The mammalian small intestine is a major site (second in total activity only to the liver) for the synthesis and secretion of plasma apolipoproteins, and contributes significantly to overall whole-body lipid dynamics. A prominent feature of the small intestine is its exposure to periodic loads of meals often containing dramatically varying amounts or types of food components, including lipids such as tri-acylglycerols, cholesterol and cholesteryl esters. Since the trans-epithelial transport of most of these latter materials requires the elaboration of particles partially covered by apolipoproteins, the regulation of the biosynthesis or, more correctly, the availability of these proteins is an important and as yet little-understood problem. Previous studies have been conducted on systems which, for one or the other reason, have not permitted the following questions to be satisfactorily or coherently answered: Does the ingestion of fat-containing meals, either acutely or chronically, increase the rate of biosynthesis of intestinal apolipoproteins such as apo B-48, and is this the principal method of matching the "demand" with the supply of this "packaging material" needed for fat transport across the intestinal epithelial cells? Alternatively, does the maintenance of a large steady-state intracellular pool in the face of variations in intracellular apolipoprotein degradation, controlled by acute or chronic lipid ingestion, produce the required "match" between supply and demand for these proteins (as has recently been suggested in studies on liver cells)? An in vitro system was therefore devised whereby sheets of intestinal epithelial cells (enterocytes) were freshly isolated from the jejuna of adult male Syrian golden hamsters and incubated for several hours in a medium supporting steady-state protein synthesis, in a manner which was assumed to be similar to the activity just before the killing of the donor animals. (Hamsters appear on various grounds to be a better small-animal model of human lipoprotein metabolism than the more commonly studied rats). The isolated epithelial cell sheets produced primary apolipoprotein products that could be extracted from the cells or detected in the incubation media, free from the subsequent modifications that they are known to undergo in vivo. Hamsters maintained on a low-fat chow were either studied as such or subjected to a variety of dietary treatments designed to maximize (over short or long time periods) intracellular apolipoprotein requirements for the "packaging" of tri-acylglycerol-rich lipoproteins, especially chylomicrons: acute bolus administration of lipid into the gut; overnight feeding of fat-enriched food; and chronic (six week) fat feeding. Using specific antisera and immuno-precipitation techniques, apo B-48 and two other principal intestinal apolipoproteins were shown to be synthesized in the steady state by intestinal cell sheets derived from control animals and from those subjected to acute or chronic fat-containing diets. Secretion took place, however, only when prior fat exposure of the donor intestines had occurred. Pulse-chase labelling was used to compare the rates of apolipoprotein synthesis, degradation and secretion in the same cell sheet preparations. The rates of apolipoprotein B-48 synthesis did not vary significantly under conditions of low or high trans-epithelial lipid flux, supporting findings derived from in vivo experimental systems. In contrast with data from other systems, however, the biosynthesis of apolipoprotein A-IV was not reproducibly increased on fat challenge. The rates of apo B-48 degradation varied significantly and were markedly reduced under conditions of fat feeding. The experiments permit a choice between the two alternatives mentioned above: Ingestion of fatty foods, either acutely or over long periods of time, does not increase the rates of biosynthesis of apolipoproteins such as apo B-48; but variations in the rate of intracellular degradation of this and probably other apolipoproteins allows the intestinal cells to match their requirements for lipid-transporting molecules to the demands of any given situation, relying in each case on a large steady-state intracellular pool maintained by "constitutive" biosynthesis. Importantly, there seems also to be a specific, possibly related effect of fat feeding on the secretion of lipoproteins into the intestinal extracellular fluid. These conclusions coincide with those obtained by other workers from studies of apolipoprotein B dynamics in isolated hepatocytes and in the hepatoma-derived liver cell line, Hep G2. The mechanisms underlying these phenomena are as yet unresolved.

To further understand the ATCase/DHOase bifunctional complex formed in Streptomyces, the genes encoding these and other pyrimidine enzymes were identified and characterized. Polymerase chain reaction (PCR) was utilized in this effort. Primers were constructed by selecting conserved regions of pyrimidine genes from known gene and protein sequences of a wide variety of organisms. These sequences were then optimized to Streptomyces codon usage. PCR products were obtained from internal sites within pyrimidine genes and also from primer combinations of different genes. The size, orientation, and partial sequence of the resulting products shows that Streptomyces has a gene organization of pyrR followed by pyrB, pyrC, carA, carB, and pyrF in an operon similar to that found in other Gram-positive bacteria.

No description available.

No description available.

