Browsing by Subject "Microbiology"
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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 Amplified Fragment Length Polymorphism Analysis of White Oak Tree Leaves(2005-07-01) Patel, Kaajal Devendra; John Planz; Joseph Warren; Arthur EisenbergThe AFLP technique at first seems to be a remarkable new technology that can be applied to the growing area of non-human DNA testing. The ability to identify organisms without prior genetic knowledge would be an asset to a field such as non-human DNA testing since not enough research in the area is being conducted. With any new technique or theory in science, intense scrutiny must be used to examine the applicability of the new technology. In the area of forensic science, the severe consequences of a false result extend far beyond the realm of scientific error. Errors make in forensic casework could result in life changing occurrences for the families of not only the victim, but the defendant as well. From this study it can be seen that AFLP as a technique may not stand up to the high expectations of reliability, and reproducibility required for a technique to be adopted into the field of forensic science. Several problems occurred through this study that may prevent this technology from becoming a widely accepted technique in non-human DNA testing. The initial problems with the technique were associated with reproducible results. The first several attempts were conducted under the same conditions, by the same analyst but yielded results that were no comparable. The RFUs of each experiment were inconsistent, not only between samples examined at different times, but samples examined within the same tiral as well. AFLP as a technique is supposedly insensitive to template concentrations however, it has been previously shown to produce differences in the electropherogram when the template is excessively diluted (26). Vos et al. (1995) determined that high dilutions yielding template DNA concentrations below 1 pg could result in irreproducible fingerprints. In this study 27.5 ng of template DNA was added to each digestion-ligation reaction, yet the resulting quantity of amplified fragments varied. These variations in quantities of amplified product could be due to PCR inefficiencies when comparing samples from different trials, but it does not explain instances where duplicate trials were inconsistent with each other (10, 22). When new ligase was introduced the resulting electropherograms did produce considerably higher RFUs for each peak, but the lack of interpretable peaks observed previously may not have been solely due to inefficient ligase. In an inter-laboratory study, Jones et al. (1997) noted that several laboratories encountered problems in obtaining complete AFLP profiles. For several groups, up to 50% of the bands were missing during the preliminary testing. Though this problem subsided with successive attempts, this approach to achieving successful results may not be feasible in a forensic setting. Often the evidence received from a crime scene may be insufficient to allow for multiple testing. In addition, multiple attempts to obtain results may open up areas for scrutiny and attack by the defense counsel. Repetitive testing may appear to be a biased search for condemning evidence against the questioned party, rather than the production of reliable results. Repetitive testing may also not be possible since laboratory reagents and time involved in the production of these results may not be within the constraints of a crime laboratory. In this study, capillary electrophoresis was used to visualize the fluorescent dyes attached to each fragment however, laboratories could use radioisotopes and polyacrylamide gels instead. This method of visualizing AFLP fingerprints is not only costly, but time consuming as well. Conducting repetitive tests in order to obtain a sample with sufficiently intense bands for analysis may not be feasible. These limitations may therefore restricts the use of the AFLP technique from only being conducted in laboratories with sufficient time and funds to conduct repetitive testing as is needed (10). Despite the potential cost in time and funds, the technique was able to produce AFLP fingerprints that were consistent with each other when the electropherograms were compared. The major source of error resulted from the method used to determine the presence of peaks within the designated categories. Since not all peaks crossed the 50 RFU detection threshold, they were not identified by the Genotyper macros. However, when the actual electropherograms were compared, these peaks were present. It has been suggested that to verify whether each peak is present in the pre-designated categories a scan of the electropherogram should be done and any peaks that were not called by the macro should be manually entered into the binary table or should be reanalyzed (Heather Coyle, personal communications). Although this method could potentially aid in the correct genotyping of each sample, it requires a considerable amount of user intervention. A considerable amount of time is needed to examine each electropherogram for the presence of peaks that are below the 50 RFU threshold. Without a redefined interpretation threshold, the analysis of each electropherogram can be highly subjective. Peaks that are relatively low need to be distinguished from peaks that may be associated with background noise. Therefore, in order to eliminate analyst bias a peak detection threshold must be established. Generally the interpretation threshold is established by a validation study of the analysis technique. In this study the lower threshold was previously established at 50 RFU for the instrument being used, but this threshold was insufficient for the recognition of all peaks present during the AFLP analysis. The question then becomes