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Yasuyuki Igarashi - One of the best experts on this subject based on the ideXlab platform.
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Isolation of Sphingoid Bases from Starfish Asterias amurensis Glucosylceramides and Their Effects on Sphingolipid Production in Cultured Keratinocytes
Journal of Oleo Science, 2019Co-Authors: Daisuke Mikami, Shota Sakai, Kohei Yuyama, Yasuyuki IgarashiAbstract:Starfish Asterias amurensis produces Sphingoid bases d18:3, 9-methyl-d18:3 (9Me-d18:3), and d22:2, which possess unique structural features. In this study, Sphingoid bases prepared from A. amurensis glucosylceramides displayed unexpected elution behaviors from a general octadecyl silyl high-performance liquid chromatography (HPLC) column. For separation and isolation, Sphingoid bases were fractionated by octadecyl silyl HPLC after N-acetylation, yielding d18:3, 9Me-d18:3, and two d22:2 isomers. To compare the biological activities of individual Sphingoid bases, their effects on sphingolipid production in normal human keratinocytes were evaluated. Treatment with Sphingoid bases increased the content of ceramides, glucosylceramides, and sphingomyelins in keratinocytes. Moreover, ceramides, which contain saturated ultra-long-chain fatty acids (C30-34), were significantly increased by treatment with d18:3, but not with other A. amurensis Sphingoid bases. The mRNA level of the early differentiation marker keratin 10 was markedly decreased and sphingolipid synthesis-related genes were slightly increased in keratinocytes exposed to A. amurensis-derived d18:3, 9Me-d18:3, and d22:2 isomers. These results suggest that A. amurensis-derived Sphingoid bases induce differentiation to varying degrees, sphingolipid production depends on their chemical structures, and d18:3 is the most promising functional Sphingoid base.
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highly efficient preparation of Sphingoid bases from glucosylceramides by chemoenzymatic method
Journal of Lipid Research, 2016Co-Authors: Siddabasave Gowda B Gowda, Yasuyuki Igarashi, Seigo Usuki, Mostafa A S Hammam, Yuta Murai, Kenji MondeAbstract:Sphingoid base derivatives have attracted increasing attention as promising chemotherapeutic candidates against lifestyle diseases such as diabetes and cancer. Natural Sphingoid bases can be a potential resource instead of those derived by time-consuming total organic synthesis. In particular, glucosylceramides (GlcCers) in food plants are enriched sources of Sphingoid bases, differing from those of animals. Several chemical methodologies to transform GlcCers to Sphingoid bases have already investigated; however, these conventional methods using acid or alkaline hydrolysis are not efficient due to poor reaction yield, producing complex by-products and resulting in separation problems. In this study, an extremely efficient and practical chemoenzymatic transformation method has been developed using microwave-enhanced butanolysis of GlcCers and a large amount of readily available almond β-glucosidase for its deglycosylation reaction of lysoGlcCers. The method is superior to conventional acid/base hydrolysis methods in its rapidity and its reaction cleanness (no isomerization, no rearrangement) with excellent overall yield.
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4,8-Sphingadienine and 4-hydroxy-8-sphingenine activate ceramide production in the skin
Lipids in Health and Disease, 2012Co-Authors: Yoshiyuki Shirakura, Kanako Kikuchi, Susumu Mitsutake, Katsuyuki Mukai, Kenji Matsumura, Yasuyuki IgarashiAbstract:Background: Ingestion of glucosylceramide improves transepidermal water loss (TEWL) from the skin, but the underlying mechanism by which a small amount of dietary glucosylceramide can vastly improve skin conditions remains unclear. In a previous report, glucosylceramides were shown to be digested to Sphingoids, which were shown to be absorbed through the intestinal epithelium. Based on these observations, we hypothesized that Sphingoids are the key molecules facilitating endogenous ceramide production. In this study, we assessed the effect of 4,8-sphingadienine (d18:2) and 4-hydroxy-8-sphingenine (t18:1), derived from konjac glucosylceramide, on stimulating ceramide production. Methods: Konjac glucosylceramide acidolysis was performed using hydrochloric acid; the resulting d18:2 and t18:1 were fractionated by column chromatography. Real-time quantitative RT-PCR was performed to assess the effect of d18:2 and t18:1 on gene expression in normal human epidermal keratinocytes, while their effect on the nuclear receptor, peroxisome proliferator-activated receptor (PPAR)γ, was measured using a receptor-cofactor assay system. The effect of d18:2 and t18:1 on stimulating ceramide production was evaluated using HPTLC analysis in a 3-dimensional human skin model. Results: We noted the upregulation of genes related to de novo ceramide synthesis as well as of those encoding the elongases of very long-chain fatty acids by d18:2 and t18:1, but not by glucosylceramide and 4-sphingenine. Both these Sphingoids also facilitated the expression of PPARβ/δ and PPARγ; moreover, they also demonstrated ligand activity for PPARγ. These results indicated that d18:2 and t18:1 promote the differentiation of keratinocytes. Analysis of the lipids within the 3-dimensional human skin model indicated that treatment with d18:2 and t18:1 not only upregulated gene expression but also increased ceramide production. Conclusions: The Sphingoids d18:2 and t18:1 activated genes related to de novo ceramide synthesis and increased ceramide production, whereas glucosylceramide and 4-sphingenine could not. These results suggest that the effect of dietary glucosylceramides on the skin is mediated by d18:2 and t18:1.
