Novel imprinted polymers as artificial enzymes

Derivatives of L-histidine were investigated as suitable models for the Asp-His couple found in the catalytic triad of serine proteases. A combination of molecular dynamics and IH NMR spectroscopy suggested that the most populous conformations of N-acetyl-L-histidine and the N-acetyl-L-histidine anion were predominated by those in which the carboxylate group was gauche to the imidazole ring overcoming steric and electrostatic repulsion, suggesting there is an interaction between the carboxylate group and the imidazole ring. Kinetic studies, using imidazole, N-acetyl-L-histidine and the N-acetyl-L-histidine anion showed that in a DMSO/H20 9: 1 v/v solution, the N-acetyl-L-histidine anion catalysed the hydrolysis of p-nitrophenyl acetate at a greater rate than using either imidazole or N-acetyl-L-histidine as catalyst. This indicates that the carboxylate group affects the nucleophilicity of the unprotonated imidazole ring. 31P MAS NMR spectroscopy was investigated as a new technique for the study of the template molecule environment within the polymer networks. It was found that it was possible to distinguish between template associated with the polymer and that which was precipitated onto the surface, though it was not possible to distinguish between polymer within imprinted cavities and that which was not. Attempts to study the effect of the carboxylate group/imidazole ring interaction in the imprinted cavity of a molecularly imprinted polymer network were hindered by the method used to follow the reaction. It was found though that in a pH 8.0 buffered solution the presence of imprinted cavities increased the rate of reaction for those polymers derived from L-histidine. Some preliminary investigations into the design and synthesis of an MIP which would catalyse the oxy-Cope rearrangement were carried out but the results were inconclusive.

Studies on the composition and nutritional value of the cultivated mushroom, agaricus bisporus (Lange) Sing

The effects of various cultural conditions on the composition and nutritional quality of Agaricus bisporus (Lange) Sing. were investigated. Variation in composition was found between different classes of sample. Sampling techniques were standardised to allow for major variations in the different developmental stages and culture ages. Fruitbodies were found to be of low calorific value but contained protein of high digestibility and quality, containing all the essential amino acids required by man. Quantitative estimates of the sulphur-containing amino acids indicated that fruitbodies were deficient in methionine and cysteine. The extent of water application and the supplementation of conventional substrates with various nitrogen-containing substances, influenced yield and composition, establishing the importance of these two factors in the physiology of fruitbodies and cultural management. Storage conditions influenced composition, high temperatures being deleterious to the nutritional value of fruitbodies. Submerged culture techniques were used to investigate the effects of various nutrients on growth and composition of mushroom mycelium, with special reference to the sulphur-containing amino acids. Yield and composition were greatly affected by the carbon:nitrogen ratio of the medium and by the nitrogen source. Significant increases in mycelial methionine content were observed on the addition of inorganic sulphate, the methionine derivative N-acetyl-L-methionine, and L-methionine. A greater increase in methionine content was obtained when the biomass of a thermophilic bacterium isolated from compost was used as a nitrogen source.

Generation of nitric oxide and its protective and toxic actions in the gastrointestinal tract

