Pressure application of gamma-aminobutyric acid (GABA) to mouse spinal and rat DBG neurones maintained in culture evoked transient membrane currents. Using the whole-cell patch clamp technique, these currents were shown to primarily involve the flow of Cl-. The GABA-evoked whole-cell currents in both types of neurones were reversibly suppressed by the GABAA antagonist bicuculline. The barbiturate phenobarbitone reversibly potentiated GABA-evoked whole-cell currents in mouse spinal neurones. Attempts to look at the unitary currents activated by GABA in outside-out patches, revealed spontaneous unitary currents. The I-V relationships of the spontaneous currents were linear and had a reversal potential of OmV in symmetrically distributed Cl solutions. Changing the monovalent cation concentrations on one or both sides of the membrane patch had no effect on the amplitude or the reversal potential of the spontaneous currents. Replacing some of the Cl- in the patch pipette with the impermeant anion SO42- shifted the reversal potential to more negative values. These spontaneous currents in both types of neurones were blocked by bath perfusion of bicuculline. GABA-activated unitary currents in outside-out patches, the main conductance state in both types of neurones was 30pS. However, GABA could occasionally also activate other conductance levels. Spontaneous Cl- currents did not occur in cell-attached patches from mouse spinal and rat DRG neurones, suggesting that the spontaneous events in the outside-out patches did not represent the activity of voltage dependent Cl- channels. Alphaxalone, a steroid anaesthetic, potentiated GABA-evoked whole cell currents in both spinal and DRG neurones. At high (muM) concentrations, pressure application of alphaxalone evoked a membrane Cl- current; this current was reversibly suppressed by blcuculline and potentiated by phenobarbitone. Pregnanolone (5beta-pregnane-3x-ol-20-one) a progesterone metabolite at low (nM) concentrations reversibly enhanced GABA currents in spinal neurones. Pregnanolone at higher concentrations pressure applied to spinal neurones had a weak direct agonist action on the GABAA receptor. Pregnanolone prolonged the burst duration of GABA-activated unitary currents in outside-out patches from spinal neurones. Some of the actions of the steroids on the GABAA receptor were very similar to the barbiturates, bemegride, a respiratory stimulant was formerly used clinically to counteract barbiturate poisoning in man. Experiments were conducted to see whether bemegride could be used as a specific barbiturate antagonist. Bemegride reduced phenobarbitone enhanced GABA currents in mouse spinal neurones. However, bemegride alone also reduced GABA and pentobarbitone evoked currants to a similar extent. This is suggestive of a noncompetitive action on the GABAA receptor, therefore it was not used to elucidate the site of action of steroids.

Master of Agribusiness / Department of Agricultural Economics / Allen M. Featherstone / Micronutrient machines have been used successfully in the beef industry, however, their use was mostly for the addition of antibiotics into the rations. Their use in the dairy industry has been very limited. Feed cost is over 50% of the total cost on a typical dairy farm, thereby creating an area where minor changes in cost per cow can impact the bottom line. Because of the high feed cost on dairy farms, income over feed cost (IOFC) is one of the bench marks as to the overall farm financial health. The feed rations also impact animal health incidences and reproduction efficiencies. Micro machines can add small amounts of a desired nutrient or product, generally less than 56 grams (± 2 oz) into the cattle's daily total mixed rations (TMR). These micronutrients are generally expensive, and their inclusion into the rations of only cows that need that particular micronutrient is one benefit of a micro machine. Micro machines also take out the human error of mixing small accurate amounts and can easily track inventories. Benefits also include the control of on-farm shrink through dust control, and environmental stewardship of resources. Lastly, by creating options to accurately add micronutrients, milk production may be increased and health incidences reduced. The dairy industry is a virtually an untapped field for this technology and this research will explore if there is a benefit from their use. As feeding systems have evolved and milk production has continued to climb, innovative technologies will continue to be implemented. Increased financial pressures will also continue to cause producers to become more efficient with their resources. As production increases in any field, fine tuning of inputs becomes more exact. The rumen inner workings and how feedstuff blends affect rumen micros and the pH levels is an area in which there is much research completed, however, much more is still needed. The addition of micro machines to fine tune rations for dairy farms TMR rations can be a profitable way to manage income over feed cost, not only by saving money spent on micronutrients but by increasing production and reducing herd health incidences.

