No description available.

No description available.

No description available.

No description available.

The role of butyrate in the metabolism and development of a healthy colonic epithelium and thus in the prevention of colonic diseases is well documented, but the ecology of butyrate-producing bacteria is little understood. Dietary polysaccharides that are not digested in the small intestine are a major source of energy for the commensal bacteria resident in the large intestine. Resistant starch makes up a large proportion of dietary substrate reaching the human colon and is also reported to be butyrogenic. Recent studies have shown that low G+C% Gram-positive bacteria, which include butyrate-producing isolates, are among the most abundant components of the adult colonic microbiota. Here, in vitro growth experiments demonstrated that certain new butyrate producing isolates (Roseburia spp./Butyrivibrio fibrisolvens) were able to utilise starch as a sole energy source, with a growth preference for high amylopectin starches. Active starch-degrading enzyme were cell-associated in these strains and were visualised using zymogram analysis and found to be of high molecular weight. An amylase gene from Butyrivibrio fibrisolvens 16.4 was sequenced following polymerase chain reaction (PCR) amplification with degenerate oligonucleotides and chromosome walking. The putative amylase consists of an open reading frame of 4002 bp encoding a protein of 1333 amino acids with a calculated Mr of 144,470. Analysis of the multidomain organisation of this enzyme indicated that it is bound to the cell wall, with its putative catalytic domain and repeated starch-binding modules protruding into the extracellular matrix. This work also describes the use of an in vitro continuous culture fermentor system and human microbiota-associated mice (in vivo) to assess the potential of certain butyrate-producing anaerobes for probiotic and/or prebiotic applications.

The c-myc gene contains an internal ribosome entry site (IRES) within its 5' untranslated region. The IRES was shown to have different activities between cell lines suggesting a requirement for protein trans-acting factors that are present in these cell lines in varying amounts. In addition a number of proteins have been shown to interact with the IRES by north-western and UV cross-linking analysis. Investigation of the protein factors involved in c-myc IRES translation identified PCBP1 (Poly (rC) binding protein 1), PCBP2, HnRNPK (heterogeneous nuclear ribonucleoprotein K), UNR (upstream of N-ras) and UNRIP (unr interacting protein) as having a role in c-myc IRES translation, PCBP1, PCBP2, HnRNPK and UNR were found to directly interact with the IRES RNA by UV cross-linking and electrophoretic mobility shift assays (EMSAs). Investigation of the proteins effect on c-myc IRES activity showed stimulation of IRES activity in HeLa cells by PCBP1 and PCBP2. The factor HnRNPK was found to have a slight stimulatory effect in vivo. In addition PCBP1 and PCBP2 were found to stimulate IRES activity in vitro in combination with UNR and UNRIP. Using the yeast three-hybrid system a number of additional proteins were found to interact with the c-myc IRES RNA. A novel Fibrillarin-like protein was identified and shown to strongly interact with the IRES by EMSA. Studies to determine a direct role of this factor in c-myc IRES translation were inconclusive. The study of translation of the c-myc gene identified an IRES within its 5'UTR. Investigation of the role of trans-acting factors in its translation showed a possible role of the factors PCBP2, HnRNPk and ITAF45 (IRES trans-acting factor 45).

This project aimed to explore factors that modulate neurolipofuscin accumulation, using two experimental arthropod species, the locust, Locusta migratoria, and the freshwater crayfish, Pacifastacus leniusculus. The pattern of age-related accumulation of neurolipofuscin in L. migratoria was found to differ from that previously reported for crustaceans, with little accumulation prior to maturity and an exponential increase with high variability thereafter. It was shown, for the first time in any crustacean, that unilateral eyestalk ablation reduces neurolipofuscin accumulation rate in the contra-lateral eyestalk of P. leniusculus. It is hypothesized that this represents either reduced lipofuscinogenesis due to neurohormonal effects on oxidative catabolism or increased lipofuscin degradation by accelerated proteolysis following CNS damage. This finding means that longitudinal measurements of lipfuscin, i.e. in the same individual, by eyestalk biopsy, cannot be used to assessa natural lipofuscin accumulation rates in individuals of unknown age. Neurolipofuscin accumulation rate in ablated signal crayfish was found to be strongly inversely correlated with physiological age, with old individuals generally losing lipofuscin after ablation. Although this pattern is likely to be an artefact of ablation, it is the first quantitative evidence of in vivo reversibility of lipofuscin accumulation for any species and has important gerontological implications. Annual cohorts were detected by modal analysis of a neurolipofuscin concentration histogram for a pond population of P. leniusculus for the first time. Growth curves fitted to the length-at-age data obtained from this analysis were compared with results of three conventional methods for growth curve estimation: size-frequency analysis, anniversary tag-recapture and laboratory rearing. This analysis highlighted problems with extrapolation of growth rates from laboratory rearing to the field. Neurolipofuscin methodology is the only approach that can give age-length data for older individuals in the wild population and measurements of longevity.

