Four experiments were conducted to evaluate effects of arginine (ARG), vitamin E (VE) and mannanoligosaccharide (MOS) on immune response and clearance of Salmonella in broiler chickens. There were 4 groups in Exp.1 (E.1) and E.2: antibiotic-free diet (CTL-); diet with antibiotic (CTL+); ARG and VE (AVE); ARG, VE and MOS (AVM). Birds were infected with 10^6 CFU/ml of a novobiocin and nalidixic acid resistant Salmonella enteric Serovar Typhimurium (ST) at d 7 (E.1) or at d 3 (E.2). In E.3 and E.4, there were three groups: CTL, AVE and AVM; at d 3, birds in E.3 were infected with 10^2 CFU/ml ST and birds in E.4 were infected with 10^6 CFU/ml ST. The following were analyzed for four experiments: Oxidative burst heterophils response (OBHR) and monocytes response (OBMR); Lymphocyte proliferative response (LP); antibodies concentrations in serum; chicken body weight (BW), cecal Salmonella concentration; gut parameters. In E.1, both AVM and AVE diet decreased OBHR; birds fed AVM showed the highest IgA level, birds fed AVM had higher IgM than CTL+ and had the lowest Salmonella population. Birds fed AVE showed higher LP than CTL- in E.1 and E.2. From E.1 and E.2, AVM decreased chicken innate immune response at a younger age and improved adaptive immune response when birds were older than 16 d. AVM reduced ST when administrated later (d 7) versus early (d 3). In E.3, no BW and Salmonella counts differences were found; birds fed AVM had a higher IgA than AVE but similar to CTL at d 17. Chickens fed CTL had the highest OBHR at d 17, chickens fed AVM had the highest LPR at d 17. In E.4, chickens fed AVM showed higher BW than CTL at d 17 and d 24. Also no Salmonella counts differences were found. Chickens fed AVE had higher LPR than chickens fed AVM at d 17. These results are consistent with results from E.1 and E.2. Overall, diet AVM has different effects on chicken immune response when chicks were infected at different age; higher level of ARG and VE improved immune response and improve gut health.

Thesis (Ph.D.)--University of Western Australia, 2005.

Thesis (Ph. D.)--Oregon Graduate Institute, 2000.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Includes bibliographical references. Also available online.

Thesis (Ph. D.)--University of Wyoming, 2007. / An interdisciplinary thesis in psychology and neuroscience. Title from PDF title page (viewed on Dec. 5, 2008). Includes bibliographical references.

Thesis (M. S.)--Florida State University, 2006. / Advisor: W. Ross Ellington, Florida State University, College of Arts and Sciences, Dept. of Biological Science. Title and description from dissertation home page (viewed Dec. 12, 2006). Document formatted into pages; contains xii, 77 pages. Includes bibliographical references.

Methylation is a post-translational modification which occurs on lysine and arginine residues. Methylation is difficult to detect due to its low abundance and lack of charge. Our laboratory previously developed a novel enrichment approach, ProMENADe, for lysine and arginine methylation in the human embryonic kidney (HEK) 293T cell line which is coupled with mass spectrometry. Simplifying a lysate with subcellular fractionation prior to enrichment increased the identification of methylation sites by 39.5% while using multiple proteases for digestion increased identification by 27%. Combining these methods yielded a 47.2% increase. Analysis at the 1% methylation level FDR filtered for C-terminal methylation identified 169 sites and further analysis revealed 74 of these sites overlap with the PhosphoSite database. This ProMENADe enrichment strategy yielded 95 novel methylation sites to the field and can be a key tool in the field of methylation allowing for the enrichment and identification of methylated proteins.

