Matching Items (13)

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Role of Metabolism in Antibiotic Resistance

Description

Each year, more and more multi-drug resistant bacterial strains emerge, thus complicating treatment and increasing the average stay in the intensive care unit. As antibiotics are being rendered inefficient, there

Each year, more and more multi-drug resistant bacterial strains emerge, thus complicating treatment and increasing the average stay in the intensive care unit. As antibiotics are being rendered inefficient, there is a need to look into ways of weakening the internal state of bacterial cells to make them more susceptible to antibiotics. For this, we first need to understand what methods bacteria employ to fight against antibiotics. In this work, we have reviewed how bacteria respond to antibiotics. There is a similarity in response to antibiotic exposure and starvation (stringent stress) which changes the metabolic state. We have delineated what metabolism changes take place and how they are associated with oxidative stress. For example, there is a common change in NADH concentration that is tied to both metabolism and oxidative stress. Finally, we have compared the findings in literature with our research on an antibiotic-resistant RNA polymerase mutant that alters the gene expression profile in the general areas of metabolism and oxidative stress. Based on this thesis, we have suggested a couple of strategies to make antibiotics more efficient; however, as antibiotic-mediated killing is very complex, researchers need to delve deeper to understand and manipulate the full cellular response.

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  • 2020-05

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Genistein-mediated diet tends to increase oxidative stress in the vasculature of female ob/ob mice

Description

Morbid obesity is associated with cardiovascular and metabolic disorders. A major contributor to the pathogenesis of these diseases is impaired vasodilation resulting from elevated reactive oxygen species (ROS). This is

Morbid obesity is associated with cardiovascular and metabolic disorders. A major contributor to the pathogenesis of these diseases is impaired vasodilation resulting from elevated reactive oxygen species (ROS). This is because certain ROS such as superoxide are raised with obesity and scavenge the endogenous vasorelaxant nitric oxide, resulting in hypertension. The objective of this study was to measure the ability of genistein to quench superoxide in the vasculature of ob/ob mice, an animal model of obesity and type 2 diabetes. Genistein is an isoflavonic phytoestrogen naturally found in soy products. While genistein has documented antioxidant and anti-inflammatory properties, it is not known whether this protects the vasculature from oxidative stress. Genistein was hypothesized to reduce superoxide in arteries from female ob/ob mice. The superoxide indicator dihydroethidium (DHE) [2µL/mL HEPES buffer] was added to isolated aortae and mesenteric arteries from mice fed either a control (standard rodent chow containing 200-300 mg genistein/kg) or genistein-enriched (600mg genistein/kg rodent chow) diets for 4 weeks. Frozen tissues sections were collected onto glass microscope slides and examined using confocal microscopy. Contrary to the hypothesis, a diet containing twice the amount of genistein found in standard chow did not significantly reduce superoxide concentrations in aortae (p=0.287) or mesenteric arteries (p=0.352). Superoxide dismutase, an antioxidant enzyme that breaks down superoxide, was significantly upregulated in the genistein-enriched diet group (p=0.004), although this elevation did not promote the breakdown of superoxide. In addition, the inflammatory marker iNOS was not downregulated in the genistein-enriched diet group (p>0.05). The results indicate that high amounts of isoflavones, like genistein, may not exhibit the purported antioxidant effects in the vasculature of obese or diabetic subjects. Further studies examining arteries from ob/ob mice fed a genistein-free diet are needed to elucidate the true effects of genistein on oxidative stress.