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phytoceramide and Sphingoid bases derived from brewer s yeast saccharomyces pastorianus activate peroxisome proliferator activated receptors
Lipids in Health and Disease, 2011Co-Authors: Itsuo Murakami, Susumu Mitsutake, Yukari Wakasa, Shinji Yamashita, Toshio Kurihara, Kota Zama, Naoyuki Kobayashi, Yukiko Mizutani, Tatsuro Shigyo, Yasuyuki IgarashiAbstract:Background Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that regulate lipid and glucose metabolism. PPARα is highly expressed in the liver and controls genes involved in lipid catabolism. We previously reported that synthetic sphingolipid analogs, part of which contains shorter-length fatty acid chains than natural sphingolipids, stimulated the transcriptional activities of PPARs. Sphingosine and dihydrosphingosine (DHS) are abundant Sphingoid bases, and ceramide and dihydroceramide are major ceramide species in mammals. In contrast, phytosphingosine (PHS) and DHS are the main Sphingoid bases in fungi. PHS and phytoceramide exist in particular tissues such as the epidermis in mammals, and involvement of ceramide species in PPARβ activation in cultured keratinocytes has been reported. The purpose of the present study is to investigate whether natural sphingolipids with C18 fatty acid and yeast-derived Sphingoid bases activate PPARs as PPAR agonists.
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identification and characterization of a saccharomyces cerevisiae gene rsb1 involved in Sphingoid long chain base release
Journal of Biological Chemistry, 2002Co-Authors: Akio Kihara, Yasuyuki IgarashiAbstract:Abstract Sphingoid long-chain bases (LCBs) and long-chain base phosphates (LCBPs) act as signaling molecules in eukaryotic cells. Accumulation of LCBPs results in cell growth inhibition in yeast, although the mechanism is unknown. Here, we identified a novel yeast gene, RSB1 (resistance to Sphingoid long-chain base), by screening a multicopy suppressor of the LCB-sensitive phenotype of the LCBP lyase mutant. RSB1encodes a polypeptide of 354 amino acids with a molecular mass of 40.4 kDa. Rsb1p is predicted to be an integral membrane protein with seven transmembrane-spanning domains. We demonstrated that cells overproducing Rsb1p showed a decrease in accumulation of exogenously added sphingosine and dihydrosphingosine because of their increased release. This release was ATP-dependent, and a mutant of the predicted ATP binding motif had no activity. Substrate specificity analysis of Rsb1p demonstrated that it is active on LCBs but not on LCBPs or other hydrophobic compounds. These results suggest that Rsb1p is a transporter or flippase that translocates LCBs from the cytoplasmic side toward the extracytoplasmic side of the membrane.
Naoto Suzuki - One of the best experts on this subject based on the ideXlab platform.
