No description available.

Nanoporous alumina is a rather newly characterized material that so far has found limited use in the construction of bioreactors. The material has many advantages compared to conventional immobilization matrices. I have investigated its use in flow-through bioreactors. The rigidity and porous structure of the material makes it an excellent choice for multienzyme reactor construction. The total activity in a reactor is easily controlled by the number of membranes since the porosity makes the material less prone to increase flow system pressure. This bioreactor is suitable for characterization of new enzymes since the amount of immobilized enzyme is standardized and the enzyme may be reused many times. We designed a simple stepwise technique for covalent immobilization on this matrix in a monolayer to minimize mass transfer effects in the reactor function. The kinetic parameters for ten different substrates were investigated for immobilized alcohol oxidase and, as a second step, a two-step reactor was also designed by addition of horseradish peroxidase. This bienzymatic reactor was, in turn, employed for measuring injected alcohol concentrations. The use of the matrix for substrate specificity screening was proven for two new epsilon-class glutathione transferases from Drosophila melanogaster. Immobilized trypsin showed a substantially prolonged lifetime and its potential use as an on-line digestion unit for peptide mass fingerprinting was also demonstrated. Finally, I investigated the immobilization of the model enzyme lactate dehydrogenase by adsorption mediated by metal ion chelation similar to IMAC. Regeneration was here possible multiple times without loss of capacity. In conclusion, immobilization of enzymes on nanoporous alumina is a convenient way to characterize, stabilize and reuse enzymes.

The main site for protein degradation along the endosomal pathway is believed to be the late endosome. Lysosomes are thought to be storage organelles that, when necessary, inject proteases into the late endosome. It was hypothesised that differences in the lumenal redox environments between the two organelles could be responsible for their functional differences. In an attempt to quantify this potential difference, the lysosomal cysteine protease cathepsin B was isolated by an improved purification procedure. Several intracellular reducing agents were used to activate cathepsin B, the most effective being cysteine. Cysteine was used to activate cathepsin B under various pH conditions in order to model endosomal conditions. An inverse relationship was found between the pH and the concentration of cysteine required to activate cathepsin B. This suggested that cathepsin B may have an optimal redox potential. In order to determine this potential, cysteinexystine redox buffers were made up and used in determination of the activity of the enzyme against a synthetic and a whole protein substrate (haemoglobin). No distinct redox potential could be determined using either substrate, but it was found that cystine stimulated proteolysis of haemoglobin. A similar stimulatory effect was observed for cathepsin D and papain hydrolysis of haemoglobin. This effect is possibly due to the ability of cystine to promote substrate structure, effectively increasing the substrate concentration. These findings and other results obtained from the literature have been used to create a model of how proteolysis may be regulated along the endosomal system. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1999.

L’agent phytopathogène Streptomyces scabiei est une bactérie du phylum actinobactérie présent dans les sols à travers le monde. S. scabiei est l’agent principal responsable de la gale commune de la pomme de terre. Cette maladie est coûteuse pour les producteurs de la pomme de terre. En effet, la pomme de terre va perdre sa valeur commerciale lorsque la bactérie attaque le périderme du tubercule et provoque des lésions liégeuses en surface ou plus ou moins en profondeur. C’est dans le périderme de la pomme de terre que la subérine va se déposer pour protéger le tubercule des agressions biotiques. Des études antérieures ont mis en évidence le rôle de la subérine comme inducteur de la production de thaxtomine A (phytotoxine essentielle à la virulence) chez S. scabiei. De même, la subérine va induire chez S. scabiei, la sécrétion d’enzymes dégradant les parois végétales, en particulier des glycosyl hydrolases, dont des cellulases. Cette thèse vise à élucider les mécanismes impliques dans la production de cellulases chez la souche S. scabiei EF-35. Dans un premier temps, le sécrétome de S. scabiei ayant poussé en présence de subérine et cellulose, soit avec un seul des deux polymères, a été analysé. Ces analyses du sécrétome ont révélé que l’addition de subérine dans un milieu contenant de la cellulose induisait une surproduction de glycosyl hydrolases. L’induction des enzymes cellulolytiques par la subérine, correlait avec la présence d’un inhibiteur de subtilase (SCAB_8801) qui pourrait jouer un rôle dans la différenciation cellulaire et le métabolisme secondaire. Ces résultats ont permis d’avancer un modèle dans lequel la subérine et le cellobiose jouent conjointement un rôle pour activer les mécanismes de virulence de la bactérie. Dans un deuxième temps, nous avons voulu savoir si le cellobiose (la molécule résultante de la dégradation de la cellulose) induisait des enzymes cellulolytiques chez S. scabiei, mais aussi chez deux autres espèces de Streptomyces pathogènes et une espèce de Streptomyces non pathogène. L’activité cellulolytique de S. scabiei en présence de subérine est beaucoup plus importante que lorsque la bactérie est en présence de cellobiose (de cinq à dix fois supérieure). De même, la présence de subérine dans le milieu de culture de S. scabiei augmente l’expression rélative des gènes des cellulases. Les deux autres Streptomyces pathogènes (S. acidiscabies et S. turgidiscabies) exhibent un profil contraire à S. scabiei. En effet, S. acidiscabies et S. turgidiscabies affichent une activité cellulolytique et une expréssion rélative des gènes de cellulases plus importante dans le milieu supplémenté de cellobiose que dans le milieu supplémenté de subérine. Streptomyces scabiei semble donc mieux adaptée que les autres espèces de Streptomyces à dégrader le matériel cellulosique encastré dans les parois subérisées du périderme. Les résultats présentés dans cette thèse visent à apporter des éléments de réponse pour mieux comprendre les interactions S. scabiei – pomme de terre. Ainsi, les biopolymères retrouvés dans la pomme de terre (subérine et cellulose) vont jouer un rôle crucial dans la virulence de l’agent phytopathogène. Cependant, il semblerait que S. scabiei s’est spécialisé au cours du temps à coloniser son hôte, la pomme de terre. La subérine induit la production de cellulases chez S. scabiei mais pas chez les autres deux Streptomyces pathogènes testés (S. acidiscabies et S. turgidiscabies). Ceci nous laisse penser que la subérine est impliquée dans des mécanismes qui restent encore à élucider.

