The process of respiration is a pattern of spontaneity and automatic motor control that originate in the brainstem. The mechanism by which the brainstem detects CO2 is termed central CO2 chemoreception (CCR). Since the early 1960’s there have been tremendous efforts placed on identification of central CO2 chemoreceptors (molecules that detect CO2). Even with these efforts, what a central CO2 chemoreceptor looks like remain unknown. To test the hypothesis that inward rectifier K+ (Kir) channels are CO2 sensing molecules in CCR, a series of experiments were carried out. 1) The first question asked was whether the Kir4.1-Kir5.1 channel is expressed in brainstem chemosensitive nuclei. Immunocytochemistry was performed on transverse medullary and pontine sections using antibodies raised against Kir4.1 and Kir5.1. Positive immunoassays for both Kir4.1 and Kir5.1 subunits were found in CO2 chemosensitive neurons. In the LC the Kir4.1 and Kir5.1 were co-expressed with the neurokinin-1 receptor that is the natural receptor for substance P. 2) The second question asked was whether the Kir4.1-Kir5.1 channel is subject to modulation by neurotransmitters critical for respiratory control. My studies demonstrated that indeed the Kir4.1-Kir5.1 channel is subject to modulation by substance P, serotonin and thyrotropin releasing hormone. 3) I performed studies to demonstrate the intracellular signaling system underlying the Kir4.1-Kir5.1 channel modulation by these neurotransmitters. The modulation by all three neurotransmitters was dependent upon the activation of protein kinase C (PKC). The Kir4.1-Kir5.1 but not the Kir4.1 channel was modulated by PKC. Both the Kir4.1 and Kir5.1 subunits can be phosphorylated by PKC in vitro. However, systematic mutational analysis failed to reveal the phosphorylation site. 4) The fourth question asked was whether Kir channels share a common pH gating mechanism that can be identified. Experiments were performed to understand the gating of the Kir6.2+SUR1 channel as specific sites for ligand binding and gating have been demonstrated. I identified a functional gate that was shared by multiple ligands that is Phe168 in the Kir6.2. Other Kir channels appear to share a similar gating mechanism. Taken together, these studies demonstrate the modulation of Kir channels in central CO2 chemoreception.

Metal toxicity is implicated in neurotoxicity, nephrotoxicity, aging and cancer. Protein oxidation resulting from oxidative stress is now known to be involved in metal toxicity. However, proteomic responses to metal induced oxidative stress have not been characterized. By using the yeast as a model, we characterized these changes occurring in response to sub-lethal doses of metals. Several proteins involved in protein synthesis, ribosome assembly decreased while antioxidant defenses, proteins involved in sulfur metabolism, and glutathione synthesis and ubiquitin increased following metal exposure. We also show that metals induced temporal and targeted protein oxidation independent of protein abundance. Among the targets were glycolytic enzymes and heat-shock proteins. As a consequence, glycolytic enzyme activities decreased whereas the levels and activities of the enzymes of the alternative pathway for glucose metabolism, pentose phosphate pathway (PPP) increased. True to prediction, we also found increased flow through the PPP as measured by elevated levels of NADPH and glutathione. NADPH and glutathione are crucial for maintaining the redox balance in the cell. Thus, rerouting of glucose metabolism into PPP is considered to be beneficial to the organism. Among the oxidation targets is a glycolytic protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that is required for apoptosis in neuronal cells. We show that not only is GAPDH required for metal induced apoptosis in yeast but also the levels of GAPDH transcript and protein increase in the cytosol and the nucleus in an isoform specific fashion. Such changes strongly implicate the role of GAPDH in yeast apoptosis. This work provides evidence for the involvement of targeted protein oxidation in metal toxicity, shows the overlaps and differences in the mechanism of copper and cadmium toxicity, allows comprehension of how metabolic processes respond to metal stress and explores the potential of GAPDH as a sensor of oxidative stress and mediator for apoptosis.

