β-arrestin2, previously recognized as a facilitator for G-protein associated 7 TMR desensitization/ internalization, has now been appreciated as an independent signal transducer that regulates multiple cellular responses including inflammation. Cecal ligation and puncture procedure (CLP) induced septic shock is an acute inflammatory response characterized by uncontrolled systemic inflammation. Myocardial ischemia/reperfusion is a chronic sterilize inflammation that requires the reaction of macrophages, fibroblasts and cardiac stem cells for regeneration and remodeling of the infarcted myocardium. Restrained chronic stress is an immune suppression model in which the inactivation of macrophages may be involved. Here we showed β-arrestin2 overexpression inhibited CLP-induced heart dysfunction in septic shock, stabilized the cardiovascular system, and eventually promoted survival. Inhibition of the activation of p38 that downstream of the IL-6 pathway may be a key regulatory target for β-arrestin2. To rescue cardiomyocytes from ischemia and reperfusion injury, Sca-1+ CSC from Wide-type or β-arrestin2 Knockout mice were delivered to the risked area before reperfusion; β-arrestin2 was shown to be a required factor and a promoter for the differentiation of the cardiac stem cells. A β-arrestin2/miR-155/GSK3β pathway was identified in this study. TLR-9 is an important part of the innate immune system which has been shown to be regulated by β-arrestin2 in various inflammatory models. Here we found, the immune suppression induced by restrained stress is mediated by Toll-like receptor 9 (TLR-9). TLR-9 facilitated the elevation of IL-1β, IL-10 and IL-17 levels in serum and decrease of the levels of plasma IFN-γ. Furthermore, macrophage apoptosis was alleviated in TLR-9 deficiency mice. In summary, β-arrestin2 and associated proteins like TLR-9 are important regulators of the immune response in a variety of disease conditions. Therapeutic strategies should be generated to balance the inflammation and anti-inflammation response by modulating β-arrestin2 expression and functions.

The 1839 bp cDNA for rat pulmonary carboxylesterase was cloned by reverse transcription polymerase chain reaction (RT PCR) from total rat lung RNA using specific primers derived from the 5' and 3' untranslated regions of rat hepatic cholesteryl ester hydrolase (CEH). The unique cDNA was sequenced and found to have 99% homology with hepatic CEH. This homology extends to the predicted amino acid sequences which show only six amino acid residue differences in the coding region: three conserved and three nonconserved changes. However, the catalytic activitites of the two proteins are dramatically different. While CEH hydrolyzes cholesterol oleate, the pulmonary carboxylesterase has no activity towards this substrate. The active recombinant lung carboxylesterase was purified using a baculovirus expression system. The substrate specifities were determined using p-nitrophenyl acetate, p-nitrophenyl caprylate and cholesterol oleate. Also, the KM, Vmax and pH optima were determined for each substrate. Comparison of the substrate specificities of the recombinant pulmonary carboxylesterase with the recombinant CEH further establish the critical role of the six amino acid residues in determining the differences in the catalytic activities of these two proteins. Cumulative mutations were made in the lung carboxylesterase sequence to those of the hepatic CEH sequence, in order to determine the role(s) of these six amino acid residues in conferring cholesterol oleate hydrolytic activity. These studies showed that GIn186 is vital for activity towards hydrophilic substrates, while the region around amino acid residue 500 consisting of Ser491, Lys492, Asn506 and Asn504 may be responsible for the absence of catalytic activity towards hydrophobic substrates.

No description available.

Magnetic, gravity and bathymetry data were collected on an extended cruise of the R/V Yaquina in 1969. The last set of data was obtained from those track lines leaving the Panama Basin. The area covered is mainly the Cocos plate (Molnar and Sykes, 1969). The data is analyzed and compared with results of previous workers and the geophysical implications considered. Generally speaking, from the magnetic part of the data, both direct and indirect methods show support of Vine and Matthew's (1963) hypothesis of sea-floor spreading and the subsequent principles of new global tectonics. The most northern magnetic anomaly profile across the East Pacific rise (at 18.3°N) shows a spreading rate about 3 cm/yr. and the most southern one (at 12.8°N) shows a rate about 5.2 cm/yr. The Cocos plate has been assumed to move in a northeast-southwest direction (N30°E to N45°E), and rotate with respect to the Pacific plate about a pole at 40°N, 110°W with an angular velocity of 19.6x10⁻⁷ deg./yr. (Larson and Chase, 1970). New material comes up from the west boundary - the East Pacific rise, and the south boundary - the Galapogos rift, causing the Cocos plate to underthrust the Americans plate at the middle American arc. Some of the points of new global tectonics can not be detected from this set of data; further detailed study of more track lines and sea bottom core samples are needed. The results of both analytical methods for determining the magnitude of induced and remanent magnetization in the second layer shows some consistence with the work of Schaeffer and Schwarz (1970), and Irving et al. (1970) at the Mid-Atlantic ridge near 45°N, in which a thinner magnetization layer at the ridge and the attenuation of magnetization intensity away from the ridge axis are suggested. Free-air gravity anomaly profiles have been employed to determine the crustal structure of two sections1 a ridge section at 12.8°N and a trench section at 14°N. For the ridge section, if the anomalous mantle was converted from normal mantle, the extension of anomalous mantle into the normal mantle requires some uplift and/or lateral expansion in the rise crest area. The tensional configuration suggested in the trench crustal section agrees with the model proposed by Elsasser (1968) for the differential movement between two lithospheric blocks. This work gives some speculations that evidence which supports the present new global tectonics theory is limited to a certain degree of accuracy. Further study of the theory based upon physics, its mechanism, and measurement techniques that would give more reliable evidence have to be developed before it can be ascertained what really happens beneath this wild, wild world. / Graduation date: 1971

