Browsing by Subject "Metabolism"
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Item A study of the influence of season on basal metabolism of normal college women(Texas Tech University, 1940-08) Bavousett, Neva DeenNot AvailableItem An investigation of the metabolic cost of tasks involving isometric pull(Texas Tech University, 1964-05) Petruno, Michael JamesNot availableItem Compartmentalization of metabolism in Bacillus thuringiensis(Texas Tech University, 1985-05) Akinwunmi, Oluwatoyin OluwafeyikemiThe compartmentalization of metabolism and the influence of amino acids and various substrates on the growth of Bacillus thuringiensis were studied. Carbon and nitrogen metabolism are compartmentalized only during vegetative growth when glucose is present in the medium, and it is pH-dependent. Organic nitrogen sources or compounds which can generate NADPH for incorporation of ammonium into glutamate were required for growth, sporulation, and crystal formation in minimal glucose-salts medium, designated BM. Abundant growth occurred when organic nitrogen sources such as glutamate, glutamine, arginine, proline, citrulline, ornithine, histidine, aspartate, and asparagine were utilized as the sole sources of carbon and (or) nitrogen at O.OIM concentration. Other organic nitrogen sources able to support growth as carbon and (or) nitrogen sources at equimolar concentrations were alanine, valine, leucine, serine, methionine, lysine, and isoleucine, but growth was less. Serine and gamma-aminobutyric acid (an NADPH generator) supported growth only as the sole sources of carbon. Also pyruvate, alphaketoglutarate, and citrate (an NADPH generator) supported growth of the organism as sole sources of carbon. Succinate, isocitrate, betahydroxybutyrate, and glyoxylate did not support growth as carbon and (or) nitrogen sources. A proposed scheme of the pathway for breakdown of these metabolites in B. thuringiensis is presented.Item The effects of acute exercise on postprandial metabolism(2012-05) Trombold, Justin Ross; Coyle, Edward F., 1952-; Tanaka, Hirofumi; Brothers, Robert M.; Nunez, Nomeli; Hursting, StephenThese studies determined the role of carbohydrate deficit from acute exercise on postprandial triglyceride elevation (PPTG). In Study 1, when energy expenditure was held constant in the exercise trials, both acute moderate (~50% VO₂ peak; MIE) and high intensity endurance exercise (90% VO₂ peak intervals; HIE) were effective to lower PPTG compared to a non-exercise control [CON; 54.9 (13.5) % and 75.2 (15.5) %, respectively, relative to CON, p<0.05], with HIE significantly lower than MIE (p=0.03). Total postprandial fat oxidation was increased in both MIE [83.3 (10.6) %] and HIE [89.1 (9.8) %] compared to CON [69.0 (16.1) %, p<0.05), with HIE significantly greater then MIE (p=0.012). These effects occurred in the absence of any change in glucose tolerance. In Study 2, when an isoenergetic meal was provided immediately after an acute exercise session (80 min; 60 min at ~65% VO₂peak and 10, 2 min intervals) consisting of either low carbohydrate (EX+LCHO) or high carbohydrate content (EX+HCHO), PPTG was siginificantly higher in EX+HCHO compared to EX+LCHO [449 (118) mg/dL/4h and 325 (63) mg/dL/4h, respectively, p=0.03], despite similar energy balance. Furthermore, postprandial fat oxidation was higher in EX+LCHO compared to EX+HCHO [256.7 (57.6) kcal/4h and 209.4 (56) kcal/4h, respectively, p=0.002]. PPTG was significantly related to fat oxidation (r=-0.61), fasting plasma [beta]-hydroxybutyrate (r=-0.62) and carbohydrate deficit (r=0.51), but not energy deficit (r=0.25). In summary, these data suggest that post-exercise carbohydrate balance from both increasing carbohydrate oxidation during exercise (i.e., exercise intensity) or by reducing post-exercise carbohydrate intake, is an important determinant of PPTG-lowering effects of exercise and that this may result from changes in fat oxidation.Item Effects of prolonged sitting and walking for two days on postprandial triglycerides in men : interaction with energy intake(2011-05) Park, Sanghee; Coyle, E. F. (Edward F.), 1952-; Brothers, Robert M.Postprandial hypertriglyceridemia (PPHT), an independent risk factor for atherosclerosis (Smyth and