to what extend the peaks can or should be called in order to correctly identify each organism without errors. The exclusion of some peaks could lead to discrepancies, such as those observed during the blind study, which could result in an initial false match or exclusion. The interlaboratory study by Jones et al. found only one scoring difference associated with the absence of one band out of a total of 172 in the AFLP profiles. This error was later associated with experimental errors that incurred during the AFLP procedure. Discrepancies such as this can lead to an erroneous identification of samples that could have severe consequences in a criminal case. At this time, the utilization of AFLP technique for further testing of other organisms such as Cannabis sativa does not seem feasible. A variety of adjustments in the technique need to be addressed before this technology should be further applied to organisms in forensic casework. In order for AFLP typing to be used for forensic casework, major improvements in the technique need to be made. Consistency in obtaining reliable electropherograms with peaks well above the RFU detection threshold must be resolved in order to allow for accurate sample interpretation. This will not only allow for greater consistency between replicates, but will also help in establishing new databases for organisms that are being tested. As with any type of forensic DNA analysis, a database must be established for each organism being tested. Without a reliable database, accurate identification of crime scene evidence cannot be established. A major improvement that is required for the utilization of AFLP typing is the process by which genotypes are identified. Utilizing the macros to identify control and variable peaks to create the binary table was a quick and easy method, however it was not always able to identify the correct genotype. The overlapping of electropherograms in GeneScan ultimately was the best method for accurate identification of the blind samples, but in a real case scenario it would not be feasible to compare each evidentiary electropherogram with those in a database. Advancements in technology will continually introduce new techniques and procedures that could be applicable to the field of forensic science. As with any new technique, the methods and theories must be validated in order to determine whether they can be used in a criminal case. The field of non-human DNA testing is growing and with the advent of new technology such as AFLP, the possibility for establishing a non-human DNA identification method may be on the horizon.Item Anionic Ligand-Gated Ion Channels: The Convulsive Site and Mechanism of Action(2001-08-01) Dibas, Mohammed I.; Hriday Das; Thomas Yorio; Neeraj AgarwalDibas, Mohammed, Anionic Ligand-Gated Ion channels: The Convulsive Site And Mechanism of Action. Doctor of Philosophy (Biomedical Sciences), August 2001, pp153, 1 table, 24 illustrations, 76 titles. Picrotoxin, a CNS convulsant inhibits all anionic ligand gated ion channels. The mechanism and the binding site for picrotoxin and its related ligands are still undefined. The second transmembrane (TMII) domain of these ligand gated ion channels is found to play a key role in the mechanism of block by picrotoxin. It has been shown that the incorporation of a phenylalanine residue in place of threonine at position 6’ within the TMII domain of B2 subunit conferred high resistance toward picrotoxin in GABAA a3B2(T6’F)y2 receptors. Mediating their blocking effect through the PTX-site, PTZ, TBPS, and U-93631 lost their inhibitory effects due to the same mutation B2(T6’F). Interestingly, this mutation uncovered a low affinity, highly efficacious stimulatory site for PTZ. PTZ seems to mediate its stimulatory effect through a novel distinct site different from that for benzodiazepine. The effect of varying subunit configuration of GABAA receptors dramatically affected the ability of the mutation B2(T6’F) to abolish the inhibitory effect of picrotoxin. While picrotoxin failed to block the current induced by GABA in a3B2(T6’F)y2 receptors, picrotoxin partially blocked the current in a3B2(T6’F)y2 receptors. In B2(T6’F)y2 receptors, picrotoxin restores its full efficacy. When phenylalanine was incorporated at position 6’ in the a1 subunit, picrotoxin completely blocked the current induced by GABA in a1(T6’F)B2y2 receptors. The combined results showed that the ability of (T6’F) mutation to regulate the inhibitory mechanism of picrotoxin as dependent on the subunit configurations and at which subunit is mutated. In addition, picrotoxin is known to inhibit GABAA receptors in use-facilitated mechanism, while it inhibits the glycine receptor in a non-use facilitated fashion. The molecular determinant behind the use-facilitated mechanism was modulated by the nature of the amino acid at position 15’ within the second transmembrane domain. The mutation of serine 15’ to either glutamine or asparagine in the glycine a1 receptors converted picrotoxin from a non-use facilitated blocker to a use-facilitated one. The latter finding suggested that this residue might residue within the PTX binding site or play a key role in the transduction pathway for picrotoxin mechanism. The overall results further support the fact that TMII domain plays a key role in the picrotoxin mechanism.Item Changes in the Microbial Community as a Potential Indicator of Clandestine Drug Operations.