by Kwok-cheong Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1983. / Bibliography: leaves 194-221.

Murine small intestinal CD103+ dendritic cells (DCs) produce retinoic acid (RA) through retinaldehyde dehydrogenase (RALDH) activity, thereby inducing ‘gut-homing’ α4β7 and CCR9 on T cells they activate, enhancing TGF-β-mediated induction of Foxp3+ regulatory T cells and suppressing induction of pro-inflammatory TH17 cells. RALDH activity of CD103+ DCs is reduced in mouse models of inflammatory bowel disease (IBD) but the role of RALDH activity in human intestinal DCs in the pathogenesis of IBD is undefined. This project aimed to determine the influence of inflammation on RALDH activity of antigen presenting cell (APC) subsets including CD103+ DCs within human distal intestinal mucosa. RALDH activity was identified by Aldefluor assay in intestinal DCs (CD103+ and CD103- subsets) alongside ALDH1A2 expression in healthy controls. In contrast with mouse models, RALDH activity was not reduced in CD103+ DCs from IBD patients. An increased frequency of CD14+ macrophages (MФ) of IBD patients displayed ALDH1A1-associated RALDH activity compared with healthy controls. Blood CD14+ monocytes, putative precursors of intestinal CD14+ MФ, of healthy controls and IBD patients displayed ALDH1A1-associated RALDH activity indicating RALDH is systemically acquired by monocytes and upregulated within the mucosa of IBD patients, or alternatively that RALDH+ monocytes are selectively recruited in IBD. In vitro, inhibition of RA receptor-α signalling blocked GM-CSF-mediated differentiation of TNFα-producing pro-inflammatory RALDH+ CD14+ MФ from monocytes, consistent with enhanced RALDH activity of intestinal CD14+ MФ in IBD supporting a pro-inflammatory phenotype. Soluble intestinal mediators including prostaglandin E2 suppressed RALDH activity of MoDCs in vitro, whilst mediators from inflamed IBD mucosa conditioned MoDCs to imprint enhanced levels of α4β7 expression on naive CD4+ T cells independent of RALDH activity. This study provides the first systematic analysis of RALDH activity in human intestinal APCs and indicates important distinctions between mouse models and human IBD.

Thesis (Ph.D.)--Boston University / Ehrlich ascites carcinoma lactic acid dehydrogenase was isolated from an eleven-day old tumor by acid precipitation, ammonium sulfate fractionation, and chromatography on DEAE cellulose. Electrophoretic analysis indicated that the final enzyme preparation and the ammonium sulfate fraction contained a single isoenzyme, that is, one of the five possible forms of lactic acid dehydrogenase two of which are tetramers of a single but different protein while the other three are tetramer mixtures of both proteins (i.e. hybrid enzymes). Ultracentrifugal analysis indicated that the final enzyme preparation was composed of two major components with sedimentation rates of 7.3 S and 1.9 S. The enzymatic activity was associated only with the 7.3 S component. The apparent loss of enzymatic activity in 0.1 M phosphate buffer pH 7.0 and the magnitude of the value of the 1.9 S component indicated that this represented the subunit of the enzyme. [TRUNCATED]