Heron 2006; Nordestgaard, Benn et al. 2007), is strongly associated with metabolic syndrome and cardiovascular diseases (CVD) (Kolovou, Anagnostopoulou et al. 2005). It has been proposed that elevated triglycerides after a high-fat meal may be a postprandial phenomenon (Zilversmit 1979). PPHT are commonly concurrent with sedentary behaviors, such as extended sitting, which amplify PPHT (Levine, Vander Weg et al. 2006). The purpose of this study was to examine the effects of prolonged sitting with or without a balanced caloric diet and walking with a balanced diet on postprandial triglycerides (PPTG). Seven healthy, young men (age, 25.6 ± 3.7 y; height, 174 ± 5 cm; weight, 71.4 ± 6.2kg; VO2max, 49.3 ± 7.7 ml/kg/min) were recruited from a college and from within the Austin community. After 2 days of food and activity control (D1and D2), subjects performed one of three trials in a randomized, cross-over design for 2 days (D3 and D4); (1) active walking with a balanced diet (WB), (2) prolonged sitting with a hyper-caloric diet (SH), and (3) prolonged sitting with a balanced diet (SB). High fat tolerance tests (HFTT) were conducted on the following day, (D5), after 13 hour over-night fasting. Blood samples were obtained in the fasting state and every hour for 6 hours after subjects had eaten a high fat test meal consisting of 1.2 g fat, 1.1 g CHO, 0.2 g protein/kg body mass. All food was provided during the 5-day duration of the study. Body postures, heart rate, and daily steps were monitored. In both sit trials (SH and SB), subjects sat ~320 minutes longer and took 10 times fewer steps than WB. In WB, the total area under the curves for plasma triglycerides (AUC[subscript T] TG) was lower, compared to SH by 21.3% (p<0.001) and to SB by 19.7% (N.S.; p = 0.055), respectively. In WB, the incremental AUC TG (AUC[subscript I] TG), an index of postprandial response, was lower than both SH by 17.4% (p <0.005) and SB by 20.1% (p <0.05), respectively. Postprandial plasma insulin concentration was lower in WB, compared to SH by 19.4% (p <0.005) in AUC[scubscript T] and 18.8 % (p < 0.05) in AUC[subscript I]. No differences were shown in the metabolic responses between SB and SH despite the diet modifications. These findings indicate that two days of prolonged sitting significantly amplifies PPTG and suppresses insulin action.Item Glycerol-3-phosphate acyltransferase regulates T cell effector function and metabolism(2013-08) Faris, Robert Allen, Jr.; Jolly, Christopher A.The aged T cell is characterized by decreased responsiveness to stimulation. Aging is associated with reduced membrane glycerophospholipid (GPL) to cholesterol ratios so it is interesting that deletion of mitochondrial glycerol-3-phosphate acyltransferase-1 which catalyzes the first step in de novo GPL synthesis induces an aged T cell phenotype in otherwise healthy mice. GPAT-1 could regulate T cell function through three possible mechanisms: maintenance of membrane GPL ratios and membrane based signaling, providing a specific substrate for downstream signaling, or direct regulation of cellular metabolism. Therefore, the goal of this project was to determine whether these mechanisms contribute to the dysfunctional T cell phenotype observed with decreased GPAT-1 activity. T cell stimulation requires significant upregulation of metabolic processes to drive clonal expansion and cytokine production. T cell dysfunction in GPAT-1 knockout mice may be partially explained by altered metabolic function. We found that GPAT-1 KO T cells have significantly reduced basal respiration rates and spare respiratory capacity which is not compensated for by increased glycolytic metabolism suggesting an inherent metabolic defect in GPAT-1 KO T cells. To better understand mechanistically how GPAT-1 regulates T cell function we moved into the Jurkat T cell line and found that shRNA mediated knockdown of the human isoform of GPAT-1 (GPAM) recapitulated key aspects of the dysfunctional T cell phenotype we observed in the mouse including highly significant reductions in IL-2 production and altered membrane GPL to cholesterol ratios. Phosphatidic acid addition was not capable of rescuing these deficiencies suggesting that GPAT-1/GPAM activity is required for