The present work has investigated the diverse interactions of selected methylenedioxyphenyl compounds with hepatic components, both in vivo and in vitro. Many methylenedioxyphenyl compounds were found to be activated via the hepatic monooxygenase system, resulting in the formation of "active metabolites" capable of binding in a 1:1 ratio with the haem of cytochrome P-450. These methylenedioxyphenyl metabolite-cytochrome P-450 complexes were found to be very stable and could be formed both in vivo and in vitro. However, safrole or isosafrole metabolite-cytochrome P-450 complexes, generated in vivo, were found to be dissociated with relative ease in the presence of certain alternative Type I substrates of the mixed function oxygenase system. This time-dependent dissociation of the complexes was observed after their formation in vivo, but not in vitro. Safrole and isosafrole appeared to induce the formation of a novel haemoprotein of the cytochrome P-448 type. The spectral properties of the safrole- or isosafrole-induced haemoproteins were found to be similar to but not identical with those of cytochrome P-448. Isosafrole-related material was found to become bound to other hepatic cellular components in an apparently irreversible manner. Results obtained using a novel approach, utilizing isolated hepatocytes, were compared with those found in vivo and in vitro using microsomes. The data obtained indicated that the use of the isolated hepatocyte system is more reliable than the usual in vitro microsomal screen for covalent binding, which has many inherent pitfalls.

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The aim of this thesis is to provide information concerning the control of the concentrations and the physiological roles of cyclic nucleotides and adenosine in animal tissues. For this purpose the activities, kinetics and some properties of adenyl cyclase, cyclic AMP and cyclic GMP phosphodiesterase, 5'-nucleotidase, adenosine kinase and adenosine deaminase have been studied in muscles, nervous tissues and livers from a number of vertebrates and invertebrates. Chapter 1 Metabolic events within an organism are controlled and coordinated at a number of levels of complexity. Cyclic nucleotides and adenosine are messenger molecules concerned with the coordination of metabolism. In Section A some aspects of cyclic nucleotide metabolism are considered and certain points are made which are relevant to the interpretation of the data presented in later chapters. Some factors which modulate the activities of adenyl cyclase and phosphodiesterase in vitro are described and the relevance of their effects to the in vivo situation is discussed. The significance of the basal and fluoride-stimulated activities of adenyl cyclase and the unusual kinetics of phosphodiesterase is considered. Cyclic AMP, and possibly cyclic GMP, transmit hormonal stimuli to the interior of the cell but it is pointed out that they may have further roles. In particular, cyclic AMP may play an important role in nervous tissue. Some of the effects of cyclic AMP depend on the phosphorylation of enzymes or other proteins by a cyclic AMP-dependent protein kinase. The mediation of protein kinase means that there may be a low concentration of cyclic AMP in the cell but nevertheless a small change in the concentration of cyclic AMP may produce a large and rapid effect on the flux through a metabolic pathway. The low concentration of cyclic AMP means that a high rate of turnover of cyclic AMP is associated with only a low utilization of energy. The high rate of turnover of cyclic AMP is necessary for a rapid response of the cell to hormonal stimulation of adenyl cyclase. It has frequently been claimed that the concentration of cyclic AMP available to protein kinase or phosphodiesterase is very different from that measured in the whole cell. It is shown that it is often possible to provide different explanations for the data which have led to this claim. In Section B adenosine metabolism and the role of adenosine are discussed in some detail. It is pointed out that it does not now appear that adenosine is a metabolite on a main pathway of intermediary metabolism. There have been few measurements of tissue concentrations of adenosine and there is no consensus of opinion concerning the primary location of adenosine intra- or extracellular. The simplest hypothesis is that adenosine equilibrates across the cell membrane. Evidence is presented to show that in vivo adenosine is produced by 5'-nucleotidase and utilized by adenosine kinase and adenosine deaminase. The regulation of the activities of these enzymes for the purpose of controlling the concentration of adenosine is discussed. A theory of the control of the concentration of adenosine by changes in the phosphorylation state of the cell is presented. This theory predicts variations in the rate of output of adenosine from the cell which are consistent with data in the literature. The output and uptake of adenosine by the cell are discussed in detail. Low concentrations of adenosine within a tissue are mostly phosphorylated within the cell; deamination of adenosine, which may be partly extracellular, becomes relatively more important when the concentration of adenosine is high. Exogenous adenosine has a number of effects on the physiology of mammalian tissues. For example, it relaxes most smooth muscles. Adenosine produces a large increase in the content of cyclic AMP in mammalian brains and it is suggested that a number of the physiological effects of exogenous adenosine are mediated by a change in the concentration of cyclic AMP. In the final section of Chapter 1 it is argued that adenosine plays a role in the regulation of blood flow in some vertebrate tissues. It may also play a role in neurotransmission. Chapter 2 For comparative studies it is necessary that enzyme assays are applicable to enzymes in homogenates of very different tissues. In some cases very small quantities of tissue are available. Moreover, when maximal activities are being measured, optimal conditions for the assays are required. Radiochemical assays were developed to meet these requirements; they are described in Section A of Chapter 2. In the succeeding sections the development of each assay is described. A number of preliminary results are presented, including pH curves. Finally, the graphical methods which were used to analyze the results and the significance of the data obtained are discussed. Chapter 3 As a preliminary to the discussion of the results of Chapter 3, it is reported that the extraction of adenyl cyclase using a hypotonic medium and a ground-glass homogenizer and pestle leads to higher basal activities and a greater degree of stimulation of enzyme activity by fluoride than extraction in isotonic medium using an ordinary glass homogenizer. The former method was used for a comparative study of basal and fluoride-stimulated activities of adenyl cyclase. The activities and K_m values of adenyl cyclase and cyclic AMP and cyclic GMP phosphodiesterase are reported for a number of animal tissues. In general the activities decrease in the order nervous tissues> hearts > muscles with a high oxidative capacity and livers > muscles with a low oxidative capacity. The kinetic data for phosphodiesterase are analyzed on the assumption that at least two enzymes (high and low K_m) contribute to the kinetics. It is argued that the analysis supports this assumption. It is also argued.that, in vertebrate tissues, one high K_m enzyme hydrolyzes both cyclic AMP and cyclic GMP. The data are applied to a simple model of the turnover of cyclic AMP in the cell. Maximum and minimum tissue concentrations of cyclic AMP are predicted from the model. The predictions are consistent with much reported data. It is suggested that tissue concentrations of cyclic AMP do not vary greatly in vivo. The results, as interpreted by the model, indicate that the extrusion of cyclic AMP from the cell does not play an important role in the turnover of cyclic AMP in most tissues. The roles of the high and low K_m phosphodiesterases are discussed. It is proposed that the high K_m enzyme prevents the accumulation of cyclic AMP in tissues where the activity of adenyl cyclase may be higher than the activity of low K_m phosphodiesterase. However, it is more efficient for the cell to synthesize the low K_m enzyme. Possible explanations for variations in the proportions of high and low K_m enzymes between tissues are suggested. One of these explanations is consistent with the present data only if the simple model of the turnover of cyclic AMP is modified. The modification of the model permits the explanation of some further data. According to the modified model adenyl cyclase and low K</sub>m</sub> phosphodiesterase are located at the cell membrane, whereas high K_m phosphodiesterase is located in the cytosol. Nevertheless, it is claimed that, unless the local activity of adenyl cyclase is much greater that that of low K_m phosphodiesterase, the compartmentation of cyclic AMP is not great. The roles of cyclic AMP in nervous tissue and muscle are discussed.

