ETD Search Results: instcode:ucin

1. Abbott, Maxwell Bret. THE STRUCTURAL MECHANISM OF Β-ADRENERGIC MODULATION OF CARDIAC TROPONIN SWITCH CALCIUM SENSITIVITY.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2001, University of Cincinnati

► Cardiac troponin is the molecular switch that activates the cardiac thin filament… (more)

Keywords: CARDIAC TROPONIN; PROTEIN-PROTEIN INTERACTIONS; NUCLEAR MAGNETIC; STRUCTURE - FUNCTION; PHOSPHORYLATION

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2. Barnes, Michael. CHARACTERIZATION OF THE COMPLEMENT RESISTANCE MECHANISM OF BORDETELLA PERTUSSIS.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2001, University of Cincinnati

► To survive within the human host, Bordetella pertussis must resist host defenses… (more)

▼ To survive within the human host, Bordetella pertussis must resist host defenses including the complement system. B. pertussis produces the BrkA (Bordetella resistance to killing) protein that inhibits complement mediated bacterolysis. We have examined the ability of each complement activation pathway to mediate killing of B. pertussis, and the influence of BrkA and lipopolysaccharide (LPS) on complement susceptibility. Wild type or BrkA mutants were not killed in serum depleted of C2 or antibody, however susceptibility to killing by complement was unchanged by depletion of factor B. These results suggest complement activation by these strains occurs exclusively by the antibody-dependent classical pathway and BrkA inhibits this pathway. LPS has been shown to affect complement resistance in other bacteria. In B. pertussis, a mutant lacking the terminal sugar of the LPS O-antigen was susceptible to killing in the absence of antibody while a LPS mutant totally lacking O-antigen displayed increased suscept ibility to antibody-mediated killing by complement. The step in this pathway at which BrkA acts was then characterized. Complement activation was monitored by ELISA, and deposition of complement components on the bacterial surface was monitored by Western blots. Production of SC5b-9 (soluble membrane attack complex) and deposition of C4, C3 and C9 showed an inverse correlation with BrkA expression but deposition of C1 was equivalent regardless of BrkA expression. These studies confirm that BrkA inhibits the classical pathway of complement and acts to prevent accumulation of C4 but not C1. We observed that complement resistance could be highly variable, so we varied growth conditions to see if growth affected resistance. Log phase bacteria were about 500-times more sensitive to killing by complement than stationary phase cultures. The effect of virulence gene regulation, BrkA expression and antigenic variation were tested to explain the fluctuation in serum resistance. None of these factors was found to influence temporal expression of complement resistance. These results suggest that log-phase susceptibility to complement may be an inherent property of rapidly growing cultures. In summary, B. pertussis is killed exclusively by the antibody-dependent classical pathway and sensitivity varies with growth phase. BrkA inhibits this pathway after C1 deposition. Advisors/Committee Members: Weiss, Alison.

Subjects: Biology, Microbiology

Keywords: BORDETELLA PERTUSSIS; COMPLEMENT; RESISTANCE; SERUM

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3. BARTON, LANCE F. PROTEASOME ACTIVATOR PA28 AND MAJOR HISTOCOMPATABILITY COMPLEX CLASS I PROCESSING IN VITRO AND IN VIVO.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2003, University of Cincinnati

► Proteasomes are large (~700kD) intracellular multicatalytic proteases found in eukaryotic cells and… (more)

▼ Proteasomes are large (~700kD) intracellular multicatalytic proteases found in eukaryotic cells and some archaebacteria and eubacteria. These proteases are responsible for degrading intracellular proteins for multiple cellular functions. In vertebrates, proteasomes also generate most of the peptide epitopes that are presented to cytotoxic T lymphocytes via major histocompatability complex (MHC) class I molecules on the cell surface. Proteasomes are important in both maintaining immunologic self-tolerance as well as facilitating the immune response towards intracellular pathogens. It is believed that the core proteasome particle is latent in cells and therefore requires activator protein complexes to enhance its activity in vivo. The PA28 family of proteasome activators contains three members: alpha, beta, and gamma. PA28 alpha and beta are ubiquitously distributed in the cell and expression is enhanced in vitro following exposure to interferon-gamma (IFNgamma). PA28gamma is localized to the nucleus and its expression is unaffected by IFNgamma. We demonstrate that the expression of PA28 alpha and beta along with other proteasome genes are enhanced by IFNgamma in vivo. These IFNgamma-inducible genes demonstrate true constitutive expression that is only partially dependent on components of the IFNgamma signaling cascade, and are completely independent of an IFNgamma stimulus. Furthermore, it has been demonstrated in vitro that PA28 may affect the specificity of proteasomes, and work in conjunction with specific catalytic sites to enhance the peptide repertoire generated by proteasomes. To investigate the functions of PA28gamma, we generated a PA28gamma-deficient mouse strain. These mice display defects in multiple proteasome-mediated pathways including MHC I antigen presentation. The effect of PA28gamma on antigen processing and presentation is antigen-specific, not global. To better understand the antigen specificity of the PA28 complexes, recombinant protein was generated and the cleavage specificities of the mouse PA28alpha/beta-proteasome complex were examined in vitro using natural peptide substrates. Advisors/Committee Members: Monaco, Dr. John J.

Keywords: proteasome; MHC; antigen processing; PA28; antigen/epitope

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4. Brzezinski, Jennifer Lynn. THE ROLE OF T CELL RECEPTOR Vβ GENE POLYMORPHISMS IN SUSCEPTIBILITY TO JUVENILE RHEUMATOID ARTHRITIS.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2001, University of Cincinnati

► Juvenile rheumatoid arthritis is the most common childhood rheumatic disease. Previous studies… (more)