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simultaneous quantitation of Sphingoid bases and their phosphates in biological samples by liquid chromatography electrospray ionization tandem mass spectrometry
Analytical and Bioanalytical Chemistry, 2012Co-Authors: Daisuke Saigusa, Kanako Shiba, Asuka Inoue, Kotaro Hama, Michiyo Okutani, Nagisa Iida, Masayoshi Saito, Kaori Suzuki, Tohru Kaneko, Naoto SuzukiAbstract:We developed a liquid chromatography/electrospray ionization tandem mass spectrometry method for the simultaneous quantitative determination of C18 sphingosine (Sph), C18 dihydrosphingosine (dhSph), C18 phytosphingosine (pSph), C18 sphingosine-1-phosphate (S1P), C18 dihydrosphingosine-1-phosphate (dhS1P), and C18 phytosphingosine-1-phosphate (pS1P). Samples were prepared by simple methanol deproteinization and analyzed in selected reaction monitoring modes. No peak tailing was observed on the chromatograms using a Capcell Pak ACR column (1.5 mm i.d. × 250 mm, 3 μm, Shiseido). The calibration curves of the Sphingoids showed good linearity (r > 0.996) over the range of 0.050–5.00 pmol per injection. The accuracy and precision of this method were demonstrated using four representative biological samples (serum, brain, liver, and spleen) from mice that contained known amounts of the Sphingoids. Samples of mice tissue such as plasma, brain, eye, testis, liver, kidney, lung, spleen, lymph node, and thymus were examined for their Sph, dhSph, pSph, S1P, dhS1P, and pS1P composition. The results confirmed the usefulness of this method for the physiological and pathological analysis of the composition of important Sphingoids.
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Simultaneous quantitation of Sphingoid bases and their phosphates in biological samples by liquid chromatography/electrospray ionization tandem mass spectrometry
Analytical and Bioanalytical Chemistry, 2012Co-Authors: Daisuke Saigusa, Kanako Shiba, Asuka Inoue, Kotaro Hama, Michiyo Okutani, Nagisa Iida, Masayoshi Saito, Kaori Suzuki, Tohru Kaneko, Naoto SuzukiAbstract:We developed a liquid chromatography/electrospray ionization tandem mass spectrometry method for the simultaneous quantitative determination of C18 sphingosine (Sph), C18 dihydrosphingosine (dhSph), C18 phytosphingosine (pSph), C18 sphingosine-1-phosphate (S1P), C18 dihydrosphingosine-1-phosphate (dhS1P), and C18 phytosphingosine-1-phosphate (pS1P). Samples were prepared by simple methanol deproteinization and analyzed in selected reaction monitoring modes. No peak tailing was observed on the chromatograms using a Capcell Pak ACR column (1.5 mm i.d. × 250 mm, 3 μm, Shiseido). The calibration curves of the Sphingoids showed good linearity (r > 0.996) over the range of 0.050–5.00 pmol per injection. The accuracy and precision of this method were demonstrated using four representative biological samples (serum, brain, liver, and spleen) from mice that contained known amounts of the Sphingoids. Samples of mice tissue such as plasma, brain, eye, testis, liver, kidney, lung, spleen, lymph node, and thymus were examined for their Sph, dhSph, pSph, S1P, dhS1P, and pS1P composition. The results confirmed the usefulness of this method for the physiological and pathological analysis of the composition of important Sphingoids.
Ronald T Riley - One of the best experts on this subject based on the ideXlab platform.
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increased Sphingoid base 1 phosphates and failure of neural tube closure after exposure to fumonisin or fty720
Birth Defects Research Part A-clinical and Molecular Teratology, 2012Co-Authors: Janee Gelineauvan Waes, Kenneth A Voss, Mark A Rainey, Joyce R Maddox, Andrew J Sachs, Nicole M Gardner, Justin Wilberding, Ronald T RileyAbstract:BACKGROUND: Fumonisin B1 (FB1) is a mycotoxin produced by a common fungal contaminant of corn. Ingestion of FB1-contaminated food is associated with increased risk for neural tube defects (NTDs). FB1 induces NTDs in inbred LM/Bc mice. FB1 inhibits ceramide synthase in de novo sphingolipid biosynthesis, resulting in accumulation of sphinganine and sphinganine-1-phosphate (Sa1P). Sa1P functions as a ligand for a family of G protein-coupled S1P receptors. METHODS: Pregnant SWV and LM/Bc mice were treated with FB1 (20 mg/kg/day intraperitoneally on embryonic day (ED) 7.5–8.5) or the known S1P receptor agonist FTY720 (10 mg/kg/day oral gavage on ED 6.5–8.5). LC/MS was used to detect Sphingoid base-1-phosphates in maternal blood spots, plasma, and embryonic tissue. Strain-specific SWV and LM/Bc mouse embryonic fibroblasts (MEFs) and serum free mouse embryo (SFME) neural progenitor cells were treated with FB1 (40 μM for 24 hr) and LC/MS was used to detect Sphingoid base-1-phosphates. RESULTS: FTY720 induced NTDs in both the SWV and the LM/Bc strains of mice. Sphinganine-1-P (Sa1P) and FTY720-P were elevated in the blood spots and plasma of mice treated with FB1 or FTY720, respectively. FTY720-P was elevated in ED 9.5 exencephalic embryos. Sa1P was elevated in SFME and MEF cells treated with FB1, and Sa1P was higher in MEFs generated from the FB1-NTD–susceptible LM/Bc strain. CONCLUSIONS: Elevated Sphingoid base-1-P after FB1 or FTY720 suggest a potential role for these bioactive lipid ligands and activation of S1P receptor signaling pathways in the failure of neural tube closure after FB1 or FTY720. Sa1P may represent a biomarker for FB1-NTD risk assessment. Birth Defects Research (Part A), 2012. © 2012 Wiley Periodicals, Inc.
