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Class of 2010 Abstract / OBJECTIVES: To comprehensively review medical literature and report angiotension converting enzyme inhibitors (ACE-­‐I) adverse drug reactions including, incidences, mechanism of action, predisposing conditions, and report prevention and treatments. METHODS: This was a descriptive retrospective study of data related to ACE-­‐I adverse drug reactions other than ACE-­‐I induced cough. It was to review the ADR that accompany with the use of ACE-­‐I. Literature obtained through search engines MEDLINE and OVID SP available through the Arizona Health and Science Library at the University of Arizona. RESULTS: This comprehensive literature review looked at angioneurotic edema, orthostatic hypotension, hyperkalemia, and increased risk of bleeding and anaphylaxis with tPA and to a minor extent Elevated serum creatinine, and Teratogenicity. Angioneurotic edema (angioedema) reports initially estimated an incidence of 0.1 to 0.7%. A comprehensive review suggested the incidence was even lower at 0.1 to 0.2%, but the OCTAVE trial that specifically looked at angioedema as an endpoint estimated an incidence of ~0.7% although the study only had a 24-­‐week follow up. Most patients that discontinued treatment due to angioedema experienced symptom relief within 72 hours. The incidence of orthostatic hypotension from a study that followed patients on lisinopril was only 0.25%;moreover, a meta-­‐analysis by Agusti et al included 51 RCT that reported a relative risk of developing OH on an ACE-­‐I alone was 1.95. Hyperkalemia incidence reporeted varied from 1.1% to 10%; the more recent literature suggests a value near the lower end of this range. Elevated serum creatinine appears to occur early in ACE-­‐I treatment with discontinuation resolving in resolution. ACE-­‐I have been shown to be teratogenic during any trimester and should generally be avoided in pregnancy. There appears to be an increased risk of bleeding and anaphylactoid typer reactions when alteplase and ACE-­‐I are used simultaneously. Muravyov et al reported the viscosity of whole blood and plasma to be decreased after only three weeks of ACE-­‐I administration. CONCLUSIONS: With the continued increasing use of ACE-­‐Is and the drug class' ability to achieve therapeutic outcomes in a wide array of patient populations, it is important to better understand the processes and mechanisms behind the ADRs associated with ACE-­‐I therapy. A basic understanding of incidence rates and physiologic mechanisms will allow clinicians to properly assess the probability of causation and better treat patients who have experienced an ACE-­‐I induced ADR. However, an in-­‐depth level of understanding can help guide clinicians in making decisions that will hopefully decrease the amount of ADRs their patients experience or prevent their patients from developing ACE-­‐I related ADR altogether. It is important to note that, in most of the aforementioned ADR situations, treatment consists of ACE-­‐I discontinuation and avoidance of future exposures.

Malic enzyme from the rat tapeworm, Hymenolepis diminuta, has been purified 320-fold to a final specific activity of 29.4. The purification procedure included heat treatment, followed by column chromatography with Sephadex G-20, two phosphocellulose columns, and Sephadex G-200, respectively. The final purified enzyme appeared to be homogeneous on disc gel electrophoresis and G-200 gel filtration.

Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in the reductive catabolism of the pyrimidine bases uracil and thymine. The clinical relevance of this enzyme is illustrated in individuals presenting with the inherited metabolic disorder thymine uraciluria. This syndrome is characterised by high plasma concentrations of thymine and uracil, and may result in clinical features including mental retardation and dysmorphia. DPD is also clinically relevant in the metabolism and subsequent inactivation of the chemotherapeutic agent 5- fluououracil (5FU). DPD activity has been shown to be highly variable in populations of healthy volunteers and cancer patients, but the mechanisms of regulation of DPD activity are as yet poorly understood. The extent of this variation may determine the efficacy or the severity of the side effects of this treatment. The aim of this research was to evaluate DPD in terms of mRNA expression, protein expression, and activity in a variety of normal and tumour tissues in an attempt to gain an insight into the regulation of DPD. Protein expression and catalytic activity were measured using the well-characterised techniques of Western blotting, and the HPLC separation of 5FU metabolites respectively. However, the method evaluating DPD mRNA expression needed to be developed and validated. After the appraisal of various mRNA detection and quantitation methodologies, competitive polymerase chain reaction (cPCR) was selected as the most suitable method for evaluating DPD transcription in these studies. The RNA samples are reverse transcribed into cDNA which then undergoes PCR amplification in the presence of known amounts of a synthetic template ('competitor') and competes for PCR primers with the target of interest. In each PCR reaction different quantities of target and competitor PCR product will be of both PCR products the concentration of the target template in the cDNA sample can be determined. Competitive PCR was demonstrated to be a highly sensitive and specific method for quantitating DPD mRNA expression, and could be used for tissues with both high and low levels of DPD (liver colon respectively). The technique was also found to be highly reproducible and reliable and was deemed to be suitable for use in further studies. To gain an understanding of the regulation of DPD in colorectal tumour, and the effect it may have upon the activity of 5FU in a specific location, the expression/activity profile of DPD was assessed in colorectal tumour, matched normal colorectal tissue, colorectal metastases to liver, and matched normnal liver. DPD activity, mRNA, and protein levels were all significantly higher in the normal liver than colon, and in the normal liver compared to liver metastases. In the colorectal tissues, mRNA levels were significantly lower in the colorectal tumour than normal colonic mucosa, however no significant difference could be determined between tissues for DPD protein and activity. A good relationship was determined between DPD activity and protein expression in colorectal tumour tissue (rs=0.61, p=0.01), whereas a weaker relationship was determined between DPD mRNA and activity for all colorectal tumour, metastases, and normal tissues (0.43, p 0.1). DPD activity has been detected in most tissues tested to date but appears to be tissue specific with higher levels observed in liver and peripheral blood mononuclear cells than other tissues. In these studies, DPD mRNA, protein, and activity were all found to be higher in the human liver tissue than normal colon.

