Browsing by Subject "Characterization"
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Item Analysis of a Tn917 Transposon Mutant and Preliminary Characterization of NonHemolytic, Catalase-Deficient Variants of Staphylococcus aureus(1999-06-01) Crum, Russell M.Crum, Russell M., Analysis of a Tn917 Transposon Mutant and Preliminary Characterization of Nonhemolytic, Catalase-Deficient Variants of Staphylococcus aureus. Masters of Science (Microbiology). June 1999. Pages-101. Tables-15. Figures-10. A Tn917 transposon mutant of Staphylococcus aureus S6C was isolated and analyzed due to its deficiency in hemolysin and lipase activities. The transposon insertion did not occur in any of the known genetic regulators, which suggested the insertion occurred in a novel regulator of at least, hemolysin and lipase activities. One end of the region where the insertion occurred was isolated, sequenced, and compared with known DNA databases. Sequence comparisons revealed the insertion occurred in one of six rRNA DNA operons, which was confirmed by Southern analysis. Transduction of the transposon insertion back into the parental strain did not result in a mutant phenotype thereby indicating that the transposon insertion into a rRNA DNA operon was not responsible for the observed mutant phenotype. Further analysis of the parent strain, S. aureus S6C, revealed a population of four relatively stable variants differing in their hemolysin and catalase activities. These data suggest that the Tn917 mutant was one of these four S6C variants.Item C. DIFFICILE: A CHARACTERIZATION OF VIRULENCE FACTORS AND GROWTH BETWEEN EPIDEMIC VERSUS NON-EPIDEMIC STRAINS(2014-03) Vitucci, John C.; Pulse, Mark; Simecka, JerryClostridium difficile (C. difficile) is a spore-forming, gram positive bacterium found naturally within a human’s intestinal flora, as well as in the environment. This organism is safe in small numbers as a part of the natural intestinal flora, but can pose problems when broad-spectrum antibiotics, such as clindamycin, kill off other natural flora. Presently, there are two different, over-arching categories of C. difficile, toxigenic strains, which cause disease, and non-toxigenic, non-disease causing strains. These toxigenic strains can be broken into sub-categories: non-epidemic and epidemic. These labels are given based on the prevalence of an isotype in a geographical location. The most prevalent isotype is classified as epidemic and the less prevalent isotypes are classified as non-epidemic. Previous research has produced in vitro studies of epidemic and non-epidemic C. difficile strains, and there have been no conclusive patterns determined from these studies to determine the difference of virulence factors between strains. Therefore, is there a difference with the growth characteristics and virulence factors contributing to the severity of infection between epidemic and non-epidemic C. difficile strains both in vitro and in vivo? In vitro characterization of five non-epidemic and five epidemic strains, starting with virulence factor characterization of viable and germinated cell counts, as well as Toxin A and B production quantification were found to be statistically different between strains. In contrast, observed differences were slight and suggest that, in vitro, C. difficile can be treated as a group from an non-epidemic versus epidemic standpoint. The next group of in vitro studies including Growth Curves, Minimum Inhibitory Concentration studies, and Inhibition Curves also showed little observed differences between the non-epidemic and epidemic strains continuing to support the observations from the first set of studies stating that: C. difficile can be characterized and treated as a group instead of individual strains during infection. Future in vivo studies will help to determine if these trends between non-epidemic and epidemic strains will remain viable when the environment contains additional factors during infection, such as p.H. shifts, nutrition influx, and treatment with antibiotics within a system. Purpose (a): Clostridium difficile (C. difficile) is a spore-forming, gram positive bacterium found naturally within a human’s intestinal flora capable of causing severe disease. Other research has focused on in vitro studies of epidemic and non-epidemic C. difficile strains, and these studies concluded no conclusive patterns between the difference in virulence factors between the individual strains. Therefore, an important question to ask is: for C. difficile, is there a difference with the growth characteristics and virulence factors contributing to the severity of infection between epidemic and non-epidemic C. difficile strains