Hypoxoside, a phenolic diglucoside, with a diarylpentane-type structure, is thought to be the medicinally active constituent of corm extracts of Hypoxis hemerocallidea Fisch. & Mey. which are reputed to alleviate the symptoms of prostate hypertrophy and urinary infections. The biosynthes is and production of this unique phytochemical were investigated in H. hemerocallidea using both in vivo and in vitro systems. It was found, in root-producing callus, that [l4]C-phenylalanine and [14]C-t-cinnamic acid were efficient precursors for hypoxoside in comparison to [14]C-sodium acetate and [14]C-acetyl coenzyme-A, which were not incorporated into the phenolic compound. Thus, at least one aryl moiety of hypoxoside was derived, via phenylalanine and t-cinnamic acid, from the shikimate pathway. The acetate pathway did not appear to be involved in the biosynthetic process. The data supports the hypothesis that the molecule is formed from two cinnamate units with the loss of a carbon atom, in opposition to the proposal that the molecule is derived from head-to-tail condensation of acetate units onto a propenylic moiety. Despite the structural similarities between hypoxoside and caffeic and p-coumaric acids, these two hydroxycinnamic acids were not efficient precursors for hypoxoside in vivo or in vitro. A number of reasons are put forward to explain this finding. It was found that the greatest concentration of hypoxoside was located in the corms of intact plants. The major biosynthetic site of the molecule was also found to be located in this organ. Since the roots did accumulate the phytochemical to a small extent, the biosynthetic potential of these organs has not been disregarded. That of the leaves has been, however. The report by PAGE (1984) that the upper region of the corm contained a greater con cent ration of hypoxoside than the lower portion, is substantiated in this study, where this region was found to be more biosynthetically active than the lower half. Light microscopic and electron microscopic studies revealed that starch storing cells, which accumulated phenolics in their vacuoles, contained seemingly synthetically active tubular endoplasmic reticulum in their cytoplasm. A greater number of these cells were concentrated in the upper region as opposed to the lower half of the corm. It is postulated that these cells are the site for biosynthesis and accumulation of hypoxoside. The shikimate pathway, from which the precursors for hypoxoside are derived, was found, through the exposure of intact plants to [14]C-carbon dioxide, to be located mainly in the leaves. It is postulated from the above study and one in which [14]C-phenylalanine, [14]C-t-cinnamic acid, [14]C-p-coumaric acid and [14]C-caffeic acid were applied to intact plants, that phenylalanine and/or cinnamic acid are the transported form of the shi kimate derivatives. p-Coumaric and caffeic acids, which are metabolically more stable, are envisaged to be the sequestering forms. The investigation of the seasonal production of hypoxoside revealed that most of the synthesis and accumulation occurred after the corms had broken winter dormancy and after the flush of leaf growth had slowed down. During dormancy the production of hypoxoside appeared to cease. The in vjtro studies, where the effects of light, temperature, nutrients, plant growth regulators and supply of potential precursors, on hypoxoside production by root-producing callus were investigated, indicate that this metabolite is not simply a "shunt" metabolite. A number of factors other than precursor availability enhanced, or reduced the jn vjtro production of this phytochemical. Furthermore, production of the phytochemical and growth were not always antagonistic. Hypoxoside, the biosynthesis of which requires a more thorough investigation, is, however, according to this investigation, a typical secondary metabolite in many respects. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1989.

Storage starch is synthesized in sub-cellular organelles called amyloplasts in higher plants. The synthesis of the starch granule is a result of the coordinated activity of several groups of starch biosynthetic enzymes. There are four major groups of these enzymes, ADP-glucose pyrophosphorylase (AGPase), starch synthases (SS), starch branching enzymes (SBE), and starch debranching enzymes (SDE). Starch phosphorylase (SP) exists as both dimeric and tetrameric forms in plastids in developing cereal endosperm and catalyses the reversible transfer of glucosyl units from glucose-1-phosphate to the non-reducing end of α-1-4 linked glucan chains, although the precise role in the pathway remains unclear. The present study was conducted to investigate the role and regulation of SP and SSIV in starch biosynthesis in developing maize endosperm. The results of this study showed that the tetrameric form of SP accounts for the majority of measurable catalytic activity, with the dimeric form being barely active and the monomer catalytically inactive. A catalytically active recombinant maize SP was heterologously expressed and used as an affinity ligand with amyloplast lysates to test protein-protein interactions in vitro. Results showed that the different multimeric status of SP influenced interactions with other enzymes of starch synthesis. Tetrameric SP interacted with SBEI and SSIIa, whilst the dimeric form of the enzyme interacted with SBEI, SBEIIb. All of these interactions were enhanced when amyloplasts were pre-treated with ATP, and broken following treatment with alkaline phosphatase (APase), indicating these interactions are regulated by protein phosphorylation. In addition, the catalytic activity of SSIV was reduced following treatment with APase, indicating a role for protein phosphorylation in the regulation of SSIV activity. Protein-protein interaction experiments also suggested a weak interaction between SSIV and SP. Multimeric forms of SP regulated by protein-protein interactions and protein phosphorylation suggested a role for SP in starch biosynthesis in maize endosperm.