Pseudomonas aeruginosa secretes several proteases associated with pathogenesis, but the most abundant and active is elastase (M4 metalloendopeptidase). Elastase (lasB), is first synthesized as a preproenzyme, with a signal peptide, an 18-kDa N-terminal propeptide, and a 33-kDa mature domain. The propeptide functions as an intramolecular chaperone that is required for the folding and secretion of elastase, but ultimately is proteolytically removed and degraded. Previous research has identified the conserved residues in the propeptide of elastase as compared to other M4 protease precursors and showed some among them to be important for the production of active elastase. In this project, the ability of the propeptide alone to fold into a defined secondary structure was explored and a molecular model was created. Furthermore, the effects of substitutions on conserved residues in the propeptide of plasmid-encoded lasB pro alleles were assessed by expressing them in a lasB propeptide mutant. The kinetics of elastase activity in culture supernatants was quantitated using a fluorescent substrate, Abz-AGLA-p-Nitro-Benzyl-Amide, to provide an accurate assessment of the effects of mutant propeptides. In vitro refolding studies were also performed to determine the effects of specific substitutions on foldase activity of the propeptide. When wild-type propeptide and mature elastase were denatured as separate proteins in guanidine-HCl buffer and renatured together, restoration of activity of the refolded elastase was measured, which was propeptide-dependent. Several mutant propeptides have now been shown to have defects using this in vitro foldase assay. Additional mutants were near wild-type activity level suggesting their role in recognition by the secretion apparatus. Residue locations were determined on a molecular model of the complex and confirmed the role of the secretion mutants as residues on the exterior. Residues that had diminished ability to refold in the in vitro assay were found to be in the interior parts of the complex, confirming their ability to be critical residues at the interface of the proteins or important in the stability of the propeptide’s intrinsic structure. The goal was to perform a series of comprehensive analyses of the propeptide and its conserved residues in order to determine its role as an intramolecular chaperone.

The recently proposed bioassay procedure that is based on the substrate induced quenching (SIQ) of an indicator fluorescence for the measurement of analyte concentrations is evaluated. In this type of assay a enzynatic reaction and a fluorescence quenching interaction are coupled together. Typically, an appropriate dehydrogenase enzyme reduces or oxidises the nicotinamide adenine dinucleotide cofactor. The change in the concentration of NADH results in variations in the excited fluorophore population as observed through fluorescence intensity. This latter aspect is used to monitor substrate (analyte) concentrations. Results on the investigation of the substrate induced quenching bioassay method and possibilities of using it as the basis of (i) a novel enzyme bioassay technique and (ii) a novel bioprobe format are presented. Ethanol was chosen as the model analyte, and a new assay procedure for its measurement was developed. A generic theoretical relation is discussed for the observed assay kinetics of substrate induced quenching (SIQ) and a model is described that includes the effects due to dynamic/static quenching of the fluorophore by either the enzyme substrate or product. The validity of the derived model is shown by comparison with experimental results for a SIQ based ethanol assay. The option of running the dehydrogenase reaction so as to consume NADH rather than generate it is also investigated. In order to demonstrate this approach acetaldehyde was chosen as the model analyte, and a assay procedure for its measurement was developed. The potential of the SIQ technique for incorporation into biosensor based upon a 'reservoir' format was demonstrated through the development of custom optical instrumentation and resevoir flowcell. Applicability of the SIQ technique to other biosensor formats such as flow-injection analysis and 'dry reagent' technology is discussed. The overall applicability of the SIQ technique is assessed through the generation of a number of SIQ assays on the following substrates: ethanol, glucose, glucose-6- phosphate, L-glutamic acid, isocitric acid, acetaldehyde, pyruvic acid, ot-ketoglutaric acid, and oxalacetic acid.