proper T cell function. This was the first time that GPAT-1 activity has been shown to be important for T cell function in a non-murine model system and strongly suggests that GPAT-1/GPAM deficiency regulates T cell function at the cellular level. We further demonstrate that phosphorylation of ZAP-70 a proximal effector of T cell activation is significantly reduced in GPAM knock down Jurkat T cells, suggesting that membrane based signaling is dysfunctional. Taken together these data suggest that GPAT-1 is necessary for regulating cellular energy demands in T cells and essential for optimal T cell activation following stimulation.Item In vivo and in vitro metabolism of clavine alkaloids(Texas Tech University, 1970-08) Hsu, Joan Chung-Ying Chen,Not availableItem Increased temperature effects on fish-mediated nutrient cycling in an East Texas stream(2017-11-07) McWilliams, Jessica Lynn; Hargrave, Chad W.The unprecedented rate of global warming is an inevitable outcome of anthropogenic CO2 release into the atmosphere and complex climatic feedbacks. In ectotherms, increasing temperature may increase metabolic rates, which could enhance the energy demands of individuals and should accelerate resource acquisition. Population size and fish biomass were measured seasonally in a small second order stream over a 10-year period to examine seasonal variation in these parameters. I examined effects of increased temperature on nitrogen and phosphorus excretion in the four most abundant fish in this stream system. These fishes represent three functional feeding guilds common to many temperate stream ecosystems and comprise approximately 80-90% of the fish community. I developed temperature dependent nitrogen and phosphorus excretion models for fishes and applied these models to daily average temperatures in the stream. I then simulated climate warming (+2, +4, & +6°C) to examine the potential effects of increased temperature on fish-mediated nutrient dynamics in a southern temperate stream ecosystem. I found that increased temperature does increase nutrient cycling and nutrient flux within aquatic ecosystems; however, these effects appear to be tied to population size, biomass in addition to seasonal temperature. With increased temperature effects in spring and autumn having the greatest effect, when temperatures are cool and fish abundance and biomass is also greatest.Item Investigation of the proteomic interaction profile of uncoupling protein 3 and its effect on epigenetics(2014-08) Yan, Xiwei; Mills, Edward MichaelUncoupling proteins (UCPs) are localized on the inner mitochondrial membrane (IMM) and “uncouple” the electrochemical proton gradient formed by the electron transport chain (ETC) from ATP production. Though the prototypical uncoupling protein 1 (UCP1) is known to mediate the cold-induced thermogenesis in rodents and human neonates, the physiological and biochemical functions of the homologs UCP2-5 are still under debate. Our research focuses on UCP3, the homolog prevalently expressed in skeletal muscle (SKM), the most important metabolic organs. UCP3 has long been speculated to have a pivotal role in maintaining the mitochondrial metabolism. Several biochemical roles have been attributed to UCP3, including the regulation of fatty-acid transport and oxidation, reactive oxygen species (ROS) scavenging and calcium uptake. And several proteins have been identified to directly bind with UCP3 and facilitate its function. But to further understand how UCP3 relates to different aspects of mitochondrial functions, a more comprehensive profile of the UCP3 interaction partners is needed. We performed a mass spectrometry-based experiment and successfully identified a list of over 170 potential proteins that may directly or indirectly interact with UCP3, and several novel functions of UCP3 are implied by these protein-protein interactions. Additionally, researches have shown that the metabolic defects are important contributing factors to the epigenetic changes. Considering the roles of UCP3 in sustaining the normal mitochondrial metabolism, we hypothesized that UCP3 has a novel function in regulating the genomic DNA methylation processes. The data we obtained from the pilot study confirms that loss