Dexmedetomidine is α2-adrenergic agent and commonly used in the intensive care setting. It is used primarily to sedate critically ill patients in various surgical, endoscopic and radiologic procedures. This medication gained superiority with other sedatives with a distinct advantage of less depression of the respiratory system. Although dexmedetomidine is often administered to septic patients, the vascular effect has not been fully studied in this clinical setting. In this thesis, rats were administered saline or bacterial lipopolysaccharide (LPS) 1, 10 and 20 mg/kg. Aortic rings were obtained after two, 24 and 72 hours exposure and dexmedetomidine-induced contractions were investigated. Rats could not tolerate prolonged exposure to 20 mg/kg LPS and died during the process. Systolic, diastolic and mean arterial pressures were reduced after LPS exposure while heart rates were elevated. Body temperatures were elevated after LPS administration (all doses and time), except a reduction at two hours with 1 mg/kg LPS. LPS increased the plasma levels of tissue necrosis factor-α and interleukin-6 after two hours LPS administration but not at 24 and 72 hours. In aortic rings with functional endothelium from rats with or without LPS exposure, dexmedetomidine had no effect on resting tension. Dexmedetomidine produced concentration-dependent contractions (10 nM to 10 μμM) after incubation with endothelial nitric oxide synthase (NOS) inhibitor L-NAME or removal of endothelium in rings without and with exposure to LPS for two, 24, 72 hours. LPS administration dose-dependently attenuated dexmedetomidine-induced contractions. In the presence of 1400W (inducible NOS inhibitor) the contractile response to dexmedetomidine was potentiated suggesting a role of NO produced by iNOS. Treatment with MnTMPyP attenuated dexmedetomidine-induced contractions indicating that the superoxide dismutase mimetic might increase NO availability by reducing superoxide. A significant role of iNOS was further supported by the detection of iNOS expression in aortic rings after LPS exposure at two, 24, and 72 hours when compared to non-LPS exposed group. Use of selective α2A and α2B receptor antagonists (BRL44408 and ARC239 respectively) showed that dexmedetomidine-induced contractions were mediated mostly via α2A receptor subtype as the former but not the latter agent significantly reduced contractions. Serotonin (5-HT, 10 nM to 100 μμM) and phenylephrine (1 nM to 100 μμM) produced concentration-dependent contractions in aortic rings with and without LPS exposure. The maximal responses to 5-HT and phenylephrine were increased at 10 mg/kg LPS as compared to a reduction in contractions by dexmedetomidine with LPS exposure at 1 and 10 mg/kg supporting alterations in α2 receptors occurred after LPS administration. In conclusion, the present study demonstrated that the vascular contractile responses of dexmedetomidine in the absence of endothelium or in the presence of eNOS inhibition were reduced in the presence of LPS at different time points and at different doses in aortic rings while two other receptor mediated vasoconstrictors, 5-HT and phenylephrine were affected. Our findings suggest that LPS may preferentially reduce the vascular contractile responses of dexmedetomidine and it is essential to exercise caution when the drug is administered to critically ill patients or with endothelial dysfunction. / published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

The lipopolysaccharide (LPS) translocation process was studied by a pulse-chase experimental procedure designed to follow the fate of newly synthesized LPS through the cell wall fractions of the marine bacterium Alteromonas haloplanktis, strain 214, variant 3 (ATCC 19855). It was determined that newly synthesized LPS I (the prominent LPS species under the conditions employed) initially enters an LPS fraction which can be released from the cell wall along with periplasmic material. This is followed by rapid entry of the newly synthesized LPS into a loosely bound LPS fraction (released from the cell wall by washing with NaCl) before final insertion into the outer membrane. The inhibition of protein synthesis with chloramphenicol was observed to have little or no effect on the rate of translocation of newly synthesized LPS. In contrast, both the respiratory inhibitor NaCN (10 mM) and the proton ionophore 3,3',4',5-tetrachlorosalicylanilide (TCS, 30 (mu)M), if added during the chase period, resulted in an immediate and complete inhibition of further LPS translocation. The results suggest an independence of the LPS transloction process from continued protein synthesis, but a requirement for an energy source. It was observed that newly synthesized LPS sedimented to a lower density position than previously formed LPS on sucrose density gradients, and displayed a faster migration rate during electrophoresis on sodium dodecyl sulfate polyarylamide gels (SDS-PAGE). This indicated compositional differences between the two LPS populations. Extended electrophoresis on SDS-PAGE separated both newly synthesized and previously formed LPS I into a number of distinct radiolabelled peaks and shoulders, indicating compositional microheterogeneity within each species. The fate of added radiolabelled galactose was also investigated. A one minute pulse with {('14)C} galactose resulted in a preferential labelling of components associated with the cell wall such that 57.3% of the total w

Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1985. / Bibliography: leaves 82-92. / Microfilm. / vii, 92 leaves, bound ill. 29 cm

Thesis (M. Sc.), Memorial University of Newfoundland, 1998. / Restricted until November 1999, Bibliography: leaves 91-107.

