Sialic acid is the terminal sugar found on most glycoproteins and is crucial in determining serum half-life and immunogenicity on glycoproteins. The scarce supply of sialic acid hinders its advancement in basic research, diagnostic development and therapeutic production. In this work, the recombinant E. coli BRL04 (pBRL89) producing sialic acid was studied by some batch and fed batch runs of high cell density cultivation using a 3-L fermentor. Some cultivation conditions including carbon source, induction time, dissolved oxygen were optimized and different feeding strategies were compared to enhance sialic acid production. The results may be helpful to the further scale-up of sialic acid production and the production of other recombinant proteins by high cell density cultivation of E. coli.

Both intermittent oral gavage tube placement(0G) and indwelling nasogastric tube placement (NG) are acceptable methods for feeding preterm infants. A randomized controlled pilot study was conducted to examine the impact of OG versus NG placement on premature infant feeding transition. Twenty healthy premature infants were enrolled and thirteen completed the study. The results were not statistically significant. Age at last tube feed averaged 35 weeks gestational age (GA) for the intermittent group and 35+4 weeks GA for the indwelling group (p=0.181). Infants in both groups were discharged at an average of 36 weeks GA (p=.836) and averaged suckled volumes at 35 weeks GA was 134.4cc/kg/day for the intermittent group versus 111.8cc/kg/day for the indwelling group (p=0.240). Infant feeding patterns were analyzed descriptively and found to be consistently variable. The lack of consistency in feeding development has implications for feeding plan development and feeding transition care.

When administered systemically, benzodiazepine receptor agonists have been shown to increase food intake in a number of species. Conversely, benzodiazepine receptor inverse agonists bring about reliable decreases in feeding. The aim of the experiments reported in this thesis was to investigate the brain and behavioural mechanisms involved in the effects of benzodiazepines on ingestion. The effect on food intake of microinjection of the benzodiazepine receptor agonist midazolam into the brainstem of the rat was investigated. A reliable hyperphagic response was elicited following injection of midazolam into both the IVth ventricle and the parabrachial nucleus (PEN). This increase in intake was reversed by pretreatment with the selective benzodiazepine receptor antagonist flumazenil. These results suggest that benzodiazepine receptors located in the brainstem, specifically in the PEN, may be responsible for the effects of benzodiazepines on ingestion. In further experiments, a microstructural approach was adopted which involved analyzing the effects of benzodiazepine ligands on the detailed pattern of licking for both a carbohydrate and a fat in the rat. The effects of midazolam were similar to the effects of increasing concentration. The effects of the benzodiazepine receptor inverse agonist Ro 15-4513 were similar to the effects of decreasing concentration. These results suggest that benzodiazepines influence ingestive behaviour by modulating palatability. The proposal that benzodiazepines may interact with opioids to influence feeding behaviour was examined in Chapters 7 and 8. Although the effects of the opioid agonist morphine and the opioid antagonist naloxone on licking behaviour were not the same as the effects of benzodiazepine ligands, naloxone blocked the effects of midazolam. These results suggest that the effects of benzodiazepine on palatability may depend on release of endogenous opioid peptides. This work has implications for understanding the neural control of ingestive behaviour and may help in developing new therapies for clinical disorders such as anorexia and bulimia.

Fermented silage was prepared from a mixture of minced tiiapias (Oreochromis spp. ), different carbohydrate substrates (molasses, corn flour, tapioca flour) and Lactobacillus plantarum as inoculum, incubated anaerobically for 30 days at 5°-35°C. The pH and protein solubilization were temperature-dependent, and the source of carbohydrate substrate did not affect non-protein nitrogen (NPN) content or proximate composition of tilapla silage. During storage at 30°C for 180 days, NPN content increased and there was 8-11% loss of tryptophan. Moist diets containing tilapla silage (stored up to 60 days) were fed to Clarias gariepinus and differences in growth and protein utilization were demonstrated, but there were no effects on body composition. Partial replacement of fish meal with co-dried tilapla silage and soybean flour blend (FSS: BF) in dry diets supported growth and protein utilization similar to the control treatment. Fish growth and protein utilization were reduced with total replacement of fish meal. Apparent protein digestibility decreased with Increasing dietary level of co-dried FSS: BF. Carcass composition was not affected and morphological defects were not observed. Co-dried tilapla silage blended with soybean meal, poultry by-product meal, hydrolysed feather meal or meat and bone meal (FSS: BM, FSP: BM, FSH: FM, FSM: BM) (providing 50% of the dietary protein) In dry diets fed to Oreochromis niloticus and Clarias gariepinus gave differences In growth, protein utilization and digestibility, and apparent energy digestibility. Carcass composition was not affected by silage blend and histological examination of exocrine pancreas, liver and Intestine tissues did not show any lesions suggestive of nutritional imbalance. Haematocrit, haemoglobin content and mean cell haemoglobin concentration values showed no differences among the treatments. The results indicated that fermented tilapla silage is a suitable protein supplement in moist or dry diets for Oreochromis niloticus and Clarias gariepinus, without affecting feed efficiency, fish growth or health.

No description available.