of UCP3 will lead to aberrant DNA methylation changes. But further experiment is still needed to investigate the regulatory pathway between UCP3 and DNA methylation. The physiological role of UCP3 in defending against cancer, diabetes and obesity has been investigated, but the mechanisms how UCP3 protect the organism from these diseases have not been elucidated. Our research sheds light on the understanding of UCP3 functions and may be of significant therapeutic benefit in the prevention and treatment of these diseases.Item Mitochondrial uncoupling links lipid catabolism to Akt inhibition and blockade of skin tumorigenesis(2014-08) Nowinski, Sara Marie; Mills, Edward MichaelIn order to support rampant cell growth, tumor cells must reprogram metabolism to simultaneously drive macromolecular biosynthesis and energy production. Mitochondrial uncoupling proteins (UCPs) oppose this phenotype by inducing futile mitochondrial respiration that is disengaged from ATP synthesis. We found that uncoupling protein 3 (UCP3) was normally expressed in follicular and epidermal keratinocytes and that its levels were augmented by calcium-induced differentiation in vitro. Over-expression of a UCP3 transgene targeted to the basal epidermis by the keratin-5 promoter (K5-UCP3) led to increased differentiation of both epidermal and bulge stem cells, the progenitors of most squamous carcinomas. Consistent with this phenotype, K5-UCP3 mice were completely protected from chemically induced skin carcinogenesis. To define the mechanisms by which UCP3 conferred such strong tumor resistance, we interbred K5-UCP3 mice with a “pre-initiated” mouse model, and found that UCP3 over-expression blocked tumor promotion. Uncoupled epidermis displayed reduced proliferation after treatment with tumor promoter, along with diminished activation of Akt signaling. This effect corresponded to decreased Akt activation by epidermal growth factor (EGF) in K5-UCP3 cells, along with UCP3 overexpressing primary human keratinocytes. Mechanistic studies revealed that uncoupling drove global lipid catabolism, along with impaired recruitment of Akt to the plasma membrane. Over-expression of wild type Akt rescued tumor promoter-induced proliferation and two-stage chemical carcinogenesis in bi-transgenic mice. Collectively, these findings demonstrate that mitochondrial uncoupling is an effective strategy to limit cell proliferation and tumorigenesis through inhibition of Akt, and suggest a novel mechanism of crosstalk between mitochondrial metabolism and growth signaling.Item NGFI-B redox sensitivity and regulation of mitochondrial bioenergetics(2011-08) Abramson, Ellen M.; Mills, Edward Michael; Bratton, Shawn; Wright, Casey; Kline, Kimberly; Nunez, NomeliChanges in intracellular redox homeostasis are implicated in both normal cell signaling and as pathophysiological mechanisms contributing to a variety of age-related diseases, including diabetes, atherosclerosis, neurodegenerative conditions, and cancer. Though a variety of well described mechanisms exist to counterbalance the overproduction of cellular oxidants and maintain optimal intracellular redox poise, the understanding of the mechanism(s) through which cellular redox homeostasis regulates cell signaling functions is less well understood. Here, we demonstrate that signaling by the immediate early gene / orphan nuclear hormone receptor NGFI-B (Nur77, TR3), which functions pleiotropically in the regulation of cell growth, metabolism, differentiation and death in diverse tissues, is redox-regulated at both the level of induction and NGFI-B-dependent gene transcription. Using co-immunoprecipitation experiments in cells, we also identified a novel interaction between NGFI-B and the cytoplasmic thiol-reducing catalyst thioredoxin1 (Trx1), that, similar to DTT, blocks NGFI-B-dependent gene expression in a manner that depends on the Trx1 active site cysteines. Together these observations add NGFI-B-dependent gene expression to a growing portfolio of transcription factor pathways that are redox-regulated. NGFI-B, in addition, appears to regulate the mitochondrial membrane potential in L6 skeletal myoblasts. NGFI-B is indispensible for T-cell receptor-mediated apoptosis and induces cell death