The activities of acetyl-CoA hydrolase and acetyl-CoA synthetase and the influence of diet and feeding level on them were investigated in various ovine tissues and used to determine both the potential rate of substrate cycling between acetate and acetyl-CoA and the contribution of this cycle to energy expenditure in the ruminant. Two experiments, using lambs, were conducted in an attempt to further understand biochemical pathways which may influence the efficiency of utilisation of ME and explain why this is lower for fibre (sugarbeet pulp) when compared to starch (barley) based diets. The influence of carbohydrate source, feeding level and protein level on plasma concentrations of acetate, glucose and insulin and the rates of acetate incorporation into CO<SUB>2</SUB> and lipid were studied. ATP-stimulated acetyl-CoA hydrolase is present in rumen epithelium, muscle and the cytoplasm of ovine liver but not in perirenal adipose tissue, and it is not inactivated by cold. 'Mitochondrial' acetyl-CoA hydrolase was detected in all tissues investigated. The activities of acetyl-CoA hydrolase and acetyl-CoA synthetase tended to be higher in perirenal adipose tissue of lambs fed on sugar beet diets and their activities decreased with increasing level of feeding (P< 0.05). Variation in plasma characteristics were more easily observed in lambs fed semi-continuously compared to lambs which received their ration once daily. Higher levels of crude protein in the diet resulted in lower plasma acetate concentrations (P< 0.05). The rates of acetate incorporation into CO<SUB>2</SUB> and lipid were influenced by acetate (P< 0.01), glucose (P< 0.0001) and insulin (P< 0.01). Higher levels of crude protein in the diet resulted in greater rates of acetate incorporation into lipid (P< 0.05) whereas feeding sugarbeet pulp resulted in increased rates of acetate incorporation into CO<SUB>2</SUB> (P< 0.05). A technique involving open column ion exchange chromatography, freeze drying and HPLC was developed for the concentration and separation of plasma organic acids. Organic acid recoveries were 43-69%. An experiment was conducted to investigate the activity of the substrate cycle between acetate and acetyl-CoA in calf liver <i>in vivo</i>. Several problems were encountered, notably huge variation in blood flows (including negative rates). This introduced large variation into the calculated fluxes and no meaningful conclusions were made. It was calculated from enzyme measurements made <i>in vitro</i>, that the substrate cycle in ovine liver may potentially account for 2.5% of basal heat production. It is suggested that the efficiency of utilisation of ME is related to glucose homeostasis, involving VFA and protein metabolism.