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translocation of Sphingoid bases and their 1 phosphates but not fumonisins from roots to aerial tissues of maize seedlings watered with fumonisins
Journal of Agricultural and Food Chemistry, 2010Co-Authors: Nicholas C Zitomer, Samantha K Jones, Charles W Bacon, Anthony E Glenn, Thomas T Baldwin, Ronald T RileyAbstract:In an earlier study using maize seedlings grown from kernels inoculated with Fusarium verticillioides, fumonisin B1 (FB1) was preferentially accumulated in leaf tissue compared to FB2 and FB3. The present study tested whether maize seedlings preferentially translocate FB1 when plants are watered with FB1 and/or FB2, without the fungus present. The results show that neither FB1 nor FB2 was translocated when administered in the watering solution, and although both FB1 and FB2 were taken up by the roots, the accumulation of FB2 in roots was significantly less than expected, indicating that FB1 was preferentially accumulated. In addition, there was clear evidence of ceramide synthase inhibition in the roots and Sphingoid base and Sphingoid base 1-phosphates accumulated in leaf tissue presumably due to translocation from the roots. These findings suggest that the fungus−plant interaction is necessary for FB1 translocation in maize seedlings infected with F. verticillioides.
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persistence and reversibility of the elevation in free Sphingoid bases induced by fumonisin inhibition of ceramide synthase
Toxicological Sciences, 2002Co-Authors: E N Enongene, Raghubir P Sharma, Neetesh Bhandari, J D Miller, Filmore I Meredith, Kenneth A Voss, Ronald T RileyAbstract:These studies determined (1) the time course for Sphingoid base elevation in the small intestines, liver, and kidney of mice following a single 25 mg/kg body weight (bw) oral dose (high dose) of fumonisin B(1) (FB(1)), (2) the minimum threshold dose of FB(1) that would prolong the elevated Sphingoid base concentration in kidney following the single high dose, and (3) the importance of the balance between the rate of Sphingoid base biosynthesis and degradation in the persistence of Sphingoid base accumulation. Following the high dose of FB(1), there was an increase in sphinganine in intestinal cells and liver that peaked at 4 to 12 h and declined to near the control level by 48 h. In kidney, sphinganine peaked at 6-12 h but remained elevated until 72 h, approaching control levels at 96-120 h. Oral administration of 0.03 mg FB(1)/kg bw (low dose) for 5 days had no effect on the Sphingoid bases in kidney. However, following an initial high dose, daily administration of the low dose prolonged the elevation in kidney sphinganine compared to mice receiving a single high dose. Thus, a single exposure to a high dose of FB(1) followed by daily exposure at low levels will prolong the elevation of sphinganine in kidney. In cultured renal cells FB(1) was rapidly eliminated, but elevated sphinganine was persistent. This persistence in renal cells was rapidly reversed in the presence of the serine palmitoyltransferase inhibitor (ISP-1), indicating that the persistence was due to differences in the rates of sphinganine biosynthesis and degradation. The in vivo persistence in kidney may be due to similar differences.