No description available.

Gefährdete Spezies in Menschenobhut zu reproduzieren und zu erhalten soll dem weltweiten Rückgang zahlreicher Papageienarten entgegenwirken. Der Erfolg solcher Zuchtprogramme wird unter anderem durch begrenzte Kenntnisse über physiologische und pathologische Vorgänge im Fortpflanzungssystem dieser Vogelordnung erschwert. Ziel der vorliegenden Arbeit war die Etablierung aussagekräftiger Parameter zur Einordnung des Reproduktionsstatus von männlichen Papageienvögeln. Dabei wurde ein Probenumfang fixierter, männlicher Reproduktionsorgane acht verschiedener Gattungen mit standardisierten histologischen und immunzytochemischen Methoden untersucht. Im Vordergrund stand die morphologische Beurteilung der untersuchten Gonaden im Bezug auf Fortpflanzungsaktivität und -status. Gleichzeitig sollten die immunzytochemischen Analysen Aufschluss über die beteiligten Hormone und Enzyme geben. Für die Etablierung vogel-spezifischer Marker wurde als Vertreter der Psittaciformes der Wellensittich (Melopsittacus undulatus, n=45) als Modellspezies ausgewählt. 15 verschiedene Antikörper aus der Gruppe der Steroidrezeptoren, steroidogenen Enzyme, Relaxinpeptide und Proliferationsmarker wurden an dieser Art getestet. Anschließend erfolgte der Transfer der erarbeiteten Methodik auf sieben weitere Papageiengattungen (Nymphicus, Eolophus, Cacatua, Psittacus, Amazona, Ara, Cyanopsitta). Anhand der Histologie konnten alle untersuchten Gonaden den drei verschiedenen Reproduktionsstadien aktiv, intermediär und inaktiv zugeordnet werden. Hierbei wurden Kriterien wie die Ausdehnung von Samenkanälchen und Interstitium, Morphologie des Keimepithels, Vorhandensein von Lipofuszin in den Samenkanälchen sowie die Teilungsaktivität von Keimzellen herangezogen. Aktive Hoden zeigen ausgedehnte Tubuli und ein schmales Interstitium, ein Keimepithel mit allen Keimzellstadien, wenig Lipofuszin und eine hohe Teilungsaktivität bei den Keimzellen. Inaktive Hoden hingegen besitzen schmale Tubuli und ein breites Interstitium, ein Keimepithel bestehend aus Sertoli-Zellen und Spermatogonien, Massen an Lipofuszin im Lumen der Samenkanälchen und eine geringe Proliferationsrate der Keimzellen. 14 der 15 getesteten Marker konnten mittels Immunzytochemie erfolgreich am Wellensittich etabliert werden. Hinsichtlich der Einordnung des Reproduktionsstatus war in erster Linie ein Absinken der steroidogenen Enzymaktivität von 3β-Hydroxysteroid-Dehydrogenase (HSD) und 17β-HSD-2 bei sexuell inaktiven gegenüber aktiven und intermediären Tieren zu verzeichnen. Auch der Androgenrezeptor (AR) wurde im Ruhestadium nicht mehr exprimiert. Die übrigen Steroidrezeptoren, steroidogenen Enzyme und Relaxinpeptide zeigten variable zelluläre Verteilungsmuster, die keine klare Aussage zum Fortpflanzungsstatus zuließen. Dennoch konnten anhand der Lokalisation dieser Faktoren in Keimzellen, somatischen Zellen des Hodens und Zellen des Nebenhodenepithels funktionelle Gesichtspunkte geklärt werden. Beispielsweise zeigte die Koexistenz des Östrogenrezeptors ERα und des steroidogenen Enzyms Aromatase in Hoden und Nebenhoden, dass nicht nur androgene Einflüsse in die Steuerung der Gonaden involviert sind. Auch der erstmalige Nachweis von Relaxin, Relaxin-like factor und ihren Rezeptoren in testikulären und epididymalen Zellen deutet darauf hin, dass diese die Funktion der beim Vogel nicht vorhandenen Prostata übernehmen. Zudem ist der Transfer der etablierten immunzytochemischen Methoden auf sieben weitere Papageiengattungen (Nymphicus, Eolophus, Cacatua, Psittacus, Amazona, Ara, Cyanopsitta) gelungen. Auch hier konnten 14 Marker in verschiedenen Zellen von Hoden und Nebenhoden sichtbar gemacht werden. Die teilweise heterogene Verteilung der Marker in verschiedenen Zelltypen war eindeutig spezies-abhängig. Dies hat gezeigt, dass die beim Wellensittich mittels Immunzytochemie erzielten Resultate nur eingeschränkt auf andere Papageienspezies übertragbar sind. Entscheidend für die Beurteilung des Reproduktionsstatus ist daher die individuelle Auswahl der Marker in Abhängigkeit von der untersuchten Spezies. Die Resultate dieser Studie liefern die Grundlage für weitere Forschungsansätze in der Reproduktionsdiagnostik von Papageienvögeln. Zum einen können die etablierten Marker in Analyse-Systemen zum Einsatz kommen, die nicht-invasiv gewonnene Medien (z. B. Faezes) untersuchen und vor allem in Zuchterhaltungsprogrammen bedrohter Arten hilfreich sind. Zum anderen ist die immunzytochemische Untersuchung von Hodenbioptaten pathologisch veränderter Hoden (z. B. Tumoren oder Entzündungen) als eine sinnvolle Ergänzung der Diagnostik von Infertilität bei männlichen Psittaziden anzusehen.

