Biotransformation of polycylic aromatic compounds by fungi and an investigation into the oxidation of alkylbenzenes by Mortierella isabellina NRRL 1757

Incubations of several polycyclic heteroaromatic compounds and two polycyclic aromatic hydrocarbons with a series of common fungi have been performed. The fungi Cunninghamella elegans ATCC 26269, Rhizopus arrhizus ATCC 11145, and Mortierella isabellina NRRL 1757 were studied in this regard. Of the aza heteroaromatics, only dibenzopyrrole gave a ring hydroxylated product following the incubation with C. elegans. From the thio heteroaromatics studied, dibenzothiophene was metabolized by all the three fungi and thioxanthone by C. elegans and M. isabellina giving sulfones and sulphoxides. Thiochromanone was metabolized stereoselectively to the corresponding sulphoxide by C. elegans. Methyl substituted thioxanthones on incubation with C. elegans produced oxidative products, arising from S -oxidation and hydroxylation at the methyl group. Of the cyclic ketones studied, only fluorenone was reduced to hydroxyfluorene and this metabolism is compared with that reported with cytochrome P-450 monooxygenases of hepatic microsomes. A series of para-substituted ethylbenzenes has been transformed stereoselectively to the 1-phenylethanols by incubation with M. isabellina. Comparisons of the enantiomeric purities obtained from products with their respective para substituent of the same steric size but different electronic properties indicate that the stereoselectivity of hydroxylation at benzylic carbon may be susceptible to electron donating or withdrawing factors in some cases, but that observation is not va lid in all the comparisons. The stereochemistry of the reaction is discussed in terms of three possible steps, ethylbenzene ---) 1-phenylethanol ---) acetophenone ---) 1-phenylethanol. This metabolic pathway could account for the inconsistencies observed in the comparisons of optical purities and electronic character of para substituents. Furthermore, formation of 2-phenylethanol (in some cases), l-(p-acetylphenyl)ethanol from p-diethylbenzene, and N-acetylation of p-ethylaniline was observed. n-Propylbenzene was also converted to optically active 1-phenylpropanol. Acetophenone, p-ethylacetophenone, and o(,~,~-trifluoroacetophenone were transformed to 1-phenylethanol, l-(p-ethylphenyl)ethanol, and 1-phenyl-2,2,2-trifluoroethanol, respectively, with high chemical and excellent optical yields. The 13 C NMR spectra of several substrates and metabolic products have been reported and assigned for the first time.

Icewine fermentation by Saccharomyces cervisiae : fundamental stress responses and comparative fermentation dynamics

Please consult the paper edition of this thesis to read. It is available on the 5th Floor of the Library at Call Number: Z 9999.5 B63 P54 2007

Metabolic, biochemical and molecular profiling of Catharanthus roseus flower cultivars and transformed hairy roots for monoterpenoid indole alkaloid accumulation /

Catharanthlls rosellS (L.) G Don is a commercially significant flower species and in addition is the only source of the monoterpenoid indole alkaloids (MIA) vinblastine and vincristine, which are key pharmaceutical compounds that are used to combat a number of different cancers. Therefore, procurement of the antineoplastic agents is difficult but essential procedure. Alternatively, CatharanthllS tissue cultures have been investigated as a source of these agents; however they do not produce vindoline, which is an obligate precursor to vinblastine and vincristine. The interest in developing high MIA cultivars of Catharantlws rosellS has prompted metabolic profiling studies to determine the variability of MIA accumulation of existing flowering cultivars, with particular focus on the vindoline component ofthe pathway. Metabolic profiling studies that used high performance liquid chromatography of MIAs from seedlings and young leaf extracts from 50 different flowering cultivars showed that, except for a single low vindoline cultivar (Vinca Mediterranean DP Orchid), they all accumulate similar levels of MIAs. Further enzymatic studies with extracts from young leaves and from developing seedlings showed that the low vindoline cultivar has a IO-fold lower tabersonine-16-hydroxylase activity than those of CatharanthllS rosellS cv Little Delicata. Additionally, studies aimed at metabolic engineering ofvindoline bios}l1thesis in Catharanthus rosellS hairy root cultures have been performed by expressing the last step in vindoline biosynthesis [Dcacetylvindoline-4-0- acetyltransferase (DAT)]. Enzymatic profiling studies with transformed hairy roots have confirmed that over-expressing DAT leads to lines with high levels of O-acetyltransferase activity when compared to non-expressing hairy roots. One particular DA T over111 expressing hairy root culture (line 7) contained 200 times the OAT activity than leaves of control lines. Additional MIA analyses revealed that DAT over-expressing hairy roots have an altered alkaloid profile with significant variation in the accumulation of h6rhammericine. Further analysis of transformed hairy root line 7 suggests a correlation between the expression of OAT activity and h6rhammericine accumulation with root maturation. These studies show that metabolic and selective enzymatic profiling can enhance our ability to search for relevant MIA pathway mutants and that genetic engineering with appropriate pathway genes shows promise as a tool to modify the MIA profile of Catharanthus roseus.