Nitric oxide is a free-radical gas which can exert both protective and damaging effects. The objectives of the thesis were: (i) to investigate arginine metabolism in isolated rat gastric mucosal cells, (ii) to investigate the role of NO in the induction of ornithine decarboxylase in the rat gastric mucosa damaged by hypertonic saline in vivo, (iii) to expose primary cultures of guinea-pig gastric mucosal cells to oxidative challenge and an NO donor, and to investigate the response in terms of heat shock protein 72 (HSP 72) induction, and (iv) to investigate the induction of iNOS and the role of potential modulators of activity in gastric cell lines. Isolated rat gastric mucosal cells converted exogenous arginine to ornithine and citrulline. This metabolism of arginine was not affected by a range of NO synthase inhibitors, but was reduced by the arginase inhibitors NG-hydroxy-L-arginine and L-ornithine. Thus, the predominant pathway of arginine metabolism involves arginase and ornithine transcarbamoylase, not NO synthase. Pretreatment of rats with NG-nitro-L-arginine promoted activation of ornithine decarboxylase after intragastric hypertonic saline, but did not increase acid phosphatase release (damage). NO may therefore restrict activation of ornithine decarboxylase in response to damage. Exposure of primary cultures of guinea-pig gastric mucosal cells to S-nitroso-N-acetyl-penicillamine (SNAP) caused a concentration dependent induction of HSP 72, which was inhibited by an NO scavenger and blockade of transcription. The effect of SNAP was enhanced by decreasing the intracellular reduced thiol content with diethyl maleate, which itself also induced HSP 72 formation. Substantial amounts of NO may induce defensive responses in cells. Induction of iNOS was not detected in HGT-1 or AGS cells exposed to cytokines. Conclusions An arginase pathway may restrict availability of arginine for NO synthase in gastric mucosa or may be present to supply ornithine for polyamine synthesis. NO may modulate the response to damage of the stomach epithelium in vivo. Exogenous NO may induce a defensive response in gastric mucosal cells.

Cell surface analysis of the basidiomycete yeast cryptococcus neoformans

Cell surface properties of the basidiomycete yeast Cryptococcus neoformans were investigated with a combination of novel and well proven approaches. Non-specific cell adhesion forces, as well as exposed carbohydrate and protein moieties potentially associated with specific cellular interaction, were analysed. Experimentation and analysis employed cryptococcal cells of different strains, capsular status and culture age. Investigation of cellular charge by particulate microelectrophoresis revealed encapsulated yeast forms of C. neoformans manifest a distinctive negative charge regardless of the age of cells involved; in turn, the neutral charge of acapsulate yeasts confirmed that the polysaccharide capsule, and not the cell wall, was responsible for this occurrence. Hydrophobicity was measured by MATH and HICH techniques, as well as by the attachment of polystyrene microspheres. All three techniques, where applicable, found C. neoformans yeast to be consistently hydrophilic; this state varied little regardless of strain and culture age. Cell surface carbohydrates and protein were investigated with novel fluorescent tagging protocols, flow cytometry and confocal microscopy. Cell surface carbohydrate was identified by controlled oxidation in association with biotin hydrazide and fluorescein-streptavidin tagging. Marked amounts of carbohydrate were measured and observed on the cell wall surface of cryptococcal yeasts. Furthermore, tagging of carbohydrates with selective fluorescent lectins supported the identification, measurement and observation of substantial amounts of mannose, glucose and N-acetyl-glucosamine. Cryptococcal cell surface protein was identified using sulfo-NHS-biotin with fluorescein-streptavidin, and then readily quantified by flow cytometry. Confocal imaging of surface exposed carbohydrate and protein revealed common localised areas of vivid fluorescence associated with buds, bud scars and nascent daughter cells. Carbohydrate and protein fluorescence often varied between strains, culture age and capsule status of cells examined. Finally, extension of protein tagging techniques resulted in the isolation and extraction of two biotinylated proteins from the yeast cell wall surface of an acapsulate strain of C.neoformans.

A spectroscope study of some semiquinone radical ions

The effect of substituents on the value of the oxidation potential of quinones is reviewed and attempts to prepare substituted diphenoquinones with high oxidation potentials are reported. Attempts to characterise the mechanism of addition and substitution in diphenoquinones by identifying the products of the Thiele acetylation of diphenoquinone are reported. The reaction proved most efficient when the incoming acetylinium ion is directed by substituents in the diphenoquinone. A 1,8-addition to diphenoquinone is reported and characterised by isolating the products of the reaction between acetyl chloride and diphenoquinone, with perchloric acid as catalyst. The alternating linewidth effects observed in e.s.r.spectra are discussed and applied to account for such effects observed in the e.s.r.spectra of diphenosemiquinone anion and cation radicals. The spectra are analysed and the intramolecular processes producing these effects are discussed. A dianion diradical where intramolecular rotation about the 1 - 1' bond is restricted is produced by the oxidation of 2,2' ,4,4' -tetra hydroxybiphenyl. Previous studies of diphenosemiquinone anions are reviewed and alkylated diphenosemiquinone anion are produced by the reduction of the parent quinone with potassium hydroxide solution, the resulting radical being stabilised by the presence of pyridine. A qualitative interpretation of the solvent-ion effect in alkylated diphenosemiquinone anions is given. Diphanosemiquinone cation radicals are reviewed and previous studies are re-examined.

