Our laboratory has recently reported that lecithin:cholesterol acyltransferase (LCAT) deficient mice are hypersensitive to insulin and resistant to diet-induced obesity, particularly in the LDL receptor (LDLR) knockout background. These phenotypes are linked to hepatic endoplasmic reticulum (ER) stress, which we showed is elevated basally and highly inducible in LDLR deficient mice. While in LCAT/LDLR deficient mice, ER stress is normalized basally and its diet-induction is attenuated. Mechanistically, we show here that excess free cholesterol (FC), in part from the bile, accumulates in the ER membrane of LDLR deficient mice. In contrast, LCAT/LDLR deficient mice have reduced levels of ER membrane FC and are resistant to cholesterol diet-induced elevations, in part from increased INSIG-1 expression and cholesterol esterification by ACAT2. Our analysis has led to the first report of cholesterol-induced hepatic ER stress in vivo and the identification of ER FC levels as a critical indicator of ER stress susceptibility.

Auditory sensitivity has often been measured by identifying neural threshold in real-time (online) which can introduce bias in the audiograms that are produced. We tested this by recording auditory nerve activity of the notodontid moth Nadata gibbosa elicited by bat-like ultrasound and analysing the response offline. We compared this audiogram with a published online audiogram showing that the bias introduced can result in a difference in the audiogram shape. In the second part of our study we compared offline audiograms using spike number as threshold with others that used spike period and stimulus/spike latency, variables that have been suggested as providing behaviourally functional criteria. These comparisons reveal that functional audiograms are more flatly tuned than simple spike audiograms. The shapes of behavioural audiograms are discussed in the context of the selection pressure that maintains their shape, bat predation.

Mammalian neurons are anoxia-sensitive and rapidly undergo excitotoxic cell death when deprived of oxygen, mediated largely by calcium entry through N-methyl-D-aspartate receptors (NMDARs). This does not occur in neurons of the anoxia-tolerant western painted turtle, where a decrease in NMDAR currents is observed with anoxia. This decrease is dependent on a modest increase in cytosolic [Ca2+] during anoxia. The aim of this study was to determine if activation of the mitochondrial permeability transition pore (mPTP) decreases NMDAR currents through release of mitochondrial Ca2+. The data indicate that mPTP opening is sufficient to cause a decrease in NMDAR currents during normoxia and the anoxia-mediated rise in cytosolic [Ca2+] and depolarization of the mitochondrial membrane potential is due to opening of the mPTP. Furthermore, since a mitochondrial uncoupler releases additional calcium during anoxia we speculate that the mitochondrial membrane potential decreases in a regulated fashion to a new set-point.

Auditory sensitivity has often been measured by identifying neural threshold in real-time (online) which can introduce bias in the audiograms that are produced. We tested this by recording auditory nerve activity of the notodontid moth Nadata gibbosa elicited by bat-like ultrasound and analysing the response offline. We compared this audiogram with a published online audiogram showing that the bias introduced can result in a difference in the audiogram shape. In the second part of our study we compared offline audiograms using spike number as threshold with others that used spike period and stimulus/spike latency, variables that have been suggested as providing behaviourally functional criteria. These comparisons reveal that functional audiograms are more flatly tuned than simple spike audiograms. The shapes of behavioural audiograms are discussed in the context of the selection pressure that maintains their shape, bat predation.

Mammalian neurons are anoxia-sensitive and rapidly undergo excitotoxic cell death when deprived of oxygen, mediated largely by calcium entry through N-methyl-D-aspartate receptors (NMDARs). This does not occur in neurons of the anoxia-tolerant western painted turtle, where a decrease in NMDAR currents is observed with anoxia. This decrease is dependent on a modest increase in cytosolic [Ca2+] during anoxia. The aim of this study was to determine if activation of the mitochondrial permeability transition pore (mPTP) decreases NMDAR currents through release of mitochondrial Ca2+. The data indicate that mPTP opening is sufficient to cause a decrease in NMDAR currents during normoxia and the anoxia-mediated rise in cytosolic [Ca2+] and depolarization of the mitochondrial membrane potential is due to opening of the mPTP. Furthermore, since a mitochondrial uncoupler releases additional calcium during anoxia we speculate that the mitochondrial membrane potential decreases in a regulated fashion to a new set-point.

Our laboratory has recently reported that lecithin:cholesterol acyltransferase (LCAT) deficient mice are hypersensitive to insulin and resistant to diet-induced obesity, particularly in the LDL receptor (LDLR) knockout background. These phenotypes are linked to hepatic endoplasmic reticulum (ER) stress, which we showed is elevated basally and highly inducible in LDLR deficient mice. While in LCAT/LDLR deficient mice, ER stress is normalized basally and its diet-induction is attenuated. Mechanistically, we show here that excess free cholesterol (FC), in part from the bile, accumulates in the ER membrane of LDLR deficient mice. In contrast, LCAT/LDLR deficient mice have reduced levels of ER membrane FC and are resistant to cholesterol diet-induced elevations, in part from increased INSIG-1 expression and cholesterol esterification by ACAT2. Our analysis has led to the first report of cholesterol-induced hepatic ER stress in vivo and the identification of ER FC levels as a critical indicator of ER stress susceptibility.