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fumonisin induced tumor necrosis factor α expression in a porcine kidney cell line is independent of Sphingoid base accumulation induced by ceramide synthase inhibition
Toxicology and Applied Pharmacology, 2001Co-Authors: Quanren He, Ronald T Riley, Raghubir P SharmaAbstract:Abstract Previous studies have shown that fumonisin B 1 (FB 1 ) inhibits ceramide synthase, resulting in accumulation of free sphinganine and sphingosine. Tumor necrosis factor-α (TNFα) plays an important role in FB 1 toxicity and the expression of TNFα mRNA in liver and kidney is increased following FB 1 exposure in mice. The objective of the current study was to investigate whether these two events (Sphingoid bases accumulation and TNFα induction) are dependent on each other. An increase in expression of TNFα mRNA was detected in LLC-PK 1 cells as early as 4 h after FB 1 treatment but decreased to the control levels after 8 h. A positive linear correlation was observed between the expression of TNFα mRNA and FB 1 concentration. Increases of intracellular Sphingoid bases were also detected after 4 h of FB 1 treatment and progressively increased until 24 h. Exposure of the cells to sphinganine or sphingosine did not significantly alter the expression of TNFα. Inhibition of Sphingoid base biosynthesis by ISP-1, a specific inhibitor of serine palmitoyltransferase, the first enzyme in de novo sphingolipid biosynthesis, efficiently blocked the accumulation of free Sphingoid bases in response to FB 1 , but it did not prevent the induction of TNFα expression. Results indicate that FB 1 -induced increase in TNFα expression is independent of Sphingoid base accumulation-induced by ceramide synthase inhibition in LLC-PK 1 cells.
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elevated Sphingoid bases and complex sphingolipid depletion as contributing factors in fumonisin induced cytotoxicity
Toxicology and Applied Pharmacology, 1996Co-Authors: William P. Norred, Jency L Showker, Ronald T RileyAbstract:Abstract Fumonisin B 1 is an inhibitor of ceramide synthase, a key enzyme in de novo sphingolipid biosynthesis and reacylation of free sphingosine. The purpose of this study was to determine the contribution of increased intracellular free sphinganine and decreased complex sphingolipids on cell growth and cell death induced by fumonisin B 1 in pig kidney LLC-PK 1 cells. Fumonisin B 1 caused an increase in intracellular free sphinganine which preceded depletion of complex sphingolipids, inhibition of cell growth, and cell death. The effects on cell growth and cell death were well correlated with the increase in free Sphingoid bases and depletion of complex sphingolipids. Exogenously added sphinganine mimicked the effects of fumonisin, but β-chloroalanine, an inhibitor of serine palmitoyltransferase which is the first enzyme in de novo sphingolipid biosynthesis, also inhibited cell growth and increased cell death. When added simultaneously, β-chloroalanine reduced the fumonisin-induced sphinganine increase by approximately 90%; however, it exacerbated the decrease in more complex sphingolipids. The effects of fumonisin on cell growth and cell death were only partially prevented by β-chloroalanine (∼50 to 60%). The results suggest that both the elevation of free Sphingoid bases and the decrease in complex sphingolipids contribute to the decreased cell growth and cytolethality of fumonisin B 1 in pig kidney LLC-PK 1 cells.
Yusuf A Hannun - One of the best experts on this subject based on the ideXlab platform.
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Sphingoid bases and the serine catabolic enzyme cha1 define a novel feedforward feedback mechanism in the response to serine availability
Journal of Biological Chemistry, 2012Co-Authors: David J Montefusco, Benjamin Newcomb, Jason L Gandy, Sarah E Brice, Nabil Matmati, Ashley L Cowart, Yusuf A HannunAbstract:Targets of bioactive sphingolipids in Saccharomyces cerevisiae were previously identified using microarray experiments focused on sphingolipid-dependent responses to heat stress. One of these heat-induced genes is the serine deamidase/dehydratase Cha1 known to be regulated by increased serine availability. This study investigated the hypothesis that sphingolipids may mediate the induction of Cha1 in response to serine availability. The results showed that inhibition of de novo synthesis of sphingolipids, pharmacologically or genetically, prevented the induction of Cha1 in response to increased serine availability. Additional studies implicated the Sphingoid bases phytosphingosine and dihydrosphingosine as the likely mediators of Cha1 up-regulation. The yeast protein kinases Pkh1 and Pkh2, known Sphingoid base effectors, were found to mediate CHA1 up-regulation via the transcription factor Cha4. Because the results disclosed a role for sphingolipids in negative feedback regulation of serine metabolism, we investigated the effects of disrupting this mechanism on sphingolipid levels and on cell growth. Intriguingly, exposure of the cha1Δ strain to high serine resulted in hyperaccumulation of endogenous serine and in turn a significant accumulation of Sphingoid bases and ceramides. Under these conditions, the cha1Δ strain displayed a significant growth defect that was sphingolipid-dependent. Together, this work reveals a feedforward/feedback loop whereby the Sphingoid bases serve as sensors of serine availability and mediate up-regulation of Cha1 in response to serine availability, which in turn regulates sphingolipid levels by limiting serine accumulation.