▼ Juvenile rheumatoid arthritis is the most common childhood rheumatic disease. Previous studies have shown that null alleles of the T cell receptor Vβ (TCRBV) gene segment TCRBV6S1 are associated with some subsets of JRA, namely polyarticular JRA. Linkage disequilibrium has been identified between the null alleles of TCRBV6S1 and alleles of the neighboring TCRBV genes TCRBV5S5P, TCRBV1S1 and TCRBV13S5, and this genetic association may therefore be due to a linked susceptibility locus. In Caucasian individuals, only three of the possible 12 haplotypes were represented, and the two TCRBV6S1 null alleles are present on distinct haplotypes. However, in additional ethnic groups significant deviations in the patterns of linkage disequilibrium were observed. This needs to be taken into account in any genetic studies involving TCR polymorphisms. TCRBV repertoire analysis was undertaken in an effort to establish a functional role for TCRBV6S1 null allele haplotypes in the pathogenesis of JRA. TCRBV1S1 is the only member of this haplotype that is readily expressed in the peripheral repertoire; analysis of synovial and peripheral blood TCRBV1S1 repertoires revealed an allelic bias wherein TCRBV1S1A2 is under-represented in heterozygotes. Additionally, individuals who are carriers of TCRBV1S1A2 have lower levels of TCRBV1S1 mRNA and reduced numbers of Vβ1 + T cells. Although we have not definitively established a role for TCRBV1S1A2 in JRA, reduced numbers of Vβ1 + T cells may nonetheless be relevant to disease pathogenesis. The transmission disequilibrium test (TDT) was used to test whether TCRBV6S1 null alleles are preferentially transmitted to JRA probands. Unexpectedly, the TDT demonstrates under-transmission of TCRBV6S1 null alleles to polyarticular JRA patients, suggesting these alleles may be protective. In fact, the two null alleles do not appear to contribute equally to disease protection in all JRA subtypes. Although this data is preliminary, it suggests that caution must be observed when interpreting the results of case-control studies that are sensitive to population stratification events. Although case-control and TDT studies have produced decidedly opposing results, there is evidence nonetheless that TCRBV6S1 null alleles may have an important role in the genetic component of JRA. Advisors/Committee Members: Choi, Edmund.

Subjects: Biology, Genetics

Keywords: TCR; JUVENILE RHEUMATOID ARTHRITIS; POLYMORPHISM; HAPLOTYPE

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5. Conkright, Michael Dale. CHARACTERIZATION OF TWO MEMBERS OF THE KRUPPEL-LIKE FAMILY OF TRANSCRIPTION FACTORS.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2001, University of Cincinnati

► Lung Krüppel-like factor (LKLF) is essential during pulmonary development, single-positive T cell… (more)

▼ Lung Krüppel-like factor (LKLF) is essential during pulmonary development, single-positive T cell development, and is indispensable during mouse embryogenesis. We performed a series of experiments to define the activation domain of LKLF as a means to further advance the understanding of the molecular mechanisms underlying transcriptional regulation by this transcription factor. Using deletion analysis, it is shown that LKLF contains a transcriptional activation domain as well as a strong autoinhibitory subdomain. The inhibitory subdomain is able to independently suppress transcriptional activation of other strong activators such as VP16. Overexpression of the LKLF autoinhibitory domain alone potentiates transactivation by wild type LKLF, suggesting the inhibitory domain binds a cofactor that prevents LKLF from transactivating. In order to isolate such a co-factor, we conducted a yeast two-hybrid screen using the LKLF inhibitory domain (a.a. 111-267). This screen resulted in several proteins that interacted with LKLF. One strongly interacting protein, WWP1, is of particular interest. WWP1 is an E3 ubiquitin protein ligase that contains four WW domains that interact with proline rich regions, a characteristic of LKLF inhibitory domain. For these reasons, we feel that WWP1 potentially regulates LKLF transactivation. In addition to LKLF, we have identified an additional member of the KLF family of transcription factors, intestinal-enriched Krüppel-like factor (IKLF). IKLF is expressed in a limited number of tissues: the highest level of IKLF expression is found in the epithelial cells located at base of the crypts in the adult intestine. GKLF, a second intestinal-enriched KLF, has an expression pattern that is reciprocal to IKLF: GKLF expression is located at the apex of the villus and is decreased towards the base of the crypts. Unlike LKLF, IKLF does not contain an inhibitory domain. The findings show that we have identified a transcription factor that is expressed predominantly in the epithelial crypt cells of the gastrointestinal tract and is a member of the Krüppel-like family of transcription factors. Finally, we demonstrate that the KLF family is regulated by the Notch signaling pathway. Notch potentiates transactivation of members of the KLF family and is dependent on intact KLF binding sites to mediate its effects. Advisors/Committee Members: Lingrel, Jerry B.

Subjects: Biology, Molecular

Keywords: LKLF; Transcription; WWP1; Inhibitors Domain; Kruppel like factors

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6. Flagella, Michael. MOLECULAR AND PHYSIOLOGICAL STUDIES OF ELECTROLYTE AND FLUID TRANSPORT PERTURBATIONS IN NKCC1 AND NHE3 DEFICIENT MICE.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2001, University of Cincinnati

► Epithelial tissues mediate absorptive and secretory ion transport processes to maintain physiological… (more)

▼ Epithelial tissues mediate absorptive and secretory ion transport processes to maintain physiological equilibrium of ions and fluid. These processes are mediated in part by ion transport proteins expressed throughout the mammalian gastrointestinal tract and the renal nephron. Ion transporters regulate, either directly or indirectly, blood and extracellular fluid volume and composition, stomach acid secretion, digestion and nutrient absorption, and excretion of metabolic by-products. Many of the genes encoding ion transporters have been identified and characterized at the molecular level; however, the specific contribution of each transporter to epithelial ion transport during normal and pathophysiological states is incompletely understood. The Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) mediates the uptake of Na+, K+, and Cl- from extracellular fluid. In secretory epithelial tissues, this uptake is important for the secretion of K+ or Cl- enriched fluid. To identify the specific contribution that NKCC1 makes to epithelial secretion, I have generated and analyzed a recombinant mouse line with a targeted disruption of the NKCC1 gene. This study tests the hypothesis that NKCC1 is required for proper epithelial Cl- and K+ secretion, and that altered Na+-K+-Cl- uptake at the basolateral membrane will perturb fluid secretion. My results demonstrate the critical role of NKCC1 in hearing and refutes a previous hypothesis that it is necessary for gastric acid secretion. An unexpected finding was that NKCC1 plays an important role in the regulation of blood pressure. Na+/H+ exchanger isoform 3 (NHE3) mediates the absorption of Na+ and HCO3- from, and secretion of H+ into, the lumen of the small intestine and proximal tubule of the kidney. Mice lacking NHE3 exhibit a mild phenotype associated with perturbations in Na+-fluid volume homeostasis and elevated serum aldosterone. I have tested the hypothesis that compensation for these perturbations involves transcriptional remodeling of the renal ion transport system. Data obtained from gene expression profiling experiments of kidney mRNA, through the use of cDNA microarray technology and Northern blot analysis, suggests that the kidney responds to perturbations in Na+-fluid volume homeostasis largely through post-transcriptional regulation of ion transport and renal hemodynamics, and not through transcriptional regulation of ion transporters. Advisors/Committee Members: Shull, Gary.