Antibodies neutralize and eliminate pathogens, malignancies, and toxins by acting either alone or in association with Fc receptors which, once engaged, activate the elimination mechanisms of phagocytic cells. Based on structural differences, antibodies are divided into functionally distinct classes (IgM, IgD, IgG, IgE and IgA). Structure-function relationships within these classes are not well characterized. In addition, animal models for the assessment of potential therapeutic strategies for the modulation of the interaction between antibodies and Fc receptors are not established. Nonhuman primates are widely used to model human diseases and, represent excellent in vivo systems for this assessment. Therefore, we have studied nonhuman primate IgD as well as IgG and IgA specific Fc receptors in rhesus macaques, cynomolgus macaques, baboons and sooty mangabeys. IgD genes had not been identified in nonhuman primates nor the IgD receptors characterized in any species. We characterized IgD genes of the four monkey species, as well as chimpanzees and dogs. In contrast to other antibody classes, the IgD hinge regions are highly conserved between human and nonhuman primates, thus indicating a role in Fc receptor binding. In humans, Fc receptors CD16a (natural killer cells) and CD16b (neutrophils) bind IgG1 and IgG3, and CD89 (myeloid cells) binds IgA. To assess ligand binding and glycosylation properties of nonhuman primate CD16a, CD16b, and CD89, we sequenced, cloned, and generated recombinant molecules in a mammalian expression system. Our results verify the presence of CD16a, but not CD16b in nonhuman primates. CD16a is expressed on monocytes and a subpopulation of lymphocytes. In sooty mangabeys, CD16 is also expressed on neutrophils. Recombinant sooty mangabey/baboon CD16a binds to human IgG1 and IgG2, but not IgG3 and IgG4. Monkey CD89 has the same peripheral blood leukocyte expression profiles as humans, and binds human and recombinant macaque IgA. Blocking of N-glycans inhibited expression of CD89, but only marginally CD16a expression. Although extensive similarities of antibody/Fc receptor interactions exist between human and nonhuman primates, several differences must be considered when evaluating therapeutic strategies. However, these differences can be exploited to further characterize the structure-function relationships existing within antibody molecules and respective receptors.

Activities of the DEAD box (Asp-Glu-Ala-Asp) family of proteins- including RNA-dependent ATPase and RNA helicase- function in all organisms to sculpt RNA-RNA duplex and RNA-protein complexes, ensuring that necessary rearrangements are rapidly and properly resolved during genetic information processing. Identified as a prototypic member of the DEAD box family and documented as an ATPase and RNA helicase, p68 plays essential and diverse functions in the control of gene expression ranging from pre-mRNA/rRNA processing and mRNA decay/stability to transcriptional activation and initiation. Despite the early implied roles in organ maturation and tumor progression, the functional contributions of p68 to growth/differentiation regulation and cancer development remain undefined. Here, we show c-Abl-dependent phosphorylation of p68 markedly associates with abnormal cell growth and cancer development. Importantly, we characterize an unanticipated signaling module through which p68 functionally contributes to Epithelial-Mesenchymal Transition (EMT) and cell proliferation. p68, which appears to be phosphorylated by c-Abl at tyrosine 593, consequently promotes an EMT through its ability to recruit â-catenin into cell nucleus via a canonic Wnt/â-catenin axis independent way; accordingly, phosphor-p68 (phosphorylated at tyrosine 593 residue) also stimulates tumor cell growth, which requires the ATPase activity of the protein. These findings define a potential mechanism whereby phosphor-p68 recruits â-catenin into cell nucleus in ATP hydrolysis driven fashion and cooperatively regulates transcriptional programs that control an EMT. The dissertation thus demonstrates a tight coordination between DEAD box RNA helicase and cancer development.