A new procedure has been developed to concentrate the phosphofructokinase from muscle of Ascaris suum with minimum loss of activity. By utilizing this method, 50 ml fraction was concentrated to a final volume of 3 ml in about 1.5 h without loss in enzyme activity. The concentrated enzyme had a specific activity of 64 units per mg. Ascaris muscle-cuticle was incubated in 50 1M solutions of either acetylcholine, serotonin, y-aminobutyric acid, levamisole, or saline alone. Phosphate analysis of the isolated phosphofructokinase from each incubation revealed that the enzyme contained the following moles of phosphate per subunit: 2.9 (acetylcholine), 2.2 (serotonin), 2.0 (y-aminobutyric acid), 1.5 (levamisole), and 3.4 (salne alone). The present study did not establish a direct correlation between degree of phosphorylation and phosphofructokinase activity. Phosphofructokinase from muscle of Ascaris suum appears to contain several phosphorylation sites, and one of these sites is required to be phosphorylated in order for the enzyme to exhibit maximum activity under physiological conditions.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

1. The induction of CYP3A enzymes was investigated using a range of structurally unrelated drugs using in vivo and in vitro models. Hepatic microsomal testosterone 6(3-hydroxylation, anti-CYP3A immunoblot analysis, and molecular biology approaches were utilised in the investigation. 2. Using the rat as an in vivo model, potent induction of CYP3A enzymes was observed after administration of the synthetic glucocorticoid dexamethasone (at 150mg.kg.day for 4 days) and pregnenolone 16?-carbonitrile (at 150mg.kg-1.day-1 for four days). However, no induction was observed after administration of rifampicin (at 50 g.kg-1.day-1 for 4 days, a dose which causes potent induction in the rabbit). 3. Investigations into the effects of drug exposure on testosterone 6?- hydroxylation in cultured female rat hepatocytes revealed a positive in vivo/in vitro correlation. Cultured cells were treated with the same drugs (at 50 M concentration) for 72hrs. Dexamethasone was shown to be more potent than PCN, and rifampicin again had no effect. Dexamethasone-mediated induction of testosterone 6?- hydroxylation was dose-dependent and was shown to be maximal after 72hrs exposure. 4. The presence of the differentiating agent dimethylsulphoxide at 2% (v/v) in the cultiure medium enhanced CYP3A induction by the synthetic steroids by approximately 100% (p 0.05). 5. The potent glucococorticoid antagonist RU 486 induced testosterone 60- hydroxylation 5-fold when administered at 50mg.kg-1.day-1 for 4 days. Induction of the CYP3A protein was confirmed by immunoblot analysis of liver microsomes. Administration of RU 486 at 50mg.kg-1.day-1 did not antagonise the induction of testosterone 6?-hydroxylatiomn by dexamethasone at 150mg.kg-1.day-1. 6. Dexamethasone (0.1 to 10 M) -mediated induction of testosterone 6(3- hydroxylation in cultured rat hepatocytes was attenuated in the presence of RU 486. It is not known whether this was due to effects on CYP3 A gene expression or inhibition of enzyme mediated activity at the active site of the enzyme. 7. The lipid lowering drug SK F 98016 (150mg.kg-1.day-1) induced testosterone 6?-hydroxylation 10-fold when administered at 150mg.kg-1.day-1 for 4 days. This was confirmed by immunoblot analysis. Co-administration of RU 486 with SK F 98016 attenuated induction of CYP3A-mediated enzyme activity. The mechanism of induction of the CYP3A genes by SK F 98016 may therefore involve 'steroidal' compounds, the action of which is antagonised by RU 486. The dexamethasone- mediated increase in spectrally determined cytochrome P450 levels was also attenuated after co-administration with RU 486. As CYP3A induction was not affected by co-administration of dexamethasone with the anti-glucocorticoid RU 486, this result suggests that the glucocorticoid receptor may be involved in the induction of other P450 genes. 8. Treatment of rat hepatocytes with SK F 98016 (50 M) for 72 hours did not result in an increase in testosterone 6?-hydroxylation. In fact testosterone 6?-, 16?- and 17-oxidation activities were reduced to 50% of the activities measured in untreated hepatocytes. This pointed to some P450 inhibitory potential of SK F 98016. Investigation of the inhibitory potential of SK F 98016 on testosterone 60- hydroxylation in hepatic microsomes from PCN-treated rats showed an inhibitory effect with an IC50 of 50 M. The inhibitory effect seen in hepatocytes is similar to the effects of exposure to clotrimazole (50 .M) for 72 hours where testosterone metabolism at the 60 and 17 positions were inhibited by >90%. 9. To investigate whether the lack of inducing effect of SK F 98016 was due to the very high lipophilicity and extensive partitioning into the cultured hepatocyte, therefore resulting in a non-physiological state, cultured hepatocytes were exposed to the same drugs with albumin (from bovine serum, at the concentration present in human blood-36g/litre) in the medium in attempt to encourage an equilibrium of drug concentration between the medium and the inside of the hepatocyte. No significant induction of testosterone 60-hydroxylation was observed in the presence of albumin.

