<i>Claviceps purpurea</i>, a fungal pathogen, of ergot diseases in agriculturally important cereal crops, produces high levels of glycerides containing ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) in its sclerotia. A fatty acid hydroxylase (CpFAH) involved in the biosynthesis of ricinoleic acid was recently identified from <i>C. purpurea</i>. This research describes the biochemical characterization of a type II diacylglycerol acyltransferase (CpDGAT2) involved in the assembly of this fatty acid into triglycerides from <i>C. purpurea</i>. Expression of CpDGAT2 in a quadruple mutant <i>Saccharomyses cerevisiae</i> H1246, in which all four triacylglycerol (TG) biosynthesis genes (DGA1, LOR1, ACAT1 and ACAT2) were disrupted, restored the ability of the mutant to synthesize TGs <i>in vivo</i>. <i>In vitro</i> enzymatic assays of microsomal preparations of the transformants indicated that CpDGAT2 preferentially use ricinoleic acid over linoleic acid, oleic acid and linolenic acids as acyl donor, and 1,2-dioleoyl-sn-glycerol over 1,2-dipalmitoyl-sn-glycerol as acyl acceptor. CpDGAT2 did not show any activities for the formation of wax esters and estolides when 1-hexadecanol and triricinolein were used as acyl acceptors. Co-expression of CpFAH and CpDGAT2 in yeast resulted in increased accumulation of ricinoleic acids compared to expression of CpFAH along with the yeast native DGAT2 (ScDGA1) or expression of CpFAH alone. Northern blot analysis indicated that CpFAH is solely expressed in sclerotium cells and no transcripts of this gene were detected in mycelium and conidium cells. CpDGAT2 is more widely expressed in cell types examined except for conidiospores where the expression is low. The highest expression of CpDGAT2 was detected in 20 day-old sclerotium cells where the highest levels of ricinoleate glycerides are accumulated. Collectively, these data indicate CpDGAT2 and CpFAH are two key enzymes coordinating the biosynthesis and bioassembly of ricinoleic acid in <i>C. purpurea</i>.

Recently, the zebrafish has been considered an important animal model that can be used to investigate human diseases and drug development. Parkinson¡¦s disease (PD), an important neurodegenerative disorder, is characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra and movement defects, including bradykinesia, tremor, and postural imbalance. However, current treatments for PD are limited and mainly improve only the clinical symptoms of the disease. Thus, a neurodegenerative rat model has been widely used for a long while to search for a new treatment for PD. However, the use of rats as an animal model has certain limitations such as breeding, efficiency, and high dosage. Recently, researchers indicated that neurotoxins such as rotenone, 6-hydroxydopamine (6-OHDA), and paraquat can induce Parkinson¡¦s-like symptoms in zebrafish, and this may be a useful PD model because of the complete development of the zebrafish nervous system, low costs, and low dosage. In this study, we treated zebrafish with 6-OHDA and analyzed their locomotor activity to establish an in vivo animal model of PD. Then, we analyzed the mRNA expression of parkin and PINK1 by reverse transcription¡Vpolymerase chain reaction (RT-PCR).Moreover, we observed tyrosine hydroxylase (TH) expression by immunohistochemical (IHC) staining to confirm if this can be used as a PD model. Finally, we found that treatment with 6-OHDA significantly reduced TH expression. We observed a similar declining trend in the case of mammals. Likewise, parkin and PINK1 mRNA expressions were also decreased after treatment with 6-OHDA. In summary, our study provides a feasible in vivo Parkinson¡¦s model, and a small volume of drugs or compounds can be screened using this model.