Ischaemia-reperfusion (IR) injury is caused by the re-introduction of oxygen to organs, following periods of reduced blood flow (ischaemia). Whilst re-establishing blood flow (reperfusion) to the heart following myocardial infarction is vital for organ survival, this paradoxically leads to tissue damage. Mitochondria are at the heart of IR injury, with succinate dehydrogenase (SDH) a major player in orchestrating the damage. Succinate accumulates during ischaemia and is rapidly oxidised by SDH upon reperfusion, producing reactive oxygen species (ROS), leading to cellular death. I have investigated the development of drugs, aimed at targeting succinate metabolism to ameliorate IR injury. I firstly screened a range of compounds for their ability to inhibit SDH, having been chosen for their similar structures to succinate or the classical SDH inhibitor, malonate. Interestingly, only malonate and oxaloacetate showed potent SDH inhibition, thus were selected for further development. Malonate ester prodrugs with different properties were characterised. Hydrolysis rates of the esters differed greatly, with tuned, labile, malonate esters releasing malonate much more rapidly. Malonate esters were taken up into cells and hydrolysed to release malonate to different extents. Additionally, mitochondria-targetedmalonatemono and diesters were developed, each differing in mitochondrial and cellular uptake andmalonate release. Targeted and nontargeted malonate esters distributed into tissues in vivo, with preliminary in vivo work carried out on IR injury models, to assess for protective effects of the compounds. In addition, the physiological role of the tricarboxylic acid cycle metabolite, itaconate, was investigated. In lipopolysaccharide stimulated macrophages, itaconate has been reported to exert its effects by inhibition of SDH however, I found itaconate was a relatively poor SDH inhibitor, indicating other mechanisms of action. Current prodrugs of itaconate have many non-specific effects, not attributable to itaconate. I therefore characterised a novel itaconate prodrug and found it to be a much better surrogate, which could be subsequently used to elucidate roles for itaconate. Overall, I have shown the importance of ester selection for the prodrug delivery of dicarboxylate molecules and developed methods to improve their biological delivery.

Dihydromyricetin (DHM), also ampelopsin, is a flavonoid compound which exhibits a broad spectrum of positive effects on the human body. Herbal extracts containing this compound have been widely used in traditional Chinese medicine mainly for their hepatoprotective properties. DHM also helps with alcohol intoxication and reduces the signs of hangover or abstinence. Given the fact that the mechanism of DHM effects on the ethanol metabolism has not been clarified yet, the effect of dihydromyricetin on the expression and activity of alcohol dehydrogenase (ADH), one of the most important enzymes involved in ethanol metabolism, was therefore studied in this thesis. The cultivation conditions of primary hepatocytes which were isolated from unpretreated and ethanol-pretreated rats and subsequently exposed to EtOH and DHM were optimized. While determining the degree of cell damage caused by EtOH in the presence of DHM, no significant trend in the protective effect of DHM was found. On the other hand, the protective effect of ethanol in hepatocytes cultivated in EtOH and DHM was detected by technique of ELISA (the determination of alanine transaminase). The Western blot technique followed by immunodetection did not detect the induction of ADH expression in hepatocytes. Furthemore, the modulation effect of...

Butane-grown Pseudomonas butanovora oxidized butane by a soluble butane monooxygenase through the terminal pathway yielding 1 -butanol as the predominant product. Alcohol dehydrogenases (ADHs) involved in butane oxidation in P. butanovora were purified and characterized at the biochemical, genetic and physiological levels. Butane-grown P. butanovora expressed a type I soluble quinoprotein 1 -butanol dehydrogenase (BOH), a soluble type II quinohemoprotein 1 -butanol dehydrogenase (BDH) and an NAD���-dependent secondary ADH. Two additional NAD���-dependent secondary ADHs were also detected in cells grown on 2-butanol and lactate. BDH was purified to near homogeneity and characterized. BDH is a monomer of 66 kDa consisting of one mole of pyrroloquinoline quinone (PQQ) and 0.25 mole of heme c as the prosthetic groups. BOH was partially purified and its deduced amino acid sequence suggests a 67-kDa ADH containing a PQQ as a cofactor. BOH and BDH exhibited high activities and preference towards I -butanol and fair preference towards butyraldehyde. While BDH could not oxidize 2-butanol, BOH is capable of 2-butanol oxidation and has a broader substrate range than that of BDH. Genes encoding BOH and BDH and their deduced amino acid sequences were identified. BOH and BDH mRNAs and 1-butanol oxidation activity were induced when cells were exposed to butane. Primary C��� and C��� alcohols were the most effective inducers for boh and bdh. Some secondary alcohols, such as 2-butanol, were also inducers for BOH mRNA, but not for BDH mRNA. Insertional inactivation of boh or bdh affected unfavorably, but did not eliminate, butane utilization in P. butanovora. The P. butanovora mutant strain with both boh and bdh genes disrupted was unable to grow on butane and 1-butanol. This result confirmed the involvement of BOH and BDH in butane and 1-butanol metabolism in P. butanovora. Roles of B011 and BDH in butane and 1-butanol metabolism were further studied at the physiological level. There are no substantial differences between BOH and BDH in the mRNA expressions in response to three different 1- butanol levels tested and in their abilities to respond to 1-butanol toxicity. Different bioenergetic roles of BOH and BDH in butane and 1-butanol metabolism were suggested. A model of 1 -butanol- dependent respiratory systems was proposed where the electrons from 1 -butanol oxidation follow a branched electron transport chain. The role of BOH was suggested to function primarily in energy generation because B011 may couple to ubiquinone with the electrons being transported to a cyanide-sensitive terminal oxidase. BDH may be more important in the detoxification of 1 -butanol because the electrons from BDH may be transferred to a terminal oxidase system that is less sensitive to cyanide, which is not capable of energy generation. / Graduation date: 2003