M.Sc. / On exposure to abiotic or biotic stresses, plants initiate a cascade of metabolic reactions, some of which lead to the biosynthesis of secondary metabolites with roles in self defense. Phytoalexins are a class of secondary metabolites synthesized de novo in response to microbial attack by activation of certain biosynthetic pathways. Cruciferae phytoalexins are all indole based with a carbon, nitrogen and sulfur containing constituent on the 3’ position of the indole ring. This common similarity of all Cruciferae phytoalexins suggests that the plants all share a common indole precursor. Camalexin is the primary phytoalexin of Arabidopsis thaliana. De novo synthesis of camalexin upon infection, as well as its antimicrobial nature supports its role in disease resistance. Evidence exists that suggests the inducible biosynthesis of camalexin involves steps of the tryptophan pathway, along with an increase in transcript and protein levels of the tryptophan pathway enzymes after microbial infection. Bacterial LPS (lipopolysaccharide) has been described as one of the microbe/pathogenassociated molecular patterns (M/PAMPs) capable of eliciting the activation of the plant innate immune system. LPS is an integral component of the cell surface of Gram-negative bacteria. It is a complex which is exposed to the external environment, and is thus involved with external interactions of the bacteria. The hypothesis investigated in this dissertation is that LPS, as a lipoglycan PAMP, results in activation of signal transduction pathways involved in defense that lead to the production of the defense metabolite, camalexin. Furthermore, that the genes CYP71B15, CYP79B2 and TSB are up-regulated in response to LPS during camalexin biosynthesis via the tryptophan pathway. To test this hypothesis, camalexin production was investigated through a combination of analytical techniques including thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography (GC), ultra pressure liquid chromatography-mass spectrometry (UPLC-MS) and fluorescence spectroscopy. Genes in the camalexin biosynthetic pathway were investigated by two-step reverse transcription polymerase chain reaction (PCR), GUS reporter gene assays and quantitative real time PCR (RT-qPCR).

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Endotoxin is a part of the cell wall of gram-negative bacteria, consisting of serotype-specific polysaccharide, core oligosaccharide, and lipid A. Lipid A is responsible for an array of pathophysiologic reactions in animal hosts. Amebocyte lysate originated from Limulus polyphemus (horseshoe crab) has been used extensively in various assay systems to detect endotoxin. One of the assays, a chromogenic Limulus amebocyte lysate (CLAL) test was developed in 1978 and has been used extensively in human clinical fields for its high sensitivity and ease in quantitation. The use of the CLAL test in veterinary fields has been limited to dogs, horses and cattle. The objective of the thesis research was to determine the level of endotoxin by the CLAL assay in broiler chickens. Since gram-negative septicemia is common in broiler chickens, the detection of endotoxemia would help in understanding the pathogenesis and in developing a new treatment or prophylactic mean. By the use of a kinetic method, the CLAL assay detected the standard endotoxin (phenol-water extract from Escherichia coli, 055:B5 strain) in the range between 100 ng and 10 pg/ml. The intra-assay variation was 1.2% and interassay variation was 18.8% based on 1.0 ng standard. The control showed spontaneous release of the chromophore starting around 40 min. after the start of the reaction. This spontaneous release was found not due to contamination of pyrogen-free water (PFW) or substrate by endotoxin. With chicken plasma, various non-specific reactions were detected. Plasma alone released the chromophore in a slow, steady manner, but this reaction was virtually eliminated by heating at 100 C. Chicken plasma contained both inhibitor(s) and enhancer(s) for the test. Endotoxin-free plasma samples were prepared by absorption and reconstituted with 1.0 ng/ml of endotoxin. After 1:10 dilution in PFW, heating (10 min.) at 100 C was found most adequate to inactivate these factors as compared with heating to 70 or 85 C. With plasma samples which had been diluted and heated at 100 C, however, still some nonspecific reaction was detected; the lysate, in the absence of substrate, caused some precipitates with chicken plasma in a nonspecific manner, making it difficult to interpret the OD readings. Because of these nonspecific reactions largely inherent to the state of lysate, sensitivity was judged only to 100 pg endotoxin/ml of chicken plasma. A commercial test kit also showed 100 pg/ml sensitivity with the end point method, but found unreliable since proper controls cannot be evaluated in a similar manner as the kinetic method. Thirty chicken plasma were collected from 3 local broiler farms and was judged to contain less than 100 pg/ml in 29 birds, while one bird showed 12.0 ng/ml of endotoxin. Twenty-three per cent of the chickens showed gram-negative bacteremia without detectable endotoxemia, and the bird with endotoxemia did not have bacteremia. One microgram of the standard endotoxin was injected intravenously to 20 broiler chickens raised in the laboratory, and 5 were sacrificed at 2, 30, 60, and 90 minutes after the injection. The endotoxin was found to be cleared from the blood at the rate of 152 pg/min. To increase the sensitivity and to decrease the cost of the CLAL test, future efforts should be made; 1) to significantly decrease the nonspecific reaction between the lysate and substrate; and 2) to block the precipitation or clotting reaction between the lysate and chicken plasma. If these nonspecific reactions be controlled, the CLAL test could be run in a simple end-point method and/or in an automated manner with chicken plasma. / Graduation date: 1997

Title from title page of PDF (University of Missouri--St. Louis, viewed Feb. 10, 2010). Includes bibliographical references.