Tyrosine hydroxylase (TyrH) catalyzes the pterin-dependent hydroxylation of tyrosine to form dihydroxyphenylalanine. The enzyme requires one atom of ferrous iron for activity. Using deuterated 4-methylphenylalanine substrates, intrinsic primary and secondary isotope effects of 9.6 ± 0.9 and 1.21 ± 0.08 have been determined for benzylic hydroxylation catalyzed by TyrH. The large, normal secondary isotope effect is consistent with a mechanism involving hydrogen atom abstraction to generate a radical intermediate. The similarity of the isotope effects to those measured for benzylic hydroxylation catalyzed by cytochrome P-450 suggests that a high-valent, ferryl-oxo species is the hydroxylating species in TyrH. Uncoupled mutant forms of TyrH have been utilized to unmask isotope effects on steps in the aromatic hydroxylation pathway which also implicate a ferryl-oxo intermediate. Inverse secondary isotope effects were seen when 3,5-2H2-tyrosine was used as a substrate for several mutant enzyme forms. This result is consistent with a direct attack by a ferryl-oxo species on the aromatic ring of tyrosine forming a cationic intermediate. Rapid-freeze quench Mössbauer studies have provided preliminary spectroscopic evidence for an Fe(IV) intermediate in the reaction catalyzed by TyrH. The role of the iron atom in the regulatory mechanism has also been investigated. The iron atom in TyrH, as isolated, is in the ferric form and must be reduced for activity. The iron can be reduced by a number of one-electron reductants including tetrahydrobiopterin, ascorbate, and glutathione; however, it appears that BH4 (kred = 2.8 ± 0.1 mM-1 s-1) is the most likely candidate for reducing the enzyme in vivo. A one-electron transfer would require a pterin radical. Rapid-freeze quench EPR experiments aimed at detecting the intermediate were unsuccessful, suggesting that it decays very rapidly by reducing another equivalent of enzyme. The active Fe(II) form can also become oxidized by oxygen (210 ± 30 M-1 s-1); this increases the affinity of catecholamine inhibitors. Serine 40 can be phosphorylated to relieve the inhibition; however, results with S40E TyrH show phosphorylation does not have an effect on the rate constant for reduction of the enzyme but causes a 40% decrease in the rate constant of oxidation.

Research Doctorate - Doctor of Philosopy (PhD) / Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated acutely by a combination of phosphorylation of three key serine (Ser) residues (Ser19, Ser31 and Ser40), and feedback inhibition by the catecholamines. Phosphorylation of Ser40 directly increases TH activity by relieving feedback inhibition of the enzyme. The phosphorylation of Ser19 or Ser31 can potentiate the phosphorylation of Ser40 in a process known as hierarchical phosphorylation. The 2 major human TH isoforms, hTH1 and hTH2, are differentially regulated by hierarchical phosphorylation in vitro. In this study, the human neuroblastoma SH-SY5Y cell line has been transfected with hTH1 and hTH2, and it has been demonstrated that phosphorylation of Ser31 potentiates the phosphorylation of Ser40 in hTH1. Phosphorylation of the equivalent Ser31 residue in hTH2 was not detectable, and thus this enzyme is not subject to Ser31-mediated hierarchical phosphorylation of Ser40 in situ. This is the first study to demonstrate that hTH1 and hTH2 are differentially regulated in situ. In addition, we have examined the nature of feedback inhibition of TH by the catecholamines. In addition to the high affinity, non-dissociable dopamine binding that is relieved by Ser40 phosphorylation, we have identified a second low affinity, readily dissociable binding site which regulates TH activity both in vitro and in situ regardless of the phosphorylation state of the enzyme. This low affinity binding site responds to changes in cytosolic catecholamine levels in situ in order to regulate TH activity. This work has contributed to our understanding of the complex nature of the regulation of TH activity.

Thesis (Ph.D.)--Kent State University, 2009. / Title from PDF t.p. (viewed April 9, 2010). Advisor: John Chiang. Includes bibliographical references (p. 225-256).