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  • 2014-05

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F2-isoprostanes and F2-isoprostane Metabolites: Biomarkers for Oxidative Stress and Therapeutic Efficacy

Description

F2-isoprostanes are a series of prostaglandin-like compounds derived from the free radical-mediated lipid peroxidation of arachidonic acid, a polyunsaturated fatty acid that is ubiquitously expressed in cell membranes. F2-isoprostanes are

F2-isoprostanes are a series of prostaglandin-like compounds derived from the free radical-mediated lipid peroxidation of arachidonic acid, a polyunsaturated fatty acid that is ubiquitously expressed in cell membranes. F2-isoprostanes are biomarkers of oxidative stress, an imbalance between oxidants and antioxidants that can cause damage to DNA, proteins, lipids, and carbohydrates. Increased production of lipid peroxidation products have been implicated in the pathology of a number of conditions and diseases in humans. The objective of this thesis was to (1) optimize the LC/MS/MS F2-isoprostane method currently used in human samples for use in research animals and veterinary medicine, including the use of solid phase extraction, and (2) validate the optimized method in rodent and canine experimental studies. Our optimized method showed that Lyprinol treatment in dogs with osteoarthritis decreases F2-isoprostane levels nearly 2-fold. In addition, adjuvant alpha-tocopherol prevented tumor-induced increased F2-isoprostane levels. Finally, contrary to earlier studies using less specific ELISA F2-isoprostane methods, we demonstrate that unconditioned dogs benefit from low intensity exercise. Our data demonstrate successful optimization of the human LC/MS/MS F2-isoprostane method in rats and canines. Importantly, our results emphasize the need to use the more sensitive and specific LC/MS/MS method as compared to ELISA-based assays in order to distinguish the 15- and 5-series F2-isoprostanes, evidenced in particular by the two canine studies.

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Date Created
  • 2015-05

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LC-MS/MS Analysis of Renal Cell Carcinoma Treated with Sulforaphane

Description

Sulforaphane(SFN)isanisothiocyanate(ITC)derivedfromcruciferousvegetables,suchas
broccoli,thatisgrowinginpopularityforitsantioxidantandanti-inflammatorycapabilities.
Furthermore,SFNhasbeendemonstratedtoimproverenalcancercarcinoma(RCC)treatment
outcomesinconjunctionwithmultipleotherformsoftherapy,whichisespeciallyimportant
consideringRCC’spoortherapeuticoutcomeswithchemotherapy.Theaimofthisstudywasto
determinetheeffectsofSFNonRCC ​invitro utilizingcellviabilityanalysisandLC/MS-MS
targetedmetabolicprofilingtorevealpathwaysresponsibleforSFN’spossibleenhancementof
chemotherapytreatmentinRCC.CCK-8resultsshowthat15 ​μ​MofSFNcausedasignificant(p
<0.05)increaseinRCCproliferation.Kruskal-Wallistestsrevealed16metabolitesinourcell,
and28inthemediumtobesignificant(p<0.05).Anorthogonalpartialleastsquares-discriminant
analysis,OPLS-DA,ofsignificantmetaboliteswasusedtocomparedtreatedandnon-treated
samplesforbothdatasetsandshoweda100%predictiveaccuracy(AUC=1).Enrichment
analysisdeterminedthatatotalof7metabolicpathwaysweresignificantlyenriched(VLCFA
β-oxidation,glutamatemetabolism,theureacycle,ammoniarecycling,glycine/serine,alanine,
andglucose-alaninecycle).Pathwayanalysisshowedhistidinemetabolismtobetheonly
significantlyaffectedpathwaybetweenbothdatasets.SFN-inducedmetaboliccharacteristics
foundinRCCwereconsistentwithknownantioxidantandanti-inflammatorypathways.Ourdata
suggeststhatthetherapeuticmechanismsofSFNarelikelyduetointeractionswithTandNKT
cellsthatprotectthemfromoxidativestress.Futureexperimentsregardingantioxidantresearch
incancershouldbecompletely ​invivo​,asopposedto ​invitro, ​inordertomaintainthenatural
physiology of cancer cells in the presence of host immune cells.

