The biosynthesis of pyrimidines in Pseudomonas putida was investigated. In this study, pyrimidine requiring mutants were isolated by conventional mutagenesis and enrichment. The strains required exogenously supplied pyrimidines for growth and were found by enzyme assays to be deficient for the product of the pyrB gene encoding the enzyme aspartate transcarbamoylase. None of the intermediates of the pathway could supply the auxotrophic requirement of the strain; only preformed pyrimidines, metabolized via salvage pathways could suffice. Pyrimidine limitation in the mutant caused a slight but significant fifty per cent increase in expression of all the de novo biosynthetic enzymes. Pyrimidine starvation's effect on nucleotide pool levels was examined in the mutant and caused a marked swelling of the purine nucleotide pools.

Plasmodium falciparum causes the most severe form of human malaria, and the continual development of resistance of this parasite to current anti-malarial drugs underpins a pressing need for the discovery of novel chemotherapeutic approaches. Polyamines and their biosynthetic enzymes are present at high levels in rapidly proliferating cells, including cancer cells and protozoan parasites. Inhibition of the malaria parasite’s polyamine biosynthesis pathway causes cytostatic arrest in the trophozoite stage, but does not cure infections in vivo. This may be due to the salvage of exogenous polyamines from the host, replenishing the intracellular polyamine pool; however the mechanism(s) of polyamine uptake by the intraerythrocytic parasite are not well understood. In this study the uptake of the polyamines putrescine and spermidine into P. falciparum-infected erythrocytes (iRBC) well as into P. falciparum parasites functionally isolated from their host cell by saponin-permeabilisation of the erythrocyte membrane was investigated using radioisotope flux techniques. While the characteristics of transport of putrescine into infected erythrocytes were similar to those of transport into uninfected erythrocytes, spermidine entered iRBC in part via the ‘new permeation pathways’ induced by the parasite in the erythrocyte membrane. Both putrescine and spermidine were taken up across the plasma membrane of isolated parasites via a saturable, temperature-dependent process that showed competition between different polyamines as well as the polyamine precursor ornithine and basic amino acids. Inhibition of polyamine biosynthesis led to increased total uptake of both putrescine and spermidine. The influx of putrescine and spermidine into isolated parasites was independent of Na+ but increased with increasing pH and showed a marked dependence on the membrane potential, decreasing with membrane depolarisation and increasing with membrane hyperpolarisation. Both anthracene and polyamine derivatives have been shown to have anti-malarial activity. Anthracene-polyamine conjugates have been developed with the aim of utilising the polyamine uptake mechanisms of cancer cells to deliver the cytotoxic anthracene moieties to these cells. Here, several anthracene-polyamine conjugates showed promising anti-malarial activity. These compounds inhibited parasite proliferation with IC50 values in the nM range, and caused an arrest in the cell cycle, as well as a decrease in the mitochondrial membrane potential. Cytotoxicity could not be reversed by the addition of exogenous polyamines, nor did the conjugates have an effect on intracellular polyamine levels. This doctoral study showed that P. falciparum parasites not only synthesise polyamines, but can also acquire putrescine and spermidine from the extracellular environment and paves the way for interfering with polyamine metabolism as an anti-parasitic strategy. / Thesis (PhD)--University of Pretoria, 2012. / Biochemistry / unrestricted

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20780号 / 生博第386号 / 新制||生||51(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 佐藤 文彦, 教授 河内 孝之, 教授 福澤 秀哉 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