Previous studies in the sheep, rabbit, cat and rat have demonstrated the ability of the neuropeptide, arginine vasopressin (AVP), to suppress endotoxin-induced fever when perfused into a discrete brain locus. Fever can also be suppressed if AVP is microinjected into the cerebral ventricles of the rat. The mechanisms by which AVP mediates antipyresis are unknown. Experiments were conducted, therefore, to examine the effect of intracerebroventricular (icv) AVP on an established fever and to assess the mechanism of action using a specific, V₁-receptor antagonist (M-AVP). Studies were also conducted to elucidate the effector mechanisms utilized to accomplish antipyresis induced by icv AVP. Finally, cerebrospinal fluid (CSF) AVP concentrations were measured in febrile and non-febrile rats to determine the role of endogenously released AVP in the CSF during fever. AVP administered icv was shown to have marked antipyretic effects at very low doses. This antipyresis was elicited in rats with an established fever but the peptide had no effect on the temperature of non-febrile rats. Thus AVP both prevented and reversed endotoxin-induced fever. Furthermore, this AVP-induced antipyresis was abolished by pretreatment with the the V₁-antagonist, M-AVP. The antipyretic effects of AVP were, therefore, receptor mediated and likely to be of physiological importance. Efforts to manipulate the endogenous AVP system by icv M-AVP were also attempted. When M-AVP was injected icv, the fever height of endotoxin-treated rats was not different from endotoxin-treated controls. In addition, M-AVP did not influence the magnitude of the antipyresis induced by indomethacin. It has become clear, however, that this method of administering the antagonist is inappropriate to block endogenous AVP effects occurring within the neuropil. Subsequent experiments in another laboratory have shown that M-AVP must be microinjected into the AVP-sensitive brain locus to effectively block endogenous activity. The antipyretic response to icv AVP was further investigated at three ambient temperatures in an attempt to identify the effector mechanisms involved. Responses of non-febrile and febrile rats to icv injections of AVP and sc injections of indomethacin were observed at cold (4°C), neutral (25°C) and warm (32°C) ambient temperatures. As in the previous experiments, AVP at 25°C decreased brain temperatures of febrile but not non-febrile rats. This antipyretic effect was also observed at the warm ambient temperature and during cold exposure. Responses to sc indomethacin were qualitatively similar to icv AVP at neutral and warm temperatures. In the cold, however, indomethacin decreased the brain temperature of both non-febrile and febrile animals, although unlike AVP, brain temperature of non-febrile animals decreased somewhat more than that of febrile animals. These data showed that AVP decreased brain temperature of febrile more so than non-febrile rats at all ambient temperatures and may therefore have been acting partially on febrile set-point. It was possible that AVP affected specific effector mechanisms since antipyretic effects were of different magnitudes at different ambient temperatures. The observation that AVP and indomethacin had qualitatively similar effects on fever at three ambient temperatures suggested that they may act via a common neural pathway. Further analysis of the mechanism of icv AVP-induced antipyresis was conducted at the three ambient temperatures while measuring specific effectors: heat loss and heat production. At 25°C, AVP-induced antipyresis was mediated by tail skin vasodilation while metabolic rate was unaffected. At 4°C, the antipyresis produced by AVP was mediated exclusively by inhibition of heat production since the metabolic rate decreased markedly following AVP. This antipyresis at 4°C was accompanied by cutaneous vasoconstriction. At 32°C, neither vasomotor tone, metabolic rate nor evaporative heat loss could be shown to contribute to the small antipyretic effect elicited by AVP. These data strongly suggest that icv AVP produced antipyresis by affecting the febrile body temperature set-point mechanism since the thermoregulatory strategy to lose heat varied at different ambient temperatures and the decrease in body temperature could not be shown to be due to changes in a single effector mechanism. As an index of endogenous AVP activity, cerebrospinal fluid (CSF) concentrations of AVP were measured in febrile and non-febrile rats in order to determine the role of CSF AVP in fever and antipyresis. The results demonstrated that the AVP release pattern was not altered in endotoxin-treated febrile compared to non-febrile rats. It was concluded that CSF AVP had no role in the febrile process. In summary, icv AVP appears to induce antipyresis by its action on febrile set-point rather than on a specific effector system. This action of AVP is mediated by a V₁-like receptor mechanism which is not affected by endogenous CSF AVP. The neural/neurochemical basis for the thermoregulatory set-point has not been clearly established so the mechanism of action by which AVP affects set-point remains to be determined. These data contribute, however, to the growing body of evidence that AVP is acting centrally as a neurotransmitter or neuromodulator to regulate body temperature during the febrile process. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