(2014-05-01) Payne, Tanisha N.; Michael AllenDust is a complex mixture of inorganic and organic materials including diverse microorganisms, which if unattended, accumulates over time. In this study, the microbial content in house dust was tested to determine its forensic detection potential in a model scenario mimicking the conditions of methamphetamine manufacturing. We hypothesized that microorganisms associated with the materials exposed to vapors will respond in a reproducible way. By identifying the microbial communities and any changes that may have occurred we expected to elucidate a correlation between microorganisms and the test chemicals involved, which was supported by the results presented. These findings may provide evidence in otherwise “cold cases” of methamphetamine manufacturer as well as information on the chemistry employed.Item Effects of Serine Protease-Like F (SPLF) on Alpha-Toxin Expression in Staphylococcus aureus(2003-05-01) Pulse, Mark E.; Mart Hart; Jerry Simecka; Ming-Chi WuPulse, Mark E., Effects of Serine protease-like F (Sp1F) on Alpha-Toxin Expression in Staphylococcus aureus. Master’s of Science (Microbiology). May 2003. Pages-67. Table-5. Figures-9. Transposon mutagenesis (Tn551) was used to generate agr-suppressor mutations in the agr-null Staphylococcus aureus strain RN6911 (Δagr;;tmn). Firty-four suppressor mutants displaying changes in hemolysin, protease, and lipase activities were isolated, and only twenty-six mutants contained Tn551 within their chromosomes. Transposon insertion sites for seven mutants were determined by sequencing amplicons generated by arbitrary-PCR. One of the insertion sites was within the serine protease-like F (spʅF) gene. Alpha-toxin message levels for the spʅF mutant were similar to RN6911. However, alpha-toxin activity in spent media isolated from the spʅF mutant were similar to RN6911. However, alpha-toxin activity in spent media isolated from the spʅF mutant was increased ten-fold as compared to RN6911. Transduction of the spʅf:Tn551 mutation back into the parental strain verified the link between the phenotype and the mutation. Whole cell lysates from Escherichia coli cells containing a plasmid copy of spʅF displayed protease activity on casein. These data suggest that SpʅF may be post-translationally modifying alpha-toxin through proteolysis.Item Fecal Coliforms in the Rio Grande: A Risk to Human Health(2004-12-01) Tompkins, Erin L.; Thomas Vaughan; Claudia S. CogginTompkins, Erin L., Fecal Coliforms in the Rio Grande: A Risk to Human Health. Master of Public Health (Environmental Health), December 2004, 45 pages, bibliography, 33 titles. The Rio Grande around Laredo, Texas/Nuevo Laredo, Mexico was designated for primary contact reaction by the EPA. However, monthly sampling over a ten-year period in this section of the river may show otherwise. Fecal contamination of the Rio Grande in this area may be a source of illness to the population. Four sites in Laredo area were tested for fecal coliform density and rate of flow. Rainfall data from the USGS was used for comparisons. The rate of flow of the Rio Grande had an impact on fecal coliform density at one site measured. Rainfall in Laredo had an impact on fecal coliform density at two measured sites, and was a significant predictor of density at these sites as well. A review of the designation for this river segment is recommended. More research is needed to determine the exposed population, and effects of high coliform densities on downstream communities.Item Identification and Characterization of Caveolins in Mouse Macrophages(2002-12-01) Gargalovic, Peter; Dory, Lad; Basu, Alakananda; McConathy, WalterPeter Gargalovic, Identification and Characterization of Caveolins in Mouse Macrophages. Doctor of Philosophy (Biochemistry and Molecular Biology), December 2002, 206 pp., 3 tables, 41 illustrations, references, 296 titles. The understanding of the mechanisms which control macrophage-lipid management, and their accumulation in atherosclerotic lesions, is of significant importance. Caveolins are proteins associated with cholesterol-rich membrane domains and are intimately linked to the regulation of lipid metabolism and transport. The expression and function of caveolin proteins in three macrophage cell types: thioglycollate-elicited mouse peritoneal macrophages, resident mouse peritoneal macrophages and the J774 macrophage cell line. Data in this work establish that the primary macrophages express caveolin-1 and -2, while J774 cells express only caveolin-2. Immunofluorescence microscopy studies indicate that caveolins in primary macrophages do not colocalize, with caveolin-1 being present on the cell surface and cavelon-2 in the Golgi compartment. Analysis of macrophages also showed that caveolin-1, but not caveolin-2, is present in detergent insoluble lipid raft membranes. While caveolin expression in macrophages is not regulated by sterols, both caveolin isoforms can be secreted from cholesterol-loaded macrophages in the presence of high-density lipoprotein (HDL). Secreted caveolins are part of the complex that has a density similar to HDL, which suggests their association with HDL and potentially a role in HDL-mediated reverse cholesterol transport. The examination of caveolin expression in macrophages shows that caveolin-1, but not caveolin-2 expression is highly upregulated by agents that induce apoptosis in these cells. Induction of caveolin-1 expression precedes DNA fragmentation, is independent of caspase activation, and correlates with the exposure of phosphatidylserine on the cell surface. Importantly, immunofluorescence analysis determined that caveolin-1 in lipid rafts colocalizes extensively with phosphatidylserine present on the surface of apoptotic cells. This study thus identifies caveolin-1 as a specific and early marker of the macrophage apoptotic phenotype. Findings here strongly implicate the involvement of caveolin-1 and lipid rafts in the changes of plasma membrane lipid composition as well as involvement in efficient clearance of apoptotic cells by a phosphatidylserine-mediated mechanism.Item Interleukin-8: Baculovirus Expression and the Receptor Signal Transduction Pathway(1994-06-01) Kang, Xiaoqiang; Stephen R. Grant; Rafael Alvarez; Paula SumstomXiaoqiang, Kang., Interleukin-8: Baculovirus Expression and the Receptor Signal