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Date Created
  • 2019-05

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Synthesis of multifunctional radical quenchers (MRQ's)

Description

Mitochondria produce most of the ATP needed for the cell as an energy source. It is well known that cellular respiration results in oxidative damage to the cell due to

Mitochondria produce most of the ATP needed for the cell as an energy source. It is well known that cellular respiration results in oxidative damage to the cell due to the production of reactive oxygen species (ROS). Mitochondrial dysfunction is believed to contribute to a number of degenerative diseases; because of this the mitochondrial respiratory chain is considered as potential drug target. A few series of idebenone analogues with quinone, pyridinol and pyrimidinol redox cores have been synthesized and evaluated as antioxidants able to protect cellular integrity and, more specifically, mitochondrial function. The compounds exhibited a range of activities. The activities observed were used for the design of analogues with enhanced properties as antioxidants. Compounds were identified which provide better protection against oxidative stress than idebenone, and it is thought that they do so catalytically.

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Date Created
  • 2012

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Quinones and analogues as cytoprotectants for cultured mammalian cells

Description

It has been well established that mitochondria play a critical role in the pathology of Friedreich's Ataxia. This disease is believed to be caused by a deficiency of frataxin, which

It has been well established that mitochondria play a critical role in the pathology of Friedreich's Ataxia. This disease is believed to be caused by a deficiency of frataxin, which research suggests is responsible for iron sulfur cluster assembly. This incomplete assembly of iron sulfur clusters is believed to be linked with dysfunctional complexes in the mitochondrial respiratory chain, increased oxidative stress, and potential cell death. Increased understanding of the pathophysiology of this disease has enabled the development of various therapeutic strategies aimed at restoring mitochondrial respiration. This thesis contains an analysis of the biological activity of several classes of antioxidants against oxidative stress induced by diethyl maleate in Friedreich's Ataxia lymphocytes and CEM leukemia cells. Analogues of vitamin E α-tocopherol have been shown to protect cells under oxidative stress. However, these same analogues show various levels of inhibition towards the electron transport chain complex I. Bicyclic pyridinols containing a ten carbon substituent provided favorable cytoprotection. N-hydroxy-4-pyridone compounds were observed to provide little protection. Similarly, analogues of CoQ10 in the form of pyridinol and pyrimidinol compounds also preserved cell viability at low concentrations.

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Date Created
  • 2012

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Oxidative stress and a high fat diet in rats : an intervention study on the effects of an organometallic compound on enzyme function, inflammatory markers, endotoxins and fasting serum glucose and insulin levels

Description

Cardiovascular disease has reached epidemic proportions resulting in its ranking as the number one cause of mortality in the Western world. A key player in the pathophysiology of vascular disease

Cardiovascular disease has reached epidemic proportions resulting in its ranking as the number one cause of mortality in the Western world. A key player in the pathophysiology of vascular disease is oxidative stress due to free radical accumulation. This intervention study was conducted to evaluate any potential mediation of oxidative stress using a soil-derived organometallic compound (OMC) with suspected antioxidant properties. A 10-week study was conducted in male Sprague-Dawley rats (n = 42) fed either a high-fat diet (HFD) consisting of 60% kcal from fat or a standard Chow diet containing only 6% kcals from fat. Rats from each diet group were then subdivided into 3 subgroups (n = 6-10 each) that received 0.0 mg/mL, 0.6 mg/mL or 3.0 mg/mL OMC. Neither the diet nor OMC significantly changed protein expression of inducible nitric oxide synthase (iNOS) in isolated aortas. Plasma levels of the inflammatory marker, tumor necrosis factor alpha (TNFα) were below detection after the 10-week trial. Superoxide dismutase (SOD), a scavenger of the free radical, superoxide, was not significantly different following HFD although levels of SOD were significantly higher in Chow rats treated with 0.6 mg/mL OMC compared to HFD rats treated with the same dose (p < 0.05). Lipopolysaccharides (LPS) were significantly increased following 10 weeks of high fat intake (p < 0.05). This increase in endotoxicity was prevented by the high dose of OMC. HFD significantly increased fasting serum glucose levels at both 6 weeks (p < 0.001) and 10 weeks (p < 0.025) compared to Chow controls. The high dose of OMC significantly prevented the hyperglycemic effects of the HFD in rats at 10 weeks (p = 0.021). HFD-fed rats developed hyperinsulinemia after 10 weeks of feeding (p = 0.009), which was not prevented by OMC. The results of this study indicate that OMC may be an effective strategy to help manage diet-induced hyperglycemia and endotoxemia. However, further research is needed to determine the mechanism by which OMC helps prevent hyperglycemia as measures of inflammation (TNFα) and vascular damage (iNOS) were inconclusive.

