Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Bibliography: p. 120-129.

Plant aromatic amino acid decarboxylases (AAADs) are a group of economically important enzymes categorically joined through their pyridoxal 5'-phosphate (PLP) dependence and sequence homology. Extensive evolutionary divergence of this enzyme family has resulted in a selection of enzymes with stringent aromatic amino acid substrate specificities. Variations in substrate specificities enable individual enzymes to catalyze key reactions in a diverse set of pathways impacting the synthesis of monoterpenoid indole alkaloids (including the pharmacologically active vinblastine and quinine), benzylisoquinoline alkaloids (including the pharmacologically active papaverine, codeine, morphine, and sanguinarine), and antioxidant and chemotherapeutic amides. Recent studies of plant AAAD proteins demonstrated that in addition to the typical decarboxylation enzymes, some annotated plant AAAD proteins are actually aromatic acetaldehyde synthases (AASs). These AASs catalyze a decarboxylation-oxidative deamination process of aromatic amino acids, leading to the production of aromatic acetaldehydes rather than the AAAD derived arylalkylamines. Research has implicated that plant AAS enzymes are involved in the production of volatile flower scents, floral attractants, and defensive phenolic acetaldehyde secondary metabolites. Historically, the structural elements responsible for differentiating plant AAAD substrate specificity and activity have been difficult to identify due to strong AAAD and AAS inter-enzyme homology. Through extensive bioinformatic analysis and experimental verification of plant AAADs, we have determined some structural elements unique to given types of AAADs. This document highlights structural components apparently responsible for the differentiation of activity and substrate specificity. In addition to producing primary sequence identifiers capable of AAAD activity and substrate specificity differentiation, this work has also demonstrated applications of AAAD enzyme engineering and novel activity identification. / Ph. D.

Thesis (MSc (Chemistry and Polymer Science))--University of Stellenbosch, 2007. / Human African Trypanosomiasis (HAT) is a disease that threatens more then 60 million men, woman and children in Africa. It is known that the inhibition of the enzyme, ornithine decarboxylase (ODC) leads to cell arrest and subsequent death of Trypanosoma brucei, the parasite that causes the disease. The fluorinated ornithine analogue, DFMO (difluoromethylornithine or eflornithine) is a known inhibitor of ODC. Although various syntheses for DFMO exist they have some practical drawbacks which prevent the cost effective production of this compound as a drug for HAT treatment. This work focuses on the synthetic preparation of the fluorinated ornithine analogue DFMO as well as the fluorinated ornithine analogues 2-MFMO, 3-fluoro-ornithine and 3,3-difluoro-ornithine. Our chosen synthetic methodology focused on the introduction of the fluorine functionality using a simpler, safer and more convenient method than current direct fluorination techniques, or those that rely on the use of CFCs. Instead we decided to develop and optimise a fluorodehydroxylation method based on the transformation of hydroxylated ornithine analogues. The fluorodehydroxylation method substitutes a hydroxyl group to the corresponding fluorine and can also be used to transform an aldehyde or ketone to the corresponding difluoro group. Application of this fluorination method requires the synthesis of appropriate ...

