Biomedical subjects

L Ashley Cowart

Publications and source records attributed to L Ashley Cowart.

9 recordsLinked to original sources

PubMed2006

Regulation of endothelial nitric-oxide synthase activity through phosphorylation in response to epoxyeicosatrienoic acids.

Endothelial nitric oxide synthase (eNOS) is a key enzyme in NO-mediated cardiovascular homeostasis and its activity is modulated by a variety of hormonal and mechanical stimuli via phosphorylation modification. Our previous study has demonstrated that epoxyeicosatrienoic acids (EETs), the cytochrome P450 (CYP)-dependent metabolites of arachidonic acid, could robustly up-regulate eNOS expression. However, the molecular mechanism underlying the effects of EETs on eNOS remains elusive. Particularly, whether and how EETs affect eNOS phosphorylation is unknown. In the present study, we investigated the effects of EETs on eNOS phosphorylation with cultured bovine aortic endothelial cells (BAECs). BAECs were either treated with exogenous EETs or infected with recombinant adeno-associated virus (rAAV) carrying CYP2C11-CYPOR, CYP102 F87V mutant and CYP2J2, respectively, to increase endogenous EETs. Both addition of EETs and CYP epoxygenase transfection markedly increased eNOS phosphorylation at its Ser1179 and Thr497 residues. Inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 prevented EETs-induced increases of eNOS-Ser(P)1179 but had no effect on the phosphorylation status of Thr497. However, inhibitors of protein kinase B (Akt), mitogen-activated protein kinase (MAPK) and MAPK kinase could block phosphorylation of eNOS at both sites. Inhibition of these kinases also attenuated the up-regulation of eNOS expression by EETs. Finally, administration of viral CYP epoxygenases expression vectors into rats enhanced eNOS phosphorylation and function in vivo. Thus, in addition to up-regulating eNOS expression, EETs also augment eNOS function by enhancing eNOS phosphorylation. EETs-induced up-regulation of eNOS phosphorylation and expression appears to involve in both PI3K/Akt and MAPK pathways.

8,11,14-Eicosatrienoic Acid↗

PubMed2006

Yeast sphingolipids: recent developments in understanding biosynthesis, regulation, and function.

Sphingolipids function as required membrane components of virtually all eukaryotic cells. Data indicate that members of the sphingolipid family of lipids, including sphingoid bases, sphingoid base phosphates, ceramides, and complex sphingolipids, serve vital functions in cell biology by both direct mechanisms (e.g., binding to G-protein coupled receptors to transduce an extracellular signal) and indirect mechanisms (e.g., facilitating correct intracellular protein transport). Because of the diverse roles these lipids play in cell biology, it is important to understand not only their biosynthetic pathways and regulation of sphingolipid synthesis, but also the mechanisms by which some sphingolipid species with specific functions are modified or converted to other sphingolipid species with alternate functions. Due to many factors including ease of culture and genetic modification, and conservation of major sphingolipid metabolic pathways, Saccharomyces cerevisiae has served as an ideal model system with which to identify enzymes of sphingolipid biosynthesis and to dissect sphingolipid function. Recent exciting developments in sphingolipid synthesis, transport, signaling, and overall biology continue to fuel vigorous investigation and inspire investigations in mammalian sphingolipid biology.

Animals↗

PubMed2006

Distinct roles for de novo versus hydrolytic pathways of sphingolipid biosynthesis in Saccharomyces cerevisiae.

Saccharomyces cerevisiae produces the sphingolipid ceramide by de novo synthesis as well as by hydrolysis of complex sphingolipids by Isc1p (inositolphosphoceramide-phospholipase C), which is homologous with the mammalian neutral sphingomyelinases. Though the roles of sphingolipids in yeast stress responses are well characterized, it has been unclear whether Isc1p contributes to stress-induced sphingolipids. The present study was undertaken in order to distinguish the relative roles of de novo sphingolipid biosynthesis versus Isc1p-mediated sphingolipid production in the heat-stress response. Ceramide production was measured at normal and increased temperature in an ISC1 deletion and its parental strain (ISC1 being the gene that codes for Isc1p). The results showed that Isc1p contributes specifically to the formation of the C24-, C24:1- and C26-dihydroceramide species. The interaction between these two pathways of sphingolipid production was confirmed by the finding that ISC1 deletion is synthetically lethal with the lcb1-100 mutation. Interestingly, Isc1p did not contribute significantly to transient cell-cycle arrest or growth at elevated temperature, responses known to be regulated by the de novo pathway. In order to define specific contributions of ISC1, microarray hybridizations were performed, and analyses showed misregulation of genes involved in carbon source utilization and sexual reproduction, which was corroborated by defining a sporulation defect of the isc1Delta strain. These results indicate that the two pathways of ceramide production in yeast interact, but differ in their regulation of ceramides of distinct molecular species and serve distinct cellular functions.