Muscle wasting is a significant complication of many diseases including diabetes mellitus, renal and liver failure, HIV/AIDS, and cancer. Sustained loss of skeletal muscle can severely impair a patient's quality of life and often results in poor tolerance and responsiveness to disease treatments. The increased protein breakdown observed during muscle atrophy has been attributed to accelerated activity of the ubiquitin-proteasome pathway, but the precise mechanisms by which this activation stimulates muscle protein loss are poorly understood. Previous work showed that the deubiquitinating enzyme USP19 is upregulated in rat skeletal muscle in various forms of muscle wasting, including streptozotocin induced diabetes, cancer, and dexamethasone treatment. 1 To further explore the role of USP19 in muscle wasting, siRNA-mediated depletion of the enzyme was carried out in L6 myotubes. Knockdown of USP19 resulted in more rapid differentiation of myoblasts into myotubes, with a greater extent of myoblast fusion. It also produced tubes that were visibly larger than those formed by myoblasts transfected with a control siRNA. At the molecular level, silencing of USP19 increased the amount of myosin heavy chain (MHC) and tropomyosin proteins. It also increased levels of MHC transcript, suggesting that USP19 acts at the level of gene transcription or mRNA stability rather than protein degradation. USP19 may mediate its effects on muscle-specific gene expression through the myogenic transcription factor myogenin, since depletion of USP19 increased protein and mRNA levels myogenin but did not affect protein levels of the related transcription factor Myf5. Moreover, the increased tropomyosin and MHC observed upon USP19 knockdown could be abolished when myogenin was simultaneously depleted using siRNA. Collectively, these results suggest that USP19 functions to inhibit the synthesis of key muscle proteins and may therefore be a promising target for the treatment of muscle atrophy.

The shikimate pathway is a biosynthetic pathway that is responsible for producing a variety of organic compounds that are necessary for life in plants and microorganisms. The pathway consists of seven enzyme catalysed reactions beginning with the condensation reaction between D-erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP) to give the seven-carbon sugar DAH7P. This thesis describes the design, synthesis and evaluation of a range of alternative non-natural four-carbon analogues of E4P (2- and 3-deoxyE4P, 3-methylE4P, phosphonate analogues of E4P) to probe the substrate specificity of different types of DAH7P synthases [such as Mycobacterium tuberculosis (a type II DAH7PS), Escherichia coli (a type Ialpha DAH7PS) and Pyrococcus furiosus (a type Ibeta DAH7PS)].

