Development and characterisation of radioimmunoassays for parathyroid hormone (PTH) and calcitonin were undertaken. These assays were applied to the study of the maintenance of mineral homeostasis during pregnancy and in the neonatal period. The PTH assay developed represents one in which many of the problems encountered in applying the radioimmunoassay technique to the measurement of the circulating hormone have been overcome. Loss of immunochemical homogeneity on radioiodination of PTH was offset by the adoption of suitable procedures for purifying the labelled hormone, and the useful lifetime of 125I-BPTH in the assay system was extended by the inclusion of a simple pre-assay gel-filtration step. The analytical significance of various non-specific interferences in the assay system has been minimised. In particular non-specific adsorption of PTH has been reduced to a consistently low level. The behaviour of plasma samples in the assay system was also rigorously studied and the assay procedure finally accepted for routine use as a result of these investigations has proved a reliable analytical method for the study of circulating immunoreactive PTH. The radioimmunoassay for calcitonin was also optimised with regard to each of its components. Assay peformance was significantly improved by modifying the radioiodination procedure used to produce 125i-human calcitonin; sodium metabisulphite was shown to produce chemical damage to the calcitonin molecule and consequently was replaced by cysteamine-hydrochloride. Immunisation procedures undertaken in the rabbit resulted in the production of anti-calcitonin sera, but these compared unfavourably with an available goat anti-calcitonin serum in the radioimmunoassay system. The assay developed using the goat antiserum was capable of detecting as little as 20 ng/l human calcitonin in plasma. Specific calcitonin receptors have been demonstrated in preparations derived from rabbit kidney tissue and a radioreceptor assay for calcitonin established. Calcitonin-receptor interaction was investigated and the relationship between the hormone's immunochemical and biological behaviour is discussed. Membrane preparations isolated from human renal tissue, by the procedure used to prepare calcitonin receptors from rabbit kidney, failed to display calcitonin binding activity. The radioimmunoassays developed were used to study plasma mineral homeostasis in a series of 227 pregnancies from a Clinical Trial in which the effect of maternal vitamin-D supplementation (1000 International Units/day) was investigated. Analytical data obtained showed that there was little tendency for pregnant women to become significantly hyperparathyroid, relative to non-pregnant controls, even though PTH concentrations increased significantly from the end of the first trimester to term. There was no evidence to support the hypothesis that calcitonin is of particular physiological importance to the mother in pregnancy. Infant plasma calcitonin was raised relative to maternal concentrations throughout the first week of life, whilst plasma PTH concentrations were relatively low at delivery but increased as post-natal maturation occurred. Although maternal vitamin-D status was shown to be a significant factor influencing extracellular calcium concentration in the newborn period, it appeared to be less important with regard to both maternal and neonatal plasma PM and calcitonin concentration. The relationship between PTH and calcitonin and vitamin-D in pregnancy and the newborn period is discussed with particular reference to the pathogenesis of late onset neonatal hypocalcaemia.

No description available.

No description available.

Sterols and steroids are relatively complex molecule^ which contain a number of asymmetric centres; numerous species are able to synthesise such molecules and transform them to a wide variety of related compounds. Thus many of the enzymes concerned in steroid hormone metabolisin offer unique opportunities as model systems for the study of substrate specificity. Although steroid hormones share certain basic structural characters the possession of specific conformations and configurations provides a physico-chemical basis for the observed differences in physiological action. In this discussion only certain aspects of the enzymic transformations of l?a-oestradiol, 17P-oestradiol and oestrone will be considered. These phenolic steroids are closely related by simple oxidation-reduction reactions, viz. 17a-oestradiol oestrone 17P-oestradiol The isolation and partial purification of a human placental 17P-oestradiol dehydrogenase by Langer and Sngel (1956) and subsequent kinetic studies by Langer, Alexander and Engel (1959) showed that this enzyme 1. had an absolute steric requirement for the 17P-hydroxyl group 2. required that the steroid substrate must possess a highly planar ring A or B or both for significant reactivity 3. interacted with the entire steroid surface. These findings were largely confirmed by Adams, Jarabak and Talalay (1962) using enzyme preparations of much higher specific activity. It was therefore of interest to extend these observations using a 17a-oestradiol dehydrogenase, thereby providing a more complete system for the investigtion of effects of substrate structure upon reaction kinetics. Such a model would also facilitate the experimental approach to studies of the mechanisrn(s) of these enzyme reactions.