Gene Deletion↗

PubMed2005

Simulation and validation of modelled sphingolipid metabolism in Saccharomyces cerevisiae.

Mathematical models have become a necessary tool for organizing the rapidly increasing amounts of large-scale data on biochemical pathways and for advanced evaluation of their structure and regulation. Most of these models have addressed specific pathways using either stoichiometric or flux-balance analysis, or fully kinetic Michaelis-Menten representations, metabolic control analysis, or biochemical systems theory. So far, the predictions of kinetic models have rarely been tested using direct experimentation. Here, we validate experimentally a biochemical systems theoretical model of sphingolipid metabolism in yeast. Simulations of metabolic fluxes, enzyme deletion and the effects of inositol (a key regulator of phospholipid metabolism) led to predictions that show significant concordance with experimental results generated post hoc. The model also allowed the simulation of the effects of acute perturbations in fatty-acid precursors of sphingolipids, a situation that is not amenable to direct experimentation. The results demonstrate that modelling now allows testable predictions as well as the design and evaluation of hypothetical 'thought experiments' that may generate new metabolomic approaches.

Computer Simulation↗

PubMed2004

Local correlation of expression profiles with gene annotations--proof of concept for a general conciliatory method.

MOTIVATION: Integrated analysis of expression data and gene ontology annotations is a prime example of biological data that need co-explanatory interpretation. This particular application is used to validate a new method for integrated analysis of varied biological information. RESULTS: The proposed method consists of determining local correlation coefficients and the corresponding P-values calculated per biological entity. This measure considers the combined intensity and significance of the agreement or disagreement, between two data sources about the same biological entity. The method is applied to the integrated analysis of gene expression and annotation of two gene sets, one from yeast and other from mouse. The potential of the method to generate accurate mechanistic hypothesis is also demonstrated. Specially, negative correlation results pose a new kind of biological hypothesis. Method performance was compared with annotation enrichment methods, and optimal conditions for the superiority of local correlation results are discussed.

Algorithms↗

PubMed2003

Roles for sphingolipid biosynthesis in mediation of specific programs of the heat stress response determined through gene expression profiling.

Previous studies have demonstrated roles for de novo production of sphingolipids in Saccharomyces cerevisiae in the regulation of the transient cell cycle arrest and nutrient permease degradation associated with the heat stress response, suggesting multiple functions for yeast sphingolipids in this response. We, therefore, sought to determine the generalized involvement of sphingolipids in the heat stress response by using microarray hybridization of RNA isolated from heat-stressed cultures of the mutant strain lcb1-100, which is unable to produce sphingolipids in response to heat. Approximately 70 genes showed differential regulation during the first 15 min of heat stress in the lcb1-100 strain compared with the wild type strain, indicating a requirement for de novo sphingolipid biosynthesis for proper regulation of these genes during heat stress. Grouping these genes into functional categories revealed several pathways, including some in which sphingolipids were previously suspected to play a role, such as stress response pathways and cell cycle regulation. Hierarchical clustering analysis revealed sphingolipid involvement in regulation of tRNA synthesis and metabolic genes and transporters. Additionally, the microarray results demonstrated novel sphingolipid involvement in transcriptional regulation of pathways of translation and cell wall organization and biogenesis. Our results demonstrate a broad-reaching effect of sphingolipids in the yeast heat stress response and suggest that the mechanism of sphingolipid involvement in several cellular pathways occurs via sphingolipid-mediated regulation of message levels.

