Browsing by Subject "in vitro"
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Item A Novel sRNA Member of the Carbon Storage Regulatory System of Escherichia Coli(2002-12-01) Weilbacher, Thomas; Jerry SimeckaWeilbacher, Thomas S., A Novel sRNA Member of the Carbon Storage Regulatory System of Escherichi coli. Master of Science (Microbiology & Immunology), December, 2002, 57 pp., 2 tables, 12 illustrations, bibliography, 44 titles. Small untranslated RNAs (sRNAs) perform a variety of important functions in bacterial systems. The 245 nt sRNA of Escherichia coli K-12, CsrC, was uncovered using a genetic screen for genes that regulate glycogen biosynthesis. CsrC RNA binds multiple copies of CsrA, a protein that post-transcriptionally regulates central carbon flux, biofilm formation, and motility in E. coli. CsrC antagonizes the regulatory effects of CsrA, presumably by sequestering this protein. The discovery of CsrC is intriguing, in that a similar sRNA, CsrB, performs essentially the same function. Both of these sRNAs possess similar imperfect repeat sequences (18 in CsrB, 9 in CsrC), primarily localized in the loops of predicted hairpins, which may serve as CsrA binding elements. Transcription of csrC increases as the culture approaches the stationary phase of growth and is activated by CsrA and the response regulator UvrY. Complementation and in vitro transcription-translation experiments reveal that CsrA effects on csrC are mediated indirectly, through UvrY. Because CsrB and CsrC antagonize the activity of CsrA and are dependent on CsrA for their synthesis, a csrB null mutation causes a modest compensatory increase in CsrC levels and vice versa. An updated model for the signaling circuitry of the Csr system is discussed.Item Characterization of the Bradykinn Receptor in Human Corneal Epithelium(1997-08-01) Wiernas, Terry Kirkham; Michael W. Martin; Glenn Dillon; Michael ForsterWiernas, Terry Kirkham, Characterization of the Bradykinin Receptor in Human Corneal Epithelium. Doctor of Philosophy (Biomedical Sciences), August, 1997, 255 pp., 5 tables, 39 figures, references, 137 titles. Bradykinin (BK) is a well-established mediator of inflammation. High levels of BK in human tears following ocular allergic provocation led to the hypothesis that BK receptors may exist on the corneal epithelium and could play a role in corneal inflammation and/or wound healing, in addition to other functions. To test this hypothesis, human corneal epithelial cells were cultured and used to conduct a series of studies to evaluate and characterize the BK receptor. Due to the limited supply and high cost of primary human corneal epithelial (P-CEPI) cells, in addition to the fact that these cells do not divide and proliferate over more than a few passages, SV40 virus-immortalized human CEPI cells (CEPI-17-CL4) were used as a model system. Extensive studies confirmed that the immortalized cells faithfully represented the primary cells. This study demonstrated the presence of BK receptors on corneal epithelial cells for the first time. The receptors were characterized as the B2 subtype and were found to be represented by an apparent single binding site. Furthermore, stimulation of these receptors was found to elicit concentration-dependent increases in both inositol phosphates, via activation of phospholipase C, and intracellular calcium mobilization. The rank order affinity of BK and its analogs as determined by binding assays was found to correlate well with the rank order potency of BK and its analogs in evoking the latter functional responses, which were blocked by two B2-receptor selective antagonists. A significant, concentration-dependent stimulation of [3H]thymidine uptake in CEPI cell DNA was elicited by BK which suggests a potential mitogenic effect of BK and a role in corneal wound healing. BK did not significantly affect the release of three pro-inflammatory cytokines, prostaglandin E2 or matrix metalloproteinase-1, and seemed to have an inhibitory effect on the release of tumor necrosis factor α. In conclusion, these studies have confirmed that CEPI-17-CL4 cells represent a good in vitro model of human corneal epithelium and have contributed to a better understanding of the ocular effects of BK and characterization of its receptor within the cornea.Item Met-Enkephalin-Arg-Phe (MERF) and Metabolism of MERF Across the Canine Heart Vascular Bed(2000-08-01) Pearlman, Eric Brian; Barbara Barron; Patricia A. Gwirtz; Michael L. SmithPearlman, Eric B., Met-Enkephalin-Arg-Phe (MERF) and Metabolism of MERF Across the Canine Heart Vascular Bed. Master of Science (Biomedical Science), August, 2000, 37 pp., 3 tables, 11 figures, references, 20 titles. Methionine enkephalin arginine phenylalanine (MERF) has been shown to be co-stored with catecholamines in vesicles. The catecholamines appear to decrease the degradation rate of 3H-MERF in vitro. The aim of this study is to investigate the spillover and metabolism of MERF across the canine heart vascular bed. I hypothesize that 3H-MERF is either degraded in the plasma or taken up and degraded by the heart. I further hypothesize that the exogenous catecholamine, isoproterenol, inhibits or reduces the rate of MERF degradation. Mongrel dogs were anesthetized and instrumented to record cardiovascular parameters, infuse 3H-MERF, and obtain blood samples across the heart. Blood samples were taken before and after stopping 3H-MERF infusion to evaluate kinetics, show steady state, and test the effect of treatments. Steady state concentration of 3H-MERF was observed after 30 min of infusion. Chromatography separated intact from degraded 3H-MERF. Three experimental groups were used: control, propranolol plus isoproterenol, and propranolol only. Blockade of β-receptors was necessary to prevent changes in coronary blood flow. Propranolol bolus (0.2 mg/kg) was administered IV at 50 min. 3 μg/min isoproterenol or 0.5 ml/min normal saline was infused starting at 70 min until the end of sample collection. The 3H-MERF venous-arterial (V-A) difference prior to treatment was negative, indicating degradation in the plasma or uptake and degradation by the heart. The 75 min V-A difference was used to calculate the effect of the infusions on the degradation or uptake of the 3H-MERF; this value was unchanged by any treatment. Spillover of 3H-MERF was significantly lower in the propranolol + isoproterenol dogs (p [less than] 0.05) compared to propranolol only treatment at 75 min. Heart rate was significantly lower for the propranolol only group compared to control. Blood pressure and change in coronary flow were unchanged. In conclusion, isoproterenol does not affect the metabolism of 3H-MERF across the canine heart vascular bed. Propranolol, however, does increase the intact 3H-MERF in the plasma, but additional β adrenergic blockade agents need to be investigated to determine the mechanism by which this takes place.Item Opioid Receptors in Aging and Oxidative Stress(2007-01-01) Raut, Atul M.; Ratka, Anna; Simpkins, James W.; Dillon, GlennRaut, Atul M., Opioid Receptors in Aging and Oxidative Stress. Doctor of Philosophy (Pharmacology and Neuroscience), January 2007, 181 pp, 4 illustrations, 21 figures, 159 titles. Oxidative stress has been implicated in aging and neurodegenerative disorders. Pain sensitivity and responses to opioids change with aging. The effect of aging and oxidative stress on opioid receptor system is not yet well understood. To study the effects of aging on pain sensitivity and opioid-induced antinociception, and to determine the possible association of oxidative stress with these pain parameters, in vivo studies were conducted. To further elucidate the effects of oxidative stress on opioid receptor proteins and their function, in vitro studies were carried out. The effects of aging on pain sensitivity and opioid-induced antinociception were studied in male Fischer 344 rats. Oxidative stress markers in cerebral cortex, hippocampus, striatum and midbrain of these rats were estimated. It was concluded that sensitivity to nociceptive stimulus increases and responses to opioids decrease with aging and age-related oxidative damage is negatively correlated with opioid-induced antinociception. To characterize the effects of oxidative stress on function of opioid receptors, changes in intracellular cyclic adenosine monophosphate (cAMP) was measured in human SK-N-SH neuronal cells under oxidative stress conditions. It was found that oxidative stress decreased the function of mu opioid receptor (MOR) but not that of delta or kappa opioid receptors (DOR and KOR respectively). Antioxidant intervention preserved the function of