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Sphingoid Bases and the Serine Catabolic Enzyme CHA1 Define a Novel Feedforward/Feedback Mechanism in the Response to Serine Availability
Journal of Biological Chemistry, 2012Co-Authors: David J Montefusco, Benjamin Newcomb, Jason L Gandy, Sarah E Brice, Nabil Matmati, L. Ashley Cowart, Yusuf A HannunAbstract:Targets of bioactive sphingolipids in Saccharomyces cerevisiae were previously identified using microarray experiments focused on sphingolipid-dependent responses to heat stress. One of these heat-induced genes is the serine deamidase/dehydratase Cha1 known to be regulated by increased serine availability. This study investigated the hypothesis that sphingolipids may mediate the induction of Cha1 in response to serine availability. The results showed that inhibition of de novo synthesis of sphingolipids, pharmacologically or genetically, prevented the induction of Cha1 in response to increased serine availability. Additional studies implicated the Sphingoid bases phytosphingosine and dihydrosphingosine as the likely mediators of Cha1 up-regulation. The yeast protein kinases Pkh1 and Pkh2, known Sphingoid base effectors, were found to mediate CHA1 up-regulation via the transcription factor Cha4. Because the results disclosed a role for sphingolipids in negative feedback regulation of serine metabolism, we investigated the effects of disrupting this mechanism on sphingolipid levels and on cell growth. Intriguingly, exposure of the cha1Δ strain to high serine resulted in hyperaccumulation of endogenous serine and in turn a significant accumulation of Sphingoid bases and ceramides. Under these conditions, the cha1Δ strain displayed a significant growth defect that was sphingolipid-dependent. Together, this work reveals a feedforward/feedback loop whereby the Sphingoid bases serve as sensors of serine availability and mediate up-regulation of Cha1 in response to serine availability, which in turn regulates sphingolipid levels by limiting serine accumulation.
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involvement of Sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in arabidopsis
Cell Research, 2007Co-Authors: Jacek Bielawski, Jinye Mu, Haili Dong, Chong Teng, Jian Zhang, Xiaohui Yang, Nario Tomishige, Kentaro Hanada, Yusuf A HannunAbstract:Involvement of Sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis
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role for de novo Sphingoid base biosynthesis in the heat induced transient cell cycle arrest of saccharomyces cerevisiae
Journal of Biological Chemistry, 2001Co-Authors: Gary M Jenkins, Yusuf A HannunAbstract:Abstract The recent findings of sphingolipids as potential mediators of yeast heat stress responses led us to investigate their possible role in the heat-induced cell cycle arrest and subsequent recovery. The sphingolipid-deficient yeast strain 7R4 was found to lack the cell cycle arrest seen in the isogenic wild type. Furthermore, strain lcb1-100, which harbors a temperature-sensitive serine palmitoyltransferase, lacked increased de novogenerated Sphingoid bases upon heat stress. Importantly, this strain was found to lack the transient heat-induced G0/G1 arrest. These results indicate a role for sphingolipids and specifically those generated in the de novo pathway in the cell cycle arrest response to heat. To determine the bioactive sphingolipid regulating this response, an analysis of key mutants in the sphingolipid biosynthetic and degradation pathways was performed. Strains deleted in Sphingoid base kinases, Sphingoid phosphate phosphatase, lyase, or dihydrosphingosine hydroxylase were found to display the cell cycle arrest. Also, the knockout of a fatty acyl elongation enzyme, which severely attenuates ceramide production, displayed the arrest. These experiments suggested that the active species for cell cycle arrest were the Sphingoid bases. In further support of these findings, exogenous phytosphingosine (10 μm) was found to induce transient arrest. Stearylamine did not induce an arrest, demonstrating chemical specificity, andl-erythro- was not as potent asd-erythro-dihydrosphingosine showing stereospecificity. To investigate a possible arrest mechanism, we studied the hyperstable Cln3 (Cln3–1) strain LDW6A that has been previously shown to be resistant to heat stress-induced cell cycle arrest. The strain containing Cln3–1 was found to be resistant to cell cycle arrest induced by exogenous phytosphingosine, indicating that Cln3 acts downstream of the Sphingoid bases in this response. Interestingly, cell cycle recovery from the transient arrest was found to be dependent upon the Sphingoid base kinases (LCB4,LCB5). Overall, this combination of genetic and pharmacologic results demonstrates a role for de novoSphingoid base biosynthesis by serine palmitoyltransferase in the transient G0/G1 arrest mediated through Cln3 via a novel mechanism.