Certaines cyanobactéries photoautotrophes sont capables de fixer l’azote atmosphérique grâce à des cellules spécialisées, les hétérocytes. De plus, en aérobiose, comme ces cellules peuvent excréter de l’ammonium lorsque leurs activités glutamine synthétase sont partiellement inhibées. Elles sont considérées comme usines cellulaires potentielles pour une bioproduction d’engrais azoté. Nous utilisons une souche mutante de Anabaena variabilis PCC 7937-C9, cyanobactérie hétérocytée à taux de croissance élevé, pour étudier la capacité à produire de l’ammonium en photobioréacteurs. Les caractéristiques de croissance de cette souche ne différent pas significativement de celles de la souche sauvage, avec un taux de croissance spécifique maximal de 3.0 j–1 à 30°C. Nous montrons qu’une partie de l’azote excrété dans le milieu de culture est entrainé sous forme de NH3 par la phase gazeuse, expliquant ainsi des sous-estimations antérieures. Cette production dépend de la température, l’irradiance, le taux d’aération et la concentration en MSX. Des études cinétiques confirment que la production d’azote ammoniacal en phase liquide et en phase gazeuse est corrélée aux variations de pH. Une régulation pulsée de pH permet d’accroitre la production de NH3. Des cultures en chemostat confirment que les productions de NH3 gazeux sont maximales à pH 8.8. Une variation cyclique des teneurs en NH4 +/NH3 dissous semble réguler les teneurs en NH4 +/NH3 en dessous d’un seuil critique de 1.5 mmol L–1 via une consommation par les cellules végétatives. Ces caractéristiques physiologiques sont analysées pour une application potentielle à la fourniture d’azote à des cultures de microalgues oléagineuses. / Some photoautotrophic cyanobacteria species are able to fix dinitrogen thanks to specialized cells, the heterocyts. Moreover, these cells are known to secrete ammonia when the glutamine synthase activity is partially inhibited under aerobic conditions. They are considered as potential cell factories for fertilizer. The present study uses a mutant strain of Anabaena variabilis PCC 7937-C9, a fast-growing heterocytous cyanobacterium, to investigate the potential use of diazotrophic cyanobacteria in photobioreactors for ammonium production. The growth characteristics of this strain cultivated in chemostat cultures are not significantly different from those of the wild strain, with a maximal specific growth rate of 3.0 d–1 at 30°C. A part of the combined nitrogen excreted in the culture medium is shown to be stripped through the aeration of the cultures as NH3, indicating previous underestimation of NH4 +/NH3 excretion. This process is shown to be affected by parameters such as temperature, irradiance, gas flow rate and MSX concentrations. Kinetics study reveals that the dissolved NH4 +/NH3 as well as the gaseous NH3 productions are correlated to pH variations production; a pulse regulation of pH is used to increase the NH3 production. Chemostat cultures with pH regulation are used to confirm that maximal gaseous NH3 is produced at pH 8.8. A cyclic variation of dissolved NH4 +/NH3 seems to regulate the NH4 +/NH3 concentrations under a threshold level of 1.5 mmol L–1; uptake of NH4 + by vegetative cells seems to be involved. These physiological features are discussed in view of operative conditions for efficient nitrogen supply for production by oleaginous microalgae.

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Bibliography: leaves 359-376 / 376 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Agronomy, 1981

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The pathway representing methionine integration and excision is an increasingly important target in drug design. Methionine aminopeptidase (MetAP), a metalloprotease responsible for cleaving the N-terminal methionine from nascent peptides, has been the object of many studies aimed to produce potential anti-bacterial, anti-fungal and anti-angiogenic agents. Though clinical trials are underway for several of these compounds, like fumagillin and CKD-731, they are still flawed based on their relatively weak inhibition and their physiological side effects. Therefore, the search for novel and potent inhibitors continues. Previous work has utilized phosphinic and phosphonic acid derivatives of methionine in co-crystallization studies with Escherichia coli MetAP (eMetAP). The aim of the research presented in this work is to study and assay various phosphorus- and sulfur-containing compounds as inhibitors and substrates in an effort to learn more about the biochemical machinery underlying MetAP catalysis. As well, we outline a predictive molecular modeling approach to MetAP inhibitor design to assist in identifying lead candidates amongst a body of possible molecular inhibitors. Ultimately, we not only hope to have identified key functional properties of molecules potentially useful as MetAP inhibitors, but also to have contributed to the knowledge base of the mechanistic features involved in this enzyme’s catalysis.

