Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [73]-84).

In all mammalian species examined thus far, the ovaries produce a burst of ornithine decarboxylase (ODC) and putrescine during ovulation or after application of a bolus of human chorionic gonadotropin (hCG). Aged mice are deficient in this peri-ovulatory ODC and putrescine burst. Moreover, peri-ovulatory putrescine supplementation in aged mice increases egg quality and reduces miscarriage rates. These studies suggest that peri-ovulatory putrescine supplementation may be a simple and effective therapy for reproductive aging for women. However, putrescine has never been used in humans and, currently no pure source of putrescine is suitable for human trials. Given that ODC is highly expressed in the ovaries during ovulation but otherwise exhibits low activity in most tissues, we hypothesized that L-ornithine, the substrate of ODC, might be a better alternative. In this study, we have demonstrated that systemic application of L-ornithine increased ovarian putrescine levels; the increase was restricted to animals that had been injected with hCG. Furthermore, L-ornithine specifically increased ovarian putrescine levels without affecting putrescine levels in most other tissues. Unfortunately, thus far peri-ovulatory L-ornithine supplementation in mouse drinking water produced mixed effects on reproductive outcome in aged mice. Therefore, our studies demonstrated the potential of L-ornithine supplementation as a possible therapy for aging-related infertility, but further work is required to produce an effective application method.

Schizophrenia is a neurodevelopmental disorder affecting 1% of the general population. Dysfunction of the prefrontal cortex (PFC) is associated with the etiology of schizophrenia. Moreover, a substantial deficit of GAD1mRNA in schizophrenic PFC has been reported by different groups. However, the underlying molecular mechanisms are still unclear. Interestingly, epigenetic factors such as histone modifications and DNA methylation could be involved in the pathogenesis of schizophrenia during the maturation of the PFC. In my work, I identified potential epigenetic changes in schizophrenic PFC and developmental changes of epigenetic marks in normal human PFC. Furthermore, mouse and neuronal precursor cell models were used to confirm and investigate the molecular mechanisms of the epigenetic changes in human PFC. My initial work examined whether chromatin immunoprecipitation can be applied to human postmortem brain. I used micrococcal nuclease (MNase)-digested chromatin instead of cross-linked and sonicated chromatin for further immunoprecipitation with specific anti-methyl histone antibodies. Surprisingly, the integrity of mono-nucleosomes was still maintained at least 30 hrs after death. Moreover, differences of histone methylation at different genomic loci were detectable and were preserved within a wide range of autolysis times and tissue pH values. Interestingly, MNase-treated chromatin is more efficient for subsequent immunoprecipitation than crosslinked and sonicated chromatin. During the second part of my dissertation work, I profiled histone methylation at GABAergic gene loci during human prefrontal development. Moreover, a microarray analysis was used to screen which histone methyltransferase (HMT) could be involved in histone methylation during human prefrontal development. Mixed-lineage leukemia 1 (MLL1), an HMT for methylation at histone H3 lysine 4 (H3K4), appears to be the best candidate after interpreting microarray results. Indeed, decreased methylation of histone H3 lysine 4 at a subset of GABAergic gene loci occurred in Mll1 mutant mice. Interestingly, clozapine, but not haloperidol, increased levels of trimethyl H3K4 (H3K4me3) and Mll1 occupancy at the GAD1 promoter. I profiled histone methylation and gene expression for GAD1 in schizophrenics and their matched controls. Interestingly, there are deficits of GAD1 mRNA levels and GAD1 H3K4me3 in female schizophrenics. Furthermore, I was also interested in whether the changes of GAD1 chromatin structure could contribute to cortical pathology in schizophrenics with GAD1 SNPs. Remarkably, homozygous risk alleles for schizophrenia at the 5’ end of the GAD1 gene are associated with a deficit of GAD1 mRNA levels together with decreased GAD1 H3K4me3 and increased GAD1H3K27me3 in schizophrenics. Finally, I shifted focus on whether DNA methylation at the GAD1 promoter could contribute to a deficit of GAD1 mRNA in schizophrenia. However, no reproducible techniques are available for extracting genomic DNA specifically from GABAergic neurons in human brain. Therefore, I used an alternative approach that is based on immunoprecipitation of mononucleosomes with anti-methyl-histone antibodies differentiating between sites of active and silenced gene expression. The methylation frequencies of CpG dinucleotides at the GAD1 proximal promoter and intron 2 were determined from two chromatin fractions (H3K4me3 and H3K27me3) separately. Consistently, the proximal promoter region of GAD1 is more resistant to methylation in comparison to intron 2 of GAD1 in either open or repressive chromatin fractions. Interestingly, overall higher levels of DNA methylation were seen in repressive chromatin than in open chromatin. Surprisingly, schizophrenic subjects showed a significant decrease of DNA methylation at the GAD1proximal promoter from repressive chromatin. Taken together, my work has advanced our knowledge of epigenetic mechanisms in human prefrontal development and the potential link to the etiology of schizophrenia. It could eventually provide a new approach for the treatment of schizophrenia, especially in the regulation of methylation at histone H3 lysine 4.