Thesis (Ph.D.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (April 26, 2007) Vita. Includes bibliographical references.

Cytochrome P450 monooxygenases or CYPs comprise a large family of enzymes that are found in all classes of living organisms, from bacteria to man. These enzymes are involved in the metabolism of many endogenous and xenobiotic (foreign) compounds. In insects, CYPs confer resistance to various insecticides, and resistance-associated overexpression of multiple CYP genes in resistant insects is a common phenomenon. In Drosophila, multiple Cyp genes including Cyp6a2 and Cyp6a8 show higher level of expression in resistant strains than in the susceptible ones. To date, molecular basis of CYP gene overexpression has not been examined in detail. Barbiturate compounds such as phenobarbital and barbital induce both these genes. An unpublished observation from our laboratory showed that Cyp6a2 as well as Cyp6a8 are induced by over-the-counter caffeine tablet, Vivarin. In the present study, I used pure caffeine as a tool to better understand the mechanism of Cyp6a2 and Cyp6a8 gene regulation in Drosophila. The specific objectives of this project has been to (1) map the upstream DNA of both genes for the sequences responsible for caffeine-induction; (2) examine whether adenosine receptors and/or cAMP-specific phosphodiesterase (cPDE) are involved in transduction of caffeine signal to induce the two Cyp6 genes; and (3) investigate whether Drosophila AP-l transcription factors (D-JUN and D-FOS) playa role in caffeine induction of Cyp6a8 gene. For these objectives, several Cyp-luc reporter plasmids carrying firefly lueiferase (luc) reporter gene under the control of different regions of Cyp6a8 and Cyp6a2 upstream DNAs were constructed to transfect Drosophila embryonic cells, Schneider line 2 (SL-2). Two transgenic reporter strains carrying firefly lueiferase (luc) reporter gene under the control of O.8-kb or 0.2-kb upstream DNA of Cyp6a8 gene were also used to study the mechanism of caffeine induction in vivo. Results of Northern blot analysis showed that caffeine induces endogenous Cyp6a2 and Cyp6a8 genes at the steady-state mRNA levels both in reporter transgenic and wild-type flies. Transfection experiments with SL-2 cells showed that -983/-1 and 7611-11 upstream DNAs of Cyp6a2 and Cyp6a8, respectively, have sequences for caffeine-induced expression. Further transfection experiments with reporter plasmids carrying luc reporter gene attached to truncated upstream DNAs of Cyp6a2 and Cyp6a8 genes showed that the regions between -265/-129 of Cyp6a2 and -199/-109 of Cyp6a8 have sequences that confer caffeine-induced expression. However, the level of both constitutive and induced expression was highest with -981/-1 DNA of Cyp6a2and -761111 DNA of Cyp6a8genes. Sequence analysis identified several putative binding sites for Activator Protein -1 (AP-l) and cyclic AMP response element CRE binding protein. (CREB) motifs in the upstream DNA of both genes. Moreover, when the four core bases of the single AP-l site present in the -109/-11 DNA of Cyp6a8 were mutated, constitutive expression decreased by 8-fold, suggesting the positive role of AP-l in Cyp6a8 gene expression. To examine whether caffeine signaling is mediated via adenosine receptor (AdoR) and/or via cPDE inhibition, SL-2 cells transfected with Cyp6a2 and Cyp6a8 reporter constructs were treated with AdoR agonists or with antagonists or with cPDE inhibitors. The Cyp6a8-luc reporter transgenic lines were also treated with these chemicals. The results showed that caffeine signaling is mediated by PDE inhibition and via increase in the intracellular cAMP level. Indeed, treatment with dibutyryl cAMP induces Cyp6a2 as well as Cyp6a8 promoters. Since induction of cAMP pathway is known to upregulate AP-I transcription factors, effect of overexpression of Drosophila D-FOS and (or) DJUN (components of AP-I) on Cyp6a8 promoter activity in SL-2 cells was examined. Surprisingly, activity of Cyp6a8-1uc reporter construct was inhibited when D-FOS or DJUN proteins were overexpressed, suggesting that AP-I proteins are inhibitory for Cyp6 gene expression. In contrast, the Cyp6a8 promoter activity was upregulated, when cells were transfected with anti-D-JUN plasmid or when cells transfected with D-JUN or DFOS sense construct were treated with caffeine. When relation between caffeine and the two AP-I proteins was examined, it was found that caffeine treatment significantly lowers the D-JUN protein level both in SL-2 cells as well as in adult flies. Reporter gene assays and Northern blot analysis showed that caffeine treatment has no effect on the transcriptional activity of the D-jun and D-fos genes. Taken together, it might be concluded that caffeine induction of Cyp6a2 and Cyp6a8 genes is mediated via degradation of D-JUN that acts as a repressor for the promoter of Cyp6a8. Induction of the cAMP pathway and subsequent phosphorylation of the AP-l proteins may relieve the AP-l mediated-repression by promoting the degradation of these proteins. Further investigation is required to resolve these possibilities.