HL-60 human promyelogenous leukaemic cells were used as a model to determine the cytotoxic potential of the unsymmetrically substituted polyamine analogues CHENSpm, CPENSpm and IPENSpm with a view to their use as chemotherapeutic agents. The cytotoxicity was compared with etoposide, an established cytotoxic drug. The analogues CHENSpm and IPENSpm induced cytotoxicity over 48 h, with decreases in cell number and protein content, with CPENSpm showing a growth inhibitory effect after 96 h. The cellular content of all 3 polyamines was decreased in all treatments, and this resulted from increases in the catabolic enzyme SS AT, and polyamine export, determined as active export by the presence of acetyl-polyamines and putrescine in the culture medium. All 3 polyamine analogues were detected intracellularly, and their internalisation was essential for their toxicity, as their co-incubation with transport inhibitors provided protection against analogue toxicity. Use of the transport inhibitors also confirmed the polyamine transporter as the route of analogue uptake. Analysis of the type of cell death induced by the unsymmetrically substituted polyamine analogues confirmed it as apoptosis, through morphological determination using DAPI staining, cell cycle analysis showing a pre G0/G1 peak, increased DNA fragmentation and induction of caspase-3-like enzymes. HL-60+ cells were used in conjunction with wild-type HL-60+ cells in an attempt to determine the pathway to apoptosis induced by these analogues. HL-60+ cells contained a stable vector that overexpressed bcl-2, an anti-apoptotic protein that prevents the mitochondrial release of apoptogenic factors including cytochrome c. These cells had shown virtually no toxicity to the analogues over all exposure periods, despite the intracellular accumulation of the analogues. Western blotting was used to probe for the presence of cytochrome c, an activator of apoptosis through the mitochondrial pathway, in the cytosol of both cell lines in response to both analogue and etoposide treatment. The detection of cytochrome c in the cytosol of wild-type HL-60 cells, but not the bcl-2 overexpressors, suggested that the mitochondrial route to apoptosis was being activated, and this was further confirmed by the increased activation of caspase-9, the initiator caspase in this route, with only basal levels of caspase-8, the initiator caspase in the death effector route, detected. These data strongly suggest that the exposure of HL-60 cells to unsymmetrically substituted analogues, CHENSpm and IPENSpm, results in the activation of apoptosis through the mitochondrial release of cytochrome c. Consideration of these analogues as future chemotherapeutic agents requires that their effects are decreased in normal cells. Two normal cell lines and primary human cell cultures were exposed to these analogues and results confirmed that the analogues were selectively cytotoxic towards malignant cells. Overall, these analogues were effective cytotoxic agents in the HL-60 human leukaemic cell model, and show promise as chemotherapeutic agents through a low dose and short exposure, which is directly comparable with etoposide. The identification of the mitochondrial pathway to apoptosis as the mechanism of action of these analogues means they would not be useful chemotherapeutic agents in tumours where anti-apoptotic proteins in the mitochondrial apoptotic pathway, such as bcl-2, are overexpressed. However, these analogues could be effective cytotoxic agents in other types of cancer, particularly those where the death effector pathway is suppressed due to a similar anti-apoptotic protein overexpression, and could be used either alone, or in conjunction with existing cytotoxic drugs.

