phytochemicals produced by the plant as secondary metabolites. Antioxidants have been linked to anti-cancer and other anti-inflammatory health benefits. QPM hybrids are desirable in developing countries where subsistent agriculture is commonly practiced and quality protein cereals are non-existent. These two diverse maize categories have been the subject of little breeding research compared to normal maize and the potential for high phenolic content as well as the characterization of these QPM hybrids has not been previously investigated. We evaluated 153 maize hybrids (84 colored, 69 QPM) across three locations. High heritability estimates were found for phenolic content (0.80), tryptophan (0.46), and endosperm opacity (0.82). It was encouraging that all three traits observed little genotype by environment (GxE) interaction across diverse environments. This proved the trait analysis procedure to be robust in detecting and separating genotypes for both total phenolic content in colored maize, and amino acids in QPM. Top combiners for phenolics were the purple maize "maize morado" and red lines, with blue, yellow and white maize performing in descending order. Within the tested hybrids, high per kernel antioxidants (measured by total phenolics) may be the answer for producing the most total phenolics, with the top hybrid yielding greater than twice the total phenolics as the top yielding yellow hybrid. The top QPM hybrid out yielded the top normal hybrid by 35 and 30% for lysine and tryptophan. Additionally, QPM endosperm opacity primarily followed an additive, mid-parent trend, with some hybrids (20%) from diverse germplasm backgrounds deviating from that trend displaying the complexity and recessive nature of multiple modifier loci. Additional agronomic and composition traits were minimally correlated with phenolics.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Auburn University. / Abstract. Vita. Includes bibliographical references (p. 145-154).