in a variety of cell types in response to diverse pro-apoptotic stimuli. Like p53, translocation of NGFI-B from the nucleus to the mitochondria may be a critical aspect of its pro-apoptotic function. Interestingly, we found that enforced NGFI-B expression in L6 skeletal muscle myoblasts led to a significant decrease of MMP that peaked 48hr after transfection and did not require a cell death-inducing stimulus. Moreover, NGFI-B transfected cells had no increase in mitochondrial cytochrome C release despite loss of MMP at 48 hr. Combined, these data suggest that loss of MMP in muscle cells may be an early event in the apoptotic process regulated by NGFI-B. This, along with the redox regulation of NGFI-B, provides unique evidence of a relationship between the mitochondria, mitochondrial by-products, ROS, and the regulation of and by the transcription factor NGFI-B.Item Oxygen Deficient Metabolism in Organs: A Link to Combustion Science(2014-12-10) Miller, Jason MathewIn an attempt to better understand and model transport of oxygen, O2, from capillaries to living cells in surrounding tissue, the group combustion (O2 deficient) concept from the field of combustion science in engineering is applied to the biological field of microvascular O2 transport from capillaries to cells immersed in interstitial fluid (IF). The conventional Krogh model represents typical biological models, considering tissue cylinder with uniform oxygen source/sink term (US) (m''', g/s/cm^3) and O2 transport from capillary on axis (COA) towards the surface; engineering models consider cylinders with O2 supplied from the surface of cylinder (COS); in addition, they present i) transport (diffusion) and ii) kinetics limited sink rates and profiles for O2. Diffusion limitation causes m''' to be proportional to local O2 concentration. Thus, the present work modifies COS engineering models for COA cases and considers only diffusion limited transport of O2 to metabolic cells from IF. O2 profiles and resulting specific metabolic rates, SMRs (W/g), are generated for four models: I) COA with oxygen dependent consumption source term (O2) (COA-O2), II) COA-US, III) COS-O2, and IV) COS-US. In order to validate the current approach, the model results are verified with the following different types of experimental data: A) If SMRs (mq W/g) are given by the allometric law, kbmk^kq=am for organ k, then COS models under limiting conditions suggest -1/3Item Planning as a metabolic intervention(2015-05) Richter, Steven Michael; Young, Robert F., Ph. D.; Arima, EugenioHuman society relies heavily on flows of natural resources though a process called social metabolism. Growing populations and material throughput have become an ominous pattern as the negative feedbacks stemming from industrialized societies push the limits of the planetary life-support system. As the home for the majority of people and the demand-driver for material consumption, the city is a strategic point of intervention. City planners have historically, though perhaps unintentionally, shaped the social metabolism of cities. To address the rift between cities and nature, this paper seeks to refine and improve upon the existing planning approaches to guiding social metabolism. A review of social metabolism studies informs how existing data and analysis techniques can be integrated into comprehensive planning. A prototype of this methodology is presented for the water metabolism of Austin TX, demonstrating the promise of integrated metabolic planning.Item Postmortem regulation of glycolysis by 6-phosphofructokinase in bovine muscle(Texas A&M University, 2004-11-15) Rhoades, Ryan D.This study was conducted to assess the regulation of glycolysis by 6phosphofructokinase (PFK) during the postmortem metabolism of beef muscle. In the first experiment, M. sternocephalicus pars mandibularis samples were excised from six randomly-selected steers. Two samples were obtained from each steer immediately postmortem; one sample was quickly immersed in liquid nitrogen and the other was stored at 4oC for 4 d. Glycogen concentrations decreased 45% from d 0 to d 4, and 39.6 ?mol/g of glycogen was still present in the tissue at d 4. Concentrations of free glucose increased (P < 0.001) from 