Spinal cord injury occurs at a rate of 11.5 - 53.4 per million in developed countries with great emotional and financial consequences. The damage caused by the initial injury is followed by secondary damage, a complex cascade of mechanisms including ischemia, oxidative stress, inflammation and apoptosis. Although nothing can be done to reverse the initial damage to the spinal cord once it occurs, the secondary damage can be targeted by therapeutics to improve recovery. Following injury, concentrations of the potent antioxidant glutathione (GSH) are decreased in the spinal cord which potentiates mechanisms of secondary damage. In an attempt to maintain the GSH concentrations, the non-essential amino acid glutamine was tested as it was shown to increase GSH concentrations both in vivo and in vitro. Glutamine is being used extensively in clinical research in an expansive number of physiological and pathological conditions including brain trauma. To examine the therapeutic potential of glutamine after spinal cord trauma, two compression injury models, the modified aneurysm clip and the modified forceps, were used to induce an injury in male Wistar rats. We have demonstrated the ability of glutamine treatment (1 mmol/kg), given 1 hour after a 30 g aneurysm clip injury to increase GSH not only in whole blood samples but within the spinal tissue at the site of injury. Increasing GSH in this way also resulted in improved locomotor scores and maintenance of white matter tissue at the injury epicenter. Experiments using the forceps model were then performed to determine if the potency of glutamine treatment would be carried over to a different model and at a variety of severities. Glutamine, again, demonstrated the ability to improve maintenance of whole blood GSH, locomotor scores and tissue histology. In our experiments, glutamine has proven to be a potent therapeutic for spinal cord injury with an effect that is matched by few compounds currently being studied and well exceeding the standard therapeutic, methylprednisolone. Given the breadth of knowledge regarding the effects of glutamine clinically in numerous paradigms and the potency of the therapeutic effect seen in these studies, we believe that glutamine is fit for clinical trial and has a high potential for success.

Primary spinal cord injury (SCI) results from direct mechanical damage to the spinal cord. The resulting pathochemical and pathophysiological events, including oxidative stress and inflammation, lead to secondary injury. The ability to decrease secondary injury may lead to improved recovery. Increasing glutathione production after SCI leads to decreased secondary injury. Glutamine is an important precursor to glutathione following trauma. Skeletal muscle phenotype is strongly influenced by neuromuscular activity. SCI causes myosin heavy chain (MyHC) profiles to shift towards faster isoforms in slow muscles and slower isoforms in fast muscles. The hypothesis was that glutamine, as a precursor of glutathione, administration to SCI rats would lead to better functional recovery and a more preserved MyHC phenotype in locomotory muscles. <p> Rats were assigned to one of four groups; healthy, laminectomy only, untreated SCI, and SCI treated with an intraperitoneal injection of 1mmol/kg glutamine every 12 hours for one week after injury. SCIs were performed at T6 with a modified aneurism clip. Functional recovery was measured weekly using the Basso-Beattie-Bresnahan scale and the angle board method. Six weeks later, all rats were killed, and their extensor digitorum longus and soleus muscles excised and weighed. MyHC composition of the muscles was determined using SDS-PAGE.<p>The hypothesis that glutamine treatment following SCI would lead to better functional recovery and a more preserved MyHC profile was validated. Glutamine treated rats received significantly higher BBB scores (p<0.01) and angle board scores (p<0.001) than untreated SCI rats. Glutamine treatment also reduces muscle atrophy in the soleus muscle, but not the extensor digitorum longus (EDL). In untreated rats the soleus muscle accounted for significantly (p<0.001) less of the percentage of total body weight than the soleus muscle from glutamine treated rats. Finally, SCI rats with preserved functional abilities displayed a significantly better preserved MyHC profile compared to untreated SCI rats. In the soleus healthy rats contain 94% type 1 myosin, treated rats maintained 68% which was significantly (p<0.001) greater than 28% maintained by untreated rats. In the EDL healthy rats contain 55% type 2b myosin, treated rats maintained 32% which was greater than 26% type 2b myosin maintained by untreated rats.