Triacylglycerol (TAG) is a main storage lipid in seeds and is utilized for seed germination. Humans commonly consume TAG in the form of vegetable oil. Currently, TAG contributes to 25% of the total calories consumed in the developed countries and dependency for biofuel production from plants lipid is also increasing day by day. By 2030 the expected consumption level of vegetable oil will be double and to fulfill this demand it is necessary to increase the oil production in plants. To accomplish this goal, it is pertinent to understand the regulation of TAG accumulation in plants. Avocado (Persea americana), a basal angiosperm, stores up to 70% oil in the form of TAG in nonseed tissue (mesocarp). Here, we use it avocado as a model system to understand TAG biosynthesis in tissues other than seed. In plants, TAG biosynthesis may involve acyl-CoA–dependent or –independent pathway catalyzed by diacylglycerol acyltransferases (DGAT) and phospholipid: diacylglycerol acyltransferases (PDAT), respectively. RNA-Seq and Q-PCR analysis of developing mesocarp of avocado revealed higher expression for DGAT1 and PDAT1, coinciding with the period of TAG accumulation. Therefore, we hypothesize that DGAT1 and PDAT1 are responsible for catalyzing the terminal step in TAG biosynthesis in Avocado mesocarp. Using the transcriptome data, we identified full-length coding sequences for DGAT1 and PDAT1. These acyltransferases are being cloned and will be characterized for their enzyme activity and substrate specificity, subsequent to their expression in yeast. Complementation of Arabidopsis dgat1 and pdat1 mutants and phenotypic characterization will also be carried out using Gateway-cloning techniques and transgenic lines will be assayed for TAG content in seeds. This study is expected to provide basic understanding of TAG accumulation in avocado mesocarp tissue.

Modern society’s demand for oil has resulted in depletion of resources and caused higher oil prices. Therefore, natural oil resources of plants are gaining the spotlight and are expected to increase twice that of current use by 2030. Plants are able to accumulate up to 90% oil by dry weight in the form of triacylglycerol (TAG) and it is derived from fleshy part of the fruits, such as mesocarp of oil palm, avocado, and olive. In seed tissues, an acyl CoA-dependent enzyme, diacylglycerol acyltransferase (DGAT) participates in conversion of diacylglycerol (DAG) to TAG. However, TAG can also be formed by an acyl CoA- independent enzyme, phospholipid:diacylglycerol acyltransferase (PDAT). Avocados (Persea americana) store up to ~70% oil in mesocarp and I am interested in identifying the acyltransferase involved in oil biosynthesis in mesocarp tissues. Based on the transcriptome data available, I hypothesize that unlike in seeds, both DGAT and PDAT are associated with TAG biosynthesis in developing mesocarp of avocado. To test this hypothesis, I will determine 1) TAG content and composition and 2) expression levels for DGAT and PDAT genes in mesocarp and seed tissues of five stages of developing fruits of avocado (n=5). Total lipids will be extracted by isopropanol-chloroform method and analyzed for composition as methyl esters on GC-FID. Total RNA, for expression analysis, will be extracted by Trizol method and analyzed with gene-specific primers by real-time PCR. Statistical significance in change in oil content in association with gene expression during fruit development between mesocarp and seed tissues will be analyzed by ANOVA repeated measures. Comparison of temporal gene expression pattern of oil accumulation mesocarp, to that of seed, will allow us to differentiate the acyltransferase(s) specifically associated with TAG biosynthesis. The proposed research work will take the field of plant lipid biochemistry a step forward in understanding TAG synthesis in fruit tissue. Specifically, I will be able to clearly demonstrate the association of a particular acyltransferase to increasing lipid content in a non-seed (mesocarp) tissue. Understanding differences in oil regulation of a basal angiosperm (avocado) in relation to a monocot (oil palm) and a dicot (olive) also will provide additional insights into fundamental changes in TAG biosynthesis during the evolution of flowering plants. My research is part of a global project that includes a group in California, Florida and Israel. Data generated from my proposed work will be used to develop a joint Binational Agricultural and Research proposal. Therefore, my research work will subsequently contribute to development of ways to manipulation of extent and timing of oil accumulationa direct benefit to avocado growers. Furthermore, this study will be a pivotal step to understanding TAG synthesis that will lead to bioenergy crop.

The mechanism by which plants synthesize and store high amounts of triacylglycerols (TAG) in tissues other than seeds is not well understood. The comprehension of controls for carbon partitioning and oil accumulation in nonseed tissues is essential to generate oil-rich biomass in perennial bioenergy crops. Persea americana (avocado), a basal angiosperm with unique features that are ancestral to most flowering plants, stores ~ 70 % TAG per dry weight in its mesocarp, a nonseed tissue. Transcriptome analyses of select pathways, from generation of pyruvate and leading up to TAG accumulation, in mesocarp tissues of avocado was conducted and compared with that of oil-rich monocot (oil palm) and dicot (rapeseed and castor) tissues to identify tissue- and species-specific regulation and biosynthesis of TAG in plants.