Call number: LD2668 .T4 1986 V47 / Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program

BACKGROUND: APOBEC3H (A3H) is a member the Apolipoprotein B mRNA-editing catalytic (APOBEC) polypeptide seven cytidine deaminases family. These APOBEC3 enzymes have the innate ability to deaminate a cytidine base converting it to a uridine base. Previous research has suggested that human A3H haplotypes have similar properties like those of A3G and A3DE, in which these APOBEC3 enzymes are known to entail anti-viral activity toward viruses such as human immunodeficiency virus. METHODOLOGY: Human A3H gene was amplified by polymerase chain reaction and inserted into E.Coli plasmids. Expression tests were conducted to verify that the clones were able to express protein. Ten liters of the cloned cells were grown, and A3H protein was purified using an established protocol in Dr. Chen's lab for deamination to test the enzyme activity. An inactive mutant of A3H was also cloned to use as a negative control for the wild type A3H. PRINCIPAL FINDINGS: The isolated A3H haplotype 1 (referred to as A3H hereafter) is indeed active, but the activity is very weak in comparison to other APOBEC3 proteins, in which A3F and A3A were used as controls. A3H deamination is most optimal with an acidic environment of pH 5.5 or 6.0, and concentrated protein will produce more deamination product (darker bands). The inactive A3H produced some unexpected visible smaller DNA bands, which may be the result of contaminated protein activities. In conclusion, the wild type A3H protein may have weak activity, but additional studies are required to confirm that the inactivated A3H mutant has no activity on the DNA substrates.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. August 2014. / Human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome (AIDS), remains a topic of global concern even though great strides have been made to combat the virus. The high replicative rate of the virus and recombination of the variety of viral strains complicate the treatment of AIDS. There has been an increasing prevalence of African HIV strains in the Americas and Europe. The viral protease (PR) is vital for the propagation of the virus; and thus, is a major target in antiviral therapy. The HIV-1 PR enzyme from the subtype C strain; which predominates in sub- Saharan Africa, has been greatly under-investigated in comparison to the protease from the subtype B strain which predominates in North America and Europe. Enzyme activity data which were part of this work suggested that the South African HIV-1 subtype C protease (CSA PR) displays improved substrate turnover in comparison to the subtype B PR. Thermodynamics and inhibition kinetics of drug binding showed that the C-SA PR is less susceptible to certain clinically-used protease inhibitors when compared to the subtype B PR. A crystal structure of the C-SA PR was solved and showed no difference to the global structure of the subtype B PR. Molecular dynamics simulations showed that the C-SA PR exhibits a wider range of open conformations. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) was performed to elucidate the mechanism of reduced drug susceptibility displayed by the C-SA PR. HDX-MS data provided insights into the basis of the increased preference for open conformers displayed by the C-SA PR and the stability of the terminal dimer interface which is a target in protease inhibition.