Sphingolipidomics of drug resistant Candida auris clinical isolates reveal distinct sphingolipid species signatures.

Independent studies from our group and others have provided evidence that sphingolipids (SLs) influence the antimycotic susceptibility of Candida species. We analyzed the molecular SL signatures of drug-resistant clinical isolates of Candida auris, which have emerged as a global threat over the last decade. This included Indian hospital isolates of C. auris, which were either resistant to fluconazole (FLCR) or amphotericin B (AmBR) or both drugs. Relative to Candida glabrata and Candida albicans strains, these C. auris isolates were susceptible to SL pathway inhibitors such as myriocin and aureobasidin A, suggesting that SL content may influence azole and AmB susceptibilities. Our analysis of SLs confirmed the presence of 140 SL species within nine major SL classes, namely the sphingoid bases, Cer, αOH-Cer, dhCer, PCer, αOH-PCer, αOH-GlcCer, GlcCer, and IPC. Other than for αOH-GlcCer, most of the SLs were found at higher concentrations in FLCR isolates as compared to the AmBR isolates. SLs were at intermediate levels in FLCR + AmBR isolates. The observed diversity of molecular species of SL classes based on fatty acyl composition was further reflected in their distinct specific imprint, suggesting their influence in drug resistance. Together, the presented data improves our understanding of the dynamics of SL structures, their synthesis, and link to the drug resistance in C. auris.

Impact of Oral Intake of Glucosylceramide Extracted from Pineapple on Xerostomia: A Double-Blind Randomized Cross-Over Trial.

Background: The aim of this double-blind randomized cross-over trial was to evaluate the effect of oral intake of glucosylceramide extracted from pineapple on oral moisture and xerostomia symptoms. Methods: Sixteen participants who had xerostomia symptoms were randomly allocated into two groups. One group received, as test samples, tablets containing glucosylceramide extracted from pineapple (GCP) followed by placebo tablets. The other group received the test samples in the reverse order. Participants were instructed to take tablets of the first test sample once a day (after breakfast) for two consecutive weeks. Then, after a washout period of four weeks, participants were instructed to take the other test sample for two consecutive weeks. The oral moisture level of the lingual mucosa, xerostomia symptoms, and the number of fungiform papillae was evaluated. Results: The oral moisture significantly increased, and the visual analog scale (VAS) of "How is the dryness of your mouth?" significantly improved after GCP tablets intake and not after placebo tablets intake. The number of fungiform papillae was not significantly different following the intake of GCP tablets or placebo tablets. Conclusion: Results suggested that oral intake of GCP may improve the moisture level and xerostomia symptoms.

High-Performance Chromatographic Separation of Cerebrosides.

High-performance thin-layer chromatography (HPTLC) is a very robust, fast, and inexpensive technique that enables separation of complex mixtures. Here, we describe the analytical separation of glucosylceramide and galactosylceramide by HPTLC. This technique can be used for quantitation purposes but also with small modification for subsequent mass spectrum analyses for structural determination.

A Simple, High-Throughput Method for Analysis of Ceramide, Glucosylceramide, and Ceramide Trihexoside in Dried Blood Spots by LC/MS/MS.

A unique monophasic extraction system coupled with LC/MS/MS to reduce matrix effects for sphingolipid analysis was developed. A solvent mixture of methanol, acetonitrile, and water was identified to simultaneously extract multiple sphingolipids with broad polarity range. To reduce matrix effects, the targeted sphingolipids were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). The extraction solvent was used as an isocratic mobile phase in chromatographic separation to eliminate solvent exchange steps and enable high-throughput multiple lipid assay. The assay is linear for ceramide from 0.6 to 9 µg/mL with bias <15 %. The intra-assay coefficient of variation is less than 10 % for concentrations from 1.2 to 9 µg/mL, and less than 25 % for concentrations below 1.2 µg/mL. For glucosylceramide and ceramide trihexoside the linear range is 0.05-3 µg/mL with biases <10 % and <20 %, respectively. The intra-assay coefficient of variation for these analytes is less than 10 % at concentrations from 0.4 to 3 µg/mL, and less than 25 % for concentrations below 0.4 µg/mL.