Carbon Monoxide Dehydrogenase (CODH), also known as Acetyl-CoA synthase (ACS), is one of seven known Ni containing enzymes. CODH/ACS is a bifunctional enzyme which oxidizes CO to CO2 reversibly and synthesizes acetyl-CoA. Recently, X-ray crystal structures of homodimeric CODH from Rhodospirillum rubrum (CODHRr) and CODH from Carboxydothermus hydrogenoformans (CODHCh) have been published. These two enzymes catalyze only the reversible oxidation of CO to CO2 and have a protein sequence homologous to that of the β subunit of heterotetrameric α2β2 enzyme from Moorella thermoacetica (CODHMt), formerly Clostridium thermoaceticum. Neither CODHRr nor CODHCh contain an α-subunit as is found in CODHMt. The precise structure of the active site for acetyl-CoA synthase, called the A-cluster, is not known. Therefore, crystallization of the α subunit is required to solve the remaining structural features of CODH/ACS. Obtaining crystals and determining the X-ray crystal structure is a high-risk endeavor, and a second project was pursued involving the preparation, expression and analysis of various site-directed mutants of CODHMt. Mutational analysis of particular histidine residues and various other conserved residues of CODH from Moorella thermoacetica is discussed. Visual inspection of the crystal structure of CODHRr and CODHCh, along with sequence alignments, indicates that there may be separate pathways for proton and electron transfer during catalysis. Mutants of a proposed proton transfer pathway were characterized. Four semi-conserved histidine residues were individually mutated to alanine. Two (His116Mt and His122Mt) were essential to catalysis, while the other two (His113Mt and His119Mt) attenuated catalysis but were not essential. Significant activity was "rescued" by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. Activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or 123. Activity was also "rescued" by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway.