No description available.

No description available.

No description available.

No description available.

A reduced blood lactate concentration ([lac-]B) is commonly observed during whole-body exercise following inspiratory muscle training (IMT). However, whether the inspiratory muscles are, in part, the source of these reductions remains unknown. Accordingly, this thesis investigated: (I) the contribution of the respiratory muscles to the systemic [lac-]B and (II) the effects of IMT upon inspiratory muscle lactate exchange and clearance. In addition, the thesis also evaluated the determinants of inspiratory muscle strength (maximal inspiratory mouth pressure; MIP). All subjects were healthy, active and free of pulmonary and respiratory muscle disease. Under resting conditions, 10 min intense volitional hyperpnoea at 85% of maximal exercise minute ventilation (VE max) increased [lac-]B by 0.96 mmol.L-1. This was attenuated by 25% following 6 wks IMT. 8 min volitional hyperpnoea at 90% VE max imposed upon exercise at the maximal lactate steady state (MLSS) increased [lac-]B by 0.99 mmol.L-1. Following 6 wk IMT, the steady state and hyperpnoea-mediated increase in [lac-]B were lower by 8 and 26%, respectively. Relative to pre-IMT, loading the trained inspiratory muscles using a low-intensity pressure threshold resistance (15 cmH2O) immediately following maximal exercise accelerated both lactate exchange and clearance capacities by ~70%. Collectively these findings support the notion that the respiratory muscles are capable of net lactate production and are the first to suggest that IMT increases their capacity for lactate clearance. This thesis also demonstrates that the respiratory muscles are responsible, in part, for the reductions observed in [lac-]B during whole-body exercise following IMT. Finally, baseline MIP was positively correlated with the strength of the chest wall inspiratory muscles. The IMT-mediated increase in MIP was negatively correlated with the relative increase in chest wall muscle strength. Therefore, these findings are the first to demonstrate that the lower the initial strength of the chest wall inspiratory muscles, the lower the MIP and the greater the improvement in global inspiratory muscle strength following IMT.