The effects of TCDD on hepatic ornithine decarboxylase (ODC) activity and endocrine function in rats were investigated. Sixteen hours after partial hepatecomy, rats which had been pretreated with TCDD for one week exhibited a 3- to 4-fold increase in ODC activity, while vehicle controls exhibited an 8- to 10-fold increase. ODC induction after either aminophylline or dexamethasone administration, agents which act via cAMP-mediated and direct nuclear events, respectively, also was inhibited by pretreatment with TCDD. RNA polymerase I activity, which positively correlates to ODC activity in growth and development, decreased concomitant with decreased induction of ODC. In unstimulated liver, RNA polymerase I activity, as well as protein, DNA and RNA levels, remained unchanged one week after TCDD. However, TCDD administration resulted in decreased liver concentrations of putrescine and spermidine, but not spermine. Within 2 days following administration of TCDD (45 or 90 μg/kg), rats exhibited hypothermia, hypothyroidism and decreased growth rate compared to pair-fed controls and rats fed ad libitum. Within 2 weeks of the administration of 90 μg TCDD/kg, body temperature had fallen to below 35°C with a low mean value 34.5°C recorded on day 16. Mean body temperatures for control rats ranged from 36.8°C to 37.5°C. One week after the administration of TCDD (45 μg/kg) to rats, serum thyroxine (T₄) and triiodothyronine (T₃) levels declined to 42% and 82% of control, respectively. Mild hypoglycemia occurred subsequent to hypothyroidism and hypothermia. At 1 week after administration of 45 μg TCDD/kg to rats, serum and pancreatic insulin levels were reduced to 25% and 76% of control, respectively. Hypophagia was determined to be responsible for decreased growth rate and hypoinsulinemia, but it could not account for hypothyroidism, hypothermia or hypoglycemia following administration of TCDD. No changes in glucagon or pancreatic, hepatic or serum somatostatin levels were found. Decreased somatostatin in the gastric antrum coincided with a 29% increase in stomach weight. The delayed toxicity of TCDD may be related to these striking hormonal alterations.

Im Rahmen der vorliegenden Arbeit sollte geprüft werden ob durch Reduktion der Glutamatdecarboxylase (GAD) Expression eine Reduktion des autoimmunogenen Potenzials in insulinproduzierenden Beta-Zellen des endokrinen Pankreas erreicht werden kann. Aus der Literatur ist bekannt, dass GAD als Autoantigen eine zentrale Stellung bei der Induktion der T-Zell vermittelten Insulitis einnimmt. Der Prozess, welcher zur Beta-Zell-Apoptose des Typ 1 Diabetes führt, ist ein bislang wenig verstandener komplexer Vorgang. Ein besseres Verständnis dieses Prozesses könnte zur Prävention der Beta-Zell-Zerstörung in der frühen Phase des Typ 1 Diabetes beitragen. In den für die Untersuchungen verwendeten INS-1 Zellen werden die beiden Isoformen der GAD exprimiert. Durch einen antisense Ansatz sollte in INS-1 Zellen die GAD Expression beider Isoformen supprimiert werden. In dieser Arbeit wurden zwei Methoden zur gezielten Suppression der Expression des Autoantigens GAD65 etabliert. Es konnte ein antisense Klon identifiziert werden, bei dem die endogene GAD65 mRNA fast nicht mehr detektierbar war. Auf Protein Ebene, im Westernblot konnte dieses Ergebnis jedoch nicht bestätigt werden. Im zweiten Teil der Arbeit wurde die Funktion der INS-1 Zellen mit supprimierter GAD65 Expression charakterisiert. Dieser Punkt beinhaltet die Analyse der Expression von Genen, welche die Beta-Zell-Funktion definieren, die Glukose-abhängige Insulinsekretion sowie die Regulation der Zytokin-induzierten Apoptose. Dabei zeigte sich aus Daten der RT-PCR, dass die mRNAs von anderen Beta-Zell-spezifischen Genen wie GLUT2, Glukokinase, Proinsulin, IDX1 und Nkx6.1 in unveränderter Menge nachweisbar sind. Also bleibt die Funktion der INS-1 Beta-Zellen erhalten, da selbst durch forcierte Reduktion der Expression des Autoantigens GAD65 die Glukose-induzierte Insulinsekretionskapazität im Wesentlichen nicht beeinträchtigt wird. In vitro Untersuchungen zeigten eine unveränderte Sensitivität der Zytokin-induzierten Apoptose nach GAD65 Suppression in INS-1 Zellen. Die zuvor genannten Resultate und die Tatsache, dass die GAD wohl eines der wichtigsten Autoantigene im Rahmen der Immunpathogenese des Typ 1 Diabetes ist, stellen die Grundlage für die Generierung GAD-supprimierter transplantierbarer Beta-Zellen mit guter Transplantatfunktion dar. Im Hinblick auf eine mögliche therapeutische Anwendung bei der Behandlung dieser humanen Autoimmunerkrankung demonstrieren die vorliegenden Daten, dass im Rahmen einer Inselzelltransplantation die Verwendung von GAD-supprimierten Beta-Zellen bei der Transplantation in das endokrine Pankreas des Menschen zu einer Verminderung von Autoimmunreaktionen führen könnte. / In the present study we investigated genetic engineering approaches for the suppression of autoantigenic GAD expression in insulin producing pancreatic beta-cells. The enzyme glutamic acid decarboxylase (GAD) represents a major autoantigen in the early immunopathogenesis of T-cell-mediated destruction of pancreatic beta-cells in type 1 diabetes mellitus. The mechanisms which trigger the apoptotic destruction of insulin producing pancreatic beta-cells leading to autoimmune diabetes are incompletely understood. The exact mechanisms remain to be clarified. The enzyme glutamic acid decarboxylase (GAD), is expressed in INS-1 pancreatic beta-cells in two distinct isoforms GAD65 and GAD67. Thus, strategies to suppress GAD expression in INS-1 cell lines were tested. Two methods for suppression of autoantigenic GAD65 expression were established. One clone overexpression of GAD65 antisense mRNA yielded an almost complete suppression of endogenous GAD65 mRNA expression. In the second part of this thesis the characterization of INS-1 cells with suppressed autoantigenic GAD65 mRNA expression including beta-cell specific gene expression, glucose-dependent insulin secretion, and cytokine induced-apoptosis was tested. Expression of glucose transporter type 2, glucokinase, preproinsulin, islet/duodenum homeo box 1 transcription factor (IDX), and NK homeodomain transcription factor (Nkx6.1) were characterized by reverse transcription polymerase chain reaction. No differences in the amount of these beta-cell specific genes could be detected between GAD65 suppressed INS-1 cell clones and controls. Moreover, reduced GAD65 expression does not affect insulin secretory capacity in INS-1 cells. Suppression of GAD65 expression in INS-1 cells does not alter sensitivity to cytokine-induced apoptosis. The data presented here suggest that suppression of GAD expression may thus provide a therapeutical approach to prevent recurrence of autoimmune beta-cell destruction in transplanted pancreatic beta-cells in type 1 diabetic patients.

