65 research outputs found
Two Orchestral Anthems by John Alcock (1715-1806): A critical edition with commentary
No full textThe music of John Alcock (1715-1806) is not familiar to most musicians. However, he did produce some attractive works and is a significant figure in English church music of the eighteenth century. His writings about his life in both parish church and cathedral music are also invaluable to research of the period. The two orchestral anthems, here appearing in critical editions, display some of the flair and attention to detail characteristic of Alcock as a composer of vocal music. The commentary examines the background to the compositions, describing the sources used, the editorial practice adopted and the place of the anthems within the music of eighteenth-century England
Diagnostic and clinical value of routine polymerase chain reaction analysis of intraocular fluid specimens in the diagnosis of suspected infectious posterior uveitis
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Structure-function relationships of ricin A-chain
No full textThis thesis represents e study of the reletionships between the structure and function of the A-chain of ricin. Ricin is one member of a family of ribosome- inactivating proteins (RIPs) that inactivate protein synthesis by modification of the ribosomal RNA. The mechanism of this catalytic effect is unknown. This project was designed to investigate which amino acid residues were important in the mechanism of catalysis.
Using site-directed mutagenesis of specific ricin A- chain residues, eight mutants were created for biochemical analysis. Residues were targeted for autogenesis by analysis of the position of conserved amino acids in the ricin 3-D structure. Glul77 was mutated to Lys, Ala and Asp. and Argl8O was mutated to Gin, Ala and Met. The arginine residue at position 29 was altered to an alanine, and a deletion of residues Serl76 to Argl80 was performed.
Mutant recombinant ricin A-chain (rRTA) constructs were prepared for in vitro and in vivo expression in the vectors pGEMl and pDS5/3 respectively. Initially, mutants were translated in a cell-free wheatgerm translation system to assess the size of the mutant polypeptides. All the constructs produced polypeptides of the correct else. The N-glycosidase activity of the mutants was then assessed with two methods of analysis using rabbit reticulocyte ribosomes. It was shown that some of the mutants were devoid of detectable activity and some had reduced activity.
Mutant constructs were expressed in Escherichia coli in order to isolate protein for quantitative activity measurements. Crude E. coli extracts containing rRTA and rRTA mutants were tested for activity. It was found that the relative activities of mutant proteins produced in E. coli was similar to that seen previously with the in vitro measurements. Soluble, active protein was recovered for a few of the mutant proteins, although no expression was observed from some constructs. Various purification procedures were assessed end ion-exchange chromatography was determined to be most suitable.
Mutant D177 was tested for its N-glycosidase activity towards salt-washed yeast ribosomes and related to the activity of wild-type rRTA. It was shown that the activity of D177 was approximately 60 fold lower than that of wild- type rRTA with the majority of the difference being observed with the kcat of the reaction. It was also shown that the data produced in this study correlated with a recently published putative mechanism of action of ricin A-chain
Targeted Secretion Inhibitors—Innovative Protein Therapeutics
No full textBotulinum neurotoxins are highly effective therapeutic products. Their therapeutic success results from highly specific and potent inhibition of neurotransmitter release with a duration of action measured in months. These same properties, however, make the botulinum neurotoxins the most potent acute lethal toxins known. Their toxicity and restricted target cell activity severely limits their clinical utility. Understanding the structure-function relationship of the neurotoxins has enabled the development of recombinant proteins selectively incorporating specific aspects of their pharmacology. The resulting proteins are not neurotoxins, but a new class of biopharmaceuticals, Targeted Secretion Inhibitors (TSI), suitable for the treatment of a wide range of diseases where secretion plays a major role. TSI proteins inhibit secretion for a prolonged period following a single application, making them particularly suited to the treatment of chronic diseases. A TSI for the treatment of chronic pain is in clinical development
Engineering Botulinum Toxins to Improve and Expand Targeting and SNARE Cleavage Activity
No full textBotulinum neurotoxins (BoNTs) are highly successful protein therapeutics. Over 40 naturally occurring BoNTs have been described thus far and, of those, only 2 are commercially available for clinical use. Different members of the BoNT family present different biological properties but share a similar multi-domain structure at the molecular level. In nature, BoNTs are encoded by DNA in producing clostridial bacteria and, as such, are amenable to recombinant production through insertion of the coding DNA into other bacterial species. This, in turn, creates possibilities for protein engineering. Here, we review the production of BoNTs by the natural host and also recombinant production approaches utilised in the field. Applications of recombinant BoNT-production include the generation of BoNT-derived domain fragments, the creation of novel BoNTs with improved performance and enhanced therapeutic potential, as well as the advancement of BoNT vaccines. In this article, we discuss site directed mutagenesis, used to affect the biological properties of BoNTs, including approaches to alter their binding to neurons and to alter the specificity and kinetics of substrate cleavage. We also discuss the target secretion inhibitor (TSI) platform, in which the neuronal binding domain of BoNTs is substituted with an alternative cellular ligand to re-target the toxins to non-neuronal systems. Understanding and harnessing the potential of the biological diversity of natural BoNTs, together with the ability to engineer novel mutations and further changes to the protein structure, will provide the basis for increasing the scope of future BoNT-based therapeutics
Train Turned Death Car Into Twisted Metal.
No full textPhotograph taken for a newspaper owned by the Oklahoma Publishing Company. Caption: "Train turned death car into twisted metal. An Oklahoma City man was killed Friday night when a passenger train hit his car near NW 92 and Western. Lee Bougdon Cocklin, 75, of 1125 NW 92, was killed when his late model car was hit by a north-bound Santa Fe Railway train, police said. Cocklin was traveling west when he was hit by a northbound Texas Chief, which was making its first trip through Oklahoma since the short-lived rail strike. E.O. Chaddock, superintendent of the Santa Fe downtown depot at its normally scheduled time, 5:40 p.m. and that flashers were in operation when the train approached the intersection. The south-bound Chief had been held up in Chicago due to the Thursday strike. Lt. O.W. Ardry, traffic division, said Cocklin was dead on arrival at Baptist Memorial Hospital from internal injuries he received.
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