both in vitro and in vivo? Methods (b): For the in vitro studies, there are multiple protocols used including, viable cell counts, spore isolation and germinated cell counts, Toxin A or B ELISA assays, growth and inhibition curves, as well as minimum inhibitory concentration (MIC) determination. In vivo UNTHSC Pre-Clinical Services 21-day Recurrence Hamster Model and Next-Gen Sequencing for microbiome research will be utilized. Results (c): When non-epidemic and epidemic C. difficile strains were characterized for major virulence factors, statistically significant differences for both intrastrain and interstrain comparisons were observed. Growth curve data showed consistent growth patterns between the strains. MIC results were consistent between strains, with no more than a 100-fold difference between the MIC of any one drug for all the strains tested. Inhibition curve results also showed minimal variation between the different non-epidemic and epidemic strain behavior when growth was tested against metronidazole, moxifloxacin, and vancomycin. Conclusions (d): During characterization in vitro, between five non-epidemic and five epidemic strain’s virulence factors, the differences in results are small, yet statistically significant. Though statistically different, observed differences are minimal and not believed to affect the individual strain's overall virulence. Therefore, it is concluded, in vitro, different strains of C. difficile have similar growth patterns and have similar virulence characteristics as a group. When further study was conducted to compare growth patterns over 24-hours, MIC’s concentrations, and Inhibition Curves, interstrain comparisons once again showed small observed differences. The overall trends in antibiotic susceptibility and growth patterns when the media was without, and supplemented with, antibiotics were seen to be similar. This continues to support that C. difficile can be treated, in vitro, as a group, independent of the labels non-epidemic and epidemic.Item Characterization of Protein Kinase C in Cisplatin Sensitive and Resistant Human Cervical Cancer HeLa Cells(2000-12-01) Mohanty, Sanghamitra; Basu, Alakananda; Simecka, Jerry; Dimitrijevich, DanMohanty, S., Characterization of protein kinase C in cisplatin sensitive and resistant human cervical cancer HeLa cells. Master of Science (Microbiology and Immunology), December, 2000. 37 pp., 11 illustrations, bibliography, 27 titles. Signal transduction plays a crucial role in carcinogenesis. A defect in signaling, by evading cell death or promoting cell proliferation, may result in neoplastic transformation or protection of cells from the cytotoxicity of anticancer drugs. Therefore, in order to understand the complex mechanism of drug resistance, it is relevant to probe into the important signal transduction pathways. Protein kinase C, a key signal transducer, influences cisplatin sensitivity in many cell lines. We examined whether or not the PKC signal transduction pathway is affected during development of resistance to cisplatin by tumor cells. PKC activators increased cisplatin sensitivity in both parental and cisplatin-resistant cells. Western blot analysis showed a slight decrease in cPKCα and nPKCε, an evaluation in nPKCδ and no change in the abundance of PKCϚ in HeLa/CP cells compared to HeLa cells. Though TPA-induced translocation of PKC isoforms was identical in both cell lines, down regulation of PKCδ was defective in resistant cells. Therefore, a deregulation in PKCδ was associated with cisplatin resistance.Item Characterization of Recombinant Lecithin: Cholesterol Acyltransferase, Secreted by a Human Lung Cell Line (1069-111) and by Pichia Pastoris Yeast Cells(2004-05-01) Tchedre, Kissaou T.; Caffrey, James L.; Harris, Ben G.; Wu, Ming-ChiTchedre, Kissaou T., Characterization of Recombinant Lecithin: Cholesterol Acyltransferase, Secreted by a Human Lung Cell Line (1069-111) and by Pichia pastoris Yeast Cells (Biomedical Sciences), May, 2004, Lecithin: cholesterol acyltransferase (LCAT) is a key enzyme in mammalian lipoprotein metabolism. Associated with the surface of high-density lipoproteins (HDL), LCAT contributes to the homeostasis of circulating free and esterified cholesterol via the reverse cholesterol transport pathway. The purpose of these studies was to characterize a recombinant form of LCAT, secreted by a human lung cell line (Beta gene 1069/111) and to evaluate a new expression system for LCAT using transformed Pichia pastoris cells. A human lung cell line (Beta gene 1069/111), transfected with pBIISK (Stratagene)+ vector was used as the source of recombinant (rLCAT) for the first stage of characterization studies. Human lung