0.84 ?mol/g at d 0 to 6.54 ?mol/g at d 4. Fructose-6-phosphate (F6P) and glucose-6-phosphate (G6P) increased (P < 0.001) from d 0 to d 4 (2.8-fold and 4.7-fold, respectively). Lactate began accumulating immediately (3.33 ?mol/g) and was elevated to 45.9 ?mol/g by d 4. Glycolytic potential was 34.4 ?mol/g higher (P < 0.05) when measured at d 0 than at d 4. The greatest activity of PFK was measured in fresh muscle extracts, between pH 7.4-7.8; by reducing the pH to 7.0, PFK activity was depressed by nearly 50% at 1 mM F6P. In a second experiment, M. longissimus lumborum samples were excised at the 13th thoracic rib location from six randomly-selected steers. Samples were obtained at intervals ranging from 40 min to 24 h postmortem. Glycogen concentrations decreased 45% between 40 and 100 min, and tended (P ≤ 0.10) to decrease between 100 min and 24 h (from 47 to 32 ?mol/g). Concentrations of free glucose increased (P ≤ 0.009) from 1.0 ?mol/g at 40 min to 5.0 ?mol/g at 24 h. Concentrations of F6P and G6P increased dramatically after 100 min (muscle pH ≤ 6.5), whereas glycogen depletion appeared to halt by 100 min. Lactate began accumulating almost immediately and tripled in concentration by 24 h. The elevation of G6P and F6P, coupled with the pH sensitivity of PFK, indicate that the postmortem decline in pH ultimately inactivates PFK prior to glycogen depletion.Item Predicting oxygen consumption from pulmonary ventilation under low to moderate work loads(Texas Tech University, 1982-08) Calisto, George WNot availableItem Pyrimidine nucleotide de novo biosynthesis as a model of metabolic control(Texas A&M University, 2006-10-30) Rodriguez Rodriguez, MauricioThis manuscript presents a thorough investigation and description of metabolic control dynamics in vivo and in silico using as a model de novo pyrimidine biosynthesis. Metabolic networks have been studied intensely for decades, helping develop a detailed understanding of the way cells carry out their biosynthetic and catabolic functions. Biochemical reactions have been defined, pathway structures have been proposed, networks of genetic control have been examined, and mechanisms of enzymatic activity and regulation have been elucidated. In parallel with these types of traditional biochemical analysis, there has been increasing interest in engineering cellular metabolism for commercial and medical applications. Several different mathematical approaches have been developed to model biochemical pathways by combining stoichiometric and/or kinetic information with probabilistic analysis, or deciphering the comparative logic of metabolic networks using genomic-derived data. However, most of the research performed to date has relied on theoretical analyses and non-dynamic physiological states. The studies described in this dissertation provide a unique effort toward combining mathematical analysis with dynamic transition experimental data. Most importantly these studies emphasize the significance of providing a quantitative framework for understanding metabolic control. The pathway of de novo biosynthesis of pyrimidines in Escherichia coli provides an ideal model for the study of metabolic control, as there is extensive documentation available on each gene and enzyme involved as well as on their corresponding mechanisms of regulation. Biochemical flux through the pathway was analyzed under dynamic conditions using middle-exponential growth and steady state cultures. The fluctuations of the biochemical pathway intermediates and end products transitions were quantified in response to physiological perturbation. Different growth rates allowed the comparison of rapid versus long-term equilibrium shifts in metabolic adaptation. Finally, monitoring enzymatic activity levels during metabolic transitions provided insight into the interaction of genetic and biochemical mechanisms of regulation. Thus, it was possible to construct a robust mathematical model that faithfully represented, with a remarkable predictability, the nature of the metabolic response to specific environmental perturbations. These studies constitute a significant contribution to the fields of quantitative biochemistry and metabolic control, which can be extended to other cellular