Tatsuya Sugawara - One of the best experts on this subject based on the ideXlab platform.
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Selective Absorption of Dietary Sphingoid Bases from the Intestine via Efflux by P-Glycoprotein in Rats
Journal of Nutritional Science and Vitaminology, 2020Co-Authors: Aoi Fujii, Takashi Hirata, Kazuhiko Aida, Tsuyoshi Tsuduki, Yuki Manabe, Tatsuya SugawaraAbstract:Various physiological functions of dietary sphingolipids, such as preventing inflammation and improving the skin barrier function, have been recently demonstrated. The sphingolipid most commonly used as a foodstuff is glucosylceramide from plant sources, which is composed of Sphingoid bases that are distinctive from those found in mammals. Although the structure of Sphingoid bases in higher plants is more complicated than the structure of those in mammals, the fate of dietary sphingolipids of plant origin is still not understood. In the present study, we investigated the absorption of 4,8-sphingadienine that originated from maize glucosylceramide in the rat intestine by using a lipid absorption assay of lymph collected from the thoracic duct. The cumulative recovery of 4,8-sphingadienine in the lymph was lower than that of sphingosine. Verapamil, a P-glycoprotein inhibitor, significantly increased the absorption of 4,8-sphingadienine but did not affect the absorption of sphingosine. Plant-derived Sphingoid bases were detected in the ceramide fraction of lymph fluid by using liquid chromatography-mass spectrometry analysis. These results indicate that 4,8-sphingadienine that originates from the glucosylceramide of higher plants is poorly absorbed in the intestine because of efflux by P-glycoprotein and can be incorporated into a ceramide moiety, at least in part, in intestinal endothelial cells.
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Sphingoid bases of dietary ceramide 2 aminoethylphosphonate a marine sphingolipid absorb into lymph in rats
Journal of Lipid Research, 2019Co-Authors: Nami Tomonaga, Tsuyoshi Tsuduki, Yuki Manabe, Tatsuya SugawaraAbstract:Various functions of dietary sphingolipids have been reported; however, little is known about marine sphingolipids. Ceramide 2-aminoethylphosphonate (CAEP), an abundant sphingolipid in marine mollusks, frequently has a unique triene type of Sphingoid base [2-amino-9-methyl-4,8,10-octadecatriene-1,3-diol (d19:3)]. We previously reported that dietary CAEP prepared from the skin of squid was digested in the intestinal mucosa of mice via ceramides to yield free Sphingoid bases. How dietary CAEP is then used in the body remains unclear. Here, we investigated the absorption of dietary CAEP using a lipid absorption assay on the lymph collected from rats with thoracic duct cannulation. Our results reveal that Sphingoid bases derived from CAEP, including d16:1, d18:1, and d19:3, were detected in the lymph after administration of CAEP. Lymphatic recovery of d19:3 was lower than that of other Sphingoid bases. A large fraction of the absorbed Sphingoid bases was present as complex sphingolipids, whereas a smaller portion was present in the free form. Fatty acids in ceramide moieties found in the lymph were partially different from dietary CAEP, which indicates that Sphingoid bases derived from CAEP could be, at least in part, resynthesized into complex sphingolipids. Future studies should elucidate the metabolism of Sphingoid bases derived from CAEP.