cells were expanded in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum for the expression of the recombinant LCAT. At 80 – 90% confluency, the medium was changed to a serum free preparation and the flasks were incubated for 48 hrs at 37°C to facilitate the secretion of the enzyme. Beta gene (1069/111) LCAT was purified from the conditioned medium using phenyl sepharose chromatography. The purified enzyme was characterized according to: carbohydrate composition, and enzyme kinetic parameters. The enzymatic characteristics, of the human lung cell line LCAT had similar Km and Vmax values to other LCAT preparations, isolated from other expression systems and human plasma. Deglycosylation reduced the molecular weight of the enzyme from about 67,000 to about 43,000 suggesting a carbohydrate component of 25-32% of the enzyme’s total mass. Detailed analysis of the carbohydrate structures revealed N-glycan structures in a complex pattern of sialylated and fucosylated tri and tetra-antennary glycosides (8). In addition to the Beta gene expression, a Pichia pastoris yeast expression system was also developed consisting of human LCAT cDNA cloned into pPICZαA vector along with a removable amino-terminal polyhistidine tag. The Pichia pastoris cells were transformed with a vector containing the LCAT gene cDNA and transformants were selected on agar plates containing zeocine (100μg/ml). Polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) were used to confirm the correct integration of the LCAT gene cDNA into the pPICZαA vector. The recombinant LCAT produced by the yeast cultures was purified by Talon affinity chromatography, taking advantage of the removable histidine tag. The enzymatic activity was determined using proteoliposome vesicles. The Yeast expression system yielded ~18 mg of enzyme protein/500 ml and thus may provide an appropriate enzyme source for characterization studies via NMR analysis and x-ray crystallography.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 Characterization of the Serotonin Receptors in the Long Posterior Ciliary Artery of the Bovine Eye(2000-08-01) Landry, Theresa A.; Quist, Eugene; Martin, Michael; Pang, Iok-HouLandry, Theresa A., Characterization of the Serotonin Receptors in the Long Posterior Ciliary Artery of the Bovine Eye. Doctor of Philosophy (Biomedical Science), August 2000, 14 pp., 5 tables, 29 illustrations, bibliography, 104 titles. Vascular disease and vasospasm are implicated in the etiology of glaucoma. The long posterior ciliary (LPCA) is the major blood supply for the ciliary body including the ciliary processes that produce aqueous humor. Information about the pharmacological control of this vessel would be helpful in understanding its normal and pathologic function. Serotonin (5-HT) is a neurotransmitter that effectively constricts the LPCA. The objective of this research is to identify the serotonin receptor subtype responsible for the 5-HT induced vasoconstriction of the LPCA and to characterize the cellular mechanisms that mediate that contraction. Ring segments of the LPCA were dissected from bovine eyes and mounted on tungsten triangles attached to a force transducer. Changes in vascular tension were measured and recorded using a physiography recorder. Dose response curves with 5-HT, 5-HT 1-like agonist, 5-CT, and 5-HT2 agonist, α-methyl-5-HT, indicate that the 5-HT 1-like receptor contributed about 15.13% to the contraction and the 5-HT2 receptor contributed to 61.61%. The EC50 for the three agonists were 283 nM (5-HT), 336 nM (5-CT), and 1.7 μM (α-methyl-5-HT). Inhibition curves with selective antagonists indicate that the IC50 is (5-HT 1-like antagonist) and ketanserin (5-HT2 antagonist). Following incubation of the rings with diltiazem 10 μM or nifedipine 10μM, the response to 5-HT was reduced 65.*% and 61.7% respectively. Incubation in calcium free PB produced similar results. Ryanodine inhibited the 5-HT contraction by 58.1% and caffeine inhibited the response 100%. PKC inhibitors bisindolymaleimide II 1 μM, disindolylamalemide II 10 μM, chelerythrine 25 μM and H-7 5 μM decreased the 5-HT response by19.8%, 55.7%, 31.1% and 61.5% respectively. Incubation of the ring segments with one of three PLC antagonists, 2-NCDC 70 μM, U73122 0.5μM, or neomycin 5 mM, prior to the addition of 1 μM serotonin, significantly reduced the contraction of each vessel, p [less than] 0.0001. The 5-HT-induced vasoconstriction of the LPCA of the bovine eye is mediated through activation of both 5-HT2 and 5-HT 1-like receptors. The contraction is dependent on the mobilization of calcium and is mediated in part through PLC activated intracellular calcium release from IP3 sensitive stores.