MSc (Med), Faculty of Health Sciences, University of the Witwatersrand / The hepatitis B virus remains a major public health problem even after decades of its discovery. Horizontal transmission during early childhood is the predominant mode of transmission in highly endemic regions such as sub-Saharan Africa. Infection exhibits a wide spectrum of clinical manifestations, from an asymptomatic stage to severe liver disease which may result in hepatocellular carcinoma (HCC). The HBV X protein (HBx) has been implicated in carcinogenesis, which often has a poor prognosis, consequently the use of highly specific monoclonal antibodies (mAbs) directed against HBx in an enzyme-linked immunosorbent assay (ELISA) could lead to early identification of HBV carriers at risk of developing liver cancer. A variety of mixed hybridoma cell cultures secreting anti-HBx antibodies were cloned and sub-cloned by “limiting dilution”. Clonal supernatants were assessed for anti-HBx antibody production by Indirect ELISA and Western/Immunoblotting. Monoclonal antibodies were then characterized according to their relative binding affinity (Indirect ELISA) and relative epitope specificity (Competitive ELISA). One of our monoclonal antibodies was found to bind to the same epitope on HBx as the commercial anti-HBx antibody and with the same high affinity. In the developed Sandwich ELISA, our monoclonal antibody proved effective as the „detecting‟ antibody when the commercial anti-HBx antibody was deployed as the „capture‟ antibody. This Sandwich ELISA will be further developed in our laboratory with the object of applying it to patient sera.

Thesis advisor: Evan R. Kantrowitz / The regulatory mechanism of allostery is exhibited by certain proteins such as Escherichia coli aspartate transcarbamoylase (ATCase), and is defined as the change in shape and activity (of enzymes) resulting from the binding of particular molecules at locations distant from the active site. This particular enzyme and the property of allostery in general have been investigated for several decades, yet the molecular mechanisms underlying allosteric regulation remain unclear. Therefore in this thesis we have attempted via several biophysical methods, along with the tools of molecular biology and biochemistry, to correlate the changes in allosteric structure with presence of the allosteric effectors and enzymatic activity. We created a double mutant version of ATCase, in which the only native cysteine residue in the catalytic chain was mutated to alanine and another alanine on a loop was mutated to cysteine, in order to lock the enzyme into the R allosteric state by disulfide bonds. This disulfide locked R state exhibited no regulation by the allosteric effectors ATP and CTP and lost all cooperativity for aspartate, and then regained those regulatory properties after the disulfide links were severed by addition of a reducing agent. This double mutant was then chemically modified by covalent attachment of a fluorescent probe. The T and R allosteric states of this fluorophore-labeled enzyme had dramatically different fluorescence emission spectra, providing a highly sensitive tool for testing the effects of the allosteric effectors on the allosteric state. The changes in the fluorescence spectra, and hence quaternary structure, matched the changes in activity after addition of ATP or CTP. This fluorophore labeled enzyme was also encapsulated within a solgel, changing the time scale of the allosteric transition from milliseconds to several hours. The fluorophore labels allowed monitoring the allosteric state within the sol-gel, and the physically trapped T and R states both showed no regulation by the allosteric effectors ATP and CTP, and no cooperativity for aspartate. The trapped T state had low-affinity for aspartate and low activity, and the trapped R state had high-affinity for aspartate and high activity. Timeresolved small-angle x-ray scattering (TR-SAXS) was used to determine the kinetics of the allosteric transition, and to monitor the structure of the enzyme in real time after the addition of substrates and allosteric effectors. These TR-SAXS studies demonstrated a correlation between the presence of the allosteric effectors, the quaternary allosteric state, and activity, suggesting like the previous studies in this thesis that the behavior of ATCase is well explained by the twostate model. However, the effector ATP appeared to destabilize the T state and CTP to destabilize the R state, suggesting a different allosteric molecular mechanism than that of the two-state model. This thesis demonstrates the validity of many of the concepts of the two-state model, while suggesting minor modifications to that elegantly simple model in order to conform with the complex structure and function of ATCase. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.