This thesis is concerned with studies on acetylcholinesterase. The problem of the role and fate of the protein has been studied using a variety of techniques. <strong>1. Biochemical experiments</strong>. When homogenates, in isotonic media (0.3M sucrose), of bovine splanchnic axons and adrenal medullae were subfractionated in a centrifuge, it was found that the acetylcholinesterase did not give the expected distribution. Much of the early work in this field had resulted in the conclusion that acetylcholinesterase was a uniquely membrane-bound enzyme (see e.g. Boell and Nachmansohn, 1940: Toschi, 1959) but my results suggested that a considerable proportion of the activity in both tissues was soluble. Thus, 47% of the acetylcholinesterase activity present in the splanchnic axons and 31% of the enzyme in the adrenal medullae could, not be sedimented by prolonged high-speed centrifugation. The possibility that the preparation of the tissue for the centrifugation experiments might have "solubilized" the acetylcholinesterase from its normal, membrane-bound localization was investigated in several ways. It was found: that different strengths of homogenization, while having the expected effect of breaking up the tissue into smaller pieces, had no effect on the proportion of the enzyme which was not sedimentable; that suspension and "homogenization" of the membranes themselves did not result in a solubilization of their acetylcholinesterase; that incubation, at 25°, of a washed fraction containing only sedimentable acetylcholinesterase activity did not result in any solubilization of the enzyme. It was concluded that the soluble enzyme was not artifactually derived from the membrane-bound form during the preparation of the tissues for the earlier experiments. Because acetylcholinesterase is an enzyme which will hydrolyze a variety of substrates, it can be localized by histochemical methods. In addition to using this technique on tissues (see below) the method has also been used to locate the enzyme on polyacrylamide gels after different tissue extracts had been subjected to electrophoresis. The results of this type of analysis showed that the acetylcholinesterase of the two tissues was separable into several isoenzymes (see Webb, 1964 for definition of term isoenzyme). The simplest case was found in splanchnic nerve axons, where there was only one membrane-bound form of the enzyme (revealed by an extraction of the enzyme activity with Triton X-100 prior to electrophoresis) and another, different, form in the high-speed supernatant. The membrane-bound isoenzyme WRS always unique but occasionally there was more than one soluble isoenzyme. However, even in these rare cases, it was qualitatively estimated that more than 95% of the total activity on the gel was due to the one normal isoenzyme. The adrenal medullae presented a more complex picture. Again, there was only one membrane-bound isoenzyme, which had the same electrophoretic mobility as the axonal form, but there was a greater number of soluble forms of the acetylcholinesterase. One of these appeared to be the same form as was soluble in the axons, but in addition there were usually four others unique to the medulla. These latter isoenzymes had higher electrophoretic mobilities than either of the two isoenzymes of the splanchnic nerve. The different isoenzymes were numbered from 1, the fastest migrating, to 6, the slowest. Thus, AChE₆ and AChE₅ represent, respectively, the membrane-bound and the slowest migrating of the soluble isoenzymes. These are the two forms which are common to both axons and medullae. The relationship between AChE₅ and AChE₆, was investigated in two ways. Electrophoresis over a range of polyacrylamide gel concentrations showed that they each changed their mobility in an identical fashion, i.e., a plot of log₁₀(R_m andtimes; 100) v. polyacrylamide concentration yielded two essentially parallel lines. This, according to Hedrick and Smith (1968), indicates that the two isoenzymes have very similar molecular sizes but different electrical charges. Supporting evidence for this suggestion was obtained by centrifugation of the acetylcholinesterase on stabilizing sucrose gradients; both the soluble and, after extraction with Triton X-100, the membrane-bound acetylcholinesterase sedimented to the same area of the gradient. Thus, when it was found that AChE₅ behaves as though it has a molecular weight of 240,000, this also indicated a similar size for AChE₆. Experiments of this type were also used to analyze the relationship between the soluble isoenzymes of the adrenal medulla. These results showed that whereas medullary AChE₅ was identical to the soluble isoenzyme in the axons, the faster migrating forms all differed from it in size at least, and possibly also in charge. Because of the ease with which AChE₅ and AChE₆ can be separated, a few properties of each were compared without prior and extensive purification of the proteins. It was found that they had an identical apparent K_m (7.5 andtimes; 10^-5M, acetylthiocholine as substrate), identical Q₁₀'s of 1.2 and identical heat denaturation properties. Thus, the only differences between them were in their relative solubilities and their electrophoretic mobilities. To try to distinguish whether the soluble isoenzyme in the axons was contained within the cytoplasm, or whether it was within a labile particle which homogenization broke to liberate the soluble internal constituents, we investigated the rate at which acetylcholinesterase accumulated in constricted splanchnic axons and analyzed the types of isoenzyme involved in the movement. Both AChE₅ and AChE₆. were found to accumulate at an identical, relatively rapid rate in these nerves. It was concluded that the AChE₅, and at least some of the AChE₆, was contained within a fragile particle and that the soluble enzyme was liberated from this particle during the homogenization procedure. <strong>2. Studies on the release of acetylcholinesterase from the isolated adrenal gland</strong>. A rapid flow rate of the acetylcholinesterase is a strong indication that there is an equally rapid removal of the enzyme from, presumably, the nerve endings. One way in which such a rapid removal could take place is by a release of the protein from the nerve. To investigate this possibility, we stimulated the isolated, perfused bovine adrenal gland with a variety of secretagogues to try and induce release of the acetylcholinesterase. It was found that the enzyme could be released by administering either a high concentration of K⁺, Dimethylphenylpiperazinium Iodide or Carbachol to the gland. The release was dependent upon the presence of Ca²⁺ ions in the perfusing fluid. Electrophoretic analysis of the perfusate showed that only AChE₅ was released from the glands. The amount of enzyme appearing in the perfusate was related to the amount of catecholamines, probably reflecting the efficacy of the stimulation, but not to the amount of acetylcholinesterase in the adrenal gland. <strong>3. Cytochemical studies</strong>. Because only one of several soluble isoenzymes of acetylcholinesterase was released, and since we were unable to analyze the storage characteristics of the protein by the normal methods (centrifugation) we resorted to a cytochemical analysis of the bovine adrenal medullae and splanchnic axons in an attempt to find how, and in what structure, the acetylcholinesterase was stored.