Transduction Pathway. Doctor of Philosophy (Biomedical Sciences), June, 1994, 150 pp., 4 tables, 36 illustrations, bibliography, 212 titles. The cDNA for human interleukin-8 (IL8) was subcloned from a bacterial source into the eukaryotic baculoviral vector expression system. Recombinant human IL-8 (rhIL-8) was synthesized and secreted from SF9 cells following infection of a recombinant virus harboring the full-length IL-8 structural gene. Recombinant human interleukin-8 was purified ([greater than] 600 fold) to homogeneity using preparative HPLC. The rhIL-8 preparation retained all of the physical, immunological, and biochemical properties of the natural product (monocyte-derived IL-8). Baculovirus vector expression coupled to preparative HPLC proved to be a very efficient method for large-scale recombinant interleukin production. Biochemical mechanisms that mediate IL-8 receptor-stimulated activities are poorly understood. In this study, I have explored the intracellular mechanism(s) induced by IL-8 in differentiated HL-60 cells. IL-8 induced a rapid and transient activation of phospholipase A2 in differentiated HL-60 cells. A consequence of phospholipase A2 activation was the release of arachidonic acid and the generation of lysophospholipids from membrane phospholipids. The IL-8 stimulated-arachidonic acid release was pertussis toxin and phospholipase A2 inhibitor sensitive, and protein kinase C independent. In contrast to another neutrophil chemotactic factor, fMLP, IL-8 did not stimulate the activation of phospholipase C and phospholipase D. When comparing the phosphorylation events induced by IL-8 and fMLP, I found that these two chemotactic factors triggered different protein phosphorylation profiles. Tyrosine phosphorylation of proteins was not detected following IL-8 stimulation in HL-60 cells. However, IL-8 stimulated the rapid autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaM kinase II). These results strongly suggest that the IL-8 receptor is closely coupled to the activation of PLA2 and that CaM kinase II is an integral component of IL-8 receptor signal pathway.Item Involvement of Caspase-2 in Cisplatin-Induced Cell Death in 2008 Ovarian Cancer Cells(2008-04-01) Adkins, Brett T.; Basu, Alakananda; Berg, Rance E.; Gryczynski, ZygmuntAdkins, B., Involvement of caspase-2 in cisplatin-induced cell death in 2008 ovarian cancer cells. Master of Science (Molecular Biology and Immunology) April, 2008, 59 pp., 12 illustrations, bibliography, 73 titles. Cisplatin, one of the most effective anticancer drugs in the treatment of ovarian cancer, causes DNA damage and leads to apoptosis. Caspases, a family of cysteine proteases, are essential for the induction of apoptosis. Initiator caspases activate effector caspases to trigger apoptosis. Caspase-2 can function as both an initiator and effector caspase although there are controversies regarding its role in DNA damage-induced apoptosis. Caspase-2 is the only caspase constitutively located in the nucleus, although its function there is unknown. In the present study we have investigated if caspase-2 is important during cisplatin-induced apoptosis and whether cisplatin treatment affects the localization of caspase-2. Caspase-2 depletion suggested that caspase-2 acts upstream of caspase-2 acts upstream of caspase-9 in cisplatin-induced apoptosis. We also made a novel observation that rottlerin, an inhibitor of DNA damage-induced apoptosis, specifically downregulates caspase-2 via the ubiquitin proteamose-mediated pathway. We further show that cisplatin induces caspase-2 translocation out of the nucleus. Moreover, translocation of caspase-2 is more important for cisplatin-induced cell death.Item Isotope Partitioning and Initial Velocity Studies with 6-Phosphofructo-1-kinase from Ascaris suum(1996-05-01) Gibson, Grant E.; Robert EasomGibson, Grant E., Isotope Partitioning and Initial Velocity Studies with 6-Phosphofructo-1-kinase from Ascaris suum. Doctor of Philosophy (Biomedical Sciences), May, 1996, 91 pages, 2 tables, 18 figures, 2 schemes, 1 reaction, 2 mechanisms, 1 diagram, bibliography, 61 titles. The natives Ascaris suum 6-phosphofructo-1-kinase (nPFK) and a chemically modified form (dPFK) which is desensitized to allosteric behavior have been studied using isotope partitioning and initial velocity techniques to determine the kinetic mechanism as well as the effects of fructose 2,6-biphosphate (F26P2) and Mg2+ on the mechanism. At 8 mM Mg2+, complete trapping (P*max≈100%) of E:MgATP* complex as fructose 1-(32P), 6-biphosphate for both enzyme forms is consistent with the previously proposed steady-state ordered mechanism ((Rao, G.S.J., Harris, B.G., and Cook, P.F. (1987) J. Biol. Chem. 262, 14074-14079) with MgATP binding before fructose 6-phosphate (F6P). A saturating amount of F26P2 causes no change in the trapping parameters for nPFK but causes a decrease in both P*max and K’F6P for dPFK. The partial trapping of E:MgATP* in the presence of F26P2 for dPFK at high MG2+ suggests that the activator changes the kinetic mechanism from an ordered to a random binding of substrates. Initiial velocity studies at 8 mM Mg2+ confirm the change in mechanism. Uncompetitive inhibition by arabinose 5-phosphate (Ara5P), a dead-end inhibitory analog of F6P, versus MgATP for nPFK in the absence and presence of F26P2 is consistent with an ordered mechanism with MgATP adding to enzyme prior to F6P. An uncompetitive pattern is also obtained with dPFK for Ara5P versus MgATP in the absence of F26P2, but the pattern becomes noncompetitive in the presence of F26P2, consistent with a change to a random mechanism. No trapping of the dPFK: (14C)F6P complex could be detected 8mM Mg2+, indicating either that dPFK:14C-F6P complex does not form or that the off-rate for F6P from enzyme is much faster than the net rate constant for formation of the first product, FBP. Initial velocity