The experiments were made using serial samples from individuals and by injecting directly to their blood stream. Alloxan injeoted at near lethal doses caused substantial elevation of blood glucose in the adapted fish. However evidence against high insulin levels in the adapted fish imcluded: a) an improved response to a repeated glucose load, and b) an improved response to a single glucose load following pretreatment with insulin. Insulin (1 IU/Kg) was effective in reducing the high blood glucose post capture to low adapted levels. She space in which glucose is diluted following injection to the blood was estimated as 29.4 - 3.4% of the body weight and this space was significantly depleted of glucose by insulin infection. 3. The concentration of lactic acid formed in the lateral muscles during exercise or capture by trawl is related to the concentration of the glycogen stores of the muscle cells. The total store is normally between 200-400 mg lactate or glucose per 100 g muscle. The lactic acid levels of blood do not necessarily reflect the elevated muscle levels and a condition where the lactate is not released from the exhausted muscle tissue to the blood is described. Measurements of blood and lymph circulation in the muscle indicate that this release or non-release of lactate is independent of circulation. adrenergic stimulation promotes the expansion of muscle blood capillaries yet inhibits passage of lactate from muscle to blood. The evidence suggests that the non-release mechanism will be found in either a property of the Sarcolemma or in the chemical form of the lactic acid within the cell. The three phases; of blood glucose change and the release and non-release of lactic acid are discussed in relation to a general adaptation syndrome. Evidence for the existence in the fish of the stress apparatus is summarised and the time scale of the changes observed in the present study is compared with that recorded in other experiments examining change following stress in teleosts. During the study investigating release arid non-release of lactic acid a previously undescribed lymph circulation was discovered and these findings are described in part 2. The anatomy of the teleost lymph system is considered in relation to the observed flow of lymph. The vessels described by anatomists as longitudinal ventral, dorsal and lateral lymph ducts do not pass lymph to the head but act as collecting sinuses; the lymph flows from these sinuses to the neural lymph duct via the interspinal ducts. At the junction of each interspinal duct and the neural lymph duct there is a simple valve. lymph drains through this system as a result. ;of a negative pressure developed in the neural lymph canal by the respiratory movements Of the gill apparatus. The rigid nature of the neural lymph duct and the valves where the interspinal ducts join it allow the negative pressure to develop from a negative/ positive gill generated pressure cycle in the cephalic lymph sinuses. Valves linking cephalic lymph sinuses aid the return of lymph to the duct of Cuvier using the same pressure cycles. The drainage system is different in the salmonidae where the neural lymph duct is degenerate and the network of peripheral ducts serve as lymph return ducts. This can prohably be explained by the occurrence of higher circulation pressures in the salmonidae. Lymph from the neural lymph duct is a clear fluid having a composition similar to that of blood plasma. like blood, lymph clots contains normal blood levels of mobile leucocytes and thrombocytes and lymph reflects blood levels of lactate. 'Lymph contains nearly no erythrocytes and the protein concentration of neural duct lymph is 80.4% that of blood plasma, 131I iodinated human serum albumin (131I HSA) passes from the vascular space and appears in the neural lyijtph canal at 80/S plasma levels.51Or labelled erythrocytes are limited to the vascular space. The volume of blood in plaice muscle and skin is normally 0.07-0.1 M1 131 per 100 g muscle and 0.4-0.7 ml per 100 g skin. She I HSA labelled extra vascular space (interpreted as lymph) measured 4-7 ml per 100 g muscle and 60-74 ml per 100 g skin. She function and significance of the lymph system is discussed.