Rhodnius prolixus is a medically-important hemipteran that serves as a vector of Chagas disease. The distribution and physiological effects of three sequence-related neuropeptides, adipokinetic hormone (AKH), corazonin (CRZ) and AKH/corazonin-related peptide (ACP) have been investigated in R. prolixus. Immunohistochemistry revealed that AKH, CRZ and ACP are not co-localized but are found within a different subset of cells and processes within the central nervous system. Physiologically, CRZ significantly increased the heartbeat rate whereas AKH and ACP did not. AKH significantly increased haemolymph lipid levels whereas CRZ and ACP did not. There is no known function for ACP. Preliminary studies indicated that CRZ may be involved in gating of ecdysis.

Rhodnius prolixus is a medically-important hemipteran that serves as a vector of Chagas disease. The distribution and physiological effects of three sequence-related neuropeptides, adipokinetic hormone (AKH), corazonin (CRZ) and AKH/corazonin-related peptide (ACP) have been investigated in R. prolixus. Immunohistochemistry revealed that AKH, CRZ and ACP are not co-localized but are found within a different subset of cells and processes within the central nervous system. Physiologically, CRZ significantly increased the heartbeat rate whereas AKH and ACP did not. AKH significantly increased haemolymph lipid levels whereas CRZ and ACP did not. There is no known function for ACP. Preliminary studies indicated that CRZ may be involved in gating of ecdysis.

Master of Science / Department of Animal Sciences and Industry / J. Ernest Minton / Previous research evaluated a laboratory strain of Bacillus licheniformis (BL) in a model swine epithelium and found it exerted anti-inflammatory effects on Salmonella enterica serovar Typhimurium (S)-induced secretion of interleukin-8 (IL-8). The current investigation evaluated the anti-inflammatory actions of Bacillus bacteria available commercially as feed additives for the swine industry. Three isolates were obtained from the product, two Bacillus subtilis (BS1 and BS3) and one Bacillus licheniformis (BL2). Swine jejunal epithelial IPEC-J2 cells were seeded into wells on permeable membrane supports and allowed to form confluent monolayers. Treatments included apical pretreatment with BL, BS1, BL2, or BS3 for 17 h without S, and the same Bacillus treatments but with 10[superscript]8 CFU S added in the final 1 h of Bacillus incubation. Two additional treatments included negative control wells receiving no bacteria (C) and positive control wells receiving only S. Following bacterial incubation, wells were washed and fresh media containing gentamicin was added. Cells were incubated for an additional 5 h, after which apical and basolateral media were recovered for quantitation of IL-8 and bacitracin. In addition, inserts with epithelial cells that had received S were lysed and lysates cultured to determine treatment effects on S invasion. Exposure to S alone provoked an increase in IL-8 secretion from IPEC-J2 cells compared to C wells (P < 0.001 for both the apical and basolateral directions). Pre-treatment with each Bacillus isolate followed by challenge with S reduced S-induced IL-8 secretion in both apical and basolateral compartments compared to the wells receiving only S (P < 0.001; except for BS3 apical, P < 0.01). Secretion of bacitracin could only be detected in BL2 and BL2+S. Fewer S colonies could be cultured from lysates of BL2+S than S, BS1+S, and BS3+S treatments (P < 0.001). Results suggest that Bacillus subtilis and Bacillus licheniformis have the ability to intervene in secretion of the neutrophil chemoattractant IL-8 from swine intestinal epithelial cells. This effect on chemokine secretion by gastrointestinal epithelial cells in vitro could not be explained solely by production of bacitracin or reduced invasion of epithelial cells by S.

Master of Science / Clinical Sciences - Veterinary Medicine / Laurie A. Beard / Objective: To determine the pharmacokinetic properties of oral pergolide in normal mares. Animals: 6 horses, 3-17 years of age, 355-582 kg Procedures: In a randomized, cross over design six healthy adult female horses received pergolide (PO) 0.01mg/kg or placebo after 8 hours of fasting. Samples were taken over a period of 6 day for each portion of the study (treatment or placebo) with a two week minimum wash out period between study periods. Quantification of pergolide concentration was determined by UPLC-MS. Quantification of α-MSH was determined by radioimmunoassay validated for horses. Quantification of ACTH concentration was determined by chemiluminescent enzyme immunoassay. Results: Pergolide was detected in all treated horses. The relatively short time to peak concentration (0.5 hours) indicates a rapid absorption. Mean maximum concentration measured was 4.05 ng/ml + 2.02 with a median time to maximum concentration being 0.415 hours (range:0.33-1.0). The mean half life of pergolide was determined to be 5.86 hours + 3.42. Lower limits of quantitation for the UPLC-MS assay was 0.5 ng/ml. α-MSH results were evaluated using a multiple analysis of variance assay for repeated measures comparing treatment, time, and period. There was a significant treatment to period effect with p=0.02. The effect of period appears to be more significant (p=0.06) compared to the effect of treatment (p=0.77). No effect from pergolide was noted on ACTH concentrations. Conclusions and Clinical Relevance: Horses appear to absorb and eliminate pergolide more rapidly than previously expected. Based on this pharmacokinetic data the dosing strategies of pergolide may need to be altered. However, assay sensitivity does need to be improved prior to recommendations being made.