data indicate that a second Mg2+ ion in addition to the one bound in MgATP is an essential activator of Ascaris suum PFK which decreases the off-rate for MgATP. Kact for Mg2+ is estimated to be 0.47±0.08mM. Isotope partitioning data at 0.1 mM Mg2+ indicate that dPFK is able to trap only 20% of the E:MgATP* both in the presence and absence of F26P2, consistent with a faster off-rate for MgATP at low Mg2+ than at high Mg2+. Partial trapping of MgATP* at low Mg2+ again suggests a random binding of substrates. Noncompetitive Ara5P inhibition versus MgATP at low Mg2+ confirms the random mechanism. An active site role both in binding MgATP and in facilitating catalysis is proposed for the second Mg2+. Furthermore, calculations from the isotope partitioning and initial velocity data as well as changes that are seen in the circular dichroic spectra for both nPFK and dPFK indicate that an enzyme structural isomerization occurs upon binding mgATP.Item Lethality of Staphylococcus in Murine Pneumonia is Due to Alpha-Toxin and Other Secreted Factors Regulated by AGR and SAR(2003-08-01) Overheim, Katie A.; Dan Dimitrijevich; Glenn Dillon; James CaffreyOverheim, Katie A., Lethality of Staphlococcus aureus in Murine Pneumonia is Due to Alpha-Toxin and Other Secreted Factors Regulated by agr and sar. Doctor of Philosophy (Biomedical Sciences), August, 2003, 91 pp, 6 Tables, 9 illustrations, bibliography, 106. The purpose of these studies was to determine if the S. aureus global regulators agr and sar play a role in staphylococcal pneumonia and if the virulence factors regulated by them contributed to the severity of staphylococcal pneumonia. To determine this, we established a pneumonia model in mice in order to identify if S. aureus global regulators agr and sar play a role in the pathogenesis of staphylococcal pneumonia. As well, we took steps to identify the extracellular factors responsible for the lethality in a murine model of staphylococcal pneumonia and determine if these factors involved in disease process could be used as targets for immune therapy. My work revealed that lethal pneumonia in a mouse model is dependent on the S. aureus global regulators agr and sar. This study also revealed that the lethality associated with our model is due to secreted factors, regulated by S. aureus global regulators agr and sar. Further investigation demonstrated the alpha-toxin is a major virulence factor involved in the lethality in our model. By generating an alpha-toxin deficient strain in S. aureus RN6390, we show a reduced virulence in our disease model. As well, antiserum to alpha-toxin, when administered with a lethal dose of S. aureus RN6390, we show a reduced virulence in our disease model. As well, antiserum to alpha-toxin, when administered a lethal dose of S. aureus RN6390, we show a reduced virulence in our disease model. As well, antiserum to alpha-toxin, when administered with a lethal dose of S. aureus RN6390 protected animals from death. By evaluating the role of alpha-toxin’s ability to contribute to lethality, we assessed numerous strains of S. aureus in our pneumonia model. We discovered that there was a correlation to alpha-toxin production levels and lethality in our pneumonia model. However, our study also demonstrated that alpha-toxin is not the only factor involved in the disease process.Item Mechanism of Gramicidin D-Induced Insulin Secretion From BTC3 Cells(1995-08-01) Dibas, Adnan I.Dibas, Adnan I., Mechanism of Gramicidin D-Induced Insulin Secretion From BTC3 cells. Doctor of Philosophy (Biomedical Sciences), August, 1995, 190 pp., 5 tables, 38 illustrations, bibliography, 265 titles. Gramicidin D, a sodium ionophore, was discovered to be a potent insulin secretagogue in the B-cell line BTC3 cells. Gramicidin D (1 uM) induced a 3.28-fold increase in insulin release relative to control, and when studied in a dynamic cell-perifusion system, was biphasic. Insulin secretion was accompanied by effects of gramicidin D to increase intracellular concentrations of Na+([Na+]i) and Ca2+ ([Ca2+)i) in BTC3 cells as determined by dynamic single-cell video imaging techniques, gramicidin D had no effect on cellular pH. The mechanism of gramicidin D-induced increase in [Ca2+ and suggested to be mediated by a combination of membrane depolarization-induced activation of voltage-sensitive Ca2+ channels and the activation of a Na+/Ca2+ exchanger in the reverse mode. Gramicidin D-induced increase in [Ca2+]I in the first phase correlated temporally with a profound (5.56-fold) activation of multifunctional Ca2+/calmodulin-dependent protein kinase II. While these observations are consistent with the involvement of this enzyme in gramicidin D-induced insulin secretion, further observations suggested that the kinase may play only a modulatory role in insulin secretion. A similar activation of myosin light chain kinase was not detected. In contrast to BTC3 cells, gramicidin D failed to induce insulin secretion from pancreatic islets. BTC3 cells and pancreatic islets exhibited distinct responses to ouabain, an inhibitor of the Na+/K+ ATPase, with respect to [Ca2+]I and insulin secretion suggesting that different mechanisms controlling Na+ homeostasis exist in these B-cell preparations. Furthermore, Na+/K+ ATPase activity in BTC3 cell membranes was found to be approximately fifty percent that of primary B-cells. Gramicidin D was identified as a secretagogue in BTC3 cells with a novel mechanism of action. The ability of this ionophore to induce insulin secretion from these cells and not primary B-cells is thought to be a function of different mechanisms of Na+ homeostasis and documents a functional difference in this insulinoma cell line.Item Mechanistic Studies of the Sheep Liver 6-Phosphogluconate Dehydrogenase and cDNA Cloning(1996-07-01) Price, Nancy E.; Neeraj Agarwal; Robert Easom; Stephen R. GrantPrice, Nancy E., Mechanistic Studies