Metal ions play essential roles in biological functions including catalysis, protein stability, DNA-protein interactions and cell signaling. It is estimated that 30% of proteins utilize metals in some fashion. Additionally, methods by which metal ions can be visualized have been utilized to study metal concentrations and localizations in relation to disease. Understanding the roles metals play in biological systems has great potential in medicine and technology. Chapters 1 and 2 of this dissertation analyzes the structure and function of the Mn-dependent enzyme oxalate decarboxylase (OxDc) and Chapter 2 presents a bioinformatic analysis of the cupin superfamily that provides the structural scaffold of the decarboxylase. The X-ray crystal structure of the W132F variant was determined and utilized together with EPR data to develop a computational approach to determining EPR spectra of the enzyme’s two metal-binding centers. Furthermore, a variant in which the catalytic Glu162 was deleted revealed the binding mode of oxalate, the first substrate-bound structure of OxDc. OxDc is a member of the cupin superfamily, which comprises a wide variety of proteins and enzymes with great sequence and functional diversity. A bioinformatics analysis of the superfamily was performed to analyze how sequence variation determines function and metal utilization. Chapters 3 and 4 discuss the expansion of lanthanide-binding tags (LBTs) to in cellulo studies. Lanthanide-binding tags are short sequences of amino acids that have high affinity and selectivity for lanthanide ions. An EGF-LBT construct used to quantify EGF receptors on the surface of A431 and HeLa cells. The results from the LBT quantification are consistent with previous studies of EGFR receptors in these cell types, validating the use of this method for future studies. The potential of using LBTs for X-ray fluorescence microscopy (XFM) was also investigated. LBT-labeled constructs were utilized to investigate if membrane bound as well as cytosolic LBT-containing proteins could be visualized and localized to their cell compartments via XFM; both membrane-localized and cytosolic proteins were successfully visualized. With the high resolution (< 150 Å) obtainable with new synchrotron beamline configurations LBTs could be used to study nanoscale biological structures in their near-native state.

Brucella est l'agent causal de la brucellose, une zoonose bactérienne répandue à l'échelle mondiale. Durant les dernières années, de nouvelles souches et espèces de Brucella (dont Brucella microti) ont été isolées de l’environnement et d’animaux sauvages. Ces souches, phylogénétiquement anciennes, sont plus acido-résistantes que les espèces classiques, plus récentes et inféodées aux animaux domestiques et à l’homme. Chez Escherichia coli, le système glutamate décarboxylase (GAD) et le système glutaminase (AR2_Q), basés respectivement sur la décarboxylation du glutamate et la déamination de la glutamine, sont les systèmes d’acido-résistance (AR) les plus efficaces. Notre équipe a démontré que le système GAD (GadB et GadC) est fonctionnel seulement dans les nouvelles souches et espèces de Brucella, et participe à la réussite de l’infection des souris par voie orale. Dans cette thèse, le rôle de nouveaux facteurs et les mécanismes moléculaires impliqués dans l’acido-résistance ont été explorés. Premièrement, nous avons montré que GlsA et GadC sont les deux protéines structurales du système AR2_Q qui, avec le système GAD, joue un rôle essentiel dans l’AR de ces nouvelles souches. De plus, chez ces mêmes souches, le système uréase intervient également dans la survie en milieu acide.Nos résultats suggèrent que les systèmes GAD, AR2_Q et uréase, en fonction de la disponibilité des substrats, pourraient contribuer à améliorer l’adaptation des nouvelles espèces dans les environnements acides naturels et/ou dans le tractus gastro-intestinal de leurs hôtes. / Brucella is the etiological agent of brucellosis, a worldwide bacterial zoonosis. In the last ten years, new and atypical strains of Brucella (among which Brucella microti) were isolated from the environment and wild hosts. These strains, of ancient origin, are considered more environmental and acid resistant than classical Brucella species, which are mostly pathogenic for livestock and humans. In Escherichia coli, the glutamate decarboxylase (GAD)-dependent system and the glutaminase (AR2_Q) system, based on the decarboxylation of glutamate and on the deamination of glutamine, respectively, are most efficient in conferring acid resistance (AR). Our team has previously demonstrated that in Brucella the GAD system (GadB and GadC) is functional only in new/atypical strains and contributes to murine infection by oral route. In this thesis, novel molecular mechanisms and factors involved in specific AR of new/atypical Brucella species were explored. Firstly, we have shown that in these strains, GlsA and GadC are the two structural proteins of the AR2_Q system, which, in concert with the GAD system, plays an essential role in AR. In addition, the functionality and role of the urease system in AR was also demonstrated in these strains.Our results suggest that the GAD, AR2_Q and urease systems may participate in a better adaptation of new Brucella species to certain natural acidic habitats and/or to the gastrointestinal tract of their hosts, depending on substrate availability.