The pathway representing methionine integration and excision is an increasingly important target in drug design. Methionine aminopeptidase (MetAP), a metalloprotease responsible for cleaving the N-terminal methionine from nascent peptides, has been the object of many studies aimed to produce potential anti-bacterial, anti-fungal and anti-angiogenic agents. Though clinical trials are underway for several of these compounds, like fumagillin and CKD-731, they are still flawed based on their relatively weak inhibition and their physiological side effects. Therefore, the search for novel and potent inhibitors continues. Previous work has utilized phosphinic and phosphonic acid derivatives of methionine in co-crystallization studies with Escherichia coli MetAP (eMetAP). The aim of the research presented in this work is to study and assay various phosphorus- and sulfur-containing compounds as inhibitors and substrates in an effort to learn more about the biochemical machinery underlying MetAP catalysis. As well, we outline a predictive molecular modeling approach to MetAP inhibitor design to assist in identifying lead candidates amongst a body of possible molecular inhibitors. Ultimately, we not only hope to have identified key functional properties of molecules potentially useful as MetAP inhibitors, but also to have contributed to the knowledge base of the mechanistic features involved in this enzyme’s catalysis.

Directed evolution has been explored for a long time. Various ideas, methods, have been shown to be feasible and successful in the enzyme field. We were interested in evolving enzymes for applications. Therefore, we evolved human cystathionine gamma-lyase (hCGL) and E. coli biotin ligase for therapeutic and biotechnology applications. Wild-type human cystathionine gamma-lyase does not have any methionine-degrading activity, unlike the high methionine-degrading abilities of bacterial methionine gamma-lyase (MGL) found in Pseudomonas putida. The ability to engineer hCGL to breakdown methionine can be a potential cancer treatment by targeting the methionine-dependent cancer cells. However, the methionine-degrading activity of previously engineered hCGL has only shown 1% activity compared to MGL, too low to be useful in practical cancer therapeutics. By using a combination of protein design and phylogenetic analysis, we further evolved hCGL to achieve a higher methionine-degrading activity, with one variant displaying as much as 7% activity compared to bacterial MGL, making it a more likely candidate in cancer treatment.In addition, it has been shown that new orthogonal pairs of biotin protein ligase and biotin have many biotechnology applications. Therefore, we have developed selection scheme for directing the evolution of E. coli biotin protein ligase (BPL, gene: BirA) via in vitro compartmentalization, and have altered the substrate specificity of BPL towards the utilization of the biotin analogue desthiobiotin. Following just 6 rounds of selection and amplification several variants that demonstrated higher activity with desthiobiotin were identified. The best variants from Round 6, BirA₆₋₄₀ and BirA₆₋₄₇, showed 17-fold and 10-fold higher activity, respectively, their abilities to use desthiobiotin as a substrate. Further characterization of BirA₆₋₄₀ and the single substitution variant BirA[subscript M157T] revealed that they had 2- to 3-fold higher kcat values for desthiobiotin, and 3- to 4-fold higher K[subscript M] values. The k[subscript cat]/K[subscript M] values for these enzymes were around 0.7-fold that of BirA[subscript wt-]. It is interesting the selections did not lower the K[subscript M] for desthiobiotin and actually led to a less efficient enzyme. This is an example of how "you get what you select for". Because peptide:DNA conjugates were distributed such that there was on average one template or less per emulsion compartment there was selection only for the catalytic rate (k[subscript cat]) of desthiobiotinylation and not for turnover. Given these conditions, it might be anticipated that the peptide substrate, rather than desthiobiotin, should be bound better by the winning variants, and in fact BirA₆₋₄₀ showed a reduced K[subscript M] value for BAP. / text