Part One Preparations of nucleoprotein having reproducible characteristics were obtained from calf thymus glands by a mild extraction procedure. A preliminary examination of the dissociation of the complex was made and its physico-chemical behaviour was investigated in relation to that of its nueleic acid component. The nucleoprotein was highly aggregated in solution at low ionic strength, but at high ionic strength a weight-average molecular weight lower than that of nucleic acid itself provided additional evidence for the dissociation of the nucleoprotein. Spectrophotometric studies showed that the nucleoprotein was denatured by heat and by alkali in a similar manner to nucleic acid, although denaturation by heat was slightly retarded. The spectrophotometric titration behaviour of the two substances was almost identical, but their potentiometric titration curves differed significantly. Nucleoprotein solutions exhibited the features of nucleic acid devolving from its unique helical structure, indicating that this structure is retained by the nucleic acid within the complex. Part Two A general reaction has been discovered between amino sugars and aldoses for which the essential requirements are a free amino group and an aldehyde group. Three compounds (A, B and C) were separated by ion- exchange chromatography from a mixture obtained by heating D-mannose with D-glucosamine hydrochloride. Compound B was identified as 5-hydroxymethyl furfuraldehyde (HMF) from its ultra-violet absorption spectrum, chromatographic behaviour, and distribution coefficients between two different solvent systems, and was characterised as its 2:4-dinitrophenylhydrazone. Compound A was very unstable and was hydrolysed by cold water to mannose (characterised as its p-nitranilide) and glucosamine (characterised as its carbobenzoxy derivative). On heating in aqueous solution, the hydrolysis was accompanied by degradation to HMF (characterised in the same way as compound B), and the formation of melanoidins. The HMF isolated from the reaction mixture was shown to arise from compound A and C14-HMF was obtained when the condensation was effected with a mixture containing C-mannose and glucosamine hydrochloride, showing that the HMF originated in the mannose moiety of compound A. From these and other experiments, the structure N-mannosyl-glucosamine was assigned to compound A. Compound C was stable in aqueous solution, and hydrolysis with acid gave equimolecular quantities of mannose and glucosamine hydrochloride, but was not accompanied by a browning reaction. On the basis of periodate oxidation experiments, its behaviour in the Elson-Morgan reaction, and a number of other colour tests, compound C was provisionally assigned the structure 6-O-α-D-mannosyl-Z-amino-2-deoxy-D-glucose. Certain aldehydes were found to behave as bases on a sulphonated polystyrene resin. This interesting discovery may provide the basis of a new method for the separation and analysis of aldehyde mixtures.

The present work, employing biochemical, biophysical and electrophysiological techniques, attempted to identify specific receptor sites in the vertebrate olfactory system for heterocyclic odorants. An in vitro rat preparation was developed and characterised for use in vapour-phase chemical modification experiments; the EOG responses obtained from this preparation were stable for up to 5 hours after the death of the animal. The signals to various compounds were differentially reduced when brominated odorants were employed as vapour-phase labelling reagents; the responses obtained to these derivatives and to their non-reactive analogues were preferentially diminished. The effect of concanavalin A on ECGs obtained from an in vivo frog preparation was examined. This lectin was found to preferentially inhibit the signals elicited by small, sweaty-smelling carboxylic acids; the responses to most of the non-carboxylic acid odorants tested were not significantly inhibited. The failure to identify specific receptor sites by electrophysiological techniques prompted the performance of odorant binding studies. Examinations of the interaction of [3H] 2-isobutyl-3- methoxypyrazine with 13,000 x g supernatant fractions of sheep olfactory epithelium showed that a component of the homogenate fraction exhibited high affinity saturable binding of this odorant (KD-10-8M). However, the presence of large amounts of non-specific binding, substantially decreased the sensitivity and accuracy of the assay. Non-specific binding was observed with tissue fractions of sheep respiratory epithelium, brain and liver. An investigation of binding specificity showed that other bell pepper odorants competed for the 2-iscbutyl-3-methoxypyrazine binding site. The steno requirements for the protein binding of various substituted heterocyclic odorants were examined using nuclear magnetic relaxation techniques. Model studies performed with bovine serum albumin showed that particular side chains of the odorants tested were primarily involved in the binding interaction. The methoxy group of 2-isopropyl-3-methoxypyrazine was found to be responsible for primary recognition by 13,000 x g supernatant fractions of sheep olfactory epithelium.

The purpose of this work was to study the effects of reactive oxygen species (ROS) on DNA and to investigate the relevance of ROS-induced DNA damage in systemic lupus erythematosus (SLE). Using model systems of ROS generation, it was found that DNA was damaged by ROS at all levels of its structure, causing strand breaks, base modifications and conformational changes. Hydrogen peroxide, a ROS generated during inflammation in vivo, produced a characteristic type of site-specific damage dependent on the DNA-bound metal ion catalysis of its degradation. 8-hydroxydeoxyguanosine (8OHDG), a modified DNA base, was used as a marker of oxidative damage to investigate the role of DNA damage in the aetiopathogenesis of SLE. Excretion of this adduct was detected in normal urine and is believed to arise from normal oxidative metabolic processes. In patients with active rheumatoid arthritis, this level of 8OHDG excretion was significantly elevated. In contrast, in SLE patients with inflammatory activity, 8OHDG was undetectable in the urine. Investigation of the mechanism responsible for this showed that SLE cells had aberrant removal of 8OHDG from DNA following oxidative stress in vitro compared to normal cells, and that ROS-denatured DNA accumulated in circulating immune complexes associated with the disease. SLE is also characterised by circulating anti-DNA antibodies. These antibodies were found to bind better to ROS-DNA than to native double-stranded DNA. Furthermore, ROS-DNA was able to stimulate lymphocytes to produce anti-DNA antibodies. The pattern of DNA damage seen in SLE patients was typical of that induced by hydrogen peroxide in vitro. This suggests that inflammation generates ROS which cause DNA damage. As a result of defective repair within cells, ROS-DNA is released into the circulation following cell death which can form complexes with anti-DNA antibodies. In addition, the ROS-DNA can stimulate further anti-DNA antibody production by acting directly on cells thus perpetuating the disease process and contributing to immune complex deposition, a deleterious manifestation of the disease process.

Erwinia carotovora subsp. carotovora (Ecc) secretes a variety of extracellular enzymes, namely pectinases (Pel), cellulases (Cel) and proteases (Prt). Some of these extracellular enzymes are considered to be the major pathogenicity determinants of this bacterium. Using the chemical mutagen ethyl methyl sulphonate (EMS), a range of Ecc mutants defective in extracellular enzyme production have been generated. One class was found to be pleiotropically defective in the production of Pel and Cel but unaffected for Prt production. Pel and Cel were still synthesised in this class of mutant but both enzymes accumulated within the periplasm. Mutants of the Pel-, Cel-, Prt+ class have been termed Out- mutants. A single Out- mutant, RJP190, was partially resistant to infection by two Ecc bacteriophages. Using a cosmid library of wild-type Ecc, 12 of the 14 Out- mutants were complemented to Out+. Further analysis of the complementing cosmids led to the identification of at least six out loci. A 3.7 kb region of DNA containing out genes was sequenced. This fragment of DNA overlapped with other out genes sequenced In this laboratory. The contiguous DNA (5.7 kb) encoded four proteins, OutD, OutE, OutF and OutG, which were visualised using a T7 directed expression system. The predicted Out proteins were found to share homology with other eubacterial proteins involved in macromolecular trafficking. Accumulated findings strongly suggest that this Out-type system is the major pathway used by Gram-negative bacteria for secreting proteins to the extracellular milieu.