Keywords: MICROARRAY; SODIUM/PROTON EXCHANGER 3; SODIUM POTASSIUM CHLORIDE ISOFORM 1; BLOOD PRESSURE; SECRETION

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7. GAMAGE, SHANTINI D. Shiga toxin-encoding phage from Escherichia coli O157:H7 - interactions with non-pathogenic E. coli and implications for toxin production.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2004, University of Cincinnati

► The foodborne pathogen, Eschericia coli O157:H7, causes about 73,000 cases of illness/year… (more)

▼ The foodborne pathogen, Eschericia coli O157:H7, causes about 73,000 cases of illness/year in the U.S. Life-threatening complications include hemorrhagic colitis and hemolytic uremic syndrome. A major disease determinant for O157:H7 is the production of Shiga toxin (Stx) in the intestine. Two immunologically distinct types of Stx, Stx1 and Stx2, can be produced by O157:H7, and the production of Stx2 has been epidemiologically linked to more severe disease. Regulation of the Shiga toxins by E. coli is unique. The toxin genes are encoded in the late gene region of a lambdoid bacteriophage lysogenized in the O157:H7 chromosome. The toxin genes are silent during lysogeny, and induction of the phage to enter the lytic cycle results in the production of phage and, concomitantly, toxin. We examined the hypothesis that during infection, toxin-encoding phage produced by O157:H7 infect intestinal E. coli , resulting in amplification of phage and toxin production. We have shown that this phenomenon does occur, both in vitro and in vivo. Clinical O157:H7 strains were further studied. Stx2-encoding phage from seven O157:H7 isolates were found to be variable with respect to protein profiles, cross-immunity and cross-lysogeny, even when the phage originated from O157:H7 isolates that were indistinguishable by molecular typing. We also studied the interaction of these phage with normal human E. coli . Only two of the seven phage were able to amplify toxin in normal E. coli , indicating that this phenomenon, while present, is rare. Lysogeny of normal E. coli by these two phage differed, and further inspection of the results suggests that normal E. coli that belong to the phylogenetic groups A1 and B are more susceptible to lysogeny. Characterization of the interaction between Stx phage and normal E. coli lead to the discovery of FI-29, a normal E. coli isolate that neutralizes Stx2, but not Stx1, by binding the toxin. Lipopolysaccharide (LPS) was shown to be the neutralizing agent. FI-29 LPS belongs to the O107/O117 serotypes, and E. coli strains of either serotype also neutralize toxin. FI-29 may be a potential probiotic or therapeutic for the prevention and treatment of O157:H7 disease. Advisors/Committee Members: Weiss, Dr. Alison A.

Subjects: Biology, Microbiology

Keywords: E. coli O157:H7; Shiga toxin; phage; toxin amplification; toxin neutralization; Gb3

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8. Gaponeko, Vadim Viktorovich. THE USE OF PARAMAGNETIC SYSTEMS IN PROTEIN GLOBAL FOLD DETERMINATION.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2000, University of Cincinnati

► Collection and interpretation of NMR structural information is often time-consuming, while the… (more)

▼ Collection and interpretation of NMR structural information is often time-consuming, while the demand of pharmaceutical industries for structures of newly discovered proteins is high. This research demonstrates the utility of paramagnetic probes for the rapid determination of protein global folds using paramagnetic relaxation enhancements, dipolar shifts, and dipolar couplings. Attachment of nitroxide and thiol-reactive EDTA tags to unique cysteines allowed use of the relaxation effects of unpaired electrons in nitroxides and manganese ions on nuclei within the protein of interest for measurement of electron-nucleus distances. Distances obtained in nitroxide labeled mutants of barnase, H102C and Q15C, combined with secondary structure restraints permitted calculation of a barnase global fold with the 3Å root mean square deviation of backbone coordinates from the crystal structure of barnase. Introduction of EDTA tags into the same barnase mutant proteins allowed the use of manganese-amide proton distances and cobalt induced dipolar shifts for calculation of amide proton atomic coordinates onto the z-axis and the xy-plane of the paramagnetic susceptibility tensor. In addition, the anisotropic magnetic field of cobalt induced alignment of protein molecules in the external magnetic field permitting measurement of dipolar couplings. Manganese and cobalt were also incorporated into barnase by fusing a zinc finger tag to the protein C-terminus allowing determination of z-coordinates and measurement of dipolar couplings. Despite the significant amount of error introduced by the mobility of the tag, an accuracy of +/- 4.5Å in calculation of z-coordinates could be achieved. The utility of unique paramagnetic reference frames for structure determination was demonstrated on calbindin containing calcium binding sites that could be substituted with cerium, ytterbium, and disprosium. Minimization against lanthanide induced dipolar shifts produced a protein structure with 3.3Å resolution. However, only 29 out of 76 amide protons in calbindin were used for global fold determination due to extreme line-broadening. In conclusion, this research has demonstrated the utility of paramagnetic systems for rapid protein structure elucidation. It has been shown that paramagnetic probes can be incorporated into nonmetal-binding proteins. The use of structural restraints obtained using paramagnetic probes should permit rapid determination of global folds of newly discovered proteins. Advisors/Committee Members: Rosevear, Paul R.