Citrullinemia and argininosuccinic aciduria are two disorders resulting from defects in two consecutive enzymes of the urea cycle, argininosuccinate synthetase and argininosuccinate lyase. Fibroblast cell lines were derived from patients with these disorders and the diagnoses, which had been made on the basis of amino acid levels in plasma and urine, were confirmed by demonstrating that the cell lines were unable to incorporate 14 c-citrulline into protein. DNA from the argininosuccinate synthetase-deficient (ASS⁻) cells was analysed by restriction enzyme digestion and hybridisation to a cDNA probe which had been cloned from human argininosuccinate synthetase mRNA. No defect in the patient's DNA could be demonstrated, indicating that no major deletions in the argininosuccinate synthetase genes were present in this patient. Co-cultures of the ASS⁻ and argininosuccinate lyase-deficient (ASL⁻) fibroblasts were able to incorporate 14 citrulline into protein at rates comparable to normal fibroblasts. This complementation did not require cell fusion, was dependent on cell contact, and was not the result of exchange of metabolites or enzymes via the culture medium. These results indicated that complementation occurred by the exchange of metabolites via intercellular junctions between the two cell types. Co-cultures of ASS⁻ and ASL⁻ cells were used as an assay system for measuring intercellular junctional communication. This allowed quantitation of the effects of pH and extracellular divalent cations on junctional communication. Tumor promoters such as phorbol esters and organochlorine pesticides have been reported to inhibit intercellular junctional communication in other systems, and this inhibitory activity may be related to the mechanism of tumor promotion. The organochlorine pesticide 1,1,1-trichloro- 2,2-bis(p-chlorophenyl)ethane (DDT) was shown to be an inhibitor of junctional communication in ASS⁻/ASL⁻ cocultures. This inhibition was reversible, of rapid onset, and independent of extracellular calcium. The tumor-promoting phorbol ester 12-0-tetradecanoyl-phorbol-13- acetate (TPA) also rapidly induced inhibition of junctional communication. However, co-cultures between Chinese hamster V79 cells, which are deficient in ASS⁻, and ASL⁻ human fibroblasts were more sensitive to inhibition by TPA than the original ASS⁻/ASL⁻ co-cultures. Refractoriness to TPA occurred following prolonged treatment with high concentrations of TPA. Retinoic acid and other retinoids also inhibited junctional communication, and the inhibitory effects of retinoic acid and TPA were additive. The significance of these results in relation to the anti-tumor-promoting activity of retinoic acid is discussed. It is concluded that co-cultures of ASS⁻ and ASL⁻ cells constitute a useful system for providing quantitative measurements of intercellular junctional communication under a wide range of experimental conditions.

Protein arginine methylation is an important post-translational modification that is vital in regulating various cellular processes such as gene transcription, cell signaling, and RNA processing. Protein arginine methyltransferases (PRMTs) are responsible for performing this important modification. PRMT1 (protein arginine methyltransferase 1) and Hmt1 (hnRNP methyltransferase 1) are the predominant PRMTs in humans and yeast, respectively. Despite growing momentum in this field, relatively little is understood about PRMT regulation. Further work discovering how PRMTs are regulated will greatly advance our understanding of diseases where PRMTs have been implicated, such as heart disease, viral pathogenesis, and cancer. It has been discovered that a human protein called hCaf1 (human Ccr4-associated factor 1) is a regulator of PRMT1 with respect to certain substrates, and also colocalizes with PRMT1. We present data that suggest the yeast homolog of hCaf1, Pop2, may also perform a similar function on Hmt1. We provide data on the expression and purification of a truncation of Pop2 from S. cerevisiae, including the temperature sensitivity of one construct of Pop2 and its susceptibility to precipitation. We also demonstrated concentration-dependent inhibition of Hmt1-catalyzed methylation of histone H4 by Pop2 in vitro. Yeast cell lysates also showed altered patterns of methylation in the presence and absence of Pop2 in vivo. In an effort to understand the mechanism employed by Pop2 to accomplish this regulatory function, pull-downs were performed suggesting that Pop2 directly interacts with histone H4, a substrate of Hmt1. Mutagenic studies with Pop2 suggested a region that may be responsible for this interaction. Given these data, we hypothesized that Pop2 is able to inhibit the methylation of histone H4 via a substrate-sequestering mechanism. Further experimentation will determine the precise interaction surfaces of Pop2 and substrate, and continue to define the details of methylation inhibition by Pop2, including the scope of its influence in the cell.