No description available.

The transport systems for proline and potassium represent two of the active transport systems in Escherichia coli. They have further similarities that their transport may be utilized as a response to osmotic perturbations in the environment. The exact mechanism of transport had not been totally elucidated. The transport of proline had been assumed to operate as a proton symport and as such had been used as a model system when other transport systems were being investigated. This study has demonstrated that the major route of proline uptake through the proline permease 1 (PP1), operates as a Na+ - proline cotransport which may accept Li+ in the place of Na+. Unusually, Na+ stimulates the Vmax of transport with little or no effect on the Km. In addition to this transport system, there are two other proline uptake systems which function primarily for the transport of betaine. The transport of K + is also facilitated by more than one system. The Kdp system is a K+ transporting ATPase; the TrkF system is a low rate transport system which may represent leak through another pathway. The TrkA transport system is the major system but the mechanism is not known. Transport through the system is energised by ATP and a pmf, while exchange through the system requires only ATP. The role of ATP was investigated in this study by the use of metabolic inhibitors and vesicles. It was determined that the availability of ATP affected the steady state level of potassium in the cells rather than the rate of potassium upake. It was speculated that ATP would act as a regulator of the system which would be driven by the pmf. ATP may regulate TrkA through phosphorylation or by allosteric modification of the carrier.

Dans le cadre de la recherche d’un succédané à la présure, la pulpe des fruits de B. aegyptiaca a été utilisée comme source d’enzyme coagulant. Les fruits ont été assainis par lavage et traitement à l’eau de javel. Une solution de NaCl 5% a été utilisée comme milieu d’extraction. Le processus de purification est constitué des étapes suivantes: clarification au charbon actif et par diafiltration, chromatographies d’exclusion et d’échanges d’ions. Des études rhéologiques physico-chimiques et sensorielles ont été effectuées sur des fromages obtenus avec cet extrait.Les résultats de cette étude montrent qu’une protéase de 38 kDa a été isolée en milieu acide et trois protéases de 28, 70 et 85 kDa en milieu basique. Les tests sur les inhibiteurs, les analyses en MALDI-TOF couplée à la LC-MS et la digestion du gel de gélatine montrent que la protéase isolée en milieu acide est une aspartique tandis que celles isolées en milieu basique sont des protéases à sérine. Les protéases isolées sont capables d’hydrolyser les caséines α, β et κ. Le galactose, l’arabinose, le rhamnose, le xylose, le mannose, l’acide glucuronique et les hexosamines ont été détectés sur la partie glycosylée de ces protéases par GC/FID et GC/MS-EI. Cependant, la détection aux lectines a montré la présence du mannose et du fucose. Les paramètres physico-chimiques et organoleptiques montrent que le fromage obtenu avec l’extrait est comparable au fromage présure. Cette étude a montré que les protéases de l’extrait de la pulpe de Balanites aegyptiaca sont capables de remplacer la présure dans la fabrication des fromages locaux. Ce travail peut contribuer significativement au développement économique des localités agro-pastorales du Cameroun. / In the framework of the search for a substitute to calf rennet, the pulp of the B. aegyptiaca fruits was used as source of milk-clotting enzyme. The fruits were cleaned by washing and treatment with sodium hypochlorite. The NaCl 5% solution was used as medium of extraction. The purification process was made up of the following stages: clarification with activated charcoal and diafiltration, exclusion and ion exchange chromatographies. Physicochemical, rheological and sensory properties of cheese obtained with this vegetable rennet were studied. The results of this study showed that a protease of 38 kDa was extracted in acid medium and three proteases of 28, 70 and 85 kDa in basic medium. The inhibition test, MALDI-TOF analysis coupled with LC-MS and gelatin digestion by protease showed that the protease extracted in acid medium is aspartic while those isolated in basic medium are serine proteases. The isolated proteases are able to hydrolyze α, β et κ caseins. Galactose, arabinose, rhamnose, xylose, mannose, glucuronic acid and hexosamines were detected on the glycosyl part of these proteases by GC/FID and GC/ME-EI. However, detection with the lectins showed in more the presence of mannose and fucose. The physicochemical and organoleptic parameters showed that the cheese obtained with the extract is comparable with the standard cheese. This study showed that the proteases of the extract of the pulp of Balanites aegyptiaca are able to replace calf rennet int the manufacture of local cheeses. This work can contribute significantly to the economic development of the agro-pastoral localities of Cameroon.

