Browsing by Subject "Membrane Transport Proteins"
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Item Antisense Blockade of Efflux Systems in Gram-Negative Pathogens(2018-01-23) Subramanian, Naveen G.; Felder-Scott, Christina F.; Sturge, Carolyn R.; Greenberg, DavidAntibiotic resistant bacteria, aka "super bugs", are a critical threat to public health worldwide, as the medical community is running out of effective antibiotics against a growing number of bacteria. One of the ways that bacteria develop resistance to antibiotics is by utilizing efflux systems that are used to pump the antibiotic out. A strategy that is currently being investigated is to restore the susceptibility of these bacteria to antibiotics by using peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) to suppress genes within these bacteria that encode components of efflux pumps. This project studied the effectiveness of PPMOs that target the AcrAB-TolC efflux pump, which is a major component of the intrinsic antibiotic resistance mechanisms of E. coli and K. pneumoniae. Experiments tested for the effect of the PPMO targeting the acrA gene, specific sequences within the acrA gene, and the tolC gene. The effect of the PPMO was measured by a change in the minimum inhibitory concentration (MIC) of common antibiotics such as Piperacillin/Tazobactam (Pip/Tazo), Azithromycin, and Levofloxacin on strains of these two bacteria. The results show that PPMOs targeted to the acrA gene have a 4-8 fold effectiveness at lowering antibiotic MICs for the bacterial strains. PPMOs that targeted the tolC gene, on the other hand, have no synergistic effect in lowering antibiotic MICs for the bacterial strains. In addition, changing the sequence of the PPMOs targeting the acrA gene was shown to have an effect, albeit small, on susceptibility to antibiotics, which suggests that targeting specific regions of a gene of interest can induce more or less susceptibility in the bacteria to antibiotics.Item The Regulation of Cholesterol Absorption: Nuclear Hormone Receptors and Niemann-Pick C1 Like 1(2007-12-15) Valasek, Mark Andrew; Repa, Joyce J.; Tansey, MalĂș G.; Anderson, Richard G. W.; Lynch, Kristen W.; Mangelsdorf, David J.Cholesterol plays fundamental roles in cellular physiology, but it is also involved in many pathophysiological processes including atherosclerosis, cholelithiasis, and some forms of neurodegenerative disease. The ability of mammals to selectively absorb cholesterol from the diet while largely excluding plant sterols has been known for more than 75 years, but the precise repertoire of molecular events necessary for this process are just beginning to be elucidated. Recently, several candidates have been put forth as putative intestinal cholesterol "permeases" responsible for cholesterol transport across the intestinal brush border. In addition, members of the nuclear receptor superfamily of ligand-activated transcription factors are known to modulate expression of genes involved in cholesterol and bile acid homeostasis, and cholesterol absorption. Therefore, we wanted not only to clarify the essential molecular mechanisms by which cholesterol was absorbed, but also to investigate the potential role of nuclear receptors in regulating essential steps in this process. Here, we show that a candidate component of the cholesterol transport machinery, caveolin-1 (CAV1), is neither required for intestinal cholesterol transport or sensitivity to the novel cholesterol absorption blocking agent, ezetimibe. This rules out a critical role for caveolin-1 and lends further support to the contention that Niemann-Pick C1 like 1 (NPC1L1) is the bona fide intestinal cholesterol permease. Therefore to better understand new ways in which nuclear receptors could regulate cholesterol absorption, we studied nuclear receptor regulation of NPC1L1 and determined that several nuclear receptors could modulate its expression in small intestine, including peroxisome proliferator-activated receptor alpha (PPAR alpha ) and retinoid X receptor (RXR). Thus, cholesterol absorption can be regulated by nuclear receptor modulation of NPC1L1 expression.