of the Sheep Liver 6-Phosphogluconate Dehydrogenase and cDNA Cloning. Doctor of Philosophy (Biomedical Sciences), July, 1996, 124 pp., 5 tables, 28 Figures, 2 appendices, bibliography, 45 titles. A kinetic characterization of sheep liver 6-phosphogluconate dehydrogenase including product and dead-end inhibition patterns, primary deuterium isotope effects, and the pH dependence of kinetic parameters has been completed in order to determine the kinetic mechanism, and chemical mechanism of the enzyme. A rapid equilibrium random kinetic mechanism has been proposed, with product and dead-end inhibition patterns both being symmetric. Primary deuterium isotope effects were equal on V and V/K, confirming a rapid equilibrium mechanism, and indicate that hydride transfer is at least partially rate limiting in the overall reaction. The maximum velocity is pH dependent, decreasing at low and high pH with slopes of 1 and -1, respectively. The V/KNADP and V/K6PG also decrease at low and high pH with slopes of 1 and -1. The pH rate profiles are consistent with a general acid/general base mechanism where the catalytic residues are involved in binding. Reverse protonation states between the general acid and the general base is proposed where an unprotonated general base accepts a proton from the C-3 hydroxyl of 6PG concomitant with hydride transfer followed by decarboxylation of the resulting 3-keto intermediate to give an enediol which is protonated by the general acid to form ribulose-5-phosphate. The pH dependence of the pKi profile of the inhibitory analog 5-phosphoribonate decreases at low and high pH with slopes of 1, and -1 respectively, and suggests that intrinsic pKs are observed in the V/K profiles. The pKs of both the general base and general acid in the E:6PG complex appears to be perturbed such that the general base pK decreases slightly, and the pK of the general acid increases slightly, as a result of direct interaction with 6PG. Additionally, in preparation for site-directed mutagenesis, cDNA clones for sheep liver 6PHDH were obtained by RT-PCR.Item Morphological and Proliferative Changes that Occur in Rat Retinal Progenitor Cells Following Incubation with Retinoic Acid and RPE-Secreted Proteins(2006-08-01) Heath, Allison K.; Rustin E. ReevesHeath Allison K., Morphological and Proliferative Changes that Occur in Rat Retinal Progenitor Cells Following Incubation With Retinonoic Acid and RPE-Secreted Proteins. Master’s of Science (Cell Biology and Genetics), August 2006, 67 pp., 12 figures, bibliography. The principal objective of this research is to characterize virally-transformed rat retinal progenitor cells following stimulation by retinal pigment epithelial (RPE) cell secreted proteins and retinoic acid. Progenitor cells were isolated from explants of postnatal rat RPE cell in vitro. Isolated progenitor cells were cloned, analyzed by microscopy and proliferation bioassays, to determine if cell proliferation occurred. The isolated progenitor cells were analyzed for differentiation by Western blot analyses and immunocytochemistry. The rat progenitor cells cultured in RPE secreted proteins proliferated, but did not differentiate as shown by the presence of nestin and vimentin in these cells. Retinoic acid caused other progenitor cells to proliferate and differentiate, which is seen through the bioassays and Western blot analyses.Item Next-Generation Sequencing of Culture Negative Bronchoalveolar Lavage Reveals the Presence of Potentially Pathogenic Microorganisms(2015-08-01) Smith, Ashley D.; Michael Allen; Rance E. Berg; Harlan P. JonesPatients undergoing mechanical ventilation are at increased risk for developing nosocomial pneumonia. Traditionally, the diagnosis of pneumonia has relied on the identification of an etiologic agent by the hospital pathology lab via quantitative culture. A problem arises when a patient experiences clinical signs and symptoms of pneumonia, but culturing of bronchoalveolar lavage (BAL) fluid reveals only normal “respiratory tract flora” or results in “no growth”. I hypothesize that culture-negative, presumptive positive BAL is infected with pathogenic bacteria that are not being cultivated on traditional culture media. To investigate this hypothesis, culture-independent techniques were chosen to examine culture-positive and culture-negative BAL. Sanger and Ion Torrent Sequencing were used to verify that molecular techniques are able to identify the same pathogens the hospital lab finds within culture-positive BAL. Ion Torrent sequencing was then used to characterize the microbial community within culture-negative BAL. Cytokine assays were used to determine if culture-positive and culture-negative patients mount a similar immune response. By sequencing colonies picked from the same culture plates used by the hospital lab, I confirmed the presence of the same pathogens identified by the hospital. Ion Torrent sequencing was able to identify hundreds of genera in both the culture-positive and culture-negative BAL samples. However, no difference in 16S copy number was found between the groups. A group of culture-negative BAL with high diversity and similar bacterial communities were observed. These samples clustered together upon principal coordinates analysis, I believe this grouping may represent a core microbiome. Production of pro-inflammatory cytokines were found to be increased in samples that were dominated by a particular pathogen/pathogens as opposed to those that were more diverse and had lower cytokine measurements. This work highlights the advantages associated with using culture-independent techniques for the diagnosis of pneumonia specifically when traditional culturing techniques fail to identify an etiologic agent.Item Positive Regulation of Acetate Metabolism and Motility by the RNA-Binding Protein CsrA in Escherichia coli(2000-08-01) Wei, Bangdong L.; Jerry Simecka; Ming-Chi