The nerve-derived trophic factor neuregulin (NRG) is a prime candidate molecule for modulating muscle fiber growth. NRG regulates signal transduction in skeletal muscle through activation of ErbB receptors present at the neuromuscular junction. In this study, we hypothesize that NRG increases protein synthesis in maturing muscle via a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. NRG signal transduction and its ability to stimulate protein synthesis (measured by incorporation of [3H]phenylalanine into the protein pool) were investigated in differentiated C2C 12 myotubes and rat diaphragm muscle (DIAm). In C2C 12 myotubes, NRG dose dependently increased phosphorylation of ErbB3 and recruitment of the p85 subunit of PI3K. NRG also increased phosphorylation of Akt, a downstream effector of PI3K. NRG treatment increased total protein synthesis by 35% compared with untreated control myotubes. This NRG-induced increase in Akt phosphorylation and protein synthesis was completely blocked by wortmannin, an inhibitor of PI3K but was unaffected by PD-98059, an inhibitor of MEK. In DIAm obtained from 3-day-old rat pups, Akt phosphorylation increased ∼30-fold with NRG treatment (vs. untreated DIAm). NRG treatment also significantly increased protein synthesis in the DIAm by 29% after 3 h of incubation with [3H]phenylalanine (vs. untreated DIAm). Pretreatment with wortmannin abolished the NRG-induced increase in protein synthesis, suggesting a critical role for PI3K in this response. The results of the present study support the hypothesis that nerve-derived NRG contributes to the regulation of skeletal muscle mass by increasing protein synthesis via activation of PI3K.

The iron-molybdenum cofactor (FeMo-cofactor) of nitrogenase is the subject of one the most intensive biochemical/genetic detective cases of modern science. At the active site of nitrogenase, the FeMo-cofactor not only represents the heart of biological nitrogen fixation, but its synthesis also serves as a model for complex metallocluster biosynthesis. Research in the Dean Lab is focused on furthering the understanding of Fe-S cluster biosynthesis in the nitrogenase enzyme system. Throughout the years, scientists from a broad range of disciplines have focused their intellectual might on deciphering not only the chemistry of the FeMo-cofactor, but also the biosynthesis of this unique metallocluster. Recent advances in the study of FeMo-cofactor biosynthesis have produced considerable insight regarding the complex series of biological reactions necessary for the synthesis of this metallocluster. The work contained within this dissertation represents my efforts to further the understanding of FeMo-cofactor biosynthesis. The concept of a molecular scaffold in FeMo-cofactor biosynthesis is generally accepted in the field of nitrogenase. Previous work has implicated the products of nifE and nifN as providing the assembly site for FeMo-cofactor synthesis. Researchers were able to purify this molecular scaffold, commonly referred to as the NifEN complex, however, detailed characterization was precluded by the inability to obtain sufficient quantities of NifEN. In an effort to fully characterize the NifEN complex, we initiated a gene fusion approach for the high level production NifEN. In addition to gene fusion, a poly-histidine tag was incorporated into NifEN, allowing purification through the application of immobilized metal-affinity chromatography (IMAC). NifEN obtained in this way was characterized using a variety of biophysical techniques and found to contain two [4Fe-4S] clusters in each NifEN tetramer. These clusters were also shown to be completely ligated by cysteine residues. With the information obtained from this study, it is concluded that the [4Fe-4S] clusters of the NifEN complex are likely to play either a structural or a redox role rather than being transferred and becoming incorporated into the FeMo-cofactor. In addition to the biophysical characterization of the NifEN complex, a separate study was started to characterize the apo-MoFe protein. In this study we used IMAC to purify a poly-histidine-tagged apo-MoFe protein produced by a nifB-deletion mutant of A. vinelandii. Using the poly-histidine fusion approach, apo-MoFe protein was obtained in sufficient quantities for detailed catalytic, kinetic and spectroscopic analyses. This multidisciplinary approach confirmed that apo-MoFe protein contained intact P clusters and P cluster environments, as well as the ability to interact with the Fe protein. It was also shown for the first time that this tetrameric form of purified apo-MoFe protein could be activated by the addition of preformed FeMo-cofactor. The NifEN complex was further characterized to investigate the presence of bound FeMo-cofactor intermediates. NifEN purified by IMAC is produced in the absence of the nitrogenase structural genes (nifHDK). In this genetic background, it is believed that the FeMo-cofactor biosynthetic machinery will become obstructed with unprocessed FeMo-cofactor intermediates, such as the Fe-S precursors of FeMo-cofactor, NifB-cofactor. Previous work indicated that NifEN can exist in either a charged or discharged form, based on the presence or absence of the FeMo-cofactor precursor, NifB-cofactor. EPR and VTMCD spectroscopies showed the presence of a new paramagnetic signal associated with NifEN that is believed to be in the charged or precursor bound state. This represents the first spectroscopic evidence for a precursor to the FeMo-cofactor. Furthermore, an interaction of NifEN and NifX was examined by size exclusion chromatography. From this study, NifX exhibited the capacity to bind a chromophore, presumably an FeMo-cofactor precursor, from the NifEN complex. NifX was also capable of binding to isolated FeMo-cofactor and the FeMo-cofactor precursor, NifB-cofactor. Finally, preliminary investigations involving interaction between the Fe protein and NifEN were initiated. Recent findings indicate that NifEN and the Fe protein have the capacity to interact specifically with one another. The interaction of NifEN and Fe protein appears to be dependent on the association of FeMo-cofactor precursor with NifEN. The NifEN complex also has the capacity to accept electrons from the Fe protein in a MgATP dependent manner. The ability of NifEN to accept electrons from the Fe protein may be involved in the role of Fe protein in FeMo-cofactor biosynthesis. / Ph. D.