Mastitis is the most prevalent and major cause of economic losses in dairy farms. Bovine mastitis caused by strains of <i>S. aureus</i> is a major economically important disease affecting the dairy industry worldwide. <i>S. aureus</i> is one of the most common udder pathogens that cause either clinical or sub-clinical mammary gland infections. Different treatment regimes have failed to cure <i>S. aureus</i> intramammary infections. Most mastitis vaccination strategies have focused on the enhancement of systemic humoral immunity rather than strengthening local intramammary immunity. Vaccines aimed at enhancing intramammary immunity of dairy cows against <i>S. aureus</i> mastitis have had limited success. Commercially available vaccines show various degrees of success and work in research laboratories with experimental vaccines suggest that in part, the failure of these vaccines lies in the limited antigenic repertoire contained in the vaccine formulations. Moreover, not only does variation in the antigenic composition but also presence of capsular polysaccharide in most pathogenic strains and decreased activity of immune effectors in milk affect the success of vaccines. In addition to these, the ability of <i>S. aureus</i> to attach and internalize into mammary epithelial cells, enables bacteria to escape from the effect of immunity and antibiotics by being hidden in the intracellular niche and thereby causing chronic recurrent intramammary infection. <i>S. aureus</i> also has the ability to become electron-transport-defective and to form slow-growing small colonies that are non haemolytic and less virulent. These small colony variants might hide from the immune surveillance in the intracellular area and revert to the parental strain causing chronic recurrent infections. If immunization targets antigenic molecules that are conserved throughout all pathogenic strains, even the small colony variants can be controlled since the immune system will clear the parental strain which causes lethal infection. Thus, immunization trials should focus on conserved immunogenic antigen molecules among pathogenic strains formulated with an adjuvant and delivered by a route of immunization to induce maximum stimulation of the immune system. Moreover, immunization should focus on inducing Th1 responses, which is protective against <i>S. aureus</i> mastitis. It has been reported that proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity might be used as such antigens to induce protection against parasitic and microbial infections. Previous study in our laboratory on mastitis-causing streptococci indicates that GapC proteins of <i>S. uberis</i> and <i>S. dysgalactiae</i> have potential as vaccine antigens to protect dairy cows against mastitis caused by environmental streptococci. Two conserved cell wall associated proteins with iii glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, GapB and GapC have been identified from <i>S. aureus</i> isolates from bovine intramammary infections. The overall goal of this study was to improve our understanding on intramammary immunity using the GapC and GapB proteins of <i>S. aureus</i> as model antigens for mastitis and to determine the regulation of expression of <i>gapB</i> and <i>gapC</i> genes and their roles in the pathogenesis of bovine <i>S. aureus</i> mastitis. We hypothesized that strengthening local intramammary immunity using GapB and GapC proteins of <i>S. aureus</i> as antigens will protect against bovine <i>S. aureus</i> mastitis. To test this hypothesis we took the approach of using the <i>gapB</i> and <i>gapC</i> genes and constructed plasmids encoding GapB, GapC and GapB::GapC (GapC/B) chimeric proteins. We set six objectives to test our hypothesis using these proteins to enhance the intramammary immunity. In aim 1 we constructed plasmids encoding the GapB, GapC proteins and also constructed a chimeric gene encoding the GapC and GapB proteins as a single entity (GapC/B chimera) as the basis for a multivalent vaccine. In this objective the humoral and cellular immune responses to GapC/B were compared to the responses to the individual proteins alone or in combination in C57 BL/6 mice. Our results showed that the GapC/B protein elicited strong humoral and cellular immune responses as judged by the levels of total IgG, IgG1, IgG2a, IL-4 and IFN-ã secretion and lymphocyte proliferation. These results strongly suggest the potential of this chimeric protein as a target for vaccine production to control mastitis caused by <i>S. aureus</i>. In aim 2 we continued our studies on GapC/B by testing the effects of DNA vaccination with plasmids encoding the individual gapB and gapC genes as well as the gapC/B protein gene with or without a boost with the recombinant proteins. The results showed that DNA vaccination alone was unable to elicit a significant humoral response and barely able to elicit a detectable cell-mediated response to the recombinant antigens but subsequent immunization with the proteins elicited an excellent response. In addition, we found that DNA vaccination using a plasmid encoding the GapC/B chimera followed by a boost with the same protein, although successful, is less effective than priming with plasmids encoding GapB or GapC followed by a boost with the individual antigens. In aim 3 we optimized immune responses in cows by comparing route of vaccination (subcutaneous versus intradermal), site of vaccination (locally at the area drained by the supramammary lymph node versus distantly at area drained by parotid lymph node. Our results showed that both subcutaneous and intradermal immunizations with the GapC/B protein at the area drained by the supramammary and parotid lymph nodes resulted in significantly increased serum and milk titers of total IgG, IgG1, IgG2, iv and IgA in all vaccinated groups as compared to placebo. The anti-GapC/B IgG1 serum and milk titers were significantly higher in all vaccinated group as compared to the placebo group. These results indicated that vaccination at the area drained by the supramammary lymph node resulted in better immune responses. In aim 4 we tested different formulations of the GapC/B antigen with adjuvants such as PCPP, CpG, PCPP + CpG and VSA-3. We found that the VSA-3 formulation induced the best immune responses in cows. In this objective we also monitored immune responses longitudinally over one lactation cycle to determine the duration of immune responses by measuring IgG, IgG1, IgG2, and IgA on monthly blood and milk samples. We found that the duration of immune responses was about four months. In aim 5 we tested the role of GapC in the virulence of <i>S. aureus</i> mastitis using the <i>S. aureus</i> wild type strain RN6390 and its isogenic GapC mutant strain H330. Our results from both in vitro adhesion and invasion assays on MAC- T cells and in vivo infection of ovine mammary glands showed that GapC is an important virulence factor in <i>S. aureus</i> mastitis. In aim 6 we examined the role of sar and agr loci on the expression of <i>gapC</i> and <i>gapB</i> genes by qRT- PCR using <i>S. aureus</i> RN6390 and its isogenic mutants defective in agrA, sarA and sar/agr (double mutant) at exponential and stationary phases of growth. Our results showed that both <i>gapB</i> and <i>gapC</i> expression were down regulated in the mutant strains, indicating that the expression of the <i>gapB</i> and <i>gapC</i> genes is controlled by the universal virulence gene regulators, agr and sar. We also checked the role of environmental factors such as pH, growth media, and oxygen tension on the expression of <i>gapB</i> and <i>gapC</i> using q-RT-PCR. Our results showed that the expression of <i>gapB</i> and <i>gapC</i> genes in different strains of <i>S. aureus</i> was not consistent under the above-mentioned environmental conditions.