Keywords: NMR; paramagnetic systems; protein structure; global fold; labeling

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9. Hersh, Megan N. VISUALIZING GENOMIC INSTABILITY: IN SITU DETECTION AND QUANTIFICATION OF MUTATION IN MICE.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2001, University of Cincinnati

► Multi-celled organisms are mosaic in nature because somatic mutations occur in individual… (more)

▼ Multi-celled organisms are mosaic in nature because somatic mutations occur in individual cells of the body. Little is known about the frequency of mutation in most cell types of mammals. To make this phenomenon more amenable to study, transgenic mice carrying a histochemically detectable reporter gene were created, allowing for individual cells with a particular frameshift mutation to be detected. In order to enhance the chances of detecting spontaneous mutation events, the transgene was modified to contain a hypermutable region of 11 G:C basepairs. The mutation frequencies in mouse brain, heart, kidney and liver were quantified, yielding information about how often -1 frameshift mutations occur in the wildtype mouse genome. Although no particular tissue had consistently more mutation events than another from the same mouse, these frequencies tend to be, on average, 5- to 7-fold higher in brain and heart as compared to liver and kidney (i.e. ~20 events vs. ~2 events per million cells). This suggests that the tissues each have different methods for coping with mononucleotide runs. The DNA mismatch repair (MMR) system is responsible for recognizing and repairing DNA basepair mismatches that can result from recombination, chemical modification or DNA replication errors. The frameshift reporter transgene was crossed into mice lacking a functional Pms2 mismatch repair gene, the products of which are known to be involved in the repair of insertion/deletion events. Mutant frequency increased dramatically in this background, and was estimated to be at least 1000-fold greater than the respective wildtype tissues in brain, heart and kidney. However, frequency increased only 10-fold in liver. These data corroborate the evidence that Pms2 plays a significant role in the maintenance of genome stability at repetitive sequences, and suggest that Pms2 operates in a tissue-specific manner. To investigate this possibility, the effect of Mlh1-deficiency was also studied. Mlh1 is known to partner with Pms2 in mismatch repair, yet the respective knockout mice for each display slightly different phenotypes, with tissue-specific differences in tumor susceptibility. Interestingly, the Mlh1-deficient mice displayed a higher mutant frequency, but a similar tissue mutation spectrum to the Pms2-deficient mice. Advisors/Committee Members: Stringer, James.

Keywords: DNA MUTATION; DNA REPAIR; MISMATCH REPAIR; MICE

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10. INGRAHAM, SUSAN ELIZABETH. THE BALANCED, RECIPROCAL TRANSLOCATION OF CHROMOSOMAL SUBBANDS 12q15 AND 14q24 AND ALTERED GENE EXPRESSION IN UTERINE LEIOMYOMA.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2002, University of Cincinnati

► A balanced, reciprocal translocation between chromosomes 12 and 14 is frequently observed… (more)

▼ A balanced, reciprocal translocation between chromosomes 12 and 14 is frequently observed in uterine leiomyoma (UL), a common benign smooth muscle tumor. Cytogenetic evidence suggests that t(12;14)(q15;q24) is an early event and therefore may represent one of the important steps in UL pathogenesis. The breakpoints of t(12;14)(q15;q24) have been localized to unusually large genomic regions (approximately 0.5 Mb) on each of the involved chromosomes. In this study, we investigated the molecular events associated with this translocation, specifically the transcription of genes within the breakpoint regions on 14q24 and 12q15. t(12;14)(q15;q24) had previously been associated with activation of HMGA2 on chromosome 12, a gene implicated in promoting cell proliferation, particularly of lipoid tissues. We analyzed this gene and discovered several novel transcripts from 12q15 that are embedded within HMGA2 . On chromosome 14, the breakpoint in one UL was found to lie within RAD51L1 , a putative DNA repair and recombination gene. Subsequent analysis of two additional t(12;14) UL breakpoints refined the chromosome 14 breakpoint region to between 700 and 1200 kb.The large size of the breakpoint regions and the mapping of breakpoints well outside both HMGA2 and RAD51L1 suggested that the translocation may alter the structure and long-range regulatory controls of genes including but perhaps not limited to HMGA2 and RAD51L1 . To test this hypothesis, an expression map was developed which consisted of ESTs and genes within and flanking both breakpoint regions. Expression of these markers was tested in matched normal and t(12;14) ULtissue samples to identify a domain of altered expression on chromosomes 12 and 14. HMGA2 and the three novel ESTs embedded within HMGA2 ., A15, B6, and D12, were overexpressed six- to more than twenty-fold, while RAD51L1 and other ESTs on chromosome 14 were not consistently or significantly altered in UL. Positional cloning of the UL breakpoint region and mapping of the domain of altered expression in tumors sets the stage for understanding the molecular mechanism for the pathogenesis of UL. Advisors/Committee Members: Menon, Dr. Anil G.

Keywords: uterine leiomyoma; translocation; gene expression; RAD5161; HMGA2

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11. Kozel, Peter J. HEARING IMPAIRMENTS IN MICE DEFICIENT IN PLASMA MEMBRANE Ca 2+ - ATPASE ISOFORM 2.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2001, University of Cincinnati

► Calcium homeostasis is regulated, in part, by the plasma membrane calcium ATPases… (more)

▼ Calcium homeostasis is regulated, in part, by the plasma membrane calcium ATPases (PMCAs), which are encoded by a family of alternatively spliced genes. Isoform 2 (PMCA2) is found at high levels in brain and heart, with lower levels in other tissues, including the kidney and inner ear. To identify the physiological roles of PMCA2, this gene was ablated in mice via homologous recombination. Mice were born in a normal Mendelian ratio and were viable. In vivo performance measurements suggested that PMCA2 does not play a major role in cardiac muscle contraction. Analysis of urinary calcium concentration suggested that the absence of PMCA2 may impair calcium reabsorption in the renal nephron. A thinning of the molecular layer and a slight increase in the number of Purkinje cells, in which PMCA2 is highly expressed, were observed by histological analysis of cerebella of Pmca2 -/- mice. The major phenotypes of Pmca2 - deficient mice involved the inner ear. Beginning twelve days post partum, Pmca2 -/- mice displayed a balance deficit that is due, in large part, to the absence of otoconia in the utricle and saccule, gravity-sensing organs in the inner ear. Pmca2 -/- mice were deaf and exhibited a spectrum of cochlear histopathologies, including the degradation of the major cell types necessary for the detection of sound. Depending on genetic background, Pmca2 +/- mice heard normally or had hearing thresholds that were 30-50 dB greater than Pmca2 +/+ littermates. PMCA2 is highly expressed in outer hair cells, and the cytoplasmic calcium concentration in outer hair cells rises following acoustic overstimulation, suggesting that PMCA2 activity may play a role in the response to sound exposure. Pmca2 -/- mice had large, permanent increases in hearing thresholds following noise exposure, indicating that they were more susceptible to noise induced hearing loss than Pmca2 +/+ littermates. These data indicate that PMCA2 plays a critical role in balance and hearing. Advisors/Committee Members: Shull, Gary.