Constantly changing environments force animals to adapt by cycling through multiple physiological states. Plasticity in sensory, motor, and modulatory neural circuits is an essential part of these adaptive processes. Invertebrates with their accessible, identifiable neurons are excellent models for investigating the molecular and cellular mechanisms underlying state-dependent neural plasticity, and provide insight into similar processes in more complex systems. These properties have allowed highly detailed characterization of several crustacean circuits with respect to their connectivities, cellular properties, responses to various inputs, and outputs. Serotonin (5-HT) is an important neuromodulator in virtually every animal species. 5-HT signals are mediated primarily by a large family of metabotropic receptors on target cells that activate diverse intracellular signaling cascades. Although 5-HT’s effects on crustacean circuits have been studied in detail, the mediating receptors have been inaccessible until recently. Crustacean receptors had not been cloned and specific drugs for use in physiological experiments could therefore not be identified. Coupling properties of 5-HT receptor families are strongly conserved between phyla, but pharmacological profiles are not. The extent of pharmacological divergence among invertebrates is unclear, however, as no systematic functional profile of 5-HT receptors from related species has been determined. This work shows that orthologs of two 5-HT receptors, 5-HT2b and 5-HT1a, are highly conserved at the molecular, functional and pharmacological level between two distantly related decapod crustaceans, Panulirus interruptus and Procambarus clarkii. A suite of drugs was functionally characterized at Panulirus and Procambarus 5-HT2b and 5-HT1a receptors in cell culture, which were then used to investigate the roles of the receptors in pyloric cycle frequency modulation in the stomatogastric ganglion, a model central pattern generator. The two receptor subtypes were found to serve different roles in the circuit and their function depends on the initial state of the circuit. Finally, an antibody recognizing 5-HT1a was used to map the localization of this receptor within the crayfish nervous system. 5-HT1a is localized to somata and neuropil throughout the nerve cord, suggesting it may respond to synaptic, paracrine or neurohormonal 5-HT signals. The protein and mRNA expression levels are variable between individual animals, perhaps reflecting distinct physiological states.

Male rat mating is a suite of individual behaviors mediated by the actions of two metabolites of testosterone (T), dihydrotestosterone (DHT) and estradiol (E2), on the brain. Individually, neither metabolite fully maintains or restores mating in castrated males, but both combined are as effective as T. Two hormone-responsive areas of the brain, the medial preoptic area (MPO) and the medial amygdala (MEA), are crucial for mating. These studies ask: by what mechanism(s) does E2 act in the MPO and MEA? We blocked the conversion of T to E2 in the MEA of intact male rats and sexual behavior was not maintained. We then infused antisense oligodeoxynucleotides (ODNs) to estrogen receptor-alpha (ER-á) mRNA bilaterally to the MPO or the MEA of intact male rats to block ER-á expression. ODN infusion of the MPO attenuated mating but infusion of the MEA had no effect. These results suggest that ER-á is the behaviorally relevant estrogen receptor (ER) in the MPO but not in the MEA. ER was originally described in the cytoplasm and nucleus of cells. Recently plasma membrane associated ERs (mER) have been reported. We conjugated E2 to Bovine Serum Albumin (BSA-E2), a large protein that will not penetrate the plasma membrane, thus restricting the action of E2 to mER, and chronically delivered it to the MPO and MEA. BSA-E2 maintained mating if put in the MPO, but not in the MEA, suggesting a surface action of E2 is sufficient in the MPO. The MPO and MEA are reciprocally connected and probably constitute elements of a larger, steroid-responsive neural network that mediates male mating behavior. To begin to describe this purported circuit, we injected Pseudorabies virus (PRV) into the prostate gland and dually labeled PRV-immunoreactive cells for ER or androgen receptors. We found dual labeling in a forebrain diencephalic circuit that includes the MPO, the medial preoptic nucleus, bed nucleus of stria terminalis, the zona incerta, the periaqueductal gray and other areas that presumably mediate both autonomic and motor aspects of male mating. Together, the results of these studies begin to elucidate locations and mechanisms of E2 mediation of male sexual behavior.

