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Includes bibliography.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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Members of the ubiquitous GTPase superfamily regulate numerous cellular functions. A core group of eight GTPases are present in all domains of life: initiation factor 2, elongation factors Tu and G, protein secretion factors Ffh and FtsY, and the poorly characterized factors YihA, YchF, and HflX. While the first five members have well defined roles in the essential cellular process of protein synthesis, a role for YihA, YchF and HflX in this process has only recently been suggested. Here, a detailed kinetic analysis examining the interaction between HflX and its cellular partners is described. 50S and 70S ribosomal particles function as GTPase activating factors for HflX by stabilizing the nucleotide binding pocket of HflX, inducing a “GTPase activated” state. These data indicates a novel mode of GTPase activation, and suggests a role for HflX in regulating translation. / xii, 185 leaves : ill. (some col.) ; 28 cm

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Methylobacterium extorquens ATCC55366 is an interesting candidate for large-scale production of recombinant proteins. Development and optimization of this recombinant expression system were done using the green fluorescent protein (GFP) gene cloned into expression vectors (pRK310 and pCM110) as model systems. Selection of efficient GFP-expressing clones, long-term production stability without selection in flasks, effects of selection, oxygen and methanol supplies, were studied during fed-batch fermentations in a 20-l bioreactor. Sequential batch-culture cultivations in shake flasks showed that specific GFP production was constant in the presence of tetracycline. However, the GFP production decreased in the absence of this selective pressure. In fed-batch fermentations of recombinant M. extorquens ATCC 55366 (pMxaF-GFP), overall GFP yields (≈70 mg/g; GFP/cell dry weight) were not affected by the presence or absence of tetracycline, nor by oxygen and methanol concentration oscillations. The cry1Aa gene from Bacillus thuringiensis kurstaki NRD-12 was cloned in pCM110 and then transformed into M. extorquens. Heterologous expression of the cry1Aa gene in M. extorquens AM1 and ATCC 55366 was detected by immunoblot analyses. This study suggests that M. extorquens can be used as a valuable expression system for intracellular recombinant protein production.

A piglet model of gastrointestinal inflammation (INF) and protein energy malnutrition (PEM) was developed to determine the mechanisms responsible for growth retardation and muscle wasting during inflammatory stress. Acute PEM decreased liver and plasma protein fractional synthesis rates (FSR), measured by stable isotope (2H3-leucine) incorporation. Conversely, both rates increased during PEM+INF at the expense of growth and muscle FSR. INF increased plasma protein synthesis by 77% in PEM without increasing its plasma concentration, demonstrating that the measurement of plasma protein concentration alone underestimates the metabolic impact of INF. INF during PEM results in a re-prioritization of amino acids from muscle protein synthesis and growth to hepatic synthesis of plasma proteins to support the acute phase response. This underscores the critical role of adequate protein-energy nutrition during inflammation in preventing muscle wasting and growth failure. This new piglet model can be applied to investigate nutritional and therapeutic interventions in inflammatory bowel disease.

Desiccation effects on rate and pattern of protein synthesis in Brassica napus (cv westar) and Brassica juncea (cv cutlass) have been examined. Results showed that while the rate of water loss was similar in the two species, the inhibition of amino acid incorporation was greater in B. napus than B. juncea at any given level of desiccation. Electrolyte leakage increased with the degree of desiccation and was greater in B. napus than in B. juncea. In both, the increase in leakage was much sharper after 12 hours of desiccation. Quantitative changes in patterns of boiling-stable protein synthesis due to desiccation stress were observed. The control level of protein radioactivity which was boiling-stable in B. napus was 16.16% and 19.96% for B. juncea. After desiccation, the percentage of boiling-stable radioactivity increased to 23.30% for B. juncea and 16.63% for B. napus. In vitro translation of total RNA indicated that desiccation alone does not induce the synthesis of new mRNA species in either cultivar, but it may change the translation pattern resulting in different levels of abundance of proteins.

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