Amino Acids↗

PubMed2002

Acute activation of de novo sphingolipid biosynthesis upon heat shock causes an accumulation of ceramide and subsequent dephosphorylation of SR proteins.

Recent studies are beginning to implicate sphingolipids in the heat stress response. In the yeast Saccharomyces cerevisiae, heat stress has been shown to activate de novo biosynthesis of sphingolipids, whereas in mammalian cells the sphingolipid ceramide has been implicated in the heat shock responses. In the current study, we found an increase in the ceramide mass of Molt-4 cells in response to heat shock, corroborating findings in HL-60 cells. Increased ceramide was determined to be from de novo biosynthesis by two major lines of evidence. First, the accumulation of ceramide was dependent upon the activities of both ceramide synthase and serine palmitoyltransferase. Second, pulse labeling studies demonstrated increased production of ceramide through the de novo biosynthetic pathway. Significantly, the de novo sphingolipid biosynthetic pathway was acutely induced upon heat shock, which resulted in a 2-fold increased flux in newly made ceramides within 1-2 min of exposure to 42.5 degrees C. Functionally, heat shock induced the dephosphorylation of the SR proteins, and this effect was demonstrated to be dependent upon the accumulation of de novo-produced ceramides. Thus, these studies disclose an evolutionary conserved activation of the de novo pathway in response to heat shock. Moreover, SR dephosphorylation is emerging as a specific downstream target of accumulation of newly made ceramides in mammalian cells.

Biological Transport↗

PubMed2002

The CYP4A isoforms hydroxylate epoxyeicosatrienoic acids to form high affinity peroxisome proliferator-activated receptor ligands.

Cytochromes P450 of the CYP2C and CYP4A gene subfamilies metabolize arachidonic acid to 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) and to 19- and 20-hydroxyeicosatetraenoic acids (HETEs), respectively. Abundant functional studies indicate that EETs and HETEs display powerful and often opposing biological activities as mediators of ion channel activity and regulators of vascular tone and systemic blood pressures. Incubation of 8,9-, 11,12-, and 14,15-EETs with microsomal and purified forms of rat CYP4A isoforms led to rapid NADPH-dependent metabolism to the corresponding 19- and 20-hydroxylated EETs. Comparisons of reaction rates and catalytic efficiency with those of arachidonic and lauric acids showed that EETs are one of the best endogenous substrates so far described for rat CYP4A isoforms. CYP4A1 exhibited a preference for 8,9-EET, whereas CYP4A2, CYP4A3, and CYP4A8 preferred 11,12-EET. In general, the closer the oxido ring is to the carboxylic acid functionality, the higher the rate of EET metabolism and the lower the regiospecificity for the EET omega-carbon. Analysis of cis-parinaric acid displacement from the ligand-binding domain of the human peroxisome proliferator-activated receptor-alpha showed that omega-hydroxylated 14,15-EET bound to this receptor with high affinity (K(i) = 3 +/- 1 nm). Moreover, at 1 microm, the omega-alcohol of 14,15-EET or a 1:4 mixture of the omega-alcohols of 8,9- and 11,12-EETs activated human and mouse peroxisome proliferator-activated receptor-alpha in transient transfection assays, suggesting a role for them as endogenous ligands for these orphan nuclear receptors.

Animals↗

PubMed2002

Structural determinants of sphingolipid recognition by commercially available anti-ceramide antibodies.

The sphingolipid mediator ceramide is involved in cellular processes such as apoptosis, differentiation, responses to cytokines, and stress responses. Experimental evidence suggests that the intracellular location of ceramide may be a key factor in determining its ultimate cellular effects. One approach to ceramide localization is the use of recently developed anti-ceramide antibodies for immunocytochemical studies. Two such commercial preparations are now available; we sought to compare and contrast their specificity for ceramide and/or other cellular lipids. By using lipid overlay assays and a diverse panel of sphingolipids, we were able to delineate the specificity and thus, the utility of these reagents. Our results indicate that one of these anti-ceramide preparations is quite specific for ceramide and dihydroceramide, whereas the other preparation recognizes dihydroceramide, phosphatidylcholine, and sphingomyelin. Furthermore, through the use of chemically modified ceramides in similar assays, we were able to determine some structural determinants of lipid recognition by both of these reagents.

Antibody Specificity↗