Wu; Stephen R. GrantWei, Bangdong L., Positive Regulation of Acetate Metabolism and Motility by the RNA-binding Protein CsrA in Escherichia coli. Doctor of Philosophy (Biomedical Sciences), August, 2000, 118 pp., 5 tables, 19 illustrations, bibliography, 175 titles. The carbon storage regulatory (Csr) system consists of a small RNA-binding effector protein, CsrA, and non-coding RNA, CsrB. CsrA acts as a global regulator and modulates specific mRNA stability in Escherichia coli. It regulates central carbon metabolism, physiology, and cell surface properties on a broad scale. In this study, the regulatory roles of csrA in acetate metabolism and motility were examined. The csrA gene was demonstrated to positively regulate acetyl-CoA synthetase and isocitrate lyase, while it did not affect phosphotransacetylase, isocitrate dehydrogenase, or citrate synthase. As a result, growth of csrA rpoS mutant strains was very poor on acetate as a sole carbon source. Surprisingly, growth also was inhibited specifically by the addition of modest amounts of acetate to rich media. Cultures grown in the presence of ≥25 mM acetate consisted substantially of glycogen biosynthesis (glg) mutants, which were no longer inhibited by acetate. Several classes of glg mutations were mapped to known and novel loci. The TCA cycle intermediates or pyruvate, but not glucose, galactose or glycerol, restored growth and prevented the glg mutations in the presence of acetate. Furthermore, amino acid uptake was inhibited by acetate specifically in the csrA rpoS strain. Apparently, central carbon flux imbalance, inhibition of amino acid uptake, and a deficiency in acetate metabolism are combined to cause metabolic stress by depleting the TCA cycle. The csrA gene was essential for motility and flagellum biosynthesis. Further studies elucidated the molecular mechanism by which CsrA positively regulates flagellum synthesis. Purified recombinant CsrA protein, which was isolated as a ribonucleoprotein complex consisting of one single CsrB molecule and ~18 CsrA subunits, directly stimulated the coupled transcription-translation of flhDC::lacZ in S-30 extracts and bound specifically to the 5’ non-coding segment of flhDC mRNA in mobility shift assay. The steady state level of flhDC mRNA was higher and its half-life was ~3-fold greater in a csrA wild type versus a csrA::kanR mutant strain, as shown by RT-PCR. Thus, CsrA is able to stimulate flhDC gene expression by a post-transcriptional mechanism that resembles its function in repression.Item Quorum Sensing in Sinorhizobium meliloti(2008-12-01) Patankar, Arati V.; Juan E. Gonzales; Jerry W. Simecka; Stephen O. MathewPatankar, Arati V., Quorum Sensing in Sinorhizobium meliloti. Doctor of Philosophy (Microbiology and Immunology), December 2008, 170 pp., 14 tables, 23 illustrations, bibliography, 212 titles. The overall goal of this study was to elucidate the role of a series of transcriptional regulators and potential signal molecules in the coordination of gene regulation in Sinorhizobium meliloti. The agriculturally important gram-negative soil bacterium S. meliloti, forms a symbiotic association with its host legume, Medicago sativa (alfalfa); thereby serving as a good model for studying host-bacterial interactions. Often, bacteria associated with eukaryotic hosts utilize global gene regulatory systems to coordinate their behavior in order to establish pathogenic or symbiotic associations. Quorum sensing is one such form of bacterial gene regulation which is mediated by signaling molecules and regulatory proteins in a population density dependent manner. In S. meliloti, the process of quorum sensing has been shown to play an important role in the relationship with its host plant. Control of essential processes such as plant nodulation and exopolysaccharide production has been attributed to the Sin/ExpR quorum-sensing system of S. meliloti. Interestingly, S. meliloti contains four additional (SMc04032, SMc00658, Smc00878 and SMc00877) putative quorum-sensing response regulators whose regulatory network was not known. The predicted protein sequences of these genes contain features typical of the LuxR family of proteins i.e., an N-terminal signal binding domain and C-terminal helix-turn-helix DNA biding domain. In order to identify their regulatory role, mutants of the response regulators were constructed and their expression profile was determined by employing genome-wide microarray and real-time PCR expression analysis. Through these analyses, it was determined that the SMc004032 locus controls expression of genes involved in the active methyl cycle, while the SMc00658, SMc00878 and SMc00877 loci control expression of genes from the denitrification of pathway of S. meliloti. Further, through phenotypic studies it was established that SMc04032 impacts stress response adaptation, and effective competition for plant nodulation. This suggests that SMc04032 could play a role in bacterial survival in the soil as well as within the host. The ability to denitrify is highly variable in different strains of S. meliloti. Through growth and enzymatic assays, it was established that the wild-type strain of this study, S. meliloti Rm8530, is a partial dentrifier in which, the capacity to metabolize nitrate is impaired. It was further determined that SMc00658, SMc00878 and SMc0877 modulated nitrite reductase activity under aerobic conditions, implying that these genes are involved in aerobic denitrification and therefor probably play a role in detoxification in S. meliloti. Based on the sequenced-genome analysis, S. meliloti possess homologs of other mediators of quorum sensing, that might be responsible for the synthesis of novel signal molecules. Bioreporter strains and mass spectrometry analysis were employed to identify production of cyclic dipeptides in S. meliloti. These compounds have been previously reported as