INFLUENCE OF INCREASED EXTRACELLULAR LEUCINE ON THE PROTEIN METABOLIC RESPONSES DURING OSMOTIC STRESS IN SKELETAL MUSCLE

The purpose of this study was to examine the effects of increased extracellular leucine concentration on protein metabolism in skeletal muscle cells when exposed to 3 different osmotic stresses. L6 skeletal muscle cells were incubated in either a normal or supplemental leucine (1.5mM) medium set to hypo-osmotic (230 ± 10 Osm), iso-osmotic (330 ± 10 Osm) or hyper-osmotic (440 ± 10 Osm) conditions. 3H-tyrosine was used to quantify protein synthesis. Western blotting analysis was performed to determine the activation of mTOR, p70S6k, ubiquitin, actin, and μ-calpain. Hypo-osmotic stress resulted in the greatest increase in protein synthesis rate under the normal-leucine condition while iso-osmotic stress has the greatest increase under the elevated-leucine condition. Elevated-leucine condition had a decreased rate in protein degradation over the normal condition within the ubiquitin proteasome pathway (p<0.05). Leucine and hypo-osmotic stress therefore creates a favourable environment for anabolic events to occur.

Level and precision of behavioural thermoregulation in the bearded dragon, Pogona vitticeps : effects of hypoxia and environmental thermal quality /

Most metabolic functions are optimized within a narrow range of body temperatures, which is why thermoregulation is of great importance for the survival and overall fitness of an animal. It has been proposed that lizards will thermoregulate less precisely in low thermal quality environments, where the costs associated with thermoregulation are high; in the case of lizards, whose thermoregulation is mainly behavioural, the primary costs ofthermoregulation are those derived from locomotion. Decreasing thermoregulatory precision in costly situations is a strategy that enhances fitness by allowing lizards to be more flexible to changing environmental conditions. It allows animals to maximize the benefits of maintaining a relatively high body temperature while minimizing energy expenditure. In situations where oxygen concentration is low, the costs of thermoregulation are relatively high (i.e. in relation to the amount of oxygen available for metabolic functions). As a result, it is likely that exposures to hypoxic conditions induce a decrease in the precision of thermoregulation. This study evaluated the effects of hypoxia and low environmental thermal quality, two energetically costly conditions, on the precision and level of thermoregulation in the bearded dragon, Pogona vitticeps, in an electronic temperature-choice shuttle box. Four levels of hypoxia (1O, 7, 5 and 4% 02) were tested. Environmental thermal quality was manipulated by varying the rate of temperature change (oTa) in an electronic temperature-choice shuttle box. Higher oT a's translate into more thermally challenging environments, since under these conditions the animals are forced to move a greater number of times (and hence invest more energy in locomotion) to maintain similar temperatures than at lower oTa's. In addition, lizards were tested in an "extreme temperatures" treatment during which air temperatures of the hot and cold compartments of the shuttle box were maintained at a constant 50 and 15°C respectively. This was considered the most thermally challenging environment. The selected ambient (T a) and internal body temperatures (Tb) of bearded dragons, as well as the thermoregulatory precision (measured by the central 68% ofthe Ta and T b distribution) were evaluated. The thermoregulatory response was similar to both conditions. A significant increase in the size of the Tb range, reflecting a decrease in thermoregulatory precision, and a drop in preferred body temperature of ~2 °C, were observed at both 4% oxygen and at the environment of lowest thermal quality. The present study suggests that in energetically costly situations, such as the ones tested in this study, the bearded dragon reduces energy expenditure by decreasing preferred body temperature and minimizing locomotion, at the expense of precise behavioural thermoregulation. The close similarity of the behavioural thermoregulatory response to two very different stimuli suggests a possible common mechanism and neuronal pathway to the thermoregulatory response.

Lifespan, body size and oxygen : aerobic metabolism and oxidative stress in cultured fibroblasts /

The allometric scaling relationship observed between metabolic rate (MR) and species body mass can be partially explained by differences in cellular MR (Porter & Brand, 1995). Here, I studied cultured cell lines derived from ten mammalian species to determine whether cells propagated in an identical environment exhibited MR scaling. Oxidative and anaerobic metabolic parameters did not scale significantly with donor body mass in cultured cells, indicating the absence of an intrinsic MR setpoint. The rate of oxygen delivery has been proposed to limit cellular metabolic rates in larger organisms (West et al., 2002). As such cells were cultured under a variety of physiologically relevant oxygen tensions to investigate the effect of oxygen on cellular metabolic rates. Exposure to higher medium oxygen tensions resulted in increased metabolic rates in all cells. Higher MRs have the potential to produce more reactive oxygen species (ROS) which could cause genomic instability and thus reduced lifespan. Longer-lived species are more resistant to oxidative stress (Kapahi et al, 1999), which may be due to greater antioxidant and/or DNA repair capacities. This hypothesis was addressed by culturing primary dermal fibroblasts from eight mammalian species ranging in maximum lifespan from 5 to 120 years. Only the antioxidant manganese superoxide dismutases (MnSOD) positively scaled with species lifespan (p<0.01). Oxidative damage to DNA is primarily repaired by the base excision repair (BER) pathway. BER enzyme activities showed either no correlation or as in the case of polymerase p correlated, negatively with donor species (p<0.01 ). Typically, mammalian cells are cultured in a 20% O2 (atmospheric) environment, which is several-fold higher than cells experience in vivo. Therefore, the secondary aim of this study was to determine the effect of culturing mammalian cells at a more physiological oxygen tension (3%) on BER, and antioxidant, enzyme activities. Consistently, standard culture conditions induce higher antioxidant and DNA ba.se excision repair activities than are present under a more physiological oxygen concentration. Therefore, standard culture conditions are inappropriate for studies of oxidative stress-induced activities and species differences in fibroblast DNA BER repair capacities may represent differences in ability to respond to oxidative stress. An interesting outcome firom this study was that some inherent cellular properties are maintained in culture (i.e. stress responses) while others are not (i.e. MR).