The number of people living with diabetes worldwide is continually increasing. The majority of these people will eventually die of cardiovascular disease, the major underlying cause of which is atherosclerosis. Despite the efforts of many researchers, gaps in our knowledge still exist regarding the molecular mechanism(s) linking the two conditions. Current data suggests that the hexosamine biosynthetic pathway (HBP) may have a role in the development of hyperglycemia-accelerated atherosclerosis. About 2-3% of glucose entering a cell is diverted into this pathway where it is modified through a series of reactions to yield the end product, UDP-N-acetylglucosamine (UDP-GlcNAc); a substrate for both N- and O-linked glycosylation of various molecules. N-linked glycosylation occurs in the endoplasmic reticulum (ER) and is an important process in the maintenance of ER homeostasis. We hypothesized that a dysregulation in the HBP can ultimately trigger ER stress – an event associated with the development of atherosclerosis. We have established a method that allows us to monitor levels of UDP-GlcNAc both in cultured cells and mouse tissues through high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS). Using this technique, we’ve shown that both glucosamine supplementation and overexpression of the rate limiting enzyme of the HBP, GFAT, in cultured cells results in elevated UDP-GlcNAc levels. Furthermore, glucosamine was shown to trigger ER stress. In contrast, three GFAT inhibitors that were previously identified in a high throughput screen were shown to decrease UDP-GlcNAc levels and one inhibitor, dehydroiso-β-lapachone, appears to prevent ER stress induction. Finally, we use complementary methods to show that the HBP is augmented in the livers of hyperglycemic mice. This process may play a role in the accelerated development of atherosclerosis. Together, these results provide further insight into the role of the HBP in diabetic atherosclerosis and the established methods provide a platform for the further investigation of this mechanism. / Thesis / Master of Science (MSc)

Yes / Biosynthetic materials have emerged as one of the most exciting and productive fields in polymer chemistry due to their widespread adoption and potential applications in tissue engineering (TE) research. In this work, we report the synthesis of a poly(ε-caprolactone)-graft-collagen (PCL-g-Coll) copolymer. We combine its good mechanical and biodegradable PCL properties with the great biological properties of type I collagen as a functional material for TE. PCL, previously dissolved in dimethylformamide/dichloromethane mixture, and reacted with collagen using carbodiimide coupling chemistry. The synthesised material was characterised physically, chemically and biologically, using pure PCL and PCL/Coll blend samples as control. Infrared spectroscopy evidenced the presence of amide I and II peaks for the conjugated material. Similarly, XPS evidenced the presence of C–N and N–C=O bonds (8.96 ± 2.02% and 8.52 ± 0.63%; respectively) for PCL-g-Coll. Static contact angles showed a slight decrease in the conjugated sample. However, good biocompatibility and metabolic activity was obtained on PCL-g-Coll films compared to PCL and blend controls. After 3 days of culture, fibroblasts exhibited a spindle-like morphology, spreading homogeneously along the PCL-g-Coll film surface. We have engineered a functional biosynthetic polymer that can be processed by electrospinning. / The EPSRC Centre in Innovative Manufacturing in Medical Devices (MeDe Innovation; EP/K029592/1).

Malaria is a life-threatening vector borne infectious disease caused by protozoan parasites of the genus Plasmodium. More than 100 species of Plasmodium can infect numerous animal species such as reptiles, birds and various mammals. However, human malaria is caused by four Plasmodium species -Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae, and occasionally by the simian malaria parasite, Plasmodium knowlesi. Of these, P. falciparum and P. vivax are the major causative agents and P. falciparum is the most virulent. About 300-500 million malaria infections occur every year leading to over 1-2 million deaths, of which 75% occur in African children of less than 5 years infected with P. falciparum. In spite of major global efforts to eliminate this disease over the past few decades, it continues to persist as a major affliction of human-kind imposing serious health and economic burden, especially to the poor countries. In India, the present scenario is about 2 million malaria positive cases every year, with almost 50% being caused by P. falciparum. Although remarkable attempts have been made over the years to develop vaccines against sexual and asexual stages of malaria parasite, an effective vaccine is still not in sight and remains as a distant goal. Hence, highly potent, less toxic and affordable antimalarial drugs remain as a first line therapy for malaria. Unfortunately, these parasites have been evolving against every known antimalarial drug and many of these drugs have lost their potency due to rapid emergence and spread of drug resistant strains. With development of resistance against frontline antimalarials such as chloroquine and antifolates, artemisinin and its derivatives seem to be the only effective antimalarials. However, recent reports on the possible emergence of artemisinin resistant strains, have led to the implementation of artemisinin-based combination therapies as a strategy to prevent drug resistance. Also, this continuous emergence of drug resistance has necessitated the development of new antimalarial drugs to combat this disease. While, Anopheles mosquitoes transmit parasites that infect humans, monkeys and rodents, Culex and Aedes mosquitoes predominate in the natural transmission to birds, and vectors of reptilian parasites are largely unknown. Of the approximately 400 species of Anopheles throughout the world, about 60 are malaria vectors under natural conditions, and 30 of which are of major importance. Ironically, the strategies implemented for controlling Anopheles, have also been hampered by insecticide resistance and other practical difficulties that exist in the scope of their applicability. In the past few years several milestones have been achieved in parasite genome, transcriptome and proteome studies, which could be exploited for the development of new drugs and drug targets. One such promising target includes the metabolic pathways of the malaria parasite which differ significantly from its human host. This thesis entitled “Unique Features of the Heme-Biosynthetic Pathway in Human Malaria Parasite, Plasmodium falciparum” unravels the unique biochemical features of heme-biosynthetic enzymes of P. falciparum, which have the potential for being drug targets. This pathway was first identified in this laboratory over 15 years ago. In the present study, five of the 7 enzymes of this pathway have been cloned, expressed, properties studied and sites of localization identified. With the knowledge on the first two enzymes coming from earlier studies, it is now possible to depict the unique hybrid pathway for heme biosynthesis in P. falciparum with full experimental validation.

Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2009. / Aims and Background: According to the World Health Organization type 2 diabetes will constitute a major global burden of disease within the next few decades. In agreement, reports show that rapid urbanization and lifestyle changes in South Africa are major factors responsible for these projections. Therefore, any perturbations that alter the regulatory steps that control myocardial glucose uptake by the cardiac-enrich glucose transporter, GLUT4, will lead in the development of diabetic cardiomyopathy and cardiac hypertrophy. Although considerable efforts are been put into unraveling molecular mechanisms underlying this process, less is known regarding the spatio-temporal regulation of GLUT4. In light of this, our specific aim was to establish in vitro fluorescence microscopy- and flow cytometry-based models for visualization and assessment of myocardial GLUT4 translocation using H9c2 cardiac-derived myoblasts. After successful establishment of our in vitro-based model for myocardial GLUT4 translocation, our second aim was to determine the role of the hexosamine biosynthetic pathway (HBP) in this process. Here, we employed HBP modulators to alter flux and subsequently evaluate its effect on myocardial GLUT4 translocation. To further strengthen our hypothesis, we also investigated the role of the HBP in hearts of an in vivo type 2 diabetes mouse model. Hypothesis: We hypothesize that increased flux through the HBP impairs myocardial GLUT4 translocation by greater O-linked glycosylation of the insulin signaling pathway, ultimately leading to myocardial insulin resistance. Methods: Rat cardiac-derived H9c2 myoblasts were cultured until ~ 80-90 % confluent for 3 days and thereafter subcultured in Lab-Tek chamber slides (~ 15, 000 cells per well) for 24 hours. Cells were then serum starved for 3 hours by insulin administration of 100 nM for 0, 5 and 30 minutes, respectively. We employed a method to quantify the relative proportion of GLUT4 at the sarcolemma using immunofluorescence microscopy- and flow cytometry-based models for visualization and assessment of myocardial GLUT4 translocation. Using these methods we investigated the role HBP have during GLUT4 translocation. The HBP were then activated through the following: a) high glucose and glutamine concentrations; b) low glucose and glucosamine stimulation; and c) over-expression of the HBP rate- limiting enzyme, i.e. GFAT. Subsequently, cardiac-derived myoblasts were fixed and probed for ~ 24 hours with antibodies specific for intracellular- and membrane-bound GLUT4, anti-myc GLUT4 (9E10) and O-GlcNAc. To assess GLUT4 translocation and O-GlcNAcylation we employed the following secondary antibodies: FITC Green for intracellular-bound GLUT4; and b) Texas Red for membrane-bound GLUT4 (immunofluorescence microscopy) and Phycoerythrin for flow cytometry-based model. Cells were thereafter viewed by multi-dimension imaging using an inverted system microscope (Olympus IX81) and a BD FACS Aria cell sorter for flow cytometric analysis. We also assessed HBP in an in vivo context by probing heart tissue - from insulin resistant db/db mice - with a GFAT monoclonal antibody. Results: The db/db mouse represents an ideal model to confirm our hypothesis in an in vivo context. In agreement, our preliminary results show increased GFAT expression versus heterozygous db/+ controls. Our in vitro model show myocardial GLUT4 translocation at 5 minute peak response when H9c2 cardiac-derived myoblasts were stimulated with 100 nM insulin, and GLUT4 vesicles return to normal after longer insulin stimulatory times (10, 15 and 30 minutes. Myocardial Glut4 v translocation was impaired when cells were stimulated with 100 nM wortmannin. Our transfection based model (immunofluorescence microscopy- and flow cytometry-based models) confirms 5 minute peak response under real time conditions. High glucose concentration (25 mM glucose), glucosamine concentrations (2.5 mM, 5 mM, and 10 mM) and over-expression of GFAT led to an impairment of myocardial GLUT4 translocation. Employment of an HBP activator (50 μM PUGNAc) also caused impairment of myocardial GLUT4 translocation. Myocardial GLUT4 translocation was restored when cells were treated with an HBP inhibitor (40 μM DON). High glucose concentrations (25 mM glucose), glucosamine concentrations (2.5 mM, 5 mM, and 10 mM) and over-expression of GFAT resulted in an increase in O-GlcNAcylation. HBP activation (50 μM PUGNAc) showed an increase in O-GlcNAcylation, while administration of 40 μM DON reversed this effect. Discussion and conclusion: We successfully established an in vitro experimental system to assay myocardial GLUT4 translocation. Our data show that dysregulated flux through the HBP impairs myocardial GLUT4 translocation. It is likely that the HBP becomes dysregulated during the pre-diabetic/early diabetic state and that O-GlcNAcylation of members of the insulin signaling pathway occurs during this stage. This will lead to myocardial insulin resistance, and in the long term, will contribute to the onset of the diabetic cardiomyopathy. Investigations to find unique inhibitors of this maladaptive pathway should therefore result in the development of novel therapeutic agents that will lead to a reduction in the growing global burden of disease for type 2 diabetes and associated cardiovascular diseases.