Studies of mutant mice whose thalamocortical tract is disrupted illustrate that genes involved in programming thalamocortical tract navigation are required by projecting cells of the dorsal thalamus and along its route to supply axon navigation cues. Small-eye homozygotes (<i>Pax6^Sey/Sey</i>) in which the transcription factor Pax6 is inactivated exhibit a range of defects throughout the developing central nervous system, including a failure in thalamocortical tract formation, and die at birth. The <i>Pax6^sey/Sey</i> phenotype describes disruption of both the dorsal thalamus and the tissues through which the thalamocortical tract navigates so does not reveal whether axons projected by the dorsal thalamus fail to navigate due to intrinsic mis-programming or due to a lack of important navigational cues along the route. To test the hypothesis that Pax6 is required within the dorsal thalamus for tract projection I developed a novel <i>in vitro</i> assay for thalamic axon navigation and cell surface properties. First I generated lines of embryonic stem (ES) cells which ubiquitously express a tau tagged green fluorescent protein (tau-GFP) transgene. Germline transmission of ES lines produced two lines of transgenic mice designated TgTP6.3 and TgTP6.4. I show that the tau-GFP transgene is ubiquitously expressed in developing TgTP6.3 brain and that tau tagging anchors tau-GFP to the microtubule component of the cytoskeleton resulting in clear fluorescent marking of subcellular structures, including axons and the mitotic machinery. The experimental potential of the TgTP6.3 mice is demonstrated in several cell mixing paradigms, including mouse chimeras and brain cell and tissue culture systems, where tau-GFP cells and axons can easily be detected against a background of unlabelled cells and tissues. The TgTP6.4 line exhibits very low levels of tau-GFP expression in the developing brain so is unsuitable for these experiments.

The LKB1 protein kinase has previously been implicated in a number of major physiological processes including cell proliferation, polarity and energy metabolism. LKB1 is an upstream kinase for AMP-activated protein kinase (AMPK) and 12 members of the AMPK-related family of kinases. In this study, western blotting of LKB1 from various mouse tissues indicated the existence of two molecular weight bands in testis. This, along with the presence of a potential alternative exon at the 3’ end of the gene, led to the discovery that an alternatively-spliced variant of LKB1 is present in mice. This new splice variant has been termed the ‘short form’ (LKB1S) and the original protein termed the ‘long form’ (LKB1L). The proteins are identical in sequence apart from the C-terminus. The overall aim of this study was to characterise the LKB1S protein; its activity, regulation and possible physiological function, using overexpression studies in mammalian cells and analysis of mice lacking LKB1S. An LKB1S-specific antibody was used to show that LKB1S is primarily expressed in testis. It was demonstrated, using overexpression studies, that both forms can activate AMPK, form an active complex with the regulatory proteins STRADα, STRADβ and MO25 and have a similar sub-cellular localisation. The C-terminus of mouse/human LKB1L can be phosphorylated at serine 431/428 (Sapkota et al., 2001). This residue is absent from LKB1S and so the effect of phosphorylation was investigated to determine if this could lead to differences in activity between the splice forms. However, the results suggest that phosphorylation at this site does not affect the ability of LKB1L to activate AMPK in cells. Male mice lacking expression of LKB1S are infertile. The mice display severe sperm abnormalities and sperm counts are significantly reduced. Histological analysis of testis showed that although early spermatogenesis appears to progress normally, abnormalities become apparent later on, culminating in very few spermatozoa reaching the epididymis. One possible reason that has been investigated is that there is a defect in the release of mature sperm from the seminiferous epithelium (spermiation).

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No description available.

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No description available.