processes as well as different organisms.Item Regulation of Mammary cell Differentiation and Metabolism by Singleminded-2s(2013-05-21) Scribner, Kelly CDuctal carcinoma in situ (DCIS) has been shown to be a precursor to invasive ductal cancer (IDC). Though the progression of DCIS to IDC is believed to be an important aspect of tumor aggressiveness, prognosis and molecular markers that predict progression are poorly understood. Therefore, determining the mechanisms by which some DCIS progress is critical for future breast cancer diagnostics and treatment. Singleminded-2s (SIM2s) is a member of the bHLH/PAS family of transcription factors and a key regulator of differentiation. SIM2s is highly expressed in mammary epithelial cells and lost in breast cancer. Loss of Sim2s causes aberrant mouse mammary development with features suggestive of malignant transformation, whereas over-expression of Sim2s promotes precocious alveolar differentiation, suggesting that Sim2s is required for establishing and enhancing mammary gland differentiation. We hypothesize that SIM2s expression must be lost in premalignant lesions for breast cancer to develop. We first analyzed Sim2s in the involuting mammary gland, which is a highly tumorpromoting environment. Sim2s is down-regulated during involution, and forced expression delays involution. We then analyzed SIM2s expression in human breast cancer samples and found that SIM2s is lost with progression from DCIS to IDC, and this loss correlates with metastasis. SIM2s expression in DCIS promoted a differentiated phenotype and suppressed genes associated with de-differentiation. Furthermore, loss of SIM2s expression in DCIS xenografts increased metastasis likely due to an increase in hedgehog signaling and matrix metalloproteinase expression. Interestingly, we found metabolic shifts with gain and loss of SIM2s in not only DCIS cells, but also MCF7 and SUM159 cells. SIM2s expression decreased aerobic glycolysis and promoted oxidative phosphorylation through direct upregulation of CDKN1a and senescence. Loss of SIM2s, conversely, promotes mitochondrial dysfunction and induction of the Warburg effect. This is the first time CDKN1a and cellular senescence have been indicated as causative to metabolic shifts within cancer cells. These studies show a new role for SIM2s in metabolic homeostasis, and this regulation is lost during tumorigenesis. These data indicate SIM2s is at the apex where aging, metabolism, and disease meet ? regulating the delicate relationship between the three.Item Role of TGR5 in Bile Acid Metabolism(2011-08-26T17:32:23Z) Li, Tingting; Mangelsdorf, David J.TGR5 is a G protein-coupled bile acid receptor present in various tissues in the body. Its agonism increases energy expenditure and lowers blood glucose. Thus, it is an attractive drug target for treating human metabolic disease. However, TGR5 is highly expressed in the gallbladder, where its function is less well-characterized. In addition, Tgr5-/- mice are resistant to cholesterol gallstone disease (CGD), although the mechanism is poorly understood. Here, we demonstrate that TGR5 stimulates the filling of the gallbladder with bile. Gallbladder volume was increased in wild-type (WT) but not Tgr5-/- mice by administration of either the naturally-occurring TGR5 agonist, lithocholic acid (LCA), or the synthetic TGR5 agonist, INT-777. This effect did not require the presence of fibroblast growth factor-15, an enteric hormone previously shown to stimulate gallbladder filling. Ex vivo analyses using gallbladder tissue showed that TGR5 activation increased cAMP concentrations and caused smooth muscle relaxation in a TGR5-dependent manner. These data reveal a novel, gallbladder-intrinsic mechanism for regulating gallbladder contractility. Further, a markedly decreased cholic acid/muricholic acid ratio was observed in Tgr5-/- mice, indicating increased hydrophobicity in the bile acid pool. Dysregulation of the expression of genes involved in bile acid transport were also observed. Our findings further suggest that TGR5 agonists should be assessed for effects on bile acid metabolism as these agonists are developed for treating metabolic disease. Potential mechanisms for TGR5 regulation of these different