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Date Created
  • 2018

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Synthesis, biochemical and pharmacological evaluation of rationally designed multifunctional radical quenchers

Description

Mitochondria are crucial intracellular organelles which play a pivotal role in providing energy to living organisms in the form of adenosine triphosphate (ATP). The mitochondrial electron transport chain (ETC) coupled

Mitochondria are crucial intracellular organelles which play a pivotal role in providing energy to living organisms in the form of adenosine triphosphate (ATP). The mitochondrial electron transport chain (ETC) coupled with oxidative phosphorylation (OX-PHOS) transforms the chemical energy of amino acids, fatty acids and sugars to ATP. The mitochondrial electron transport system consumes nearly 90% of the oxygen used by the cell. Reactive oxygen species (ROS) in the form of superoxide anions (O2*-) are generated as byproduct of cellular metabolism due to leakage of electrons from complex I and complex III to oxygen. Under normal conditions, the effects of ROS are offset by a variety of antioxidants (enzymatic and non-enzymatic).

Mitochondrial dysfunction has been proposed in the etiology of various pathologies, including cardiovascular and neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, ischemia-reperfusion (IR) injury, diabetes and aging. To treat these disorders, it is imperative to target mitochondria, especially the electron transport chain. One of the methodologies currently used for the treatment of mitochondrial and neurodegenerative diseases where endogenous antioxidant defenses are inadequate for protecting against ROS involves the administration of exogenous antioxidants.

As part of our pursuit of effective neuroprotective drugs, a series of pyridinol and pyrimidinol analogues have been rationally designed and synthesized. All the analogues were evaluated for their ability to quench lipid peroxidation and reactive oxygen species (ROS), and preserve mitochondrial membrane potential (Δm) and support ATP synthesis. These studies are summarized in Chapter 2.

Drug discovery and lead identification can be reinforced by assessing the metabolic fate of orally administered drugs using simple microsomal incubation experiments. Accordingly, in vitro microsomal studies were designed and carried out using bovine liver microsomes to screen available pyridinol and pyrimidinol analogues for their metabolic lability. The data obtained was utilized for an initial assessment of potential bioavailability of the compounds screened and is summarized fully in Chapter 3.

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Date Created
  • 2014

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Synthesis of nitrogen heterocyclic compounds for therapeutic applications

Description

Reactive oxygen species (ROS) are a series of molecules, ions, and radicals derived from oxygen that possess remarkable reactivity. They act as signaling molecules when their concentration in cells is

Reactive oxygen species (ROS) are a series of molecules, ions, and radicals derived from oxygen that possess remarkable reactivity. They act as signaling molecules when their concentration in cells is within a normal range. When the levels of ROS increase, reaching a concentration in which the antioxidants cannot readily quench them, oxidative stress will affect the cells. These excessive levels of ROS result in direct or indirect ROS-mediated damage of proteins, nucleic acids, and lipids. Excessive oxidative stress, particularly in chronic inflammation, has been linked with mutations and carcinogenesis. One of the main targets of ROS in severe oxidative stress is mitochondrial DNA (mtDNA). The synthesis of analogues of alpha-tocopherol is described as potential compounds with the ability to remediate defective mitochondria. An interesting possibility for eradicating cancer cells is to selectively target them with oxidative species while avoiding any deleterious effects on healthy cells. To accomplish this, analogues of the beta-hydroxyhistidine moiety of the antitumor agent bleomycin (BLM) were synthesized. The first part of this thesis focuses on the synthesis of simplified analogues of alpha-tocopherol. These analogues possess a bicyclic pyridinol as the antioxidant core and an alkyl group as the lipophilic chain to mimic alpha-tocopherol. Additionally, analogues with a completely oxidized pyridinol core were synthesized. Some of these analogues showed promising properties against ROS production and lipid peroxidation. The protection they conferred was shown to be tightly regulated by their concentration. The second part of this thesis focuses on the synthesis of analogues of beta-hydroxyhistidine. BLMs are glycopeptides that possess anticancer activity and have been used to treat testicular carcinomas, Hodgkin's lymphoma, and squamous cell carcinomas. The activity of BLM is based on the degradation of DNA, or possibly RNA, caused by a Fe(II)-BLM complex in the presence of O2. The beta-hydroxyhistidine moiety of BLM contributes to metal coordination via two ligands: the N-3 nitrogen atom of imidazole and possibly the nitrogen atom of the amide. A series of beta-hydroxyhistidine analogues has successfully been synthesized.