Aspects of tissue protein metabolism were studied in different groups of Atlantic salmon (Salmo salar). Firstly, 2 groups of sea water salmon were fasted for 3 and 5 days, prior to the measurement of tissue fractional rates of protein synthesis (ks, %day), using the method of 3H-phenylalanine flooding. The sensitivity of liver and gill to a short-term fast was indicated by their reduced rates of ks, while in other tissues such as ventricle, stomach and red muscle protein synthesis was unaffected. Both liver and gill are tissues which show the greatest capacities to synthesise protein, expressed by their high RNA contents. The response of tissue protein metabolism to sexual maturation was investigated in 2 groups of salmon undergoing river migration, tissues were analysed in both July salmon (sexually maturing) and in more mature October salmon. White muscle contributed most of the amino acids required during maturation, denoted by the high loss of protein and RNA from the flesh of the salmon by October. Red muscle, gill and ventricle were tissues which were protected during maturation, showing only slight changes in rates of protein metabolism. Liver, stomach and ovary on the other hand increased their RNA and protein contents, and showed increased rates of protein synthesis. The liver however, displayed a greater increase in its RNA concentration than protein i.e. the liver by October had increased its capacity to produce large amounts of export proteins. Despite the overall loss by stomach and other visceral tissues of protein and RNA, the stomach by October showed a dramatic 5-6 fold increase in its rate of protein synthesis. It was concluded that smooth muscle may have a particular role or function in the sexually mature fish. Factors controlling these in vivo changes in protein synthesis were investigated using rainbow trout (Oncorhynchus mykiss) isolated hepatocytes and a smooth muscle preparation in vitro . Anabolic actions on hepatocyte protein synthesis were exhibited by insulin, thyroxine and in the absence of fetal calf serum by triiodothyronine. The synthetic glucocorticoid, dexamethasone, unlike its actions in muscle, also exhibited an anabolic action on hepatocytes, by raising RNA and protein levels in cells from immature fish, while increasing protein synthesis rates in liver cells of sexually mature fish. Estradiol, like dexamethasone, stimulated rates of protein synthesis over 24 hours. However, hydroxyprogesterone caused no change in protein synthesis and decreased the effect of estradiol. Estradiol also increased protein synthesis rates in smooth muscle by some 30%. However, hydroxyprogesterone, as in liver cells, caused no stimulation of protein synthesis and again decreased the estradiol stimulated ks. It was proposed that estradiol is one of the factors involved in increasing the ks in the stomach of the sexually maturing salmon, while progesterone regulates the action of estradiol towards the end of sexual maturation, when such an effect of estradiol on liver and smooth muscle ks is unnecessary.

Many marine animals accumulate large amounts of lipid in their livers, muscles and other body tissues. Alkyldiacylglycerols and wax esters are two examples of unusual lipid classes which are stored as a large percentage of the total body lipids in some marine species, these lipids being unusual only in the sense that they do not occur in such high proportions in the body lipids of terrestrial species. 2. Several roles have been proposed for the marine lipid reserves generally and the neutral lipid classes alkyldiacylglycerols and wax esters in particular. It is considered that special biochemical mechanisms operate in species which store these lipid classes in quantity. The biosynthesis of their mutual precursor, fatty alcohol, is considered to be fundamental to these mechanisms. 3. In order to study the biosynthesis of alkyldiacylglycerols and wax esters in more detail, two animals typical of marine groups which accumulate these lipids were chosen as a source of experimental material. The dogfish, Squalus aoanthias, contains alkyldiacylglycerols that comprise about 40% of the total liver lipid which itself accounts for about 60% of the wet weight of the liver. The calanoid copepod, Euchaeta norvegiaa, contains in its body an oil sac where wax esters comprise 60% of the total lipid. The body lipid of this animal accounts for about 20% of the dry weight. 4. In vivo and in vitro incorporations of radioactively labelled precursors were used to study routes of neutral lipid biosyntheses in dogfish, tissues, especially liver. It was found that liver rapidly oxidised fatty alcohol to fatty acid which, was then exported to other tissues, especially muscle. Triacylglycerols were readily formed in liver and intestinal tissue from fatty acid precursors. 5. Wax esters were also readily biosynthesised in preparations of dogfish liver tissue, such biosynthesis requiring the presence of ATP and CoA. The formation of wax esters was enhanced by the addition of fatty alcohol to incubations of liver tissue. 6. A feature of the in in vitro work with dogfish tissues was the lack of a significant rate of biosynthesis of alkyldiacylglycerols compared to the other neutral lipids. There was also a very low rate of formation of fatty alcohol from fatty acid precursor and it is postulated that biosynthesis of fatty alcohol with consequent accumulation of alkyldiacylglycerols may be dependent on suitable reducing conditions existing in the cells,not realised in these experiments. Conditions must exist in the natural environment of the animal to facilitate the biosynthesis ofalkyldiacylglycerols for storage in the liver. Although this lipid class may be used as an energy reserve by dogfish, it is thought that its accumulation in the liver of this animal may be significant in buoyancy regulation. 7. Cell-free studies with preparations of whole Euohaeta showed that precursor fatty acid and fatty alcohol were readily incorporated into wax esters. Biosynthesis of wax esters was again found to be dependent on the presence of ATP and CoA. The presence of fatty alcohol but not fatty acid greatly enhanced production of wax esters. 8. Similar experiments with, cell-free preparations from Calanus finmarchicus and Acartia sp. confirmed, the Euchaeta results and showed additionally that Biosynthesis of fatty alcohol in calanoid copepods is dependent on the presence of reduced pyridine nucleotides. Thus, in common with the dogfish, the accumulation of a major lipid class was thought to require the presence of biosynthesised fatty alcohol. 9. A mechanism is presented which allows for the efficient conversion of glucose and fatty acids into wax esters in response to an intermittent high quantity of phytoplankton food for temperate species of copepod. It is argued that the limitations, normally present in lipid biosynthesis are eliminated by this mechanism so that a very extensive conversion of phytoplankton food to lipid may take place in a relatively short time. Wax esters are looked upon as long term energy reserves in calanoid copepods in place of the more usual triacylglycerols.