Die Anzahl neurologischer Erkrankungen bei denen Autoantikörper gegen zentralnervöse An-tigene bekannt sind, hat in den letzten Jahren deutlich zugenommen. Allerdings gibt es nur für wenige dieser Erkrankungen hinreichende experimentelle Belege für eine pathogene Wir-kung der Autoantikörper. Zwei dieser Erkrankungen wurden im Rahmen dieser Arbeit näher untersucht: die Juvenile Neuronale Zeroid-Lipofuszinose (JNCL) mit Autoantikörpern gegen die 65 kD Isoform der Glutamatdecarboxylase und das Stiff Person Syndrom (SPS) mit Auto-antikörpern gegen Amphiphysin. Die phänotypische Charakterisierung der cln3 knockout-Maus, einem Mausmodell für die JNCL, zeigte eine progressive Verschlechterung der motorischen und koordinativen Fä-higkeiten, eingeschränktes reizbedingtes Lernen und gesteigertes angstähnliches Verhalten. Diese Symptome ähneln denen der humanen Erkrankung. Elektrophysiologisch konnte eine Antikörper-induzierte zerebelläre Dysfunktion identifiziert werden, die einer verminderten lokalen GABAergen Hemmung zugeordnet wird. Eine Reduktion der Antiköperproduktion im Tiermodell durch eine Depletion der Plasmazellen durch den Proteseinhibitor Bortezomib hatte einen positiven Effekt auf die Krankheitsentwicklung. Im zweiten experimentellen Teil der Arbeit wurde der Einfluss von Autoantikörpern gegen Amphiphysin von Patienten mit SPS auf die synaptische Transmission untersucht. Es zeigte sich hierbei in Patch-Clamp Experimenten eine Störung der GABAergen Übertragung v.a. bei hochfrequenter Stimulation, was im Einklang mit dem vermuteten Antikörper-induzierten Endozytosedefekt steht. Passiver Transfer von humanen Autoantikörpern gegen Amphiphysin induzierte angst-ähnliches Verhalten in Ratten, einem weiteren Kernsymptom des SPS. Aktive Immunisierung gegen Amphiphysin und anschließende Öffnung der Blut-Hirn-Schranke in Mäusen führte zu einer subklinischen Veränderung der Reflexverarbeitung von Ia Afferenzen auf Motoneurone im Rückenmark der Mäuse. Insgesamt konnten in zwei Erkrankungen des ZNS autoimmune Mechanismen identifi-ziert werden, die zu einer Antikörper-induzierten Fehlregulation der zentralen synaptischen Transmission führen. Diese Ergebnisse können wegweisend sein auch für die Erforschung der Pathophysiologie anderer Antikörper-assoziierte Erkrankungen des ZNS. / A growing number of neurological disorders are associated with autoantibodies targeting an-tigens within the central nervous system. Only in few cases experimental evidence corrobo-rates a pathogenic role of the autoantibodies. Two autoantibody associated diseases were investigated in detail in this work: the Juvenile neuronal ceroid lipofuscinoses (JNCL) with au-toantibodies against the 65kD isoform of the glutamate decarboxylase and the Stiff Person Syndrome (SPS) with autoantibodies against amphiphysin. The analysis of cln3 knockout mice, an animal model of the JNCL, revealed a phenotype resembling the human disorder, including progressive motor decline, limited cued learning and an increase in anxiety-related behavior. Electrophysiological analysis revealed an autoan-tibody mediated cerebellar dysfunction, which is best explained by diminished local GABAer-gic inhibition. A reduction of autoantibody production in the cln3 knockout mice by depletion of plasma cells after treatment with Bortezomib had a positive effect on the disease out-come. In the second experimental part, the effect of autoantibodies to amphiphysin from SPS patients on synaptic transmission was analysed. In patch-clamp experiments the GABAergic synaptic transmission was found to be disturbed primarily during high-frequent stimulation. This is in line with the hypothesized defect of synaptic vesicle endocytosis induced by autoan-tibodies. Passive transfer of human autoantibodies to rats induced anxiety-related behavior, a key symptom of SPS. The active immunization of mice against amphiphysin and subsequent opening of the blood brain barrier led to subclinical disturbances of the Ia afferent-motor neuron reflex pathway within the spinal cord. In conclusion, for two CNS disorders autoimmune mechanisms were identified leading to antibody-induced deregulations of central synaptic transmission. These findings may have implication for the research on pathomechanisms of other putative antibody mediated dis-orders.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 96-103).