Blast injuries represent a problem for civilian and military populations. The response to thoracic blast injury involves a reflex bradycardia, hypotension and apnoea. Casualties who have suffered a blast injury are likely to receive morphine as an early treatment, and may go on to suffer a haemorrhage, thus requiring fluid resuscitation. Aims of this thesis included determination of the effect of blast injury on the response to haemorrhage and whether these responses or their interaction are modified by morphine, and to compare the cardiovascular effects of early and late resuscitation with different solutions following blast injury and haemorrhage. Early cessation of the blast-induced apnoea is important if the patient is to adequately maintain arterial oxygen tensions and thus prevent the development of tissue hypoxia and a subsequent secondary inflammatory response. Therefore, the final aim of this thesis was to determine whether doxapram could shorten the duration of apnoea induced by thoracic blast. Results confirmed that the response to thoracic blast injury involves a bradycardia, hypotension and apnoea, and also a vasodilation and a reduction in blood flow in the femoral vascular bed. New findings from this thesis show that thoracic blast augments the bradycardia and hypotension seen during haemorrhage and that morphine attenuates this effect. The hypovolaemic blast-injured patient may be resuscitated early or late after haemorrhage with blood, 0.9% saline, colloids (modified gelatin and hydroxyethyl starch) hypertonic saline or hypertonic/hydroxyethyl starch. These fluids restored blood pressure and femoral blood flow to pre-haemorrhage levels for at least 30 minutes. However, resuscitation with hypertonic saline/de>ttran was shown to be deleterious following blast injury and haemorrhage as blood pressure and femoral blood flow was not maintained for longer than 5 minutes following resuscitation with this fluid. The blast-induced apnoea and hypotension can be significantly attenuated by doxapram immediately following blast injury. This respiratory stimulant may also result in an improvement in venfilation/perfusion matching in the lungs and thus better fissue oxygenation, as administration of doxapram resulted in an improvement in the indices of metabolic acidosis. The new information gained from the work covered by this thesis could potentially lead to better treatment of the blast-injured victim.

In this project, the N-acetyl-ex-D-galactosamine (GalNAc) binding lectin from the green marine alga Codium fragile ssp. tomentosoides (C. fragile) was purified and techniques developed to label the lectin for visualisation by light and electron microscopy and electrophoresis. This represents the first time a histochemical reagent has been developed from a marine alga. The new reagent was initially assessed for transmission electron microscopy using human blood group A1 erythrocytes. A novel method gave a topographical view, and showed the distribution of gold particles on the surface of erythrocytes. The pattern of C. fragile lectin binding to pig normal oral epithelium was studied in the environmental scanning electron microscope to avoid charging artefact, using paraffin wax sections of pig normal epithelium stained with C. fragile lectin-gold conjugate enhanced with silver. X-ray micro analysis demonstrated lectin binding on the plasma membrane surface of epithelial cells at cells to cell contacts suggesting binding to cellular adhesion molecules. Biotinylated lectins binding GalNAc were used to investigate, identify and compare the binding of lectins in pig normal oral epithelium and altered glycoconjugates in cultured malignant cells from human oral tumours, using lectin histochemistry in the light microscope. Lectin from C. fragile was compared with Dolichos biflorus, a lectin from plants, and Helix pomatia (HP A) from snails. Although each lectin binds GalNAc it was shown that their binding pattern to pig normal oral epithelium was different, demonstrating that these lectins could be used to identify altered cellular glycosylation in the normal cellular maturation process. Cultured human oral tumour cells from different grades of malignancy were investigated using this panel of lectins. Binding of GalNAc specific lectins to cultured tumour cells changed in relation to their level of differentiation. This discriminating capability of GalNAc specific lectins offers exciting potential as indicators of tumour prognosis in human oral epithelial tumours. The lectins from C. fragile and HP A gave very similar staining results using histochemistry. Binding of these lectins to cell membrane glycoproteins was investigated using electrophoresis to show that C. fragile lectin binds to more and different cell membrane glycoproteins than lectin from HP A, but did not bind to purified CD44, excluding this adhesion molecule as a candidate for binding these lectins.