Abstract An efficient system for expressing recombinant human collagens is expected to have numerous scientific and medical applications, but this is difficult to achieve because most systems do not have sufficient levels of activity of prolyl 4-hydroxylase, the key enzyme of collagen synthesis. A recombinant form of human type II collagen, the main structural component of cartilage, was produced here in insect cells by coinfecting them with two baculoviruses, one coding for the proα chains of human type II procollagen, and the other for both the α and β subunits of human prolyl 4-hydroxylase. The amino acid composition of the recombinant form was very similar to that of the non-recombinant protein, with the exception that the hydroxylysine content was very low. The highest expression levels obtained in suspension cultures were 50 mg/l. An additional baculovirus coding for human lysyl hydroxylase was used to express type II collagen with a high hydroxylysine content. Marked differences in the rate of fibril formation in vitro and the morphology of the resulting fibrils were found between the recombinant type II collagens having 2 and 19 hydroxylysine residues/1000 amino acids, the maximal turbidity of the former being reached within 5 min, whereas the absorbance of the latter increased up to about 10 h. In addition, the latter collagen formed thin fibrils, whereas the former produced thick fibrils on a background of thin ones. The data indicate that regulation of the extent of lysine hydroxylation, and consequently of the amounts of hydroxylysine-linked carbohydrate units, may have major effects on collagen fibril formation. In order to study the expression of recombinant human collagens in yeasts, cDNAs for the proα chains of procollagens of type I, II and III were transformed into a recombinant P. pastoris strain expressing human prolyl 4-hydroxylase subunits. All the P. pastoris strains obtained produced full-length proα chains. Cells coexpressing the proα1(I) chains and prolyl 4-hydroxylase produced homotrimeric type I procollagen molecules, whereas cells coexpressing the proα1(I) and proα2(I) chains and prolyl 4-hydroxylase produced heterotrimeric molecules with the correct 2:1 chain ratio. pCα1(I) and pCα2(I) chains lacking the N propeptides assembled into pCcollagen molecules and yielded correctly folded and fully hydroxylated collagen molecules upon pepsinization. The Tm values of recombinant type I-III collagens produced in shaker flasks were about 38°C and the degree of hydroxylation of proline residues was lower than that in the corresponding non-recombinant collagens. When the recombinant collagens were produced in a 2-litre fermentor equipped with an O2 supply system, the expression levels increased markedly to 0.2–0.6 g/l. In addition, all these collagens were identical in 4-hydroxyproline content to the corresponding non-recombinant proteins, and all of them formed native-type fibrils.

Abstract Collagen prolyl 4-hydroxylases catalyze the hydroxylation of proline residues in collagens. The vertebrate enzymes are α2β2 tetramers in which the β subunit is identical to protein disulphide isomerase (PDI). Two isoforms of the catalytic α subunit have been identified in vertebrates, forming type I [α(I)]2β2 and type II [α(II)]2β2 collagen prolyl 4-hydroxylase tetramers. This thesis reports on the cloning and characterization of a third vertebrate α subunit isoform, α(III). The recombinant human α(III) isoform associates with PDI to form an active type III collagen prolyl 4-hydroxylase tetramer, and its Km values for the cosubstrates are very similar to those of the type I and II enzymes, those for a peptide substrate and an inhibitor being found to lie between the two. The α(III) mRNA is expressed in all tissues studied but at much lower levels than the α(I) mRNA. A novel mixed tetramer PHY-1/PHY-2/(PDI-2)2 was found to be the main collagen prolyl 4-hydroxylase form produced in the nematode Caenorhabditis elegans in vivo and in vitro. However, mutant nematodes can compensate for the lack of the mixed tetramer by increasing the assembly of PHY-1/PDI-2 and PHY-2/PDI-2 dimers, these forms also being unique. The catalytic properties of the recombinant mixed tetramer were characterized, and it was shown by the analysis of mutant worms that PHY-1 and PHY-2 represent the only catalytic subunits needed for the hydroxylation of cuticular collagens. The roles of the two catalytic sites in a collagen prolyl 4-hydroxylase tetramer were studied by using the C. elegans mixed tetramer and a hybrid C. elegans PHY-1/human PDI dimer. An increase in the chain length of the peptide substrate led to an identical decrease in the Km values in both enzyme forms. It is thus clear that two catalytic sites are not required for efficient hydroxylation of long peptides, and their low Km values most probably result from more effective binding to the peptide-substrate-binding domain. Inactivation of one catalytic site in the mixed tetramer reduced the activity by more than 50%, indicating that the remaining wild-type subunit cannot function fully independently.