Keywords: MICE; HEARING LOSS; NOISE INDUCED; PMCA2; CALCIUM

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12. Langland, Gregory Todd. Interaction Between the BLM Helicase and the DNA Mismatch Repair Protein, MLH1.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2003, University of Cincinnati

► Bloom's syndrome (BS) is a rare autosomal recessive disorder that greatly predisposes… (more)

▼ Bloom's syndrome (BS) is a rare autosomal recessive disorder that greatly predisposes affected individuals to cancer. Such individuals also are small in size, sensitive to the sun, have immune dysfunction and gross genomic instability. The cytogenetics of BS cells have been extensively studied and have shown increased levels of homologous recombination, quadriradial formations, telomeric associations and chromosome breakage. The gene responsible for BS has been positionally cloned and and encodes a RecQ helicase family member with strand displacement activity that is dependent on ATP and Mg2+. In order to have a greater understanding of BLM helicase function in the cell in regards to DNA replication, recombination and repair, we identified protein-partners of BLM. The C-terminus of BLM identified the DNA mismatch repair protein MLH1 from a yeast two-hybrid screen. In vitro and in vivo immunoprecipitations confirmed the interaction between these two proteins. Using an in vitro mismatch repair assay, BS cell extracts were tested for their ability to correct a single nucleotide mismatch. The BS cell extracts were able to remove the single nucleotide mismatch from the plasmid DNA, demonstrating that the BLM-MLH1 interaction is not necessary to correct a single nucleotide mismatch substrates. Helicase assays then were performed which demonstrated that MLH1 or the mutL heterodimer modulates the enzymatic activity of BLM by stimulating BLM's strand displacement activity on the double-overhang (DO) substrate. Finally, we performed experiments with the supF20 mutagenesis system and demonstrated that extracts from BS cells are unable to utilize micro-homology elements within the supF20 gene to restore supF function following the induction of a double strand break (DSB). Additional experiments with the pUC18 mutagenesis system demonstrate that although the efficiency and fidelity of DSB repair by BS extracts are comparable to those of normal extracts when ligatable ends are present, a significant 5-fold increase in mutation rate with BS extracts is observed when terminal phosphates are removed from the DNA substrate that needs repair. Mutant plasmids recovered following DSB repair by BS extracts contain smaller deletions within the lacZ α gene not commonly recovered from normal extracts. Colorectal cancer cell lineHCT116 extracts lacking MLH1 were also examined although the efficiency and fidelity of end-joining was similar to control extracts. This suggests that the BLM-MLH1 interaction is not necessary for proper end-joining. In summary this work demonstrates that BS cells lacking the BLM helicase process DSBs differently than normal cells and strongly suggests a role for BLM in aligning micro-homology elements during recombinational events in DSB repair. Disruption of the BLM helicase may lead to replication fork collapses, improper processing of DSBs, genomic instability and ultimately cancer. Advisors/Committee Members: Groden, Dr. Joanna.

Subjects: Chemistry, Biochemistry

Keywords: Bloom's Syndrome; RecQ Helicase; DNA repair; Homologous Recombination; mismatch repair

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13. LEDOUSSAL, CLARA SIGISMONDI. ABSORPTIVE FUNCTIONS OF THE NHE2 AND NHE3 SODIUM/PROTON EXCHANGERS IN INTESTINE AND KIDNEY.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2001, University of Cincinnati

► Maintenance of sodium-fluid volume homeostasis requires the tightly regulated activities of Na… (more)

▼ Maintenance of sodium-fluid volume homeostasis requires the tightly regulated activities of Na + /H + exchangers in intestine and kidney. Expression of NHE2 and NHE3 on intestinal and renal apical membranes suggests that both isoforms might contribute to sodium absorption. Studies with knockout mice showed that the loss of NHE3, but not NHE2, causes diarrhea, thereby demonstrating that NHE3 is the major absorptive exchanger and indicating that any remaining absorptive capacity contributed by NHE2 is not sufficient to compensate fully for the loss of NHE3. It remains possible, however, that NHE2 plays an important supplementary role in sodium absorption and blunts the severity of the diarrheal state in NHE3-deficient mice. To test this hypothesis in vivo , we crossed doubly heterozygous mice carrying null mutations in the Nhe2 and Nhe3 genes and assessed the diarrheal phenotype by analyzing the viability, systemic acid-base status, aldosterone levels, and intestinal contents of the resulting offspring. The loss of NHE2 in an NHE3-deficient background caused no apparent enhancement of the diarrheal state. To study the role of NHE2 and NHE3 in sodium-fluid volume homeostasis and renal sodium conservation, mice with Nhe2 and/or Nhe3 null mutations were fed a sodium-depleted diet, and urinary sodium excretion, blood pressure, and renal function were analyzed. Under Na + deprivation, Nhe2 null mice, on either a wild-type (Nhe2-/-Nhe3+/+) or an Nhe3 heterozygous background ( Nhe2 -/- Nhe3 +/- ), exhibited no increase in urinary sodium excretion and had similar blood pressure and glomerular filtration rate (GFR) relative to control mice ( Nhe2 +/+ Nhe3 +/+ , Nhe2 +/+ Nhe3 +/- , respectively), suggesting an absence of salt-wasting. In contrast, Nhe3 null mutants maintained on a sodium-depleted diet for three days exhibited hyponatremia, hyperkalemia, severe acidosis, mild urinary salt-wasting, and sharply reduced bodyweight, blood pressure and GFR. The overall study demonstrated a major role of NHE3 in intestinal and renal Na + reabsorption and maintenance of systemic electrolyte, acid-base, and fluid volume homeostasis, with little or no contribution from NHE2. Advisors/Committee Members: Shull, Dr. Gary E.

