The nicotinic cholinergic receptor protein of the fish electric organ, Torpedo fuscomaculata, has been isolated, purified and shown to represent a true model for the nAChR from other species and higher vertebrates. It is an integral membrane protein composed of four different subunits, tightly associated with other functional, but non-specific proteins. Purification of the nicotinic cholinergic receptor by chromatofocusing demonstrates an improved method over that of affinity and ion-exchange chromatography. Gel chromatography and SDS-polyacrylamide gel electrophoresis show evidence of four subunits; a(40-44 kDa), 6(53 kDa ),'Y(63 kDa) and 6(66 kDa) despite some degradation of receptor molecules by intracellular proteases. Spectrophotometric and fluorimetric studies of receptor-ligand interactions, show the functional and chemical integrity of the receptor to remain intact after solubilisation. The effect of cholinergic ligands on purified receptor preparations indicate quenching of the intrinsic fluorescence of the receptor. Agonists, like acetylcholine, bind and cause local conformational transitions, changing the active region from a hydrophobic to a hydrophilic environment. This phenomenon is illustrated by the 10-fold increase in fluorescence when the receptor is in a desensitised state. Antagonists, such as d-Tubocurarine, block this conformational transition. In vitro rectus abdominis muscle preparations . show the nitrosamines, dimethylnitrosamine and diphenylnitrosamine, to be true agonists of the nAChR. However their low affinity and specificity for the receptor precludes them as photoaffmity labelling agents. Photoactivation of dimethylnitrosamine occurs when associated with an acidic hydrogen at the active site of the receptor, suggesting energy-transfer labelling to be more facile than photoaffmity labelling. The membrane-bound receptor, in the presence of these nitrosamines, undergoes conformational transitions regulating the opening and closing of the ion-channel. Desensitisation and receptor activation are shown to involve one and the same molecular transition.

The prevalence of allergic diseases has dramatically increased over the last three decades. Presently, it is estimated that 20-30 per cent of the developed world suffers from allergic diseases. The majority of allergic diseases are rooted in the activities of IgE; an immunoglobulin which exerts its effector functions by interacting with a network of proteins. This network includes its low affinity receptor CD23. Cross linking of membrane IgE and CD21 by soluble CD23 results in an increase in IgE synthesis. This marks the interaction between CD23 and CD21 as an attractive therapeutic target. However, details regarding this interaction are inadequate for rational drug design. To obtain a deeper understanding of the CD23-CD21 interaction recombinant human CD21 (SCR1-2 and SCR5-8) and CD23 (16 kD and 25 kDa) were produced. The cloning, expression and purification of recombinant proteins comprised a significant portion of this study. Recombinant CD23 was expressed as inclusion bodies, refolded by rapid dilution and purified by size exclusion chromatography. Conversely, recombinant CD21 was expressed as soluble MBP-fusions and purified with an amylose affinity resin. The interaction between recombinant CD23 and CD21 was analysed by flow cytometry and ELISA experiments. Flow cytometry showed that 16 kDa and 25 kDa CD23 interacted with SCR5-8 to the same extent. Semi-quantitative ELISA experiments showed that both SCR1-2 and SCR5-8 were able to interact with 16 kDa and 25 kDa CD23. This suggests that the binding sites of SCR1-2 and SCR5-8 occur on 16 kDa CD23. Furthermore, since proteins were expressed in E. coli it suggests that the CD23-CD21 interaction does not require glycosylation. Furthermore, considering what is known about the SCR1-2-CD23 interaction from previous NMR studies; i.e. that the C-terminal tail (residues residues 289-298) of CD23 is responsible for binding SCR1-2, indicates that SCR5-8 binds somewhere within the lectin domain of CD23. This indicates that the CD23-CD21 interaction involves C-terminal tail-SCR1-2 and lectin domain-SCR5-8 interactions.