A study of some constituents in human female saliva

Changes in the concentration of some constituents in women's saliva during the menstrual cycle were studied. Saliva was used because it is easier to collect than other body fluids and is continuously available for analysis. Glucose, the enzyme 17-Acetyl-D-glucosaminidase (NAG) and Calcium which are saliva constituents and belong to three different chemical groups were selected for the study. Several analytical techniques were investigated. The fluorometric assay procedure was found to be the best because of its specificity and sensitivity for the estimation of these constituents. resides the fluorametric method a spectrophotometric method was used in the NAG determination and an atomic absorption method in the calcium estimation. Glucose was estimated by an enzymatic method. This is based on the reaction of glucose with the enzymes glucose oxidase and peroxidase to yield hydrogen peroxide, which in turn oxidises a non-fluorescent substrate, p-hydroxyphenylacetic acid, to a highly fluorescent product. The saliva samples in this determination had to be centrifuged at high speed, heated in a boiling water bath, centrifuged again and then treated with a mixture of cation and anion resins to remove the substances that inhibited the enzyme system. In the determination of the NAG activity the saliva samples were diluted with citric acid/phosphate buffer, and then centrifuged at high speed. The assay was based on the enzymic hydrolysis of the non-fluorescent substrate 4-Methyl-umbelli1eryl-p-D-glucosaminide to the highly fluorescent 4-Methyl-umbelliferone• Calcium was estimated by a fluorometric procedure based upon the measurement of the fluorescence produced by the complex formed between calcein blue and calcium, at pH 9 - 13. From the results obtained from the analysis of saliva samples of several women it was found that glucose showed a significant increase in its level around the expected time of ovulation. This was found in seven cycles out of ten. Similar results were found with the enzyme NAG. No significant change in the calcium levels was observe& at any particular time of the cycle. The levels of the glucose, the activity of the enzyme NAG and the concentration of the calcium were found to change daily, and to differ from one subject to another and in the same subject from cycle to cycle. The increase observed it salivary glucose levels and the enzyme NAG activity could be monitored to predict the time of ovulation.

The metabolism of n-methyl compounds

The metabolism of compounds containing the N-methyl group is discussed with particular consideration being made to the possible role of the product of oxidative metabolism, the N-hydroxymethyl moiety, in the generation of potentially toxic, reactive electrophiles. Particular pathways which are considered are: (i), the production of formaldehyde; (ii), the generation of iminium ions or imines; and (iii), the formation of N-formyl compounds which might act as formylating agents. 4-Chloro-N-(hydroxymethyl)benzamide and 3-(4-chlorophenyl)-1-hydroxy-methyl-1-methylurea (the product of oxidative metabolism of 3-(4-chlorophenyl)-1,1-dimethylurea) are model carbinolamides which do not readily release formaldehyde. The electrophilic properties of these model carbinolamides were investigated: neither reacted with nucleophiles such as cyanide or glutathione under physiological conditions. In contrast, N-(acetoxymethyl)-4-chlorobenzamide yielded the cyanomethylamide with potassium cyanide and S-(4-chlorobenzamidomethyl)glutathione with glutathione. 4-Chloro-N-(hydroxymethyl)benzamide and 3-(4-chlorophenyl)-1,1-dimethylurea were not biotransformed to electrophilic moieties when incubated with mouse hepatic 9000 x g supernatant and Acetyl-CoA or PAPS-generating system. N-(Acetoxymethyl)-4-chlorobenzamide was non-mutagenic to Salmonella typhimurium in the short term bacterial assay; but toxicity to the bacteria was observed. 4-Chloro-N-(hydroxymethyl)benzamide and 3-(4-chlorophenyl)-1,1-dimethylurea showed no mutagenicity or toxicity in the mutagenicity assay including an Aroclor-induced rat hepatic 9000 x g supernatant. Addition of Acetyl-CoA or a PAPS-generating system did not produce a mutagenic response. 4-Chloro-N-formlbenzamide did not act as a formylating agent towards the weak nucleophile aniline. However, 4-chloro-N-formylbenzamide, N-formylbenzamide, 3-(4-chlorophenyl)-1-formyl-1-methylurea and 3-(4-chlorophenyl)-1-formylurea are all metabolised by mouse hepatic mirosomes and post-microsomal supernatant. The results demonstrate the potential for N-hydroxymethyl compounds to generate highly reactive species if these are substrates for conjugation with sulphate (or acetate). The model compounds employed here, apparently do not show any ability to be conjugated themselves, however, other N-hydroxymethyl compounds might be readily conjugated. The formation of N-formyl compounds does not appear to be toxicologically significant, as adjudged on limited experiments performed, but rather represent a detoxification pathway.