Subjects: Biology, Animal Physiology

Keywords: Na+/H+ exchanger; NHE2; NHE3; intestine; kidney

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14. OGDEN, STACEY KATHRYN. HBx-MEDIATED DISRUPTION OF p53 TUMOR SUPPRESSOR PROTEIN FUNCTION LEADING TO RE-ACTIVATION OF A SILENCED TUMOR MARKER GENE.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2002, University of Cincinnati

► Chronic infection with Hepatitis B Virus (HBV) is a predominant risk factor… (more)

Keywords: gene regulation; liver cancer; chromatin structure modification; In vitro transcription

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15. PARSONS, STEPHANIE A. THE ROLE OF CALCINEURIN IN SKELETAL MUSCLE HYPERTROPHY AND FIBER TYPE DIVERSITY.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2004, University of Cincinnati

► Calcineurin is a calcium-regulated serine-threonine protein phosphatase that controls developmental and inducible… (more)

▼ Calcineurin is a calcium-regulated serine-threonine protein phosphatase that controls developmental and inducible biological responses in diverse cell types, in part through activation of the transcription factors nuclear factor of activated T cells (NFAT) and myocyte enhancer binding factor 2 (MEF2). In skeletal muscle, calcineurin has been implicated in the regulation of myoblast differentiation, myofiber hypertrophy, and fiber-type switching in response to alterations in intracellular calcium concentration. However, considerable disagreement persists regarding the functional role of calcineurin signaling these processes. Here, we evaluated the molecular phenotypes of skeletal muscle from calcineurin-deficient mice, including calcineurin Aalpha-/-, calcineurin Abeta-/-, and calcineurin B1 LoxP/LoxP mice expressing cre recombinase specifically in skeletal muscle. Calcineurin expression appears to be important during early myogenesis in establishment of fiber number in some muscles. We also found that calcineurin Aalpha-/- and Abeta-/- mice exhibit overall growth defects, and calcineurin Abeta-/- mice have smaller relative muscle weights. These defects were not seen, however, in calcineurin B1 LoxP MLC-cre mice, in which calcineurin is down-regulated at embryonic day 12.5, indicating that these defects originate from early myogenic events. Decreased levels of calcineurin expression also inhibited the establishment of slow/oxidative fibers. This decrease in some cases occurred without any change in the nuclear occupancy of NFAT proteins, suggesting that NFATs are not involved in the initial establishment of fiber type. Loss of calcineurin expression did not affect muscle growth following IGF-1/GH administration or mechanical overload. NFAT-luciferase reporter mice, used to analyze calcineurin activity, displayed no change in activity after IGF-1/GH administration but exhibited a dramatic increase following overload, which correlated with increased NFATc1 protein expression in all mice except calcineurin B flox/flox MLC-cre. Calcineurin B flox/flox MLC-cre mice also did not undergo the normal fast-to-slow fiber type switching that normally occurs during hypertrophy. Collectively, our results indicate that calcineurin has two important roles during development, in establishment of fiber number and in mediating the slow-twitch phenotype. In adult skeletal muscle, loss of calcineurin expression negatively impacts the relative fiber number of skeletal muscle and the ability to undergo fiber type switching in response to overload, but does not affect muscle growth in response to hypertrophic stimuli. Advisors/Committee Members: Molkentin, Dr. Jeffery D.

Subjects: Biology, Molecular

Keywords: calcineurin; skeletal muscle; hypertrophy; NFAT; overload; myosin heavy chain

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16. Pieples, Kathy. THE FUNCTIONAL SIGNIFICANCE OF THE STRIATED ISOFORM OF TROPOMYOSIN 3 IN NORMAL AND PATHOLOGICAL STATES.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2001, University of Cincinnati

► Striated muscle tropomyosin, a thin filament protein of the sarcomere, has three… (more)

▼ Striated muscle tropomyosin, a thin filament protein of the sarcomere, has three isoforms: α-TM, β-TM and TPM 3. Although the α-TM and β-TM isoforms are well characterized, little was known about TPM 3. We cloned and sequenced the murine TPM 3 cDNA. It exhibits a 93% nucleotide homology and 99% amino acid homology to the human sequence. TPM 3 is not endogenously expressed in murine hearts; however, it is found in slow-twitch but not in fast-twitch musculature. Results suggest a molecular mechanism regulates the stability or production of TM RNAs and proteins, with β-TM message either not as efficiently translated or as stable as α-TM and TPM 3. In order to determine the functional significance of TPM 3, we generated six transgenic mouse lines that produce varying levels of TPM 3 message in the heart. TPM 3 protein accounts for 40-60% of the striated muscle tropomyosin. The TG mice have normal life spans and exhibit no gross morphological aberrations. Physiological assessment of TG TPM 3 mice reveals hyperdynamic effects on systolic and diastolic function, decreased calcium sensitivity in force generation and attenuation of length dependent calcium activation. This is the first work to demonstrate a correlation between α-TM and TPM 3 content and cardiac performance. A mutant TPM 3 (Met8Arg), linked to nemaline myopathy, was overexpressed in transgenic mouse hearts. Results show that high levels of transgene incorporation are lethal, and only one line of mice survived. In the surviving line, mutant RNA is expressed at levels comparable to the wild-type TPM 3 overexpressers, with the protein accounting for ~40% of the total striated tropomyosin. The mutant mice have normal life spans, but some mice have a minor heart phenotype. Rods, typically seen in nemaline patients, are not found in any of the animals. Physiologically, there is a mild hyperdynamic effect that is intermediate to NTG mice and the wild-type TG mice. If mutant TPM 3 levels are increased through TGxTG matings, the situation is lethal. The mice die before 14 days of age postpartum, with their hearts exhibiting a dilated phenotype and have thrombi formation. Thus, this mutation is only tolerated at low levels in murine hearts. Advisors/Committee Members: Wieczorek, David F.

