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

S-Adenosylmethionine (SAM) synthetases isolated from both the yeast and hyphal-phase of the dimorphic fungus, C. albicans, were partially purified using DEAE cellulose ion-exchange column chromatography. Further characterization was accomplished using enzyme kinetics and specific enzyme effectors. SAM synthetase is the enzyme responsible for synthesis of SAM which is the major methyl group donor in the methylation of macromolecules. Kinetic studies on column samples, from both phases, were performed. The yeast-phase enzyme had apparent Km ranges for L-methionine and ATP of 1.06-1.42mM and 1.11-1.69mM, respectively. The hyphal-phase enzyme had apparent Km ranges for L-methionine and ATP of 1.34-2.66mM and 3.29-6.28mM, respectively. Effector studies (in vitro) indicate that 10% (v/v) dimethyl sulfoxide (DMSO) and 5mM cycloleucine inhibit SAM Synthetase from both phases, 24% and 46%, respectively. The methionine analogues DLmethionine sulfone, DL-methionine-DL-sulfoxide and L-methioninesulfoximine and sinefungin, an analog of SAM, had no effect on SAM synthetase activity. Although the data is inconclusive with respect to the existence of isozymes, the observed Km's of the yeast and hyphal-phases are different suggesting that isozymes may exist. Additionally, the yeast-phase DEAE column profile has a shoulder prior to the main peak of activity indicating that more then one form of the enzyme may be present. / Department of Biology

We have studied the effect of S-adenosylmethionine (SAM) on tail flick latency in the rat. We also studied the effect of methionine the immediate precursor of SAM. Administration of methionine to the rat increases brain SAM, but little is known about its behavioral effects. Long-Evans rats were given SAM and methionine orally at different doses and tail-flick latency was measured at various times. Both methionine and SAM increased tail-flick latency, but methionine did so at a lower dose. A biochemical study showed that methionine was more effective than SAM in raising brain SAM probably because it is transported better into brain. The biochemical measurements were not consistent with the idea that the effects of SAM and methionine were mediated by an increase in brain 5-HT. / Folate deficiency can lower brain SAM levels and cause depression. Thus, methionine, which raises brain SAM, may overcome the effects of folate deficiency. Seven day food records were done by 26 psychiatric outpatients who were stable on lithium treatment. Eight patients had mean daily folate intakes below those recommended. Some of those with low folate intake had high methionine intake consistent with the idea that methionine could substitute metabolically for folate deficiency. Daily methionine intakes ranged from 13 to 304% of the recommended intake. As methionine had behavioral effects in the rat at doses much less than the daily dietary intake this raises the question of whether varying daily intakes of methionine in humans have behavioral implications. (Abstract shortened by UMI.)

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xvii, 176 p.; also includes graphics (some col.) Includes bibliographical references (p. 158-176). Available online via OhioLINK's ETD Center

Thesis (Ph.D.)--Ohio University, August, 2005. / Includes bibliographical references (leaves 105-110)

Malaria is considered the most prevailing human parasitic disease. Despite various chemotherapeutic interventions being available, the parasite responsible for the most lethal form of malaria, Plasmodium falciparum, is continuously developing resistance towards drugs targeted against it. This, therefore, necessitates the need for validation of new antimalarial development. Polyamine biosynthetic enzymes, particularly S-adenosylmethionine-L-decarboxylase (PfAdoMetDC), has been identified as a suitable drug target for protozoan parasitic diseases due to its essential role in cell proliferation. Furthermore, in Plasmodium polyamine biosynthesis, PfAdoMetDC is organised into a unique bifunctional complex with ornithine decarboxylase (PfAdoMetDC/ODC) covalently linked by a hinge region, distinguishing this enzyme as unique a drug target. However, inhibitors targeting this pathway have not been successful in clinical assessment, creating the need for further research in identifying novel inhibitors. This study focused on the structural and functional characterisation of protein-specific properties of the AdoMetDC domain in P. falciparum parasites, as well as identifying novel inhibitors targeting this enzyme as a potential antimalarial therapeutic intervention. In order to develop novel inhibitors specifically targeting PfAdoMetDC through a structure-based drug discovery approach, the three-dimensional structure is required. However, due to a lack of structural and functional characterisation, determination of the crystal structure has been challenging. Heterologous expression of monofunctional PfAdoMetDC was achieved from a wild-type construct of the PfAdoMetDC domain including the covalently linked hinge region. In chapter 2, deletion of a large non-homologous, low-complexity parasite-specific insert (A3) in monofunctional PfAdoMetDC resulted in an increased yield, purity and sample homogeneity, whilst maintaining protein functionality and structural integrity. However, truncation of the proposed non-essential hinge region resulted in low-level expression of insoluble protein aggregates and a complete loss of protein activity, indicating that the hinge region is essential for monofunctional PfAdoMetDC. However, in the absence of the three-dimensional PfAdoMetDC crystal structure, novel derivatives of a well-known AdoMetDC inhibitor, MDL73811, were tested for their activity against heterologous PfAdoMetDC, as well as their potency against P. falciparum parasites, in chapter 3. The compound Genz-644131 was identified as a lead inhibitor of PfAdoMetDC, however, the poor membrane permeability of the compound resulted in low in vitro activity. Drug permeability of Genz-644131 into P. falciparum infected erythrocytes and its potency was significantly improved by its encapsulation into a novel immunoliposome based drug delivery system. The results presented here provide essential information for development of a unique strategy in obtaining suffiecient levels of fully active recombinant PfAdoMetDC of sufficient purity for crystallisation studies and subsequent structure-based drug design efforts. The combination of Genz-644131 with the novel drug delivery system, which markedly improved its potency against PfAdoMetDC may proof to be a viable antimalarial chemotherapeutic strategy for future investigations. / Thesis (PhD)--University of Pretoria, 2014. / tm2015 / Biochemistry / PhD / Unrestricted