Thesis advisor: Evan Kantrowitz / Aspartate transcarbamoylase (ATCase) catalyzes the reaction between carbamoyl phosphate (CP) and aspartate to form N-carbamoyl-L-aspartate (CA) and inorganic phosphate (Pi). In Escherichia coli, it catalyzes the committed step of pyrimidine nucleotide biosynthesis. It is heterotropically activated by ATP, and is inhibited by CTP and UTP. X-ray studies have revealed valuable information regarding the catalytic mechanism, as well as insights into the homotropic and heterotropic interactions. However, localized changes and changes under biological conditions are difficult to study this way. The expression of a viable and active fluorescent mutant of ATCase containing a 7-hydroxycoumarin amino acid will allow for studies of the signal transduction process involved in the negative cooperativity observed in the binding of allosteric effectors. The fluorescent intensity should correlate to the binding of the NTPs at close proximity to the unnatural fluorescent amino acid. Furthermore, TR-SAXS will be used to observe transiently stable intermediates of ATCase formed during the T to R transition. This method’s typically observed poor time resolution caused by the dead time of the stopped-flow mixer will be addressed by the used of a caged aspartate, 4-methoxy-7-nitroindolinyl-L-aspartate, synthesized in this work. / Thesis (MS) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Digitized by Kansas Correctional Industries

No description available.

In the treatment of arrhythmias, amiodarone is a primary therapeutic agent. Cytochromes P450 (CYP) 1A1 and 1A2 facilitate biotransformation of amiodarone to the biologically active desethylamiodarone. Side effects have been reported during therapy and some are correlated with increased desethylamiodarone levels. Exposure to polycyclic aromatic hydrocarbons (PAH) like -naphthoflavone induces CYP1A1 and CYP1A2 and therefore can increase desethylamiodarone levels. Desethylamiodarone, however, was reported to inactivate human CYP1A1 and therefore can conceivably inhibit its CYP1A1-mediated formation. Our primary objective was to investigate the effect of -naphthoflavone on amiodarone disposition. Since rats were used, CYP isoenzymes involved in desethylamiodarone formation in human were compared to their rat counterparts. The effect of ketoconazole on desethylamiodarone formation, the inactivating potential of desethylamiodarone on CYP1A1 and the mechanism of -naphthoflavone-amiodarone interaction were assessed. Human CYP1A1 and rat CYP2D1 had the highest intrinsic clearance (Clint) for desethylamiodarone. Human and rat CYP1A2 had the lowest Clint. Ketoconazole (18.8 M) inhibited all isoforms except for rat CYP1A2; it potently inhibited human CYP1A1 and CYP3A4 and rat CYP2D2 and CYP1A1. After a single amiodarone dose was administered to control and -naphthoflavone pretreated rats, the plasma area under concentration-time curve (AUC) of desethylamiodarone increased. With multiple doses, amiodarone AUC(0-24h) decreased in -naphthoflavone plasma (30%), lung (35%), liver (48%), kidney (52%), heart (34%), and intestine (43%). Desethylamiodarone AUC(0-24h) increased in -naphthoflavone plasma (36%), lung (56%), liver (101%), kidney (65%), and heart (73%). Desethylamiodarone caused no inactivation of CYP1A1 when preincubations were diluted and nicotinamide adenine dinucleotide phosphate (NADPH) was added in the probe incubation samples. Evidence for reversible mixed-competitive inhibition was apparent. Addition and/or replenishment of NADPH were important factors in maintaining control activity. -naphthoflavone increased desethylamiodarone formation only in lung and kidney microsomes. Desethylamiodarone formation in liver, intestine and heart microsomes was not altered. Body-weight-normalized liver mass was significantly increased (27%) by -naphthoflavone. In conclusion, human CYP1A1 was more efficient in forming desethylamiodarone than rat isoenzyme. Exposure to PAH increased desethylamiodarone levels in vivo. Increased desethylamiodarone levels were partly caused by CYP1A1 induction, and by increased liver mass. Desethylamiodarone did not inactivate CYP1A1 activity. / Pharmaceutical sciences