The aim of these experiments was to investigate the interrelationships of fat and carbohydrate (CHO) metabolism in mammalian muscle. In particular, it was hoped to clarify the mechanisms regulating the integration of the supply and utilisation of metabolic substrates in skeletal muscle. This was achieved by studying the response to a perturbation of normal metabolic processes. Administration of a low CHO diet following exereise-induced glycogen depletion resulted in a situation where the muscle and liver glycogen stores were lower than normal, and the availability of plasma FFA was greater than normal. Administration of a high CHO diet immediately following the low CHO diet resulted in the achievement of greater than normal glycogen stores and a restricted availability of FFA. Subjects were studied at rest and during exercise of different intensities at each stage of this dietary regime Measurements were made of blood metabolites and cardiovascular and respiratory parameters. Following the low CHO phase of the diet, fat metabolism was enhanced, as evidenced by a higher than normal plasma concentration of FFA and glycerol and a lower than normal respiratory exchange ratio (R). CHO metabolism was depressed; the blood concentrations of glucose, lactate and pyruvate were lower than normal. Following the high CHO phase of the diet, plasma FFA and glycerol concentrations were lower than normal, and the R value was higher than normal, indicating a decreased contribution of fat to oxidative metabolism. Blood glucose, lactate and pyruvate concentrations were higher than normal under these conditions. In a second series of experiments, subjects were studied before and after a twenty-four hours' fast. Pasting is known to result in a decreased rate of CHO utilisation accompanied by a compensatory increase in fat availability and oxidation, but has little effect on the muscle glycogen store. Blood and muscle metabolite concentrations were measured in resting and exercising subjects before and after fasting. In addition, skeletal and cardiac muscle as well as blood metabolite concentrations were measured in fed and fasted rats. In fasted human subjects and rats, the blood glucose concentration was lower than normal; an elevation of circulating FFA and ketone concentrations occurred in response to fasting. It appears that the inter-relationships of fat and CHO metabolism may be regulated by several different mechanisms. An increased rate of fat oxidation may result in an intra-cellular accumulation of citrate, resulting in turn in an inhibition of glycolysis at the level of phosphofructokinase; an increased rate of fat oxidation may also decrease glucose utilisation by an inhibition of pyruvate dehydrogenase activity. There was also some evidence to support the proposal that high lactate concentrations may exert an inhibitory influence on fat metabolism. Finally, although hormone levels were not measured, it appears that, during fasting, a decreased circulating insulin level may result in a decreased rate of glucose uptake and utilisation by human skeletal muscle.

Data available prior to this thesis had shown that, of all bovine organs examined, the adrenal cortex exhibited the second highest level of glutathione S-transferase (GST) expression behind the liver. This finding, along with increasing evidence implicating the importance of GST in endogenous detoxification processes, formed the basis for a further extensive investigation of the GST isoenzymes expressed by the adrenal cortex. Investigation of the GST isoenzymes expressed by a number of different bovine organs using affinity chromatography on S-hexylglutathione-Sepharose 6B (S-hexG-Ag) revealed a marked organ-specific distribution of these enzymes. Bovine adrenal cortex, in particular, expressed isoenzymes from each GST class, as determined by immunoblotting experiments. GST activity determinations of these enzyme pools using a number of model substrates revealed the bovine enzymes to possess a specificity distinct to that of rat and human GST. Isoelectric focusing of the bovine adrenal cortex isoenzymes showed them to possess pl values similar to those found in other species. The affinity-purified mu- and pi-glass isoenzymes were resolved using anion-exchange chromatography, followed by reverse-phase hplc. Using this approach, at least 3 mu-class GST subunits and 1 pi-class GST subunit were identified. Ion-exchange chromatography failed to resolve the affinity-purified alpha-class GSTs, and reverse-phase hplc analysis resolved 2 polypeptides, designated Ya<SUB>1</SUB> and Ya<SUB>3</SUB> respectively. Analysis of the protein that failed to bind to the S-hexG-Ag column revealed that about 35% of GST activity remained in this fraction. Application of this material to glutathione-Sepharose 6B (GSH-Ag) resulted in the purification of an abundant alpha-class GST (1.3% total cytosolic protein). This GST was found to exhibit marked peroxidase and Δ5-ketosteroid isomerase activities, in addition to high activity with 4-hydroxynonenal. SDS/PAGE analysis revealed 2 distinct polypeptides of Mr 25900 and 26500, the former being equivalent to the Ya<SUB>3</SUB> subunit purified on S-hexG-Ag, and the latter named Ya<SUB>2</SUB>. Ion-exchange chromatography of the GSH-Ag purified alpha-class GST isoenzyme pool resulted in a complex picture, suggesting there to be at least 3 distinct subunits in this pool.