Malaria presents a global health risk that is becoming increasingly difficult to treat due to increased resistance of both the parasite and mosquito to all known drugs. Identification of novel drug targets are therefore essential in the fight against malaria. Polyamines are small flexible polycations that are represented by three basic polyamines. The interaction of polyamines with various macromolecules may lead to stabilisation of DNA, regulation of transcription, replication, and also have an important role in cellular differentiation, proliferation, growth and division. Therefore, disruption of polyamine biosynthesis presents a unique drug target worth exploiting. Polyamine biosynthesis in P. falciparum is regulated by a unique bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase (AdoMetDC/ODC) complex, which is unique to P. falciparum and differs completely from human polyamine biosyntehsis. The inhibition of AdoMetDC induces spermidine and subsequent spermine depletion within the parasite that ultimately results in cell cycle arrest. A functional genomics approach was used within this study to identify a global response of the parasite due to the inhibition of AdoMetDC with the irreversible inhibitor, MDL73811. The proteomics approach was optimised for conditions specific to our laboratory with regard to protein extraction, Plasmodial protein quantification, spot detection and finally protein identification by mass spectrometry (MS). This methodology resulted in reliable spot detection and achieved a 95% success rate in MS/MS identification of protein spots. Application of this methodology to the analyses of the Plasmodial ring and trophozoite proteomes ultimately resulted in the identification of 125 protein spots from the Plasmodial ring and trophozoite stages, which also confirmed stage specific protein production. Various protein isoforms were present which may be of significant biological importance within the Plasmodial parasite during development in the intraerythrocytic developmental cycle. Subsequent application of the 2-DE methodology to the proteome of AdoMetDC inhibited parasites resulted in the identification of 61 unique Plasmodial protein groups that were differentially affected by the inhibition of AdoMetDC in 2 time points. The transcriptome of AdoMetDC inhibited parasites were also investigated at 3 time points. Investigation into the transcriptome revealed the differential regulation of 549 transcripts, which included the differential regulation of polyamine specific transcripts. Inhibition of AdoMetDC provided a unique polyamine specific transcriptomic signature profile that demonstrated unique interactions between AdoMetDC inhibition and folate biosynthesis, redox metabolism and cytoskeleton biogenesis. The results presented provide evidence that the parasite responds to AdoMetDC inhibition by the regulation of the transcriptome and proteome in an attempt to alleviate the effects of AdoMetDC inhibition. Further analyses of the metabolome also provided evidence for the tight regulation of the AdoMet cycle. Overall, this study demonstrated important functional consequences as a result of AdoMetDC inhibition. / Thesis (PhD)--University of Pretoria, 2010. / Biochemistry / unrestricted

No description available.

Infection with "Pneumocystis" causes a ≥ 99% depletion of plasma S-adenosylmethionine (AdoMet) levels in both "Pneumocystis" pneumonia (PcP) animal models and patients. AdoMet is a critical cellular metabolic intermediate, with a pivotal role as methyl donor in a myriad of biochemical processes and necessary for the synthesis of the essential polyamines spermidine and spermine. In the target tissue of "Pneumocystis", the lung, levels of AdoMet were previously shown to be depleted experimentally using nicotine. Here we show that chronic administration of nicotine in an animal model of PcP resulted in decreased lung AdoMet content. Since "Pneumocystis" is dependent on this metabolite, PcP burden was also relived. We hypothesized that the underlying mechanism behind nicotine-induced AdoMet depletion was an increased consumption of AdoMet through the polyamine pathway where the increased activity of N-1-spermidine/spermine acetyl transferase raises the catabolic / anabolic cycling of polyamines, a process that utilizes AdoMet. In a critical test of our hypothesis, we found that blockage of polyamine metabolism via inhibition of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) hinders the effect of nicotine on lung AdoMet levels. Further support is provided by metabolite analyses showing nicotine to cause a strong diversion of AdoMet toward polyamine synthesis and away from methylation reactions; these shifts are also reversed by inhibition of ODC. Because the nicotine effect on "Pneumocystis" is so striking, we considered the possibility of tissue specificity. Using laser capture microdissection (LCM), we collected samples of lung alveolar regions (site of infection) and respiratory epithelium for controls. We found nicotine to cause increased ODC activity in alveolar regions but not airway epithelium; we conclude that tissue specificity likely contributes to the effect of nicotine on "Pneumocystis" pneumonia. Our studies demonstrate the feasibility of pharmacological manipulation of the polyamine pathway in order to reduce AdoMet levels in the lung and prompted the assessment of compounds alternative to nicotine with the potential to achieve a comparable effect. In vitro evaluation of the polyamine analog DENSPM along with putrescine in type II alveolar cell lines, indicates that although such a combination has the potential to induce polyamine flux, an apparent competition for the same polyamine transport system impairs simultaneous uptake of both compounds at effective concentrations. In conclusion, we showed that chronic nicotine administration causes reduction of AdoMet levels in rat lung following 21 days of treatment, by a mechanism involving the induction of polyamine flux, which is responsible of increased AdoMet utilization for polyamine biosynthesis. According to LCM-based analysis, this effect seems to be confined to the alveolar regions of the lung. / Biochemistry

No description available.