Keywords: TROPOMYOSIN; TPM3; NEMALINE; MUSCLE; RODS

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17. RAMBOW-LARSEN, AMY ALISON. ASSEMBLY AND SECRETION OF PERTUSSIS TOXIN BY BORDETELLA PERTUSSIS.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2003, University of Cincinnati

► Bordetella pertussis is the causative agent of whooping cough. Pertussis toxin, one… (more)

▼ Bordetella pertussis is the causative agent of whooping cough. Pertussis toxin, one of the major virulence factors of B. pertussis , is an AB 5 toxin comprised of protein subunits S1 through S5. The individual subunits are secreted to the periplasm where the toxin is assembled. The Ptl secretion system secretes assembled toxin past the outer membrane. In this study, we examined toxin expression, assembly, and secretion. Cultures followed a typical bacterial growth cycle. A one-hour lag phase was followed by logarithmic growth until the cultures entered stationary phase around 24 hours. Secreted toxin was first observed at 3 hours. Secretion continued throughout logarithmic growth phase, decreasing as the culture entered stationary phase. Toxin secretion occurred at a constant rate of 3 molecules/minute/cell from two to eighteen hours. More toxin subunits were produced than secreted, resulting in periplasmic accumulation. Periplasmic subunits were detected in both soluble and membrane-associated cellular fractions, with about half of the periplasmic subunits incorporated into holotoxin. Secretion component PtlF was present at a low level at time zero, and increased between 2 and 24 hours, from 30 to 1000 molecules per cell. However, the initial amount of PtlF supported maximal secretion. The accumulation of both periplasmic toxin and secretion components suggests translation rates exceed the rate of secretion, and that secretion, not toxin and Ptl-complex assembly is rate limiting. Peptidoglycan acts as a barrier for transport through the periplasm of large folded molecules. Assembled pertussis toxin, and most secretion component proteins are too large to diffuse through intact peptidoglycan. Therefore, we hypothesized that the Ptl system would contain a peptidoglycanase. PtlE possessed a sequence match to the active site of glycohydrolases, suggesting this protein might cleave the sugar backbone of peptidoglycan. A polyhistidine tagged PtlE fusion protein possessed peptidoglycanase activity. Fusion proteins with alanine substitutions at one or both of the putative active site residues (D53 and E62) lacked peptidoglycanase activity. B. pertussis strains expressing PtlE alleles with the amino acid substitutions were deficient for pertussis toxin secretion. Based on these results, we conclude that PtlE is a peptidoglycanase responsible for the local removal or re-arrangement of the peptidoglycan layer during Ptl-secretion complex assembly. Advisors/Committee Members: Weiss, Dr. Alison.

Subjects: Biology, Microbiology

Keywords: pertussis; toxin; secretion; bacteria; pathogen

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18. SCHAEFFER, LYNDSAY MORGAN. THE ROLE OF PHAGOCYTIC DEFENSES AND INNATE IMMUNITY IN THE CLEARANCE OF BORDETELLA PERTUSSIS INFECTIONS.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2004, University of Cincinnati

► Bordetella pertussis is the causative agent of the human respiratory disease whooping… (more)

Subjects: Biology, Microbiology

Keywords: Bordetella, innate immunity, SP-A, SP-D, phagocytosis

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19. SCHECKELHOFF, MARK ROBERT. SPECIFIC T CELL REPERTOIRES MEDIATE PROTECTIVE IMMUNITY TO HISTOPLASMA CAPSULATUM.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2003, University of Cincinnati

► Histoplasma capsulatum (Hc) is a dimorphic fungus that causes a wide spectrum… (more)

▼ Histoplasma capsulatum (Hc) is a dimorphic fungus that causes a wide spectrum of disease. The interaction of T cells with macrophages and the subsequent production of cytokines are essential for protection. This thesis examines the importance of the T cell receptor (TCR) repertoire during the generation of immunity to infection with Hc and vaccination with a protective antigen (Ag). During primary infection, Vβ4 + T cells expand in the lungs and depletion of these cells increases the fungal burden in mice. To examine the Ag-specificity of lung Vβ4 + cells, they were isolated from infected mice and used to generate hybridomas. These cells were screened for Ag reactivity using an extract derived from the cell wall and cell membrane of Hc yeast. T cell immunoblotting with hybridomas indicate that the majority of lung-derived cells respond with an antigen at 110-130 kDA. Mass spectrometry identified this antigen as a homolog of Sec31 from Saccharomyces cerevisiae . In parallel, we examined the TCR repertoire following immunization with the protective Ag, Hsp60. A majority of T cell clones derived from Hsp60 immunized mice expressed Vβ8.1/8.2. Depletion of Vβ8.1/8.2 + cells abrogated the efficacy of Hsp60 immunization. Among these cells, those that generate IFN-γ and reacted to F3 were able to confer protection in IFN-γ -/- and TCR α/β -/- mice. The protective response to Hsp60 also relies on the presence of IL-10, IL-12, and IFN-γ. To determine if the absence of IL-10 and IFN-γ disrupt the generation of immunity to Hsp60 by altering the TCR repertoire, T cells were isolated from IL-10 and IFN-γ deficient mice following immunization with Hsp60. The IL-10 -/- -derived TCR repertoire resembled that previously observed in wildtype animals, while IFN-γ -/- repertoire had a smaller proportion of Vβ8.18/2 + cells. Together, the data presented here demonstrate that specific subsets of Ag-specific T cells mediate the protective response to Hc during both the respond to primary infection as well as the challenge subsequent to vaccination. The activity of these cells is associated with production of IFN-γ, which also influences the repertoire during initial stages of the immune response. Advisors/Committee Members: DEEPE, JR., DR. GEORGE S.

Keywords: HISTOPLASMA CAPSULATUM; T CELL RECEPTOR; IMMUNITY; FUNGUS; VACCINE

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20. Spicer, Zachary. THE ROLE OF H,K-ATPASE ISOFORMS IN GASTROINTESTINAL FUNCTION.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2001, University of Cincinnati

► The two known isoforms of the mammalian H,K-ATPase are the gastric and… (more)