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Sphingoid bases from sea cucumber induce apoptosis in human hepatoma hepg2 cells through p akt and dr5
Oncology Reports, 2013Co-Authors: Zakir Hossain, Tatsuya Sugawara, Takashi HirataAbstract:Abstract Biofunctional marine compounds have recently received substantial attention for their nutraceutical characteristics. In this study, we investigated the apoptosis-inducing effects of Sphingoid bases prepared from sea cucumber using human hepatoma HepG2 cells. Apoptotic effects were determined by cell viability assay, DNA fragmentation assay, caspase-3 and caspase-8 activities. The expression levels of apoptosis-inducing death receptor-5 (DR5) and p-AKT were assayed by western blot analysis, and mRNA expression of bax, GADD45 and PPARγ was assayed by quantitative RT-PCR analysis. Sphingoid bases from sea cucumber markedly reduced the cell viability of HepG2 cells. DNA fragmentation indicative of apoptosis was observed in a dose-dependent manner. The expression levels of the apoptosis inducer protein Bax were increased by the Sphingoid bases from sea cucumber. GADD45, which plays an important role in apoptosis-inducing pathways, was markedly upregulated by Sphingoid bases from sea cucumber. Upregulation of PPARγ mRNA was also observed during apoptosis induced by the Sphingoid bases. The expression levels of DR5 and p-AKT proteins were increased and decreased, respectively, as a result of the effects of Sphingoid bases from sea cucumber. The results indicate that Sphingoid bases from sea cucumber induce apoptosis in HepG2 cells through upregulation of DR5, Bax, GADD45 and PPARγ and downregulation of p-AKT. Our results show for the first time the functional properties of marine Sphingoid bases as inducers of apoptosis in HepG2 cells.
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isolation of Sphingoid bases of sea cucumber cerebrosides and their cytotoxicity against human colon cancer cells
Bioscience Biotechnology and Biochemistry, 2006Co-Authors: Tatsuya Sugawara, Shota Sakai, Nobuhiro Zaima, Akiyo Yamamoto, Ryoko Noguchi, Takashi HirataAbstract:Sea cucumber is a health-beneficial food, and contains a variety of physiologically active substances including glycosphingolipids. We show here the Sphingoid base composition of cerebrosides prepared from sea cucumber and the cytotoxicity against human colon cancer cell lines. The composition of Sphingoid bases prepared from sea cucumber was different from that of mammals, and the major constituents estimated from mass spectra had a branched C17–19 alkyl chain with 1–3 double bonds. The viability of DLD-1, WiDr and Caco-2 cells treated with sea cucumber Sphingoid bases was reduced in a dose-dependent manner and was similar to that of cells treated with sphingosine. The Sphingoid bases induced such a morphological change as condensed chromatin fragments and increased the caspase-3 activity, indicating that the Sphingoid bases reduced the cell viability by causing apoptosis in these cells. Sphingolipids of sea cucumber might therefore serve as bioactive dietary components to suppress colon cancer.
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efflux of Sphingoid bases by p glycoprotein in human intestinal caco 2 cells
Bioscience Biotechnology and Biochemistry, 2004Co-Authors: Tatsuya Sugawara, Takashi Hirata, Mikio Kinoshita, Teruo Miyazawa, Masao Ohnishi, Tsuyoshi Tsuzuki, Junichi Nagata, Morio SaitoAbstract:The aim of this study was to determine whether Sphingoid bases that originated from various dietary sources, such as mammals, plants, and fungi, are substrates for P-glycoprotein in differentiated Caco-2 cells, which are used as a model of intestinal epithelial cells. In Caco-2 cells, the uptake of sphingosine, the most common Sphingoid base found in mammals, was significantly higher at physiological temperatures than those of cis/trans-8-sphingenine, trans-4, cis/trans-8-sphingadienine, 9-methyl-trans-4, trans-8-sphingadienine, or sphinganine. Verapamil, a potent P-glycoprotein inhibitor, increased the cellular accumulation of Sphingoid bases, except for sphingosine, in a dose-dependent manner. Incubation with 1 μM digoxin for 48 h caused up-regulation of murtidrug-resistance (MDR)1 mRNA and decreased the accumulation of Sphingoid bases in Caco-2 cells, except for sphingosine. Thus P-glycoprotein probably contributes to the selective absorption of sphingosine from dietary sphingolipids in the digestive t...