Arginine utilization in P. aeruginosa as the source of carbon, nitrogen, and energy is controlled by a complicated regulatory mechanism. While ArgR in the presence of exogenous arginine is required for auto-induction of the aotJQMOP-argR operon for arginine uptake and regulation, this operon is subjected to carbon catabolite repression via an unknown mechanism. This study demonstrated that succinate exerted a stronger repression effect than glucose or pyruvate on arginine induction of an aotJ::lacZ fusion in wild type PAO1. Expression of the aotJ::lacZ fusion was analyzed against three different backgrounds, cbrAB, crc, and vfr, that have been suggested to play a role in carbon catabolite repression. These mutations exerted a negative effect on the arginine-responsive induction to different extents, with the order of cbrAB > vfr > crc. A series of aotJ::lacZ fusions with deletions in the aotJ regulatory region were constructed and the effect of exogenous arginine examined in the argR mutant and its parent strain. The results indicated that a 250-bp sequence upstream of the ArgR operator is required for the optimal induction of the operon by exogenous arginine, and revealed the presence of a cryptic promoter (P0) in this region. Electromobility shift assays with crude cell-free extracts of PAO1 revealed that a DNA-binding protein other than ArgR binds to the aforementioned 250-bp region. Through reverse genetics, two regulatory proteins MvaT and MvaU were identified and specifically interacted with the aotJ-argR regulatory region. The MvaT/MvaU are involved in the regulation of the P0 promoter. The importance of MvaT and MvaU for bacterial growth was supported by the notion that no true mvaT mvaU double knockout mutant can be constructed. This is the first case to characterize the growth phenotypes of quasi-mvaT mvaU double mutants complemented with fusions for arginine-inducible expression of mvaT or mvaU. Further analysis of the physiological significance of MvaT and MvaU revealed their involvement in swarming response and pyocyanin synthesis. The defect in pyocyanin synthesis was correlated to the diminished level of PQS, an important chemical signal compound in the quorum sensing network of P. aeruginosa.

HIV-1 protease (PR) inhibitors (PIs) are important anti-HIV drugs for the treatment of AIDS and have shown great success in reducing mortality and prolonging the life of HIV-infected individuals. However, the rapid development of drug resistance is one of the major factors causing the reduced effectiveness of PIs. Consequently, various drug resistant mutants of HIV-1 PR have been extensively studied to gain insight into the mechanisms of drug resistance. In this study, the crystal structures, dimer stabilities, and kinetics data have been analyzed for wild type PR and over 10 resistant mutants including PRL24I, PRI32V, PRM46L, PRG48V, PRI50V, PRF53L, PRI54V, PRI54M, PRG73S and PRL90M. These mutations lie in varied structural regions of PR: adjacent to the active site, in the inhibitor binding site, the flap or at protein surface. The enzymatic activity and inhibition were altered in mutant PR to various degrees. Crystal structures of the mutants complexed with a substrate analog inhibitor or drugs indinavir, saquinavir and darunavir were determined at resolutions of 0.84 – 1.50 Å. Each mutant revealed distinct structural changes, which are usually located at the mutated residue, the flap and inhibitor binding sites. Moreover, darunavir was shown to bind to PR at a new site on the flap surface in PRI32V and PRM46L. The existence of this additional inhibitor binding site may explain the high effectiveness of darunavir on drug resistant mutants. Moreover, the unliganded structure PRF53L had a wider separation at the tips of the flaps than in unliganded wild type PR. The absence of flap interactions in PRF53L suggests a novel mechanism for drug resistance. Therefore, this study enhanced our understanding of the role of individual residues in the development of drug resistance and the structural basis of drug resistance mechanisms. Atomic resolution crystal structures are valuable for the design of more potent protease inhibitors to overcome the drug resistance problem.