Thesis(M.Sc. (Chemistry)) -- University of Limpopo, 2019 / Imidazolyl-ethanamine Schiff base ligands of the N^N type were prepared by condensation reaction of histamine dihydrochloride with para-substituted aldehyde derivatives to yield: (E)-N-benzylidene-2-(1H-imidazol-4-yl)ethanamine 119a, 4-((E)(2-(1H-imidazol-4-yl)ethylimino)methyl)phenol 119b, E)-N-(4-fluorobenzylidene)-2(1H-imidazol-4-yl)ethanamine 119c and (E)-N-(4-nitrobenzylidene)-2-(1H-imidazol-4yl)ethanamine 119d, which were characterised by 1H and 13C-NMR, FTIR specroscopy and HRMS. 2D-NMR experiments (1H-1H COSY and 2D-HMBC) for representative ligand 119b were performed to qualify success in the condensation reaction. An attempted reaction to coordinate Schiff base ligand 119c to zinc chloride was carried out in an NMR tube and traces of the product were observed between 12 and 24 h monitoring using 1H-NMR. Iodine promoted cyclocondensation reaction of anthranilamide and para-substituted aldehyde derivatives afforded 2-aryl-quinazolin4(3H)-ones 120a-e and subsequent chloro-aromatisation reaction in SOCl2 afforded electrophilic C4-(Cl) 2-aryl-4-chloro-quinazolines 121a-e and the compounds were characterised by 1H and 13C-NMR and FTIR spectroscopic techniques. The 2-aryl-4chloro-quinazolines served as prerequisites for de-chloro amination on the C4-(Cl) position by 2-amino-3-nitropyridine to yield 2-aryl-N-(3-nitropyridin-2-yl)quinazolin-4amine derivatives 123a-e in good yield and the derivatives were characterised by 1H and 13C-NMR, FTIR and HRMS spectroscopic techniques. The C4-(Cl) position further allowed for Sonogashira cross-coupling with ethynylpyridine to yield 2-aryl-4(ethynylpyridine)quinazoline derivatives 125a-e which were characterised by 1H and 13C-NMR, FTIR and HRMS spectroscopic techniques. The 2-aryl-4(ethynylpyridine)quinazoline served as ligands for coordination to monomeric pcymene ruthenium(ll) which yielded (ɳ6-p-cymene)RuCl2-2-aryl-4(ethynylpyridine)quinazoline derivatives 126a-e in good yield. Compounds 126a-e were characterised by 1H and 13C-NMR, FTIR and HRMS spectroscopic techniques. 2D-HMBC NMR of representative ligands 126c and 126e showed long range couplings from 1JCH to 9JCH and this was confirmed by coordination induced shifts (CIS) ranging from 1 ppm to 11 ppm. Compounds 119a-d, 123a-e and 125a-e were inductively docked into the active receptors of tyrosine kinase (PDB:2SRC), glutamine synthetase (PDB:1HTO) and oxidoreductase (PDB:3F8P). The docking scores obtained gave hits ranging from -5 to -10 Kcal/mol. Compounds 119a-d, 121a-e, 123a-e, 125a-e and 126a-e were assayed employing the broth-dilution method which gave promising anti-Mycobaterium tuberculosis activity. Compound 125e gave good activity of <0.244 µg/mL over 7 day and 14 day sampling. Coordination of ligands 125a-e to Ru(ll) group resulted in loss of activity, notably for ligand 125e. / NRF and Sasol Inzalo Bursary