Inositol dehydrogenase (IDH, EC 1.1.1.18) from <i>Bacillus subtilis</i> catalyzes the reversible NAD⁺-dependent oxidation of the axial hydroxyl group of <i>myo</i>-inositol to form 2-keto-<i>myo</i>-inositol, NADH and H⁺. IDH is the first enzyme in catabolism of myo-inositol, and <i>Bacillus subtilis</i> is able to grow on <i>myo</i>-inositol as the sole carbon source. Our laboratory has previously shown that this enzyme has an unusual active site that can accommodate large hydrophobic substituents at 1L-4-position of <i>myo</i>-inositol.<p> In this dissertation, the further characterization of this IDH is described, with focus on the mechanism, inhibition, kinetics, substrate binding, and alteration of substrate specificity. A kinetic isotope effect study revealed that the chemical step of the reaction was not rate-limiting. In order to probe the inositol-binding site, five inositol analogues were synthesized and evaluated as competitive inhibitors. Recently the crystal structures of the <i>apo</i>-IDH, <i>holo</i>-IDH and ternary complex have been solved. Using structural information, as well as modeling and sequence alignment approaches, we predicted the active site structure of the enzyme. On the basis of these predictions, coenzyme specificity was converted from entirely NAD⁺-dependent to 6-fold preference for NADP⁺ over NAD⁺ by site-directed mutagenesis. The critical residues for coenzyme recognition were therefore identified. Besides coenzyme specificity alteration, eleven amino acid residues in and around the proposed <i>myo</i>-inositol active site were also modified to test their roles in order to improve our understanding of substrate binding and activation.

Inositol dehydrogenase (IDH, EC 1.1.1.18) from <i>Bacillus subtilis</i> catalyzes the reversible NAD⁺-dependent oxidation of the axial hydroxyl group of <i>myo</i>-inositol to form 2-keto-<i>myo</i>-inositol, NADH and H⁺. IDH is the first enzyme in catabolism of myo-inositol, and <i>Bacillus subtilis</i> is able to grow on <i>myo</i>-inositol as the sole carbon source. Our laboratory has previously shown that this enzyme has an unusual active site that can accommodate large hydrophobic substituents at 1L-4-position of <i>myo</i>-inositol.<p> In this dissertation, the further characterization of this IDH is described, with focus on the mechanism, inhibition, kinetics, substrate binding, and alteration of substrate specificity. A kinetic isotope effect study revealed that the chemical step of the reaction was not rate-limiting. In order to probe the inositol-binding site, five inositol analogues were synthesized and evaluated as competitive inhibitors. Recently the crystal structures of the <i>apo</i>-IDH, <i>holo</i>-IDH and ternary complex have been solved. Using structural information, as well as modeling and sequence alignment approaches, we predicted the active site structure of the enzyme. On the basis of these predictions, coenzyme specificity was converted from entirely NAD⁺-dependent to 6-fold preference for NADP⁺ over NAD⁺ by site-directed mutagenesis. The critical residues for coenzyme recognition were therefore identified. Besides coenzyme specificity alteration, eleven amino acid residues in and around the proposed <i>myo</i>-inositol active site were also modified to test their roles in order to improve our understanding of substrate binding and activation.