The experimental work described in this thesis was conducted in the surgical research laboratories of Dr Francis D. Moore in the Peter Bent Brigham Hospital (now the Brigham & Women's Hospital) Boston, Massachusetts, USA, between 1978-1979. It formed part of an ongoing programme of research into protein metabolism in man; specifically to measure total body nitrogen turnover and hence protein synthesis and breakdown, initially in normal volunteers receiving various intravenous feeding regimens, and subsequently in patients. The previous year, 1977, had seen the publication of 'Substrate Interaction in Intravenous Feeding' by Bruce Wolfe et al., from the same laboratories. This was an extensive piece of work incorporating many studies and compared nitrogen balance data together with biochemical, hormonal and plasma amino acid data in normal men fed intravenously with a variety of regimens. Shortly afterwards a series of protein turnover studies was embarked upon, using the uN glycine method, and in collaboration with Dr Vernon Young of the Massachusetts Institute of Technology. The first experiments were essentially a repeat of the studies described by Wolfe et al. (vide supra) but in addition nitrogen turnover, protein synthesis and breakdown were estimated using a continuous 60 hour infusion of uN glycine and measuring enrichment of urinary urea with uN when a plateau was reached. Normal volunteers were studied firstly on normal oral diet and then on a iv succession of intravenous regimens:- amino acids alone (AA), amino acids plus 'high dose' glucose (AA + HOG), amino acids plus fat emulsion (AA + FE), amino acids plus 'low dose' glucose (AA + LOG), amino acids, fat emulsion and low dose glucose (AA + LDG+ FE), and finally 'low dose' glucose alone (LOG). The studies on normal diet, AA and AA+HOG were conducted by Andrew Sim (a Glasgow/Harvard exchange fellow) and Bruce Wolfe. The author took no practical role in these experiments, but was responsible for analysis of the data and the protein metabolism calculations, and was a co-author when the work was published in 1979 (Sim et al., Glucose Promotes Whole-Body Protein Synthesis from Infused Aminoacids in Fasting Man, Lancet i, 68-71). Subsequently, the author did the experiments using AA + LOG + FE, AA + FE, and AA + LOG and LOG. The results on these four regimens were incorporated in a paper presented in 1979 at the Tripartite Meeting of the Surgical Research Society at Oxford under the title 'Isotope Studies of substrate interaction in parenteral nutrition', and also at the 2nd European Congress on Parenteral and Enteral Nutrition at Newcastle upon Tyne in 1980, and later published as 'The Effect of Fat Infusion on Protein Metabolism' (Acta. Chir. Scand., Suppl. 507, 475-484, 1981). When the studies on the various intravenous feeding regimens were completed, attention was turned to the possible distorting effects of variables such as exercise and diet v on the behaviour of the isotope equilibrium curve and plateau. Such effects, if present, might have significance when studies were carried out on patients at a later stage in the research programme. Because each study lasted 48-60 hours, changes might occur either unintentionally or as a result of the needs of clinical management, and if they affected the plateau would alter the resultant calculations of turnover, synthesis and breakdown. Such a potential source of error clearly needed investigation. A pilot study was done in two subjects, later repeated on each, to observe any effects on the curve and plateau of both doubling protein intake and bicycle exercise. Subsequently, more extensive studies were done varying the protein and energy intakes, both orally and intravenously, allowing a detailed analysis of curve perturbation, and introducing the concept of basal catabolic rate. Finally, protein turnover, synthesis and breakdown were estimated seven times in four seriously ill patients. All of the studies mentioned above form the basis of the thesis. Unfortunately, the gestation period of this thesis has been long. There are two main reasons for this. Firstly, the work done was part of a five-year programme of research, with the intention of publishing a paper in a scientific journal at the completion of each stage. This was done vi with the first three regimens (normal diet, AA and AA + HDG) but not with the last four (AA + FE, AA + LOO+ FE, AA + LOG, LDG), although the results were presented at two scientific meetings. Shortly after returning to the United Kingdom the author was appointed a consultant surgeon and this career move assumed priority. Secondly, although it was intended to publish the perturbation studies, it proved impossible to reduce the size of the text to a manageable level suitable for publication in the form of a scientific paper. However, despite the long interval since the experiments were done, no similar work has been published. In particular, virtually no attention has been paid to intentional perturbation. Also, whereas there was a spate of interest in protein turnover studies in the late 1970s and early 1980s, virtually no publications have appeared since 1985. It seems that the potential applications of the method are considered exhausted, and interest has been lost, rather as it was in the 1950s following a short flurry of activity exploring the first cumbersome technique. Hence, it seemed all the more pertinent, even at this late stage, to publish the work in the form of a thesis which could describe in chronological order the continuum of studies as briefly mentioned above. In order to preserve such a progression, the following Introduction contains, with few exceptions, only references up to the time that the experimental studies were commenced, 1978, but the subsequent Discussion(s) in the various sections will attempt to include the relevant literature up to the present time.