▼ The two known isoforms of the mammalian H,K-ATPase are the gastric and colonic H,K-ATPases. Gene targeted mice were used to elucidate the in vivo roles of these H,K-ATPases. The gastric H,K-ATPase is expressed in the gastric parietal cell; mediates gastric acid secretion; and may play an important role in the development, maintenance and, function of the gastric mucosa. To explore these possibilities, mice carrying a targeted deletion in the gastric H,K-ATPase gene were generated. Homozygous mutant ( Atp4a -/- ) mice were born at the expected Mendelian ratio, were fertile, exhibited no overt pathology or behavioral abnormalities, and had normal systemic electrolyte and acid-base status. As expected, they were achlorhydric and hypergastrinemic. Surprisingly, chief cells and parietal cells were present in normal numbers in the Atp4a -/- stomach. However, parietal cells from these stomachs had dilated canaliculi, atypical canalicular microvilli and tubulovesicles, and subsets of these cells contained abnormal mitochondria and/or massive glycogen stores. Stomachs of adult Atp4a -/- mice were also metaplastic, with large cysts, abnormal mucus cells, and ciliated cells. These results indicate that the ablation of the gastric H,K-ATPase in the mouse stomach causes achlorhydria, hypergastrinemia, perturbations in the secretory membranes of the parietal cell, perturbations in the parietal cell energy metabolism, and metaplasia of the gastric mucosa, without affecting parietal cell viability or chief cell differentiation. Up regulation of the colonic H,K-ATPase (cHKA) during hyperaldosteronism suggests that cHKA may function in K + conservation and/or electrogenic Na + absorption in the colon when Na + -conserving mechanisms are activated. To test this hypothesis, wild-type ( cHKA +/+ ) and cHKA-deficient ( cHKA -/- ) mice were fed Na + -replete and Na + -restricted diets. Both genotypes exhibited similar responses to Na + -restriction. As expected, cHKA -/- mice had elevated fecal K + excretion on the Na + -replete diet. Dietary Na + -restriction led to increased K + excretion in cHKA -/- mice, and the amiloride-sensitive, ENaC-mediated short-circuit current in the distal colon was significantly reduced in the knockout. These results demonstrate that cHKA plays an important role in K + conservation during dietary Na + -restriction, and that cHKA-mediated K + recycling across the apical membrane is required for maximum electrogenic Na + absorption. Advisors/Committee Members: Shull, Gary E.

Subjects: Biology, Molecular

Keywords: Atp4a; parietal cell; cHKA; K-ATPase; gastric; parietal; mice

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21. Straughen, Joel E. BLM Is a Suppressor of DNA Recombination.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2002, University of Cincinnati

► Bloom’s syndrome (BS) is a rare, recessive chromosome breakage disorder characterized by… (more)

▼ Bloom’s syndrome (BS) is a rare, recessive chromosome breakage disorder characterized by small stature, sun sensitivity, facial erythema, immunodeficiency, female subfertility, male infertility, and a predisposition to a variety of cancers. When this body of work was started, the gene for Bloom’s syndrome (BLM) had yet to be identified. This work presents characterization of the genomic region at BLM and the identification of BLM. With the cloning of the gene, the answers to a number of questions could be investigated. Additional chapters present data that demonstrate that increased genomic instability and recombination are the result of loss of function of the Bloom’s syndrome gene product. Somatic cells from BS individuals are characterized by a high frequency of chromatid exchanges both between and within chromosomes, as well as by a high mutation rate at specific loci. DNAs from BS and normal clonal cell lines were first examined for alterations at microsatellite repeat loci. Alterations in size microsatellite repeats were observed at a 10-fold increase in frequency in BS clones compared to normal clones. A contiguous representation of 2-Mb region that contains the BLM gene was generated. YAC and P1 clones from the region were identified and ordered using genetic markers in the region along with newly developed sequence tagged sites from radiation-reduced hybrids, polymorphic dinucleotide repeat loci, and end-sequences of YACs and P1s. The physical map, and DNA markers derived from it, was instrumental in identifying BLM. With the gene identified, the genomic structure was determined and a rapid DNA screening test was developed for the identification of Blm Ash, the most common mutation in BS. To determine whether BLM can suppress recombination we over-expressed BLM in separate cell lines capable of identifying recombination or frameshift events. However, no significant difference was noted between cells transfected with BLM and those transfected with vector alone. Finally, we established a mouse model of BS using homologous recombination to disrupt mouse Blm. Genotyping offspring from heterozygous parents did not identify any offspring homozygous for the knockout allele, suggesting embryonic lethal phenotype. In long-term studies, heterozygosity for Blm increases tumor formation compared to wild-type littermates. Advisors/Committee Members: Groden, Dr. Joanna.

Keywords: Bloom's Syndrome; recombination; cancer

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22. Towne, Jennifer E. CYTOKINE REGULATION OF AQUAPORIN 5 MEDIATED LUNG FLUID HOMEOSTASIS.

Degree: PhD, Medicine : Molecular Genetics, Biochemistry and Microbiology, 2001, University of Cincinnati

► Aquaporins (AQPs) are water channel proteins which function to increase plasma membrane… (more)

▼ Aquaporins (AQPs) are water channel proteins which function to increase plasma membrane water permeability in secretory and absorptive cells in response to osmotic gradients. In the lung, AQP1 and AQP5 are expressed at the site of gas exchange. Water movement through the air space-capillary barrier in the distal lung is facilitated by AQP1 in the capillary endothelium and AQP5 in the alveolar epithelium. The overall goal of this thesis was to examine the regulation of distal lung AQPs. First, the regulation of AQP1 and AQP5 expression in the lung was examined under conditions of aberrant fluid handling induced through intratracheal infection of mice with adenovirus. The expression of AQP1 and AQP5 mRNA and protein were significantly decreased in the lungs after adenovirus infection. Immunohistochemical analysis demonstrated that the decreases in AQP1 and AQP5 expression were not localized to regions of overt inflammation but were found throughout the lung. Decreased AQP1 and AQP5 levels during adenoviral infection suggest a role for AQP1 and AQP5 in the abnormal fluid fluxes detected during pulmonary inflammation. The mechanisms underlying this decrease in AQP levels are therefore of considerable interest. We next examined the possible regulation of AQP5 by the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). Treatment of murine lung epithelial (MLE-12) cells in culture with TNF-α resulted in a concentration- and time-dependent decrease in AQP5 mRNA and protein expression. Activation of the p55 TNF-α receptor (TNFR1) with an agonist antibody was sufficient to cause decreased AQP5 expression, demonstrating that the TNF-α effect is mediated through TNFR1. Inhibition of nuclear factor κB (NF-κB) translocation to the nucleus blocked the effect of TNF-α on AQP5 expression, indicating activation of NF-κB is required, while inhibition of extracellular signal-regulated (ERK) or p38 MAP kinases had no effect. These data show that TNF-α decreases AQP5 mRNA and protein expression, and that the molecular pathway for this effect involves TNFR1 and activated NF-κB. The ability of inflammatory cytokines to decrease aquaporin expression may help explain the connection between inflammation and edema and contribute to the development of novel treatments for diseases such as asthma. Advisors/Committee Members: Menon, Anil G.

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