Spinal cord injury occurs at a rate of 11.5 - 53.4 per million in developed countries with great emotional and financial consequences. The damage caused by the initial injury is followed by secondary damage, a complex cascade of mechanisms including ischemia, oxidative stress, inflammation and apoptosis. Although nothing can be done to reverse the initial damage to the spinal cord once it occurs, the secondary damage can be targeted by therapeutics to improve recovery. Following injury, concentrations of the potent antioxidant glutathione (GSH) are decreased in the spinal cord which potentiates mechanisms of secondary damage. In an attempt to maintain the GSH concentrations, the non-essential amino acid glutamine was tested as it was shown to increase GSH concentrations both in vivo and in vitro. Glutamine is being used extensively in clinical research in an expansive number of physiological and pathological conditions including brain trauma. To examine the therapeutic potential of glutamine after spinal cord trauma, two compression injury models, the modified aneurysm clip and the modified forceps, were used to induce an injury in male Wistar rats. We have demonstrated the ability of glutamine treatment (1 mmol/kg), given 1 hour after a 30 g aneurysm clip injury to increase GSH not only in whole blood samples but within the spinal tissue at the site of injury. Increasing GSH in this way also resulted in improved locomotor scores and maintenance of white matter tissue at the injury epicenter. Experiments using the forceps model were then performed to determine if the potency of glutamine treatment would be carried over to a different model and at a variety of severities. Glutamine, again, demonstrated the ability to improve maintenance of whole blood GSH, locomotor scores and tissue histology. In our experiments, glutamine has proven to be a potent therapeutic for spinal cord injury with an effect that is matched by few compounds currently being studied and well exceeding the standard therapeutic, methylprednisolone. Given the breadth of knowledge regarding the effects of glutamine clinically in numerous paradigms and the potency of the therapeutic effect seen in these studies, we believe that glutamine is fit for clinical trial and has a high potential for success.

Poly-Q expanded exon 1 of huntingtin (Q103) fused to GFP is toxic to yeast cells containing endogenous yeast prions, [PIN+] ([RNQ+]) and/or [PSI+], which presumably serve as aggregation nuclei. Propagation of yeast prions is modulated by the chaperones of Hsp100/70/40 complex. While some chaperones were reported to influence poly-Q aggregation in yeast, it was not clear whether they do it directly or via affecting yeast prions. Our data show that while dominant negative Hsp104 mutants antagonize poly-Q aggregation and toxicity by eliminating endogenous yeast prions, some mutant alleles of Hsp104 decreases size and ameliorate toxicity of poly-Q aggregates without affecting prion propagation. Elevated levels of the yeast Hsp40 proteins, Ydj1 and Sis1, exhibit opposite effects on poly-Q aggregation and toxicity without influencing prion propagation. Among the yeast Hsp70s, only overproduction of Ssa4 antagonized poly-Q toxicity. We have also isolated dominant Anti-poly-Q-toxicity (AQT) mutants counteracting poly-Q toxicity only in the absence of the major ubiquitin-conjugating enzyme Ubc4. Prion forming potential of other Q-rich proteins and influence of Q and P-rich regions on prion propagation were also studied. Our data connects poly-Q aggregation and toxicity to the stress defense pathway in yeast. As many stress-defense proteins are conserved between yeast and mammals, our data shed light on possible mechanisms modulating poly-Q aggregation and toxicity in mammalian cells.

Glutamine synthetase (GS; EC 6.3.1.2; L-glutamate ammonialigase) catalyzes the ATP-dependent conversion of glutamate and ammonia into glutamine. Due to its key role in nitrogen metabolism, including nucleotide, amino acid and urea biosynthesis, the enzyme has been ascribed an extraordinarily long evolutionary history. Thus, GS has been used as a molecular clock to establish phylogenetic relationship between different species. Through the National Center of Biotechnology Information (NCBI) using Basic Local Alignment Search Tool (BLAST) programs BLASTx (translated nucleotide-protein alignment) and BLASTn (nucleotide-nucleotide alignment) system, we obtained the complete cDNA of GS from tilapia cDNA liberary. Furthermore, the results of the alignment of tilapia GS sequence with that of other species indicated a close relationship between tilapia GS and other fishes. We also found that there is 79% homology between mammal and tilapia within the open read frame (ORF) of GS. However, sequence analysis by computer software revealed the fact that the size (0.5 kb) of GS 3¡¦untranslated region (3¡¦-UTR) of tilapia GS is different from that of mammals. Moreover, there is the complete distinct sequence of the 3¡¦-UTR of tilapia GS from that of mammals. The 3'-UTR of many eukaryotic mRNAs has been implicated in the control of mRNA stability, processing, polyadenylation, and translational regulation. Accordingly, to comprehend the role of 3¡¦-UTR in GS phylogenesis, we examine whether the 3'-UTR of tilapia GS is involved in the regulation of GS expression in mammals. We first generated the construct using pEGFP-N2 carrying the ORF (1.1kb) of tilapia GS gene (ORF-GFP) or the full length (1.6kb) of tilapia GS gene (Full-GFP). Transient or stable transfection of C6 gliomal cells with ORF-GFP indicated that GS mRNA and protein was expressed. When C6 cells were stably transfected with Full-GFP, the expression of GS mRNA, but not its protein, was found. Adenine/uridine-rich sequence elements (AREs) of the 3¡¦-UTR have been known to regulate mRNA stability of certain chemokines. Four AREs are also found in the 3¡¦-UTR of tilapia GS. We further generated the constructs with tilapia ORF-GFP and its 3¡¦-UTR containing 1-4 AREs (A1-GFP, A2-GFP, A3-GFP and A4-GFP). Stable transfection of C6 cells with the different constructs indicated that tilapia GS mRNA is normally transcripted, while there was no expression of GS proteins in stable transfectants. The findings suggest tilapia GS protein expression in mammals by its 3¡¦-UTR and unidentified evolutionary role of the 3¡¦-UTR region of GS.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 180-187).