Gilvocarcin V (GV) and ravidomycin (RMV) exhibit excellent antitumor activities in the presence of near-UV light at low concentration maintaining a low in vivo cytotoxicity. Although, the exact molecular mechanism for in vivo actions of these antibiotics has yet to be determined, a [2+2] cycloaddition reaction of the vinyl side chain with DNA thymidine residues in addition to the inhibition of topoisomerase II and DNAhistone H3 cross-linking are reported for the GV’s mechanism of action. Such activities have made these molecules interesting candidates for the biosynthetic investigation to generate analogues with improved activity/solubility. Previous biosynthetic studies have suggested that the GV biosynthetic pathway involves a number of synchronously occurring transformations leading to the oxidative C-C bond cleavage and other intriguing biosynthetic reactions, such as the vinyl side chain formation, methylations, Cglycosylation and dehydrogenation. Although gene inactivation results identified many candidate genes whose corresponding enzymes are involved in these biochemical transformations, their exact functional roles and the identity of their natural substrates remained elusive. To provide more insights into these complex biochemical tranfrormations, three specific aims were set up. Specific aim 1 was to clone and characterize the RMV biosynthetic gene cluster. Through the comparison of GV cluster with the RMV cluster, the genes encoding the biosynthesis of sugar and tetracyclic aromatic moieties were identified. RavGT, the sole glycosyltransferase of the RMV cluster has demonstrated to have unprecedented sugar donor substrate flexibility, transferring an amino-pyranose sugar as well as a neutral furanose sugar. Specific aim 2 was to characterize all of the TDP-D-ravidosamine biosynthetic enzymes. The aim also included to a one-pot enzymatic synthetic protocol for the routine production of TDP-D-ravidosamine. Specific aim 3 focussed on a total enzymatic synthesis of defucogilvocarcin M (defucoGM), the polyketide-derived core of GV and RMV. This aim clearly identified the minimal enzymes required to biosynthesize the complex architecture of defucoGM from the simple building blocks acetate and malonate. In addition, the GV-pathway enzyme GilR was fully characterized. Through in vitro studies, GilR was shown to catalyze the dehydrogenation of hemiacetal moiety of the penultimate intermediate pregilvocarcin V to the lactone moiety of GV at the last step.

Gilvocarcin V (GV), the principal product of Streptomyces griseoflavus Gö 3592 and other Streptomyces spp., is the most prominent member of a distinct class of antitumor antibiotics that share a polyketide derived coumarin-based aromatic core. GV and other members of this class including polycarcin V from Streptomyces polyformus, often referred to as gilvocarcin-like aryl C-glycosides, are particularly interesting because of their potent bactericidal, virucidal and antitumor activities at low concentrations while maintaining low in vivo toxicity. Although the precise molecular mechanism of GV bioactivity is unknown, gilvocarcin V has been shown to undergo a photoactivated [2+2] cycloaddition of its vinyl side chain with thymine residues of DNA in near-UV or visible blue light. In addition, GV was shown to selectively crosslink histone H3 with DNA, thereby effectively disrupting normal cellular processes such as transcription. Furthermore, GVs ability to inhibit topoisomerase II has also been attributed as a mechanism of action for gilvocarcin V activity. The excellent antitumor activity, as well as an unprecedented structural architecture, has made GV an ideal candidate for biosynthetic studies toward the development of novel analogues with improved pharmacological properties. Previous biosynthetic research has identified several candidate genes responsible for key steps during the biosynthesis of gilvocarcin V including an oxygenase cascade leading to C-C bond cleavage, methylations, lactone formation, C-glycosylation and vinyl side chain formation. In this study, we further examined two critical biosynthetic transformations essential for the bioactivity of gilvocarcin V, namely starter unit incorporation and C-glycosylation, through the following specific aims: 1) creation of functional chimeric C-glycosyltransferases through domain swapping of gilvocarcin-like glycosyltransferases and identification and evaluation of the donor substrate flexibility of PlcGT, the polycarcin V pathway specific C-glycosyltransferase; 2) creation of a library of O-methylated-L-rhamnose analogues of polycarcin V for structure activity relationship studies; 3) identification of the role of GilP and GilQ in starter unit specificity during gilvocarcin V biosynthesis; and 4) creation of a plasmid based approach in which selective gilvocarcin biosynthetic genes were utilized to produce important gilvocarcin intermediates for further in vivo and in vitro experimentation.