Abstract Adenoviral gene transfer is a valuable tool in molecular biology research. In order to be an efficient and safe vector, adenovirus structure and infection mechanism as well as molecular biology of the used transgene need to be well studied. The aim of this study was to evaluate the role of adenovirus as a gene transfer vector from several perspectives. Adenovirus uses receptor-mediated endocytosis in order to enter the target cell. The effect of Rab5 GTPase on adenovirus entry and gene transfer efficiency was examined first. Next, adenovirus was used as an investigatory tool in the cardiovascular research, focused on clarifying the role of adrenomedullin (AM) in heart and vascular remodeling. Finally, a model of adenoviral gene transfer into skin fibroblasts was used. The role of Rab5 GTPase in the adenovirus endocytosis was examined in HeLa cells using Cy3-labeled adenovirus, and gene transfer efficiency using β-galactosidase encoding adenovirus. Rab5 increased both adenovirus uptake and gene transfer, whereas dominant negative Rab5S34N decreased both endocytosis and gene transfer. The data indicate that Rab5 is needed in mediating the adenovirus uptake into the target cell. In the rat heart, adenovirus-mediated AM gene transfer transiently improved systolic function both in vivo and in vitro. AM caused activation of translocation of protein kinases C ε and δ, whereas phosphorylation of p38 mitogen activated protein kinase was decreased in the left ventricle. AM significantly attenuated the development of angiotensin II-induced cardiac hypertrophy. In rats with myocardial infarction, AM enhanced dilatation of left ventricle and thinning of anterior wall. The role of AM in neointima formation was evaluated in rat artery after endothelial injury. Intravascular AM gene transfer decreased neointimal growth and increased neointimal myofibroblasts apoptosis. These results show that AM regulates left ventricular systolic function and remodeling in the heart, and plays a role in pathological vascular remodeling. Adenovirus-mediated lysyl hydroxylase (LH) gene transfer into skin fibroblasts of type VI Ehlers-Danlos syndrome patient and rat skin increased functional LH production, elevated LH activity, and human LH mRNA production both in vitro and in vivo. LH gene replacement therapy may thus lead to possibilities to improve skin wound healing in Ehlers-Danlos syndrome patients.

Abstract Lysyl hydroxylase (EC 1.14.11.4, procollagen-lysine 2-oxoglutarate 5-dioxygenase, PLOD) catalyses the formation of hydroxylysine in collagens and in the other collagen like proteins. Hydroxylysine participates in the formation of cross-links between collagen molecules and can bind to the carbohydrates, galactose and glucosylgalactose. Patients with the type VIA Ehlers-Danlos syndrome (EDS) have characteristically a deficiency in hydroxylysine of collagen in their skin that is caused by reduced activity of lysyl hydroxylase 1. In this work the mutations were studied in detail in four different Ehlers-Danlos VIA patients. The first patient characterized in this study had a duplication of seven exons in the lysyl hydroxylase gene 1. The mutation was caused by homologous recombination of two identical 44-nucleotide regions of Alu sequences in introns 9 and 16 in the gene. This study also suggests that uniparental isodisomy does not explain the homozygosity of the mutation. The second patient was found to have two mutations in the gene for lysyl hydroxylase 1 in a compound heterozygote state. The study resulted in the discovery of the first deletion mutation in the gene. The deletion was caused by an Alu-Alu recombination that removes about 3 kb from the gene including all the exon 17 sequences. The other mutation causes deletion of exon 16 from the mRNA. Deletion of the penultimate nucleotide of intron 15 destroys the consensus sequence of the intron/exon boundary and thus causes the deletion. The third patient was described to have a nonsense codon in exon 14 of one allele which causes a reduction in the amount of lysyl hydroxylase mRNA and leads to aberrant RNA splicing in the cell. The other allele was concluded to be operationally null. In the last work two novel null mutations were found in the gene for lysyl hydroxylase 1. The first was a one nucleotide deletion in the acceptor splice site of intron 4 and the other an insertion of a C nucleotide in exon 2. The abnormal alleles lead to markedly decreased lysyl hydroxylase mRNA levels. This work revealed many exon deleted splicing variants of lysyl hydroxylase mRNA which were first discovered in affected cells, but traces of similarly spliced mRNA species were also found in the cytoplasm of normal human skin fibroblasts. These data indicate that the splicing machinery of the cell is leaky. In this thesis, several types of stuctural mutations in the DNA were found to be responsible for lysyl hydroxylase deficiency in patients with type VIA variant of EDS. The different mechanisms causing these mutations were also studied in detail.