Proteins released into medium used to culture, for 24h, endometrium removed from guinea-pigs on Days 7 and 15 of the oestrous cycle, from ovariectomised guinea-pigs treated with progesterone and/or oestradiol, and from Day-15 pregnant guinea-pigs were separated using affinity chromatography on Blue Sepharose (BS) CL-6B into proteins with (BS+ ve) and without (BS-ve) affinity for this gel resin. The turnovers of total protein released from the endometrium were not significantly different between Days 7 and 15 of the cycle. However, the turnover of BS-ve proteins was significantly greater on Day 15 than on Day 7 of the cycle. This change in emphasis away from BS+ ve proteins to BS-ve proteins from Day 7 to Day 15 of the cycle appears to be regulated by oestradiol. Significantly greater amounts of [<SUP>3</SUP>H]-leucine were incorporated into secreted proteins synthesised by Day-15 endometrium than by Day 7 endometrium, the difference being 2.1-fold. [<SUP>3</SUP>H]-Glucosamine was also incorporated into secreted proteins synthesised by Day-7 and Day-15 endometrium in culture. However, there was no significant difference between the amounts of glycosylated protein synthesised and secreted by Day-7 endometrium compared to Day-15 endometrium, The secretion of proteins, synthesised <i>de novo</i>, was low in ovariectomised guinea-pigs treated with progesterone, higher in ovariectomised guinea-pigs treated with oestradiol, and at a maximum in ovariectomised guinea-pigs which had been treated with progesterone and then oestradiol. The secretion of proteins, synthesised <i>de novo</i>, by Day-15 pregnant endometrium was also low.

This is a study of the control of luteinining hormone (LH) secretion in the marmoset monkey, Callithrix jacchus. The way in which the hypothalamo-hypophysial-gonadal system operates to regulate LH secretion in primates is reviewed. A double antibody hetorologous radioimmunoassay for measuring LH in marmoset plasma was developed and validated. Intra-muscular administration of synthetic luteinising hormone-releasing hormone (LH-RH) induces a marked increase in plasma LH concentrations in the marmoset, suggesting that the releasing hormone has an important physiological role in controlling LH secretion in this species. The effects of steroid hormones on pituitary response to exogenous LH-RH were examined. Pituitary responsiveness to LH-RH was enhanced in long term, but, not in short term, gonadeotomised animals. Whereas oestradiol-174 implants inhibited pituitary response to LH-RH in long term castrates, implants of progesterone augmented the response. A direct action of gonadal steroids on the pituitary gland is therefore suggested. The way in which steroid hormones influence the hypothalamo-hypophysial system was further studied by examining their ability to depressor increase circulating LH concentrations. Gonadectomy resulted in an increase in plasma LH levels indicating that LH secretion is normally suppressed by the action of gonadal steroids. In the "open-loop" situation the elevated LH levels are the result of an episodic secretion of the hormone by the pituitary gland. Closure of this feedback loop with oestradiol-17f caused a chronic suppression of LH secretion, suggesting that this steroid is an important component of the negative feedback mechanism regulating tonic LH secretion. The effects of progesterone, testosterone and dihydrotestosterone on LH secretion in gonadectomised marmosets were also tested. Whereas these hormones prevented the post-castration rise in IX concentrations, they were apparently ineffective in suppressing LH levels in long term gonadectomised animals. A decrease in the sensitivity of the hypothalamic-pituitary system to negative feedback as the interval from castration increases is suggested. Positive feedback control of LH secretion was also examined. A single injection of oestradiol benzoate had a biphasic effect on LH secretion, with an initial negative feedback effect characteristically preceding the positive response. Oestrogen induced LH release was observed in castrated and intact males as well as in castrated females, suggesting that the positive feedback response to oestrogen in the marmoset is not a sexually dimorphic characteristic. The ability of progesterone, testosterone, and dibydrotestosterone to induce, or modify oestrogen induced, positive feedback was also assessed in gonadectomised animals. The effects of inhibition of LH-RH were also examined. Active immunisation against LH-RH induced a breakdown of the hypothalamo-hypophysial-gonadal system, resulting in inhibition of gonadal and pituitary function. LH-RH induced LH release was suppressed by the use of competitive antagonist analogues of LH-RH. The potential application of these procedures in fertility control was investigated.

No description available.

No description available.

No description available.