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Date Created
  • 2014

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The efficacy of nopales (Opuntia spp) on lipoprotein profile and oxidative stress among moderately hypercholesterolemic adults

Description

Background: Evidence about the purported hypoglycemic and hypolipidemic effects of nopales (prickly pear cactus pads) is limited. Objective: To evaluate the efficacy of nopales for improving cardiometabolic risk factors and

Background: Evidence about the purported hypoglycemic and hypolipidemic effects of nopales (prickly pear cactus pads) is limited. Objective: To evaluate the efficacy of nopales for improving cardiometabolic risk factors and oxidative stress, compared to control, in adults with hypercholesterolemia. Design: In a randomized crossover trial, participants were assigned to a 2-wk intervention with 2 cups/day of nopales or cucumbers (control), with a 2 to 3-wk washout period. The study included 16 adults (5 male; 46±14 y; BMI = 31.4±5.7 kg/m2) with moderate hypercholesterolemia (low density lipoprotein cholesterol [LDL-c] = 137±21 mg/dL), but otherwise healthy. Main outcomes measured included: dietary intake (energy, macronutrients and micronutrients), cardiometabolic risk markers (total cholesterol, LDL-c, high density lipoprotein cholesterol [HDL-c], triglycerides, cholesterol distribution in LDL and HDL subfractions, glucose, insulin, homeostasis model assessment, and C-reactive protein), and oxidative stress markers (vitamin C, total antioxidant capacity, oxidized LDL, and LDL susceptibility to oxidation). Effects of treatment, time, or interactions were assessed using repeated measures ANOVA. Results: There was no significant treatment-by-time effect for any dietary composition data, lipid profile, cardiometabolic outcomes, or oxidative stress markers. A significant time effect was observed for energy, which was decreased in both treatments (cucumber, -8.3%; nopales, -10.1%; pTime=0.026) mostly due to lower mono and polyunsaturated fatty acids intake (pTime=0.023 and pTime=0.003, respectively). Both treatments significantly increased triglyceride concentrations (cucumber, 14.8%; nopales, 15.2%; pTime=0.020). Despite the lack of significant treatment-by-time effects, great individual response variability was observed for all outcomes. After the cucumber and nopales phases, a decrease in LDL-c was observed in 44% and 63% of the participants respectively. On average LDL-c was decreased by 2.0 mg/dL (-1.4%) after the cucumber phase and 3.9 mg/dL (-2.9%) after the nopales phase (pTime=0.176). Pro-atherogenic changes in HDL subfractions were observed in both interventions over time, by decreasing the proportion of HDL-c in large HDL (cucumber, -5.1%; nopales, -5.9%; pTime=0.021) and increasing the proportion in small HDL (cucumber, 4.1%; nopales, 7.9%; pTime=0.002). Conclusions: These data do not support the purported benefits of nopales at doses of 2 cups/day for 2-wk on markers of lipoprotein profile, cardiometabolic risk, and oxidative stress in hypercholesterolemic adults.

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Date Created
  • 2013