Phenylketonuria and hyperphenylalanemia are autosomal recessive inborn errors of phenylalanine metabolism that are caused by mutations in the phenylalanine hydroxylase gene. Due to the stringency of the present dietary therapy, alternative treatments are being studied. Phenylalanine ammonia-lyase (PAL) is one of the potential dietary supplements for these patients. PAL is a well-studied plant enzyme which breaks down phenylalanine into trans-cinnamic acid and ammonia (Camm and Towers, 1973). It is found in the cytoplasm of the plant cells and is naturally encapsulated by plant cell walls which may protect it against the acidic pH environment in the gastrointestinal tract. It presumably degrades ingested Phe that circulates in the intestinal lumen. In this study, red spring wheat seedlings (Triticum aestivum L.) found to contain high PAL activity naturally were investigated as a potential alternative oral therapy. Specifically, the objectives were (1) to evaluate different drying methods on generating concentrated and dried preparation of wheat seedlings containing high levels of PAL activity; (2)to examine the retention of PAL activity over three months of storage under various storage conditions; (3) to determine the stability of PAL activity in simulated human digestion condition to establish if further study of using plant source enzyme in vivo is warranted. Freeze-drying (FD) was found to retained the most activity (>90 % recovery dry wt basis)compared to air-drying (AD) and vacuum-microwave drying (VMD) for both leaf and residual seed/root samples. Pre-freezing of leaf tissues at -18 °C before FD significantly retained the highest PAL activity compared to pre-freezing at -25 °C, -35 °C, and -80 °C (P<0.05). Over three months of storage, 60-80 % of PAL activity was recovered in leaf and —100 % was recovered in residual seed/root tissues after storage at -20 °C. After in vitro protein digestion, 36% and 42 % of PAL activity was recovered in fresh leaf and root tissues respectively; however, FD tissues were found to be susceptible to proteases and acidic environment and no activity was recovered after three hours of in vitro protein digestion. High performance liquid chromatography (HPLC) analysis of the residual Phe after in vitro protein digestion confirmed that fresh tissues had significantly higher conversion of Phe than that of FD tissues. Together, these results suggest that red spring wheat seedlings may have potential as a dietary supplement for phenylketonuric patients while further study to enhance PAL activity in plant preparations is required.

Chemical complementation is a general system for detecting protein-small molecule interactions, and linking that interaction to genetic selection. In this chemical complementation system, the interaction of a nuclear receptor and a ligand is essential for yeast survival. In first generation chemical complementation, a two-component assay was developed where the Gal4 DNA-binding domain is fused to the ligand binding domains of nuclear receptors, and expressed in the strain S.cerevisiae PJ69-4A. The Gal4 DNA binding domain binds to a Gal4 response element controlling transcription of a selective marker, and the nuclear receptor ligand-binding domain binds its ligand. This system was developed using the retinoid X receptor, the pregnane X receptor, and the liver X receptor and their ligands 9-cis retinoic acid, paclitaxel, and oxysterols, respectively. Yeast survive on selective plates only in the presence of both components: a nuclear receptor and the corresponding ligand. Growth was observed at the highest concentration of ligand (10-5 M) and, compared to Gal4-activated growth, the growth density was less and growth time was more. The second generation chemical complementation system is a three-component system comprising a nuclear receptor ligand-binding domain fused to the Gal4 DNA binding domain, the ligand, and a nuclear receptor coactivator fused to the yeast Gal4 activation domain. This system was developed using the retinoid X receptor and has been extended to several other nuclear receptors. The sensitivity of chemical complementation is increased 1000-fold, and growth time and density are equivalent to Gal4-activated growth. An assay was developed to provide a quantitative high-throughput assay for evaluating nuclear receptor- ligand interactions, and measuring EC50 values for the ligand-receptor pairs. Chemical complementation can be used in a variety of applications, such as drug discovery for nuclear receptor-based disease, providing a high-throughput assay for the discovery of potential nuclear receptor agonists, and with the use of the negative chemical complementation system, the discovery of nuclear receptor antagonists. Chemical complementation is used for protein engineering, specifically engineering receptors to bind and activate in response to other ligands. Chemical complementation is also used for deciphering and assembling biosynthetic pathways.

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