physiological and pathological processes are discussed.Item Studies of bacterial catabolic enzymes: implications for the evolution of enzymes and metabolic pathways(2003) Wang, Susan C.; Whitman, Christian P.The origins of metabolic pathways and the evolution of the enzymes that comprise them have provoked intense debate and spawned a number of theories. The “patchwork” theory of Jensen, in which existing enzymes are combined to give new pathways, is one. Another emerging theme is that of “catalytic promiscuity,” the ability of an enzyme to catalyze a low-level activity that differs from its physiological function. Such an activity can be amplified through mutation(s) to yield a more efficient enzyme. These ideas are often used to explain the origins of “superfamilies,” which consist of enzymes that catalyze different reactions yet share sequence and/or structural homology. The catechol meta-fission pathway, a plasmid-encoded degradation pathway for simple aromatic compounds, is rich in enzyme chemistry and replete with structural and evolutionary diversity. 4-Oxalocrotonate tautomerase (4-OT), the best characterized enzyme in this pathway, is a member of the tautomerase superfamily. Two additional enzymes, YwhB, an enzyme of unknown function, and trans-3- chloroacrylic acid dehalogenase (CaaD) are also members. One defining feature of this superfamily is the conservation of an N-terminal proline which functions as a catalytic base. CaaD catalyzes dehalogenation via a hydration mechanism. Through site-directed mutagenesis, kinetic and pH rate analysis, and irreversible inhibition with 3-halopropiolates, Pro-1 of the β-subunit of CaaD was identified as a general acid catalyst instead of a general base catalyst, thus differentiating it from the rest of the superfamily. 4-OT and YwhB also catalyze dehalogenation, but at a low level. These results suggest that one or both enzymes may be the ancestor(s) of CaaD. Both enzymes are also inhibited by 3-halopropiolates, but by a different mechanism than that observed for CaaD. 2-Hydroxymuconate semialdehyde dehydrogenase (2-HMSD) immediately precedes 4-OT in the meta-fission pathway and is a member of the aldehyde dehydrogenase superfamily. Its catalytic mechanism may utilize a cysteine to form a thiohemiacetal intermediate, which is followed by hydride transfer to NAD+. 2- HMSD has been characterized with several substrates, and site-directed mutagenesis has identified the essential catalytic cysteine. Additionally, 2-HMSD is reversibly inhibited by a product analog. This work sets the stage for further investigaton of its structure and mechanism.Item Study of pure and conjugated culture batch fermentation of Cephalosporium acremonium(Texas Tech University, 1986-12) Khang, Yong HoPenicillin N and cephalosporin C (CPC) were produced sequentially by ATCC 36225 (69). Cell-free extracts of its mutant, ATCC 52518, catalyzed the conversion of penicillin N into cephalosporin C (38). It was postulated that the yield of CPC production might be altered when these two cultures were grown together. Optimum experimental conditions were determined by examining the effect of aeration, agitation, and other parameters on growth. The effect of sample size on the analysis of cell dry weight was also examined to reduce experimental error. The proper air flow rate was found to be 50 ml/min in a 2-L glass fermenter when no antifoam agent was added. Microbial films were severely reduced when 60-70% of fermenter volume was used as working volume. The relationship between the two microorganisms was found to be neutralistic when the cellular growth of conjugated cultures (ATCC 36225 + ATCC 52518) was compared with that of each pure culture (Figure 4.14). The growth yield of ATCC 36225 was twice that of ATCC 52518 and the growth yield of conjugated cultures was the same as that of ATCC 36225 (Table 4.5). The CPC production yield of conjugated cultures, however, was less than that of the ATCC 36225 culture. On the basis of these results, it was concluded that precursor amino acids (L-(a)-aminoadipate, L-cysteine, and L-valine) produced by ATCC 52518 could not stimulate CPC synthesizing enzymes of ATCC 36225 to synthesize 3-lactam antibiotics in the conjugated cultures.