Sufficient pools of deoxyribonucleotide triphophates (dNTPs) are essential for the high fidelity replication and repair of DNA, the hereditary material for a majority of living organisms. Ribonucleotide reductase (Rnr) catalyzes the rate-limiting step of de-novo DNA synthesis, the reduction of ribonucleosides to deoxyribonucleosides. Since the cell relies primarily upon ribonucleotide reductase for its dNTPs, both the cellular levels and activity of Rnr are heavily regulated, especially when DNA damage occurs or during replication blocks in the cell cycle. If dNTP pools become too high, too low, or imbalanced, genomic instability results, leading to either the formation of cancerous cells or cell death. High levels of dNTPs are required by actively propagating cells for the replication of new DNA molecules. Therefore, Rnr makes an excellent target for anti-cancer, anti-microbial, and anti-fungal chemotherapeutic agents. Deficiencies in the cellular mismatch repair (MMR) machinery have been linked to genetic instability and carcinogenesis. Two alleles of Rnr1 were recently discovered, Rnr1S269P and Rnr1S610F, which have a mismatch repair synthetic lethal (msl) phenotype in Saccharomyces cerevisiae cells with missing or defective MMR genes. To uncover the molecular mechanism of the msl phenotype in these two mutants, recombinant Rnr1p-S269P and Rnr1p-S610F were subjected to in vitro activity assays, X-ray crystallography, and in vitro nucleoside-binding assays (Chapter 3). The Rnr1S269P allele was shown to dysregulate specificity cross talk by X-ray crystallography experiments, leading to reduced levels of dATP in the cell. A 2-fold reduction in binding of ADP substrate was observed in the Rnr1S610F allele, however reduction of the kcat is believed to cause the observed msl phenotype in this mutant. The first X-ray crystal structures of the large subunit of ribonucleotide reductase from Homo sapiens (hRRM1) are also presented here (Chapter 4). The hRRM1●TTP and hRRM1●TTP●GDP structures describe the binding of effector and substrate to the specificity and catalytic sites. In addition, the two structures hRRM1●TTP●ATP and hRRM1●TTP●dATP are the first X-ray crystal structures of Rnr from any species with the allosteric activity site occupied with the natural ligands ATP and dATP. Size exclusion chromatography data and a low resolution X-ray crystal structure of hexameric S. cerevisiae Rnr provide a model for dATP-dependent oligomerization.

