Asthma is a chronic respiratory illness where the airway occasionally constricts, becomes inflamed, and is lined with excessive amounts of mucus. There are many lipid mediators involved in this inflammatory response. Hepoxilins are biologically active hydroxyepoxy eicosatrienoic acid metabolites of arachidonic acid, formed through the 12-lipoxygenase pathway. Our goal in this study was to identify if hepoxilins had a direct effect on smooth muscle contractions, to identify the source of hepoxilins in asthmatic-like mouse model, and to determine if blocking this pathway reduced inflammation associated with ovalbumin sensitization. We found that hepoxilins had no direct effect on smooth muscle contraction. We identified the bronchiolar epithelium as a major source of hepoxilins in OVA-challenged mice. We demonstrated that baicalein inhibits total lung resistance in the OVA-treated mice, but also found that the drug in-vivo is not specific for 12-lipoxygenase.

Plasma membrane (PM) proteins are at the interface between the cell and the external environment and are therefore the most accessible to therapeutic drugs. I utilized cationic silica beads and mass spectrometry (MS)-based proteomics to enrich for PM proteins of human cardiomyocytes, coronary smooth muscle cells, and coronary endothelial cells. The enrichment of PM proteins was confirmed and 1006 proteins were specifically filtered and enriched into a set of known and novel cardiomyocyte PM-associated proteins of which 42% had PM-associated gene ontology annotations and/or predicted transmembrane helices. Two novel candidates, namely popeye domain-containing protein 2 (POPDC2) and protein kinase C and casein kinase substrate in neurons protein 3 (PACSIN3) were selected and found to have confirmed PM localization. In conclusion, silica bead membrane extraction combined with MS-based proteomics successfully enriched for PM proteins of the human heart of which two novel candidate proteins were shown to have confirmed PM localization.

Sciatic and saphenous neurectomy produces in mice a neuropathic pain-like behaviour (‘autotomy’). A/J mice express higher autotomy levels than C57BL6/J mice. A previous study used autotomy data for these strains and their 23 recombinant daughter inbred lines of the AXB-BXA set, to map a quantitative trait locus (QTL) for autotomy on chromosome 15. Since then, this QTL, named Pain1, was replicated twice. Since all three studies used a low-resolution genetic map based on microsatellites its confidence length precluded identification of candidate gene(s). To overcome this problem, I used a higher resolution SNP-based genetic map and refined Pain1’s peak location, identifying in it 80 candidate genes. But only Lynx1, Arc and Sharpin had sequence mismatches between A/J and C57BL6/J, known neural functions, and contrasting expression levels in DRGs and spinal cord of intact, sham-operated, and neurectomized mice of these lines. Meeting these criteria made them our best candidate genes for autotomy.

The incretin hormone glucagon-like peptide-1 enhances glucose-dependent insulin secretion and is therefore a most attractive therapeutic approach for the treatment of Type 2 Diabetes Mellitus. The anti-diabetic drug, metformin, has previously been shown to increase circulating levels of GLP-1, although its mechanism of action is currently unknown. Neither metformin nor AICAR (activators of AMPK) directly stimulated GLP-1 secretion from the L-cell in vitro. However, oral treatment of rats with metformin enhanced plasma levels of active and total GLP-1, independent of GLP-1 degradation. Furthermore, pre-treatment with the general muscarinic antagonist, atropine, or the M3 antagonist, 4-DAMP, decreased metformin–induced GLP-1 secretion, while M1 and M2 antagonists did not. Chronic bilateral subdiaphragmatic vagotomy had no effect, while the GRP antagonist, RC-3095, reduced metformin-induced GLP-1secretion. Therefore, I conclude that metformin-induced GLP-1 secretion occurs in part through the parasympathetic nervous system, the M3 and GRP receptors, but is independent of the vagus nerve.

DISC1 is an established susceptibility gene for schizophrenia. To gain insight on the neural mechanisms responsible for hippocampal deficits in schizophrenia, we sought to characterize place cell activity and theta rhythm in our Disc1-L100P mouse strain that we have previously shown to express deficits in spatial working memory. Our findings suggest that the rate code of place cells is intact. We found that Disc1-L100P mice have deficits in theta rhythm, increased neural noise, and lower levels of PV+ interneurons in the hippocampus. Our findings are supportive of impaired temporal coding in Disc1-L100P place cells. We found that Disc1-L100P place cell waveforms were broader than those of wild-type mice and putative interneuron waveforms were narrower. These findings suggest that ion-channel function and expression in the hippocampus is altered in Disc1-L100P mice. In schizophrenic subjects deficits in working memory are associated with aberrant oscillatory activity, increased noise, and lower PV+ interneuron expression.

