Project description:Ceramide-1-phosphate, the phosphorylated form of ceramide, gained attention recently due to its diverse intracellular roles, in particular in inflammation mediated by cPLA(2)alpha. However, surprisingly little is known about the physical chemical properties of this lipid and its potential impact on physiological function. For example, the presence of Ca(2+) is indispensable for the interaction of Cer-1-P with the C2 domain of cPLA(2)alpha. We report on the structure and morphology of Cer-1-P in monomolecular layers at the air/water solution interface in the absence and presence of Ca(2+) using diverse biophysical techniques, including synchrotron x-ray reflectivity and grazing angle diffraction, to gain insight into the role and function of Cer-1-P in biomembranes. We show that relatively small changes in pH and the presence of monovalent cations dramatically affect the behavior of Cer-1-P. On pure water Cer-1-P forms a solid monolayer despite the negative charge of the phosphomonoester headgroup. In contrast, pH 7.2 buffer yields a considerably less solid-like monolayer, indicating that charge-charge repulsion becomes important at higher pH. Calcium was found to bind strongly to the headgroup of Cer-1-P even in the presence of a 100-fold larger Na(+) concentration. Analysis of the x-ray reflectivity data allowed us to estimate how much Ca(2+) is bound to the headgroup, approximately 0.5 Ca(2+) and approximately 1.0 Ca(2+) ions per Cer-1-P molecule for the water and buffer subphase respectively. These results can be qualitatively understood based on the molecular structure of Cer-1-P and the electrostatic/hydrogen-bond interactions of its phosphomonoester headgroup. Biological implications of our results are also discussed.
Project description:ObjectiveTotal ceramide concentrations are linked with increased insulin resistance and cardiac dysfunction. However, recent studies have demonstrated that plasma concentrations of specific very-long-chain fatty ceramides (C24:0 and C22:0) are associated with a reduced incidence of coronary heart disease and all-cause mortality. We hypothesized that specific genetic loci are associated with plasma C22:0 and C24:0 concentrations.MethodsHeritability and genome-wide association studies of plasma C24:0 and C22:0 ceramide concentrations were performed among 2,217 participants in the Framingham Heart Study Offspring Cohort, adjusting for cardiovascular risk factor covariates and cardiovascular drug treatment.ResultsThe multivariable-adjusted heritability for C22:0 and C24:0 ceramides was 0.42 (standard error [SE], 0.07; p=1.8E-9) and 0.25 (SE, 0.08; p=0.00025), respectively. Nineteen single nucleotide polymorphisms (SNPs), all on chromosome 20, significantly associated with C22:0 concentrations; the closest gene to these variants was SPTLC3. The lead SNP (rs4814175) significantly associated with 3% lower plasma C22:0 concentrations (p=2.83E-11). Nine SNPs, all on chromosome 20 and close to SPTLC3, were significantly associated with C24:0 ceramide concentrations. All 9 were also significantly related to plasma C22:0 levels. The lead SNP (rs168622) was significantly associated with 10% lower plasma C24:0 ceramide concentrations (p=9.94E-09).ConclusionSNPs near the SPTLC3 gene, which encodes serine palmitoyltransferase long chain base subunit 3 (SPTLC3; part of the enzyme that catalyzes the rate-limiting step of de novo sphingolipid synthesis) were associated with plasma C22:0 and C24:0 ceramide concentrations. These results are biologically plausible and suggest that SPTLC3 may be a potential therapeutic target for C24:0 and C22:0 ceramide modulation.
Project description:Global positioning systems (GPS) have recently been shown to reliably quantify the spatiotemporal characteristics of Polo, with the physiological demands of Polo play at low- and high-goal levels also investigated. This study aims to describe the spatiotemporal demands of Polo across 0-24 goal levels. A player-worn GPS unit was used to quantify distance, speed and high-intensity activities performed. Data were divided into chukkas and five equine-based speed zones, grouped per cumulative player handicap and assessed using standardized mean differences. Average distance and speed per chukka increased in accordance with cumulative player handicap, with the magnitude of differences being trivial-large and trivial-very large, respectively. Differences between time spent in high-intensity speed zones (zones 4 and 5) show a linear increase in magnitude, when comparing 0 goal Polo to all other levels of play (Small-Very Large; 6-24 goals, respectively). High-intensity activities predominantly shared this trend, displaying trivial-large differences between levels. These findings highlight increased cardiovascular, anaerobic and speed based physiological demands on Polo ponies as playing level increases. Strategies such as high-intensity interval training, maximal speed work and aerobic conditioning may be warranted to facilitate this development and improve pony welfare and performance.
Project description:Regulation of sphingolipid metabolism plays a role in cellular homeostasis, and dysregulation of these pathways is involved in cancer progression. Previously, our reports identified ceramide as an anti-metastatic lipid. In the present study, we investigated the biochemical alterations in ceramide-centered metabolism of sphingolipids that were associated with metastatic potential. We established metastasis-prone sublines of SKOV3 ovarian cancer cells using an in vivo selection method. These cells showed decreases in ceramide levels and ceramide synthase (CerS) 2 expression. Moreover, CerS2 downregulation in ovarian cancer cells promoted metastasis in vivo and potentiated cell motility and invasiveness. Moreover, CerS2 knock-in suppressed the formation of lamellipodia required for cell motility in this cell line. In order to define specific roles of ceramide species in cell motility controlled by CerS2, the effect of exogenous long- and very long-chain ceramide species on the formation of lamellipodia was evaluated. Treatment with distinct ceramides increased cellular ceramides and had inhibitory effects on the formation of lamellipodia. Interestingly, blocking the recycling pathway of ceramides by a CerS inhibitor was ineffective in the suppression of exogenous C24:1 -ceramide for the formation of lamellipodia. These results suggested that C24:1 -ceramide, a CerS2 metabolite, predominantly suppresses the formation of lamellipodia without the requirement for deacylation/reacylation. Moreover, knockdown of neutral ceramidase suppressed the formation of lamellipodia concomitant with upregulation of C24:1 -ceramide. Collectively, the CerS2-C24:1 -ceramide axis, which may be countered by neutral ceramidase, is suggested to limit cell motility and metastatic potential. These findings may provide insights that lead to further development of ceramide-based therapy and biomarkers for metastatic ovarian cancer.
Project description:The title compound, C(43)H(45)NO(12), was prepared from the reaction of 2,6-bis-(dibromo-meth-yl)pyridine and bis-phenol in the presence of caesium carbonate as a base. The central ring makes dihedral angles of 64.83?(6), 13.48?(6), 56.96?(6) and 66.21?(6)° with the peripheral rings. In the crystal, mol-ecules are linked by weak C-H?O and C-H?? inter-actions, forming a folded structure.
Project description:Sphingolipids are structural membrane components that also function in cellular stress responses. The serine palmitoyltransferase (SPT) catalyzes the rate-limiting step in sphingolipid biogenesis. Its activity is tightly regulated through multiple binding partners, including Tsc3, Orm proteins, ceramides, and the phosphatidylinositol-4-phosphate (PI4P) phosphatase Sac1. The structural organization and regulatory mechanisms of this complex are not yet understood. Here, we report the high-resolution cryo-EM structures of the yeast SPT in complex with Tsc3 and Orm1 (SPOT) as dimers and monomers and a monomeric complex further carrying Sac1 (SPOTS). In all complexes, the tight interaction of the downstream metabolite ceramide and Orm1 reveals the ceramide-dependent inhibition. Additionally, observation of ceramide and ergosterol binding suggests a co-regulation of sphingolipid biogenesis and sterol metabolism within the SPOTS complex.