Sister chromatid cohesion is essential for proper chromosome segregation during meiosis. However, the mechanism of meiotic cohesion in Drosophila is unclear. We describe a novel protein, SOLO (Sisters On the LOose) that is essential for meiotic cohesion in Drosophila melanogaster. solo mutations cause high nondisjunction of sister and homologous chromatids of sex chromosomes and autosomes in both sexes. In solo males, sister chromatids separate prematurely and segregate randomly during meiosis II. Although bivalents appear intact throughout meiosis I, sister centromeres lose cohesion prior to prometaphase I and orient nearly randomly on the meiosis I spindle. Centromeric foci of SMC1 are absent in solo males at all meiotic stages. SOLO and the cohesin protein SMC1 co-localize to meiotic centromeres from early prophase I until anaphase II in wild-type males but both proteins are removed prematurely from centromeres at anaphase I in mei-S332 mutants, coincident with premature loss of cohesion in those mutants. solo mutations in females cause reduced frequency of homologous recombination between X chromosomes and autosomes, partially due to the loss of inhibition of sister chromatid exchange. Synaptonemal complex assembly is severely disrupted in early meiotic stage in solo females. SOLO colocalizes with SMC1 and C(3)G in meiosis. Additionally, SOLO is required for stabilizing chiasmata generated from residual recombination events. The data about the phenotypes of solo males and females and colocalization patterns of SOLO strongly suggest SOLO is a component of potential cohesin in Drosophila meiosis. Drosophila males undergo meiosis without recombination. However, the underlying mechanism is not known. Mutations of vasa cause high frequency of X-Y exchange in meiosis. Chromatin bridges at anaphase I and II, due to dicentric recombination events, were observed in vasa males. vas and solo double mutant showed precocious segregation of homologs at metaphase I besides chromatin bridge at anaphase I and II. Our data thus for the first time demonstrate that inhibition of meiotic recombination during male meiosis requires vas function and interactions between vas and solo regulate chromosome dynamics in male meiosis.

R67 dihydrofolate reductase (DHFR) catalyzes the transfer of a hydride ion from NADPH to dihydrofolate (DHF) to produce tetrahydrofolate (THF). The enzyme is a homotetramer and its 222 symmetry allows for binding of both ligands to a single active site pore. A productive ternary complex is formed by the binding of one molecule of DHF and NADPH and inter-ligand cooperativity has been suggested to be essential for binding and catalysis. To gain further insight into the thermodynamics involved in the ground state and the transition state, temperature dependent studies on DHF binding and catalysis were performed. It was observed that binding of both NADPH and DHF is enthalpy driven. From van’t Hoff plots, the change in enthalpy, entropy and free energy for NADPH binding to R67 DHFR in the ground state were determined. Similarly, the thermodynamics of DHF binding to the R67 DHFR-NADPH complex in the ground state were determined. Arrhenius plots were also employed to study the energetics of the transition state. A comparison of TdeltaS values (for DHF binding to R67 DHFR-NADPH complex) in both ground state and transition state indicates that TdeltaS is more negative in the transition state (–11.3 kcal/mol) as compared to the ground state (–5.4 kcal/mol). This indicates a reorientation of the substrate in the transition state. The role of water in DHF and NADPH binding to R67 DHFR was also investigated. For this, the effect of osmotic pressure on the Ka /Km of ligand binding, as well as the kcat of the reaction was studied. It was observed that the kcat of the reaction was not significantly affected, while the binding of ligands was affected with increasing osmolality. Specifically, binding of NADPH tightened as osmolality increased, while binding of DHF weakened with increasing osmolality, suggesting release of water upon NADPH binding and an uptake of water on DHF binding. Results from in vivo experiments on E.coli cells containing wild type and mutant clones of R67 DHFR were also consistent with in vitro experiments, suggesting that water is involved in ligand binding to R67 DHFR.

The dynamic nature of proteins in solution is often an indispensable factor in biological function such as enzymatic catalysis. Complementary to the conventional structural analysis, computational simulations have the advantage to reflect the dynamic nature of proteins or enzymes. One of the computational simulation methods, the quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations, has been widely applied to the research in structural analysis, ligand-receptor binding and enzymatic catalysis. In this dissertation, QM/MM MD simulations were applied to the studies on cytidine deaminase (CDA), yeast cytosine deaminase (yCD), and kumamolisin-As, as well as two protein lysine methyltransferases (PKMTs), DIM-5 and SET7/9. In the simulations of the transition state analogue (TSA) binding of zebularine 3, 4-hydrate to CDA and of 4-[R]-hydroxyl-3,4-dihydropyrimidine (DHP) to yCD, proton transfers were observed between the TSA and a catalytic Glu residue in both cases. Such general acidbase mechanism was also observed in the stabilization of the tetrahedral intermediate by a critical Asp residue during the acylation of kumamolisin-As. Moreover, dynamic substrate-assisted catalysis (DSAC) involving the His of the substrate at P1 site was proposed. It was suggested that DSAC may contribute to the transition state stabilizations and substrate specificity of kumamolisin-As. The origin of the product specificities of PKMTs was studied by comparison of QM/MM MD simulations on the first, second and third methyl transfers in the trimethylase DIM5 and the monomethylase SET7/9. The product specificities of the enzymes can be well explained by population distributions of well-aligned reactive structures and the relative free energy barriers for the methyl transfers. The structural and energetic reasons for the product specificities were discussed and a triplet code based on the relative free energy barriers for the three methyl transfers was proposed in the determination of product specificities of PKMTs.