The thesis deals with the creation of the biosynthetic pathways of selected alkaloids of opium poppy. Then is following the design primers for selected genes and optimization of PCR reaction of these genes for amplification and sequencing of the longest fragments. The PCR reaction was optimized for the 7OMT, TNMT and CODM genes. These genes are involved in biosynthetic pathways of morphine, codeine, papaverine, noscapine and sanguinarine. Each gene was split in half. The primers were designed separately for each half of the gene. Altogether were designed 13 pairs primers. 5 pairs primers were optimized by gradient PCR and gel electrophoresis. These primers were used for sequencing analysis.

Triacylglycerols (TAGs) are stored in variable amounts (1-90 % by dry weight) in seed and non-seed tissues of various plant species. We analyzed the deep transcriptional profiling data obtained for eight species (brassica, castor, nasturtium, euonymus, oil palm, date palm, and avocado), to gain insights into tissue- and species-specific regulation and biosynthesis of TAG in plants. In all tissues analyzed, an increased expression was noted for genes mostly associated with fatty acid biosynthesis in plastid, but much less increase in those for TAG assembly in the endoplasmic reticulum. In most oil-rich tissues, transcripts associated with hexose metabolism in plastid also showed higher expression, relative to cytosol; this is likely associated with the need for high pyruvate flux directed toward plastid fatty acid synthesis. Additionally, expression of WRINKLED1 transcription factor, a regulatory element associated with oil biosynthesis in seed and non-seed tissues of monocot and dicot plants, was observed in most oil-rich tissues. Transcripts for other regulatory factors that are candidates associated with TAG synthesis in seed and non-seed tissues are also identified. In summary, our studies point to distinctive modes of regulation of fatty acid biosynthesis and TAG assembly that are conserved in both seed and non-seed oil-rich plants.