Abstract Rhodiola rosea is a medicinal plant, mainly used in Asia and Scandinavia. It is characterized as an adaptogen and is reported to have many pharmacological properties, which are ascribed to the glycosides of cinnamyl alcohol and tyrosol. As natural habitats are already overharvested and the cultivation of this plant needs 4–6 years, the production of the pharmacologically important compounds in in vitro cultures could be an alternative. In the work presented here, the production of these glycosides in compact callus aggregate cultures of roseroot was addressed. Biotransformation of exogenously added cinnamylalcohol and tyrosol was studied. Glucosylation of the precursors yielded high amounts of rosin and salidroside and low amounts of rosavin. During the course of this work, four new glycosides of cinnamyl alcohol were found and identified. The optimal concentration of the precursors and the time needed for the biotransformation was also determined. For enhancing the biotransformation rate, glucose was added to the culture medium alongside with sucrose, which doubled the production of cinnamyl alcohol glycosides but did not affect the production of salidroside. A pilot experiment using air-lift bioreactor was performed. A cDNA fragment encoding tyrosine decarboxylase was isolated and described. The expression of this gene was analysed in the leaves and roots of two chemotypes. The results demonstrate the important role of tyrosine decarboxylase in the production of salidroside. The results revealed production of the pharmacologically important glycosides of Rhodiola rosea; however the successful pilot bioreactor experiment remains to be scaled-up. New information was obtained on the biosynthesis of salidroside, which substantiate the metabolic engineering of roseroot.

Malaria is one of the most serious tropical infectious diseases affecting mankind. The prevention of the disease is hampered by the increasing resistance of the parasite to existing chemotherapy and -prophylaxis drugs. The need for novel therapeutic targets and drugs is therefore enormous and the understanding of the biochemistry of the parasite is imperative. The aim of this study was the identification and molecular characterisation of the eDNA of one such metabolic target protein, ornithine decarboxylase (ODC), in the human malaria parasite P. falciparum. The P. falciparum ODC eDNA was isolated by means of a modified RT-PCR technique, RACE. No sequence data were available and the primers used were based on consensus areas identified in the protein sequences from other related organisms. The isolation and identification of the eDNA with degenerate primers was successful in 3' -RACE, but necessitated the optimisation of the eDNA synthesis protocol and the use of total RNA as starting material. The sequence obtained facilitated the application of 5' -RACE with ODC-specific primers based on the 3' -RACE sequence data. The full-length ODC eDNA sequence was obtained by overlap-alignment of various segments. A novel suppression PCR technology was applied during the 5' -RACE in order to create an uncloned eDNA library of amplified cDNAs representing only the mRNA population. The P. falciparum ODC eDNA contains an open reading frame of ---2847 bp and translates to a large 939 amino acid protein. The protein contained large internal insertions and was extended by '""273 N-terminal residues compared to ODCs from other organisms. Several possible signature motifs were identified for phosphorylation, glycosylation and transamidation. The P. falciparum ODC protein seems to contain more hydrophilic and a-helix forming residues. These characteristics should be further investigated after expression of the recombinant protein. The isolation of the P. falciparum ODC eDNA facilitates the validation of this protein as an antimalarial target. / Dissertation (MSc)--University of Pretoria, 1998. / gm2014 / Biochemistry / unrestricted