quorum-sensing signal molecules in several bacteria. The results presented in this study provide a better understanding of S. meliloti’s metabolic and physiological properties and will be fundamental in future studies of bacterial interaction with its host and survival within its ecological niche.Item Resistance of Bacillus Subtilis Spores Lacking Either Nucleotide Excision Repair or Spore Photoproduct Lyase to Ultraviolet (UV) Radiation from Artificial or Natural Sources(1996-06-01) Xue, Yaming; Tony Romeo; Ming-Chi Wu; Wayne L. NicholsonXue, Yaming, Resistance of Bacillus subtilis spores lacking either nucleotide excision repair or spore photoproduct lyase to ultraviolet (UV) radiation from artificial or natural sources. Master of Science (Biomedical Sciences), June 1996, pp., 4 tables, 12 illustrations, references, 38 titles. Exposure of bacterial spores to UV radiation causes the accumulation of a unique pyrimidine dimer in the DNA, “spore photoproduct” (SP). In Bacillus subtilis, two distinct DNA repair pathways are used for removal of SP: general nucleotide excision repair (the uvr parthway), or the SP-specific enzyme SP lyase (the spl pathway). Spores of four strains of Bacillus subtilis differing in their repair capabilities were irradiated under either artificial or solar UV. To determine the biologically-relevant cumulative UV dose under each irradiation condition, a sporocidal dosimeter was constructed. The results showed: (i) Both uvr and spl pathways contributed to the survival of spores under all tested conditions. The spl pathway was more efficient than uvr pathway in repairing the DNA damage caused by UV-C and solar UV-A, but no significant difference was noted in repairing DNA damage caused by UV-B or full-spectrum solar UV. (ii) Exposure of spores to solar UV can cause cellular lethal damage which is reparable by neither repair pathway.Item Responses of Myocardial Antioxidant Systems to Intermittent Hypoxia and Ethanol Withdrawal(2008-05-01) Schulz, Diana R.; Robert Mallet; H. Fred DowneySchulz, Diana R., Responses of Myocardial Antioxidant Systems to Intermittent Hypoxia and Ethanol Withdrawal. Master of Science, May 2008, 50 pp., 3 tables, 14 figures, bibliography: 38 titles. Introduction: The effects of ethanol intoxication on the heart have been extensively studied over the past century; however the consequences of ethanol withdrawal on the heart have not been documented. Myocardial adaptations to hypoxia can improve contractility, enhance antioxidant defense mechanisms, decrease lipid peroxidation induced by oxidative stress, and bolster cardiac resistance to ischemia-reperfusion. This study was conducted to determine if ethanol intoxication-withdrawal can harm the myocardium, and if intermittent hypoxia conditioning (IHC) can induce antioxidant enzymes and proteins that blunt these effects. Hypothesis: IHC increases myocardial antioxidant enzymes and other stress proteins, which could protect myocardium from ethanol intoxication-withdrawal. Methods: Four month old Sprague Dawley rats (n=61) were divided into 8 groups. Four groups were fed a 6.4% ethanol enriched diet, and the other four were fed an isocaloric dextrin diet. IHC was initiated two weeks after the diets began. The antioxidant N-acetylcysteine (NAC) was injected throughout the IHC and sham conditioning programs to interrogate the role of reactive oxygen species in the effects of ethanol intoxication-withdrawal and IHC on myocardial proteins. Proteins were extracted from snap-frozen myocardium for assays of nitric oxide synthase (NOS), gluthathione peroxidase (GPx) and superoxide dimustase (SOD) activities, and immunoblot analyses of endothelial NOS (eNOS) and heat shock protein 70 (Hsp70). Results: Ethanol withdrawal inactivated myocardial SOD, but did not affect activities of GPx and NOS, or contents of Hsp70 and eNOS. IHC lowered SOD activity by 55% in dextrin-fed rats, but partially protected SOD activity from ethanol intoxication-withdrawal. IHC did not affect myocardial GPx, NOS, and Hsp70 in dextrin and ethanol-withdrawn rats. NAC alone increased SOD activity in dextrin-fed and ethanol withdrawn rats and, when combined with IHC, NAC fully protected SOD activity from ethanol intoxication-withdrawal. Conclusions: Ethanol intoxication-withdrawal had statistically significant effects on the antioxidant enzyme SOD. IHC and NAC selectively protected SOD activity from ethanol intoxication-withdrawal. This specificity suggests that the oxidative stress induced by ethanol intoxication-withdrawal is discrete in comparison to the massive oxidative stress inflicted on the brain under these conditions. The differences in the intensity of oxidative stress could be one of the factors influencing the divergence of results in comparison to previous investigations of ethanol intoxication-withdrawal.Item STAT6 and Its Relationship with PSA and Annexin A2 in Human Prostate Cancer(2008-05-01) Roth, Cherice P.; Singh, Meharvan; Jones, Harlan P.; Sharma, RajendraRoth, Cherice, STAT6 and its relationship with PSA and Annexin A2 in Human Prostate Cancer. Master of Science (Biochemistry and Molecular Biology), May 2008, 49 pages, 13 illustrations, reference list, 54 titles. The increase of signal transducer and activator of transcription (STAT6) has been correlated with increased prostate tumor size as well as Gleason score. This molecule’s exact role in prostate cancer is still unknown. This research focused on the relationships of STAT6 in prostate specific antigen (PSA) expression as well as its novel interaction with annexin A2. These data show that STAT6 is involved in an alternate PSA expression pathway. It is also concluded that the interaction of STAT6 and annexin A2 increased the activated STAT6 (p-STAT) but not total STAT6. Chromatin immunoprecipitation also confirmed the novel protein-protein interaction between STAT6 and annexin A2 is nuclear.