Investigation of the metabolism of N, N-Dimethylformamide and its metabolites

The industrial solvent N, N-dimethylformamide (DMF) causes liver damage in humans. The hepatotoxicity of N-alkylformamides seems to be linked to their metabolism to N-alkylcarbamic acid thioesters. To clarify the role of metabolism in DMF hepatotoxicity, the metabolic fate of DMF was investigated in rodents. DMF was rapidly metabolised and excreted in the urine as N-hydroxymethyl-N-methyl-formamide (HMMF), N-acetyl-S-(N-methylcarbamoyl) cysteine (AMCC) and a metabolite measured as formamide by GLC. At high doses (0.7 and 7.0mmo1/kg) a small proportion of the dose was excreted unchanged. AMCC, measured by GLC after derivatisation to ethyl N-methylcarbamate, was a minor metabolite. Only 5.2% of the dose (0.1mmo1/kg) in rats or 1.2% in mice was excreted as AMCC. The minor extent of this metabolic pathway in rodents might account for the marginal liver damage induced by DMF in these species. In a collaborative study, volunteers were shown to metabolise DMF to AMCC to a greater extent than rodents. Nearly 15% of the inhaled dose (0.049mmo1/kg) was excreted as AMCC. This result suggests that the metabolic pathway leading to AMCC is more important in humans than in rodents. Consequently the risk associated with exposure to DMF might be higher in humans than in rodents. The metabolism of formamides to S-(N-alkylcarbamoyl) glutathione, the metabolic precursor of the thioester mercapturates, was studied using mouse, rat and human hepatic microsomes. The metabolism of NMF (10mM) to S-(N-methylcarbanoyl)glutathione (SMG) required the presence of GSH, NADPH and air. Generation of S-(N-methyl-carbamoyl)glutathione (SMG) was inhibited when incubations were conducted in an atmosphere of CO:air (1:1) or when SKF 525-A (3.0mM) was included in the incubations. Pre-treatment of mice with phenobarbitone (PB, 80mg/kg for 4 days) or beta-naphthoflavone (BNF, 50mg/kg for 4 days) failed to increase the microsomal formation of SMG from NMF. This result suggests that the oxidation of NMF is catalysed by a cytochrome P-450 isozyme which is unaffected by PB or BNF. Microsomal incubations with DMF (5 or 10mM) failed to generate measurable amounts of SMG although DMF was metabolised to HMMF. Incubations of microsomes with HMMF resulted in the generation of a small amount of SMG which was affected by inhibitors of microsomal enzymes in the same way as in the case of NMF. HMMF was metabolised to AMCC by rodents in vivo. This result suggests that HMMF is a major intermediate in the metabolic activation of DMF.