Elucidation of the Retinoid Signalling Pathway Involved in Axon Guidance in Lymnaea stagnalis and Xenopus laevis

The vitamin A metabolite, retinoic acid (RA), is known to play a crucial role in several developmental processes including axial patterning and differentiation. More recently, RA has been implicated in the regenerative process acting through its classical signaling pathway, the nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), to mediate gene transcription. Moreover, RA has been shown to act as a guidance molecule for growth cones of regenerating motorneurons of the pond snail, Lymnaea stagnalis. Our lab has recently shown that RA can induce this morphological response independent of nuclear transcription, however, the role of the retinoid receptors in RA-induced chemoattraction is still unknown. Here, I show that the retinoid receptors, RXR and RAR, may mediate the growth cones response to the metabolically active retinoic acid isomers, all-trans and 9-cis RA, in Lymnaea stagnalis. Data presented here show that both an RXR and RAR antagonist can block growth cone turning in response to application of both isomers. Because no prior investigations have shown growth cone turning of individual vertebrate neurons, I aimed to show that both retinoic acid isomers were capable of inducing growth cone turning of embryonic spinal cord neurons in the frog, Xenopus laevis. For the first time in Xenopus, I showed that both all-trans and 9-cis RA were able to induce significantly more neurite outgrowth from cultured embryonic spinal cord neurons and induce positive growth cone turning of individual growth cones. In addition, I showed that the presence of the RXR antagonist, HX531, blocked 9-cis RA-induced growth cone turning and the RARβ antagonist, LE135, blocked all-trans RA-induced growth cone turning in this species. Evidence provided here shows for the first time, conservation of retinoic acid-induced growth cone turning in a vertebrate model system. In addition, these data show that the receptors involved in this morphological response may be the same in vertebrates and invertebrates.

Functional Characterization of Monoterpenoid Indole Alkaloid (MIA) Biosynthetic Genes in Catharanthus roseus

The monoterpenoid indole alkaloids (MIAs) of Madagascar periwinkle (Catharanthus roseus) are known to be among the most important source of natural drugs used in various cancer chemotherapies. MIAs are derived by combining the iridoid secologanin with tryptamine to form the central precursor strictosidine that is then converted to most known MIAs, such as catharanthine and vindoline that dimerize to form anticancer vinblastine and vincristine. While their assembly is still poorly understood, the complex multistep pathways involved occur in several specialized cell types within leaves that are regulated by developmental and environmental cues. The organization of MIA pathways is also coupled to secretory mechanisms that allow the accumulation of catharanthine in the waxy leaf surface, separated from vindoline found within leaf cells. While the spatial separation of catharanthine and vindoline provides an explanation for the low levels of dimeric MIAs found in the plants, the secretion of catharanthine to the leaf surface is shown to be part of plant defense mechanisms against fungal infection and insect herbivores. The transcriptomic databases of Catharanthus roseus and various MIA producing plants are facilitating bioinformatic approaches to identify novel MIA biosynthetic genes. Virus-induced gene silencing (VIGS) is being used to screen these candidate genes for their involvement in iridoid biosynthesis pathway, especially in the identification of 7-deoxyloganic acid 7-hydroxylase (CrDL7H) shown by the accumulation of its substrate, 7-deoxyloganic acid and decreased level of secologanin along with catharanthine and vindoline. VIGS can also confirm the biochemical function of genes being identified, such as in the glucosylation of 7-deoxyloganetic acid by CrUGT8 shown by decreased level of secologanin and MIAs within silenced plants. Silencing of other iridoid biosynthetic genes, loganic acid O-methyltransferase (LAMT) and secologanin synthase (SLS) also confirm the metabolic route for iridoid biosynthesis in planta through 7-deoxyloganic acid, loganic acid, and loganin intermediates. This route is validated by high substrate specificity of CrUGT8 for 7-deoxyloganetic acid and CrDL7H for 7-deoxyloganic acid. Further localization studies of CrUGT8 and CrDL7H also show that these genes are preferentially expressed within Catharanthus leaves rather than in epidermal cells where the last two steps of secologanin biosynthesis occur.