Asthma is a chronic respiratory illness where the airway occasionally constricts, becomes inflamed, and is lined with excessive amounts of mucus. There are many lipid mediators involved in this inflammatory response. Hepoxilins are biologically active hydroxyepoxy eicosatrienoic acid metabolites of arachidonic acid, formed through the 12-lipoxygenase pathway. Our goal in this study was to identify if hepoxilins had a direct effect on smooth muscle contractions, to identify the source of hepoxilins in asthmatic-like mouse model, and to determine if blocking this pathway reduced inflammation associated with ovalbumin sensitization. We found that hepoxilins had no direct effect on smooth muscle contraction. We identified the bronchiolar epithelium as a major source of hepoxilins in OVA-challenged mice. We demonstrated that baicalein inhibits total lung resistance in the OVA-treated mice, but also found that the drug in-vivo is not specific for 12-lipoxygenase.

Sciatic and saphenous neurectomy produces in mice a neuropathic pain-like behaviour (‘autotomy’). A/J mice express higher autotomy levels than C57BL6/J mice. A previous study used autotomy data for these strains and their 23 recombinant daughter inbred lines of the AXB-BXA set, to map a quantitative trait locus (QTL) for autotomy on chromosome 15. Since then, this QTL, named Pain1, was replicated twice. Since all three studies used a low-resolution genetic map based on microsatellites its confidence length precluded identification of candidate gene(s). To overcome this problem, I used a higher resolution SNP-based genetic map and refined Pain1’s peak location, identifying in it 80 candidate genes. But only Lynx1, Arc and Sharpin had sequence mismatches between A/J and C57BL6/J, known neural functions, and contrasting expression levels in DRGs and spinal cord of intact, sham-operated, and neurectomized mice of these lines. Meeting these criteria made them our best candidate genes for autotomy.

Plasma membrane (PM) proteins are at the interface between the cell and the external environment and are therefore the most accessible to therapeutic drugs. I utilized cationic silica beads and mass spectrometry (MS)-based proteomics to enrich for PM proteins of human cardiomyocytes, coronary smooth muscle cells, and coronary endothelial cells. The enrichment of PM proteins was confirmed and 1006 proteins were specifically filtered and enriched into a set of known and novel cardiomyocyte PM-associated proteins of which 42% had PM-associated gene ontology annotations and/or predicted transmembrane helices. Two novel candidates, namely popeye domain-containing protein 2 (POPDC2) and protein kinase C and casein kinase substrate in neurons protein 3 (PACSIN3) were selected and found to have confirmed PM localization. In conclusion, silica bead membrane extraction combined with MS-based proteomics successfully enriched for PM proteins of the human heart of which two novel candidate proteins were shown to have confirmed PM localization.

The incretin hormone glucagon-like peptide-1 enhances glucose-dependent insulin secretion and is therefore a most attractive therapeutic approach for the treatment of Type 2 Diabetes Mellitus. The anti-diabetic drug, metformin, has previously been shown to increase circulating levels of GLP-1, although its mechanism of action is currently unknown. Neither metformin nor AICAR (activators of AMPK) directly stimulated GLP-1 secretion from the L-cell in vitro. However, oral treatment of rats with metformin enhanced plasma levels of active and total GLP-1, independent of GLP-1 degradation. Furthermore, pre-treatment with the general muscarinic antagonist, atropine, or the M3 antagonist, 4-DAMP, decreased metformin–induced GLP-1 secretion, while M1 and M2 antagonists did not. Chronic bilateral subdiaphragmatic vagotomy had no effect, while the GRP antagonist, RC-3095, reduced metformin-induced GLP-1secretion. Therefore, I conclude that metformin-induced GLP-1 secretion occurs in part through the parasympathetic nervous system, the M3 and GRP receptors, but is independent of the vagus nerve.

DISC1 is an established susceptibility gene for schizophrenia. To gain insight on the neural mechanisms responsible for hippocampal deficits in schizophrenia, we sought to characterize place cell activity and theta rhythm in our Disc1-L100P mouse strain that we have previously shown to express deficits in spatial working memory. Our findings suggest that the rate code of place cells is intact. We found that Disc1-L100P mice have deficits in theta rhythm, increased neural noise, and lower levels of PV+ interneurons in the hippocampus. Our findings are supportive of impaired temporal coding in Disc1-L100P place cells. We found that Disc1-L100P place cell waveforms were broader than those of wild-type mice and putative interneuron waveforms were narrower. These findings suggest that ion-channel function and expression in the hippocampus is altered in Disc1-L100P mice. In schizophrenic subjects deficits in working memory are associated with aberrant oscillatory activity, increased noise, and lower PV+ interneuron expression.