It is generally accepted that the neurotransmitter, serotonin mediates morphine-produced analgesia, however, it is not clear whether this mediation occurs at brain or spinal cord serotonin receptors. An issue that has not often been considered is the differential role that serotonin receptor types may play in morphine-produced analgesia. Paul and Phillips (1986) observed that pirenperone, a serotonin antagonist with a preferential affinity for the S2 receptor, attenuates morphine-produced analgesia. This result is particularly interesting because there are reportedly no S2 receptors in the spinal cord. The purposes of this dissertation were: to confirm the finding of Paul and Phillips, to localize the S2 receptors that mediate the anti-analgesic effect of pirenperone, and to test the hypothesis that pirenperone may exert its anti-analgesic effect through alpha-adrenergic receptors. In each of five experiments, tail-flick latencies (the time that it takes for each rat to withdraw its tail from a 52 C water bath) were measured 0, 30, 60, 90, and 120 min after drug injection. In Experiment 1, the analgesic effect of 10 mg/kg of morphine sulphate was challenged with 0.08, 0.16, and 0.24 mg/kg of pirenperone. Each dose of pirenperone attenuated morphine-produced analgesia. Moreover, each dose of pirenperone produced hyperalgesia in rats receiving no morphine. In Experiment 2, morphine-produced analgesia was challenged with 1, 3, and 10 mg/kg of ketanserin HCI. Only the very high 10 mg/kg dose ofketanserin significantly attenuated morphine-produced analgesia. Because ketanserin is pharmacologically similar to pirenperone but does not readily enter the central nervous system, this result indicates that central S2 receptors mediate the anti-analgesic effect of pirenperone and ketanserin. A third experiment demonstrated that 10 mg/kg of ketanserin did not block the analgesia produced by ketamine. Ketamine is thought to produce analgesia by a different mechanism than morphine. Thus, the attenuation of analgesia by S2 receptor blockers is not a general phenomenon, and it may be specific to morphine-produced analgesia and other analgesics that act on this system. Experiment 4 was designed to assess whether it is S2 receptors in the brain or in the spinal cord that mediate the anti-analgesic effect of S2 receptor blockade. The analgesic effect of morphine on tail-flick latencies was challenged with pirenperone in rats with spinal cords transected at the lower thoracic level and in sham-surgery comparison rats. Pirenperone attenuated morphine-produced analgesia in the sham-surgery group but not in the rats with transected spinal cords. These results indicate that brain S2 receptors mediate the attenuation of morphine-produced analgesia by pirenperone. In the fifth and final experiment, morphine-produced analgesia was challenged with 10 mg/kg of LY53857. LY53857 is an S2 antagonist which unlike pirenperone and ketanserin has no action at alpha-adrenergic receptors. Like pirenperone and ketanserin, LY53857 attenuated morphine-produced analgesia. This result supports the view that S2 receptorsmediate the anti-analgesic effects of pirenperone and ketanserin. Together, the results of these five experiments indicate that S2 receptors in the brain are important for opioid-mediated analgesia. This conclusion challenges the widely held view that only spinal cord serotonin receptors mediate morphine-produced analgesia. / Arts, Faculty of / Psychology, Department of / Graduate

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A research report submitted to the Faculty of Health Sciences, University of Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Science (MSc) in Pharmaceutical Affairs. / Lack of information has been identified as a major factor as to why patients do not take their medicines as the prescriber intends. Provision of appropriate information in a suitable form is therefore crucial. The package insert (PI) is the document that ensures the safe and effective use of the medicine under most circumstances. It presents a scientific, objective account of the medicine’s use as established by pre-clinical, clinical and often post-marketing studies. The patient information leaflet (PIL), which contains information for the consumer should be less scientific. South African legislation states that information contained in PILs must be aligned to PIs but the text must be readily intelligible for the patient. The study included a detailed comparison of prescribing information contained in the PI compared to the PIL in selected Angiotensin Receptor Blockers (ARBs). Findings of this comparative analysis revealed that key safety information was omitted from the PILs. An evaluation of the readability of the PILs was also performed by the use of Fry’s readability formula as well as applying elements of critical discourse analysis to determine if the texts in the PILs are suitable for its purpose. The results of the Fry’s readability assessments of all the PILs indicated that they had exceeded the recommended grade 7 reading level, which is in line with the adult literacy rate that qualifies anyone older than 15 years with a grade 7 qualification as being literate. Findings from the critical discourse analysis of the PILs show frequent use of medical jargon, complex sentence construction as well as ambiguity and slippage in the meaning of the texts in the PILs. The texts are not patient-friendly. Overall, the findings from this study indicate an urgent need to address the poor construction of PILs, to ensure that patients receive appropriate written prescribing information. This will ultimately ensure the safe and effective use of the medicine. / KP2020

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

No description available.

No description available.