B virus (Cercopithecine herpesvirus 1), which causes an often fatal zoonotic infection in humans, shares extensive homology with human herpes simplex virus type 1 (HSV-1). The ƒ×134.5 gene of HSV-1 plays a major role in counteracting dsRNA-dependent protein kinase (PKR) activity. HSV-1 Us11 protein, if expressed early as a result of mutation, binds to PKR and prevents PKR activation. The results of experiments in this dissertation revealed that although B virus lacks a ƒ×134.5 gene homolog, it is able to inhibit PKR activation, and subsequently, eIF2ƒÑ phosphorylation. The initial hypothesis was that B virus Us11 protein substitutes for the function of ƒ×134.5 gene homolog by blocking cellular PKR activation. Using western blot analysis, Us11 protein (20 kDa) of B virus was observed early following infection (3 h post infection). Expression of B virus Us11 protein was not blocked by phosphonoacetic acid (PAA), an inhibitor of DNA replication, confirming Us11 is not a ¡§true late¡¨ gene of B virus as it is in HSV-1. Analysis of these results suggested that B virus Us11 protein compensates for the lack of the ƒ×134.5 gene homolog and prevents PKR activation. Next, the results demonstrated that B virus Us11 recombinant protein prevented PKR activation by dsRNA in vitro. A B virus Us11 protein stable expression cell line (U373-BVUs11) was established to investigate whether Us11 protein inhibited PKR activation in vivo. Experiments revealed that B virus Us11 protein stably expressed in U373 cells prevented PKR activation and subsequent eIF2ƒÑ phosphorylation induced by the infection of these cells with ƒ´ƒ×134.5 of HSV-1. As the consequence of preventing PKR activation and subsequent eIF2ƒÑ phosphorylation, B virus Us11 protein complemented ƒ´ƒ×134.5 HSV-1 in U373 cells as evidenced by restoration of virus protein synthesis and replication in U373 cells. Furthermore, pull-down assays showed that B virus Us11 protein binds to PKR. In addition, the results demonstrated that B virus Us11 protein stably expressed in U373 cells counteracted the inhibiting effect of IFN-ƒÑ on HSV-1 replication by preventing PKR activation. These data suggested that B virus and HSV-1, two closely related viruses, use different mechanisms to counteract PKR activity.

Studies Directed to the Optimization of Fermentation of Rhodococcus sp. DAP 96253 and Rhodococcus rhodochrous DAP 96622 by GENE KIRK DRAGO Under the Direction of George E. Pierce ABSTRACT Bench- and pilot plant scale fed-batch fermentations were performed in stirred-tank bioreactors (STBR) with Rhodococcus sp. DAP 96253 and R. rhodochrous DAP 96622 in an attempt to elucidate parameters that may affect the optimization of a fermentation process for high biomass production and high inducible expression of cobalt-high-molecular-mass nitrile hydratase (Co-H-NHase. The effects of these factors on amidase (AMDase) activity were also investigated. Biomass and NHase production were inhibited by a total addition of acetonitrile and acrylonitrile (AC / AN) at 500 ppm during a 48 h run. Biomass and enzyme activity were uncoupled when the inoculum mass was increased from 4 g (wet weight) to ¡Ý 19 g. Other factors that allowed for the uncoupling of biomass production from enzyme activity were the reduction of the AC / AN feed rate from a step-addition at 2500 ¦Ìl / min to a continuous addition at 80 ¨C 120 ¦Ìl / min, and the delay to 18 h post-inoculation the time of initial inducer addition. The inhibition of both biomass production and NHase activity was relieved when both the total concentration of AC / AN was reduced to ¡Ü 350 ppm and the AC / AN feedrate was reduced. The factors with the greatest influence were shown to be the inducer, the inducer concentration, inoculum mass and source as well as the major carbohydrate and nitrogen source. In addition, this lab is the first to report high AN-specific NHase induction by asparagine (1300 ppm) in a fed-batch fermentation system. Prior to this program, 250 mg of cells (wet weight) per liter could be provided in 4 ¨C 10 days with an activity of 1 U NHase per mg of cells (dry weight). Current production is > 50 g / L in 48 h with an NHase activity > 150 U / mg of dry cell weight. INDEX WORDS: Amidase, Asparagine, Biodetoxification, Fermentation, Nitrile, Nitrile Hydratase, Rhodococcus