For centuries people have used plants as medicine or food additives with varying success to cure and prevent diseases. Written records about medicinal plants date back at least 5 000 years to the Sumerians. According to World Health Organization (WHO) around 80 % of the population in developing countries is dependent on herbal medicine for basic healthcare needs. Even at the dawn of the twenty-first century, 11 % of the 252 drugs considered as basic and essential by WHO were exclusively of flowering plant origin. The genus Helichrysum, belongs to the family Asteraceae and is represented by approximately 600 species in Africa, of which 244 species are indigenous in South Africa. In Helichrysum aureonitens, galangin is one of the flavonol compounds with good medicinal properties. H. aureonitens was targeted to be enhanced via cell suspension culture to potentially produce valuable natural products. In ethanol extracts of cell suspension cultures, galangin was not detected even though it was present in the leaves of the intact plants. Some other compounds were induced in higher amounts in the cells of H. aureonitens suspension cultures when compared to that produced in the intact plants. To find out the reasons for the absence of galangin in the cells of H. aureonitens suspension cultures, some of the intermediates of the 4’-OH biosynthetic pathway for production of flavonols were analyzed by GC-MS, including cinnamic acid, p-coumaric acid, naringenin and kaempferol. None of these were detected in the H. aureonitens cell suspension cultures. The major compound from H. aureonitens cell suspension cultures was isolated and identified as a new chlorophenol compound named 4-chloro-2-(hepta-1,3,5-triyne-1-yl)-phenol (a triyne). This triyne has previously been proposed as being an intermediate in the acetylene biosynthetic pathway in Helichrysum spp., however only the methyl ether form had previously been isolated from the roots of H. coriaceum. The triyne isolated from the H. aureonitens cell suspension cultures in the present study was detected in intact plant extracts, but at very low concentrations. Results of the anti-tuberculosis assay of the cell suspension culture extracts and the triyne showed that the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the ethanol extract of cell suspension cultures against Mycobacterium tuberculosis H37Rv were found to be 1.0 mg/ml and 2.0 mg/ml respectively and the triyne was not active at 200 µg/ml. The ethanol extract of the cell suspension cultures and the triyne were also evaluated for their cytotoxicity against monkey kidney Vero cells and human prostate epithelial carcinoma (DU145) cell lines. The inhibitory concentrations (IC50) of the crude extract and the triyne was found to be 12.11 and 1.51 µg/ml against the Vero cells respectively. The crude extract and the triyne showed similar activity in the prostate cancer cell lines by exhibiting IC50 values of 3.52 and 2.14 µg/ml respectively. The triyne therefore warrants further investigation for its potential as an anticancer drug. Flavonoids represent the major phenolic compounds which are responsible for the medicinal properties in the Helichrysum genus. Some of flavonols, including kaempferol, quercetin, and galangin are also present in H. aureonitens. In this study both galangin and kaempferol (containing a 4’-OH group) were detected in leaf samples of H. aureonitens. But GC-MS analysis of the leaf samples of H. aureonitens did not show the existence of biosynthetic intermediates such as p-coumaric acid and naringenin (compounds having a 4’-OH) while cinnamic acid and some other compounds with no OH at the 4’ position on the B ring, were detected. The chemical structure analysis of the isolated compounds showed that they are pinocembrin chalcone, pinocembrin, pinobanksin and galangin, all containing no OH group at the 4’ position. This indicates that some part of the 4’-OH biosynthetic pathway for 4’-OH flavonoids is not functional in H. aureonitens. Since the only (yet identified) enzyme responsible for hydroxylation at the 4’ position on the B ring is cinnamate 4-hydroxylase (C4H), it can be postulated that C4H might be able to hydroxylate other substrates in H. aureonitens plants. One copy of C4H was isolated and cloned from H. aureonitens. It has 1518-base pairs (including stop codon, TAA) and an open reading frame encoding a 506-amino-acid polypeptide. It showed the highest homologies to Echinacea angustifolia (Asteraceae) C4H with 83.6 % identity on the nucleotide level but 93 % identity on the amino acid level. The genomic DNA sequence of the isolated C4H from H. aureonitens indicates the presence of three introns with a longer size compared to the Arabidopsis thaliana C4H gene structure. The presence of the first intron has not been reported before in the C4H gene from other plants and it is therefore a new finding from the isolated C4H in H. aureonitens. To check the putative isolated C4H, the full length cDNA of C4H was isolated from H. aureonitens and for the first time integrated in a secreted expression vector, pPICZáC, and transformed into Pichia pastoris. After the 48 hrs induction protein was collected, precipitated by ammonium sulphate and finally column purified. The results of SDS-PAGE electrophoresis and Western blot showed the expression of a protein with a size of 50-60 kDa. The calculated mass of C4H with regarding to a polyhistidine tag is about 60.5 kDa. The secreted expression was found as an effective system for the production of a soluble C4H protein with easy purification. / Thesis (PhD)--University of Pretoria, 2009. / Plant Science / unrestricted