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

No description available.

Rapid expansion of the aquaculture industry in recent decades has resulted in infectious diseases emerging as a major constraint to fish production, causing large economical losses worldwide. Therefore, prevention practices are indispensable for maintaining the industry's profitability and sustainability. Vaccination is a proven effective strategy for disease control in aquaculture; however, improvements in vaccine efficacy are still needed. Because amino acid supplementation not only enhances fish growth but also immune responses, a series of experiments were conducted to test the hypothesis that dietary supplementation of arginine and glutamine, two amino acids with immunomodulatory roles, may promote growth and increase the efficacy of vaccination against Edwardsiella ictaluri in channel catfish. An initial experiment demonstrated that dietary arginine supplementation at 2 and 4% of diet enhanced growth and feed efficiency of channel catfish. Dietary arginine deficiency diminished plasma levels of arginine, citrulline, ornithine, glutamine and glutamate, and impaired innate performance of macrophages and neutrophils. In a separate experiment, dietary glutamine supplementation failed to enhance growth responses; however, supplementation at 2% of diet had strong positive effects on intestinal histology and enterocyte migration rate. In addition, serine, asparagine, glycine and threonine were increased in plasma of fish fed the diet with glutamine at 2%. A third experiment revealed that activated macrophages utilized large quantities of glutamine in media and to a lesser extent arginine. These two amino acids also were the most utilized by proliferating lymphocytes. Supplementing media with these amino acids positively modulated phagocytosis and bactericidal capacity of macrophages, as well as increased the proliferation rate of lymphocytes. A final experiment indicated that dietary supplementation of arginine (4%) and glutamine (2%) optimized the nutritional and immunological status of channel catfish, and enhanced responses to E. ictaluri vaccination. At the same time, this supplementation ameliorated some short-term adverse effects of vaccination on growth. Higher specific antibody titers, better lymphocyte responsiveness and survival to the bacterium were seen in vaccinated fish fed arginine- and glutamine-supplemented diets. These results support an expanded role of dietary arginine and glutamine manipulation as a tool to improve growth and vaccine efficacy of channel catfish.

Glutamine serves as a major energy source for all stages of preimplantation mouse embryo development, whether the embryos are raised in vivo or in vitro from the one-cell stage. Glutamate dehydrogenase (GDH) and glutamine synthetase (GS) are enzymes that are involved in the metabolism of glutamine. GDH catalyzes the conversion of glutamate into a-ketoglutarate, a primary component of the tricarboxylic acid cycle. GS catalyzes the conversion of glutamate to glutamine. The expression of GDH RNA and GS RNA were analyzed in preimplantation mouse embryos using reverse transcription (RT) with an oligo dT primer followed by Polymerase Chain Reaction (PCR) amplification of GDH and GS cDNAs using gene specific primers. Data show that GDH RNA is expressed in mouse embryos grown in vivo at the one-cell, two-cell, eight-cell, and blastocyst stages of development. GS RNA is not expressed at the one-cell stage, but first appears at the two-cell stage and is expressed at the eight-cell and blastocyst stages. Semiquantitative PCR analysis using a globin internal standard demonstrated that GS RNA is present at high levels at the two-cell stage and declines by 51 % by the blastocyst stage. These results suggest that, within the preimplantation mouse embryo, GDH RNA is expressed by both the maternal genome as well as the embryonic genome, while GS RNA is only expressed by the embryonic genome. This study provides an explanation for why glutamine is utilized as an energy source during preimplantation development, which allows for a better understanding of glutamine metabolism and its role during early mouse development. / Department of Biology