Dairy cows fed diets supplemented with 2.5% calcium-salt fatty acids (CSFA) (DM basis) during early lactation produced more 4% FCM than cows fed the control diet. Feeding this diet also increased whole lactation performance. Supplementing diets with 1.25% CSFA or animal fat did not increase production performance of cows. Dietary fats were used directly for greater milk production and, based on unchanged body condition scores, did not alleviate body fat mobilization. Changes in the milk fat content and composition due to fat supplemented diets were negligible. A slight decrease in milk protein and SNF percentage was observed with CSFA but not with animal fat feeding. Palatability of the diets was good and DM intake was not impaired. Feeding animal fat at 1.25% and CSFA up to 2.5% of the DM does not impair forage digestibility and nitrogen balance. Dietary fat increased plasma triglyceride (TG) and cholesterol concentration. Injection of 10.3 mg d$ sp{-1}$ or 350 mg 14d$ sp{-1}$ of recombinant bovine somatotropin (rbST) did not change 4% FCM production or milk composition at any stage of lactation.

The hypothalamus is a central regulator of energy homeostasis. Recently, proglucagon-derived peptides have emerged as potential appetite regulators. The proglucagon gene is expressed in the periphery and also in selective hypothalamic neurons. The regulation of hypothalamic proglucagon by two key regulators of energy balance, insulin and leptin, remains unstudied. Central glucagon-like peptide (GLP)-1 receptor (GLP-1R) activation by exendin-4, a long-acting GLP-1R agonist, induces anorexia; however, the specific hypothalamic neuronal populations activated by exendin-4 remain largely unknown. The role of GLP-2 as a central appetite regulator is poorly understood. In this thesis, using murine hypothalamic cell lines and mice as experimental models, mechanisms involved in the direct regulation of proglucagon gene by insulin and leptin were studied, and the actions of exendin-4 and GLP-2 on hypothalamic neuropeptides were determined. It was found that insulin and leptin regulate hypothalamic proglucagon mRNA by activating Akt and signal transducer and activator of transcription 3, respectively. Insulin and leptin did not regulate human proglucagon promoter regions, but affected proglucagon mRNA stability. In mice, intracerebroventricular exendin-4 and GLP-2 induced anorexia, activated proopiomelanocortin- and neuropeptide Y-expressing neurons in the arcuate nucleus and neurotensin- and ghrelin-expressing neurons in major hypothalamic appetite-regulating regions. In the hypothalamic neuronal models, exendin-4 and GLP-2 activated cAMP-response element-binding protein/activating transcription factor-1, and regulated neurotensin and ghrelin mRNA levels via a protein kinase A-dependent mechanism. Overall, the in vivo and in vitro findings suggest that these neuropeptides may serve as potential downstream mediators of exendin-4 and GLP-2 action. This research demonstrates direct regulation of hypothalamic proglucagon by insulin and leptin in vitro, and reports a previously unrecognized link between central GLP-1R and GLP-2R activation and regulation of hypothalamic neuropeptides. A better understanding of the regulation of hypothalamic proglucagon and central GLP-1R and GLP-2R activation is important to further expand our knowledge of feeding circuits.

The hypothalamus is a central regulator of energy homeostasis. Recently, proglucagon-derived peptides have emerged as potential appetite regulators. The proglucagon gene is expressed in the periphery and also in selective hypothalamic neurons. The regulation of hypothalamic proglucagon by two key regulators of energy balance, insulin and leptin, remains unstudied. Central glucagon-like peptide (GLP)-1 receptor (GLP-1R) activation by exendin-4, a long-acting GLP-1R agonist, induces anorexia; however, the specific hypothalamic neuronal populations activated by exendin-4 remain largely unknown. The role of GLP-2 as a central appetite regulator is poorly understood. In this thesis, using murine hypothalamic cell lines and mice as experimental models, mechanisms involved in the direct regulation of proglucagon gene by insulin and leptin were studied, and the actions of exendin-4 and GLP-2 on hypothalamic neuropeptides were determined. It was found that insulin and leptin regulate hypothalamic proglucagon mRNA by activating Akt and signal transducer and activator of transcription 3, respectively. Insulin and leptin did not regulate human proglucagon promoter regions, but affected proglucagon mRNA stability. In mice, intracerebroventricular exendin-4 and GLP-2 induced anorexia, activated proopiomelanocortin- and neuropeptide Y-expressing neurons in the arcuate nucleus and neurotensin- and ghrelin-expressing neurons in major hypothalamic appetite-regulating regions. In the hypothalamic neuronal models, exendin-4 and GLP-2 activated cAMP-response element-binding protein/activating transcription factor-1, and regulated neurotensin and ghrelin mRNA levels via a protein kinase A-dependent mechanism. Overall, the in vivo and in vitro findings suggest that these neuropeptides may serve as potential downstream mediators of exendin-4 and GLP-2 action. This research demonstrates direct regulation of hypothalamic proglucagon by insulin and leptin in vitro, and reports a previously unrecognized link between central GLP-1R and GLP-2R activation and regulation of hypothalamic neuropeptides. A better understanding of the regulation of hypothalamic proglucagon and central GLP-1R and GLP-2R activation is important to further expand our knowledge of feeding circuits.

No description available.