published_or_final_version / Biochemistry / Master / Master of Philosophy

No description available.

Xylella fastidiosa is a small, xylem-limited bacterium that causes a number of diseases in over 100 species of plants. Many of the species infected are economically important (such as coffee, grapevines, citrus, and almond) and billions of dollars worldwide are lost annually due to X. fastidiosa infection of crops. The bacterium colonises both plant and insect hosts, using the insect host to transfer it from plant to plant. Sequencing of the X. fastidiosa genome in 2000 discovered that while the genome is reduced, it contains a high number of putative bifunctional enzymes. One of these enzymes, aspartate kinase:diaminopimelate decarboxylase (AK:DapDc), occurs in only a handful of species and is predicted to catalyse the first and last steps of lysine biosynthesis. This study reports the first experimental characterisation of this enzyme. AK:DapDc was over-expressed in the pET30dSE plasmid in Escherichia coli BL21 DE3 cells. It was purified by Ni2+ His-Trap chromatography followed by size exclusion chromatography. Homology models of AK:DapDc were created in SWISS-MODEL, which indicate homology with the aspartate kinase from Arabidopsis thaliana and the diaminopimelate decarboxylase from E. coli. Circular dichroism, and analytical ultracentrifugation were used to obtain information about the secondary and quaternary structure of AK:DapDc. This data, in combination with the homology models, suggests that AK:DapDc exists as a dimer or tetramer in solution. A coupled enzyme assay to assay for diaminopimelate decarboxylase activity has been set up, and preliminary crystal screens have been carried out.