Agmatine is an endogenous amine derived from decarboxylation of arginine catalysed by arginine decarboxylase. Agmatine is considered a novel neuromodulator and possesses neuroprotective properties in the central nervous system. The present study examined whether agmatine has neuroprotective effects against repeated restraint stress-induced morphological changes in rat medial prefrontal cortex and hippocampus. Sprague-Dawley rats were subjected to 6 h of restraint stress daily for 21 days. Immunohistochemical staining with β-tubulin III showed that repeated restraint stress caused marked morphological alterations in the medial prefrontal cortex and hippocampus. Stress-induced alterations were prevented by simultaneous treatment with agmatine (50 mg/kg/day, i.p.). Interestingly, endogenous agmatine levels, as measured by high-performance liquid chromatography, in the prefrontal cortex and hippocampus as well as in the striatum and hypothalamus of repeated restraint rats were significantly reduced as compared with the controls. Reduced endogenous agmatine levels in repeated restraint animals were accompanied by a significant increase of arginine decarboxylase protein levels in the same regions. Moreover, administration of exogenous agmatine to restrained rats abolished increases of arginine decarboxylase protein levels. Taken together, these results demonstrate that exogenously administered agmatine has neuroprotective effects against repeated restraint-induced structural changes in the medial prefrontal cortex and hippocampus. These findings indicate that stress-induced reductions in endogenous agmatine levels in the rat brain may play a permissive role in neuronal pathology induced by repeated restraint stress.

In the present study, we examined the possible effect of chronic treatment with glucocorticoids on the morphology of the rat brain and levels of endogenous agmatine and arginine decarboxylase (ADC) protein, the enzyme essential for agmatine synthesis. Seven-day treatment with dexamethasone, at a dose (10 and 50 μg/kg/day) associated to stress effects contributed by glucocorticoids, did not result in obvious morphologic changes in the medial prefrontal cortex and hippocampus, as measured by immunocytochemical staining with β-tubulin III. However, 21-day treatment (50 μg/kg/day) produced noticeable structural changes such as the diminution and disarrangement of dendrites and neurons in these areas. Simultaneous treatment with agmatine (50 mg/kg/day) prevented these morphological changes. Further measurement with HPLC showed that endogenous agmatine levels in the prefrontal cortex and hippocampus were significantly increased after 7-day treatments with dexamethasone in a dose-dependent manner. On the contrary, 21-day treatment with glucocorticoids robustly reduced agmatine levels in these regions. The treatment-caused biphasic alterations of endogenous agmatine levels were also seen in the striatum and hypothalamus. Interestingly, treatment with glucocorticoids resulted in a similar change of ADC protein levels in most brain areas to endogenous agmatine levels: an increase after 7-day treatment versus a reduction after 21-day treatment. These results demonstrated that agmatine has neuroprotective effects against structural alterations caused by glucocorticoids in vivo. The parallel alterations in the endogenous agmatine levels and ADC expression in the brain after treatment with glucocorticoids indicate the possible regulatory effect of these stress hormones on the synthesis and metabolism of agmatine in vivo.

Ornithine is an amino acid of which the side chain contains three pairs of diastereotopic protons. The diastereotopic protons are chemically nonequivalent. The NOEDIF spectra of ornithine were obtained in an acid solution to assign the individual chemical shifts of the β diastereotopic protons with the aid of the theoretical calculation of conformation distribution. The β Pro-S resonance was found to be at a higher field than β pro-R proton. (S,S)-Ethylenediamine-N,N'-disuccinic acid (EDDS) was found to be a good chiral chemical shift reagent ligand in water solution. The Ytterbium complex of EDDS was used to distinguish L-ornithine and D-ornithine. For the α protons, the enantiomeric shift difference was affected by the pH of the solution and the substrate : Yb(EDDS)’ ratio. The association constants between Yb(EDDS)’ and L- or D-ornithine were found to be slightly different. Yb(EDDS)’ may also be useful to distinguish between the diastereotopic protons in ornithine and other molecules in NMR spectra.