MOR. Western immunoassays revealed that MOR but not DOR and KOR proteins were decreased under oxidative stress conditions. Thus, these findings show a selective impairment of the MOR function and reduction in MOR protein under conditions of oxidative stress. The results from the in vivo and in vitro projects demonstrate the involvement of aging and oxidative stress in modulation of pass sensitivity, opioid-induced antinociception and opioid receptor function and expression.Item Regulation of Human Macrophage Colony-Stimulating Factor Transcription(2001-05-01) Kamthong, Pisate John; Lad Dory; Richard Easom; Stephen R. GrantThe role of macrophage colony-stimulating factor (M-CSF) in hematopoiesis has been firmly established mainly by using bone marrow cell cultures. Semi-solid culture of bone marrow cells that were independently developed by Bradley and Metcalf in 1966 and Pluznik and Sachs in 1965, has been the standard method to study proliferation and differentiation of hematopoietic cells since the mid-1960s. It supports the clonal expansion of the hematopoietic colonies in vitro. Thus provides the means to functionally assay the hematopoietic colonies in vitro. Thus provides the means to functionally assay the hematopoietic progenitor cells and aides the discovery of growth factors regulating the progenitor cell differentiation. Macrophage colony-stimulating factor (M-CSF) was initially identified as a hematopoietic growth factor that stimulates the proliferation, differentiation and survival of monocytes, macrophages, and their progenitors (Robinson et al. 1969; Stanly et al. 1971). M-CSF is produced by a large variety of cells throughout the body. It can be purified from various body fluids as well as the conditioned media of several cell lines and tissues, such as leukocytes, placenta, lung, pancreatic cancer cells and spleen (Metcalf 1984; Stanley and Guilbert 1981; Yunis 1983). The sources of M-CSF recently have been extended to include liver parenchymal cells (Ezure et al. 1997) and thyrocytes (Kasai et al. 1997). It was also previously called colony-stimulating factor from human urine (CSF-HU) attributing the source of human urine growth factor that stimulated the formation of small aggregates consisting of granulocyte clusters in a soft agar culture system of human bone marrow cells (Metcalf 1974). At first without the knowledge about biochemical structures of CSFs, several colony-stimulating factors, later proven to be M-CSF, were recognized by their sources, i.e. mouse L-cell CSF, mouse uterus CSF, human lung-conditioned-medium CSF. Later, researchers in the field adopted the reclassification of CSF subtypes using the predominant colony types stimulated by the factor in semi-solid bone marrow cell cultures. M-CSF is for CSF that predominantly stimulates macrophage colony formation. Granulocyte colony-stimulating factor (G-CSF) refers to a granulocyte-active material, such as peritoneal cell-conditioned medium CSF (Horiuchi and Ichikawa 1977). CSF stimulating both types of colonies is called granulocyte-macrophage colony-stimulating factor, GM-CSF. M-CSF was also termed colony stimulating factor 1 (CSF-1), described as the first CSF to be purified (Stanley 1977). G-CSF was later called CSF-2. The existence of these two biochemically distinct CSFs was first identified in this lab (Wu et al. 1981). By the same virtue, GM-CSF sometimes was referred to as CSF-3. All of the three CSFs were later identified as distinct peptides encoded by different genes. Sachs and Pluznick group at Rehovot introduced alternative nomenclature for CSFs. The term macrophage granulocyte inducer-1M, MGI-1M, refers to M-CSF, MGI-1G is equivalent to G-CSF, and MGI-GM is used for GM-CSF. M-CSF stimulates differentiation of progenitor cells (colony-forming unit macrophage, CFU-M) to mature monocytes, and prolongs the survival of monocytes (Motoyoshi et al. 1997). It enhances expression of differentiation antigens (Hashimoto et al. 1997) and stimulates chemotactic, phagocytic and the killing activities of monocytes (Wang et al. 1988.) It also stimulates production of several cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and interleukin (IL)-6 by priming monocytes, and directly stimulates production and secretion of IL-8 and reactive nitrogen intermediates (Motoyoshi and Takaku 1991).