This thesis research started with analysis of oat fatty acids by using three different transmethylation methods. Basic sodium methoxide was compared with traditional acidic methanol for the total fatty acid analysis, while diazomethane was used to analyze free fatty acids. Epoxy FAs were readily hydrolyzed to dihydroxy fatty acids under the acidic condition, which suggest an overestimation of hydroxyl fatty acids and underestimation of epoxy fatty acids in previous analyses. The sodium methoxide method proved more reliable to quantify the oat seed fatty acid composition. CDC Dancer oat seed analyzed here was comprised mostly of palmitic acid (PA), oleic acid (OA) and the polyunsaturated fatty acid (PUFA) linoleic acid (LA) in quantities of 23%, 32%, and 37% of total seed FA, respectively. As well, the seed contained small quantities of another PUFA, α-linolenic (ALA) and several unusual oxygenated fatty acids (UFAs), Δ15-hydroxy fatty acid (15HFA) and epoxy fatty acids in quantities of 0.85%, 0.68%, and 2.3%, respectively. This thesis further aimed to identify and assemble all FAD2-like genes from an oat Expressed-Sequence Tag (EST) database using FAD2 and FAD2-like proteins from other organisms as query sequences in order to clone all putative FAD2-like genes-of-interest (GOIs) from oat. From the contig assemblies of retrieved oat ESTs, four distinct, putative genes were identified. From the Δ12-desaturase (FAD2) queries, a putative FAD2-like (AsFAD2) gene was identified; the Δ15-desaturase (FAD3) queries revealed two putative oat FAD3-like (AsFAD3-1 and AsFAD3-2) genes, while an ω-3 desaturase (FAD7) query identified a fourth putative full-length FAD6-like coding sequence of two possible lengths, AsFADX and AsFADX+. The GOIs were then subcloned into a yeast expression vector and functionally characterized. AsFAD2a and AsFAD2b both demonstrated Δ12 desaturation on 18:1-9c substrate. AsFAD3-1 had no activity on any substrates present, while AsFAD3-2 exhibited weak Δ15-desaturation activity specifically on 18:2-9c,12c. Finally, AsFADX converted 18:1-9c to 18:2-9c,12c, while AsFADX+ had no activity. Then, a comparative analysis of transcript levels of these GOIs via quantitative real-time PCR (qRT-PCR) was performed across oat germinating seed, root, leaf, and developing seed. AsFAD2 transcript abundance was generally much higher than AsFAD3-1 and AsFAD3-2 in all tissues. AsFAD3-1 mRNA level was highest in developing seed tissue, slightly lower in leaf tissue, and lowest in root. AsFAD3-2 mRNA was highest in germinating seed, and lowest in leaf tissue. In summary, the data produced from this thesis could be used to enhance breeding efforts for establishing oat cultivars with healthier oil content.

Mutations in the SLC4A1 gene result in misfolding and trafficking defects of the human erythroid (AE1) and kidney (kAE1) forms of the anion exchanger 1 glycoprotein. This affects the amount of functional protein at the cell surface, resulting in hematological and renal diseases. In this thesis, the role of the quality control system of molecular chaperones (cytosolic and ER) was examined during the biosynthesis of wild type and mutant AE1 in different cellular models. The hypothesis to be tested is that molecular chaperones are responsible for the intracellular retention of AE1 mutants. Chaperones were found to interact with AE1 and kAE1 in vitro and in vivo (HEK-293, K562, MDCK cells). Disruption of the calnexin-AE1 interaction in K562 cells did not affect the cell surface levels of wild type or mutant erythroid AE1. AE1 also trafficked to the cell surface in mouse embryonic fibroblasts completely deficient in calnexin or calreticulin. In contrast, in MDCK cells, disruption of the calnexin-kAE1 interaction allowed functional dominant (R589H, R901stop), but not misfolded kAE1 mutants (kSAO, G701D), to escape the ER and traffic to the cell surface. Calnexin is therefore not required for the cell surface expression of erythroid AE1, but can be responsible for the intracellular retention of certain kAE1 mutants in cells with the complete complement of molecular chaperones. Components involved in membrane glycoprotein folding and quality control (calnexin, ERp57, Hsc70, Hsp70), were lost at later stages during the differentiation of CD34+ erythroid progenitor cells. This suggests that the loss of molecular chaperones may facilitate the massive production of red cell glycoproteins, allowing erythroid AE1 mutants to escape quality control, traffic to the plasma membrane, and be present in mature red blood cells. These studies demonstrate that the role chaperones play varies, depending on cellular context. By understanding the cellular context and factors involved, therapeutic strategies may be tailored to deal with protein misfolding diseases, and in the case of kAE1, rescue the cell surface trafficking of misfolded, but functional, transport protein using pharmacological modulators.