Inhibition of invasion and metastasis of human liver cancer HCCLM3 cells by portulacerebroside A.

CONTEXT: Portulacerebroside A (PCA) is a novel cerebroside compound isolated from Portulaca oleracea L. (Portulacaceae), an edible and medicinal plant distributed in the temperate and tropical zones worldwide. OBJECTIVE: This study investigates the effects of PCA in human liver cancer HCCLM3 cells on metastasis and invasion. MATERIALS AND METHODS: After the cells were treated with PCA (2.5, 5, and 10 µg/ml) for 6, 12, 24, or 48 h, adhesion, transwell invasion, and scratch tests were conducted and cell functions were evaluated. Western blot and FQ-RT-PCR assays explored the mechanism of PCA-inhibited invasion and metastasis in the cells. RESULTS: The adhesion rate of the cells was suppressed at 0.5 h (79.4 ± 1.0, 68.7 ± 1.3, and 58.1 ± 1.3%, versus 100 ± 1.5% in the control), 1 h (78.2 ± 1.2, 70.9 ± 1.6, and 55.4 ± 1.9%, versus 100 ± 1.2% in the control), and 1.5 h (71.6 ± 1.1, 62.3 ± 0.9, and 50.4 ± 0.9%, versus 100 ± 1.1% in the control). The 24 h invasion ability was decreased (356.6 ± 11.2, 204.0 ± 17.6, and 113.0 ± 9.5%, versus 443.6 ± 15.4% in the control). The migration capability was also restrained by PCA for 24 h (324.8 ± 25.4, 250.4 ± 21.0, and 126.3 ± 10.1, versus 381.6 ± 30.6 in the control) and 48 h (470.3 ± 34.3, 404.0 ± 19.7, and 201.0 ± 15.4, versus 752.0 ± 63.6 in the control). There was an increase in the mRNA and protein expression levels of TIMP-2 and nm23-H1, inhibition in the mRNA expression of MTA1, MMP-2, and MMP-9, and suppression in the protein expression of MTA1, RhoA, Rac1/Cdc42, MMP-2, but not RhoC and MMP-9. CONCLUSION: PCA suppresses the invasion and metastasis of HCCLM3 cells possibly by modulation of the mRNA and protein expression of related parameters. This is the first study to reveal a new potential therapeutic application of PCA in antimetastatic therapy for liver cancer.

Effects of portulacerebroside a on apoptosis of human leukemia HL60 cells and p38/JNK signaling pathway.

Acute myeloid leukemia is known as one of the most malignant diseases. We aimed at exploring the effect of portulacerebroside A (PCA) on the apoptosis in human leukemia HL60 cells and clarify the possible mechanisms involved in. By MTT analysis, we found that PCA (1-100 µM) inhibited the cell viability in a time- and dose-dependent manner, and cell cycle was arrested at G0/G1 period. PCA treatment from 5 to 50 µM dose-dependently induced apoptosis from 12.7 ± 1.56% to 52.7 ± 6.214% of HL60 cells. Mitochondrial membrane potential (MMP) was decreased and reactive oxygen species (ROS) accumulated obviously. mRNA expressions and protein levels of Bax/Bcl-2, caspase-3 and caspase-9 were elevated significantly. ERK1/2, JNK1/2 and p38 MAPK pathway were blocked detected by western blot analysis. In conclusion, PCA can act as a new agent for leucocythemia treatment.

Structural analysis of cerebrosides from Aspergillus fungi: the existence of galactosylceramide in A. oryzae.

Glucosylceramide and galactosylceramide were detected in three Aspergillus species: Aspergillus oryzae, Aspergillus sojae and Aspergillus. awamori, using borate-coated TLC. The cerebrosides from A. oryzae were further purified by ion exchange and iatrobeads column chromatographies with or without borate, and determined the composition of sugar, fatty acid and sphingoid base by GC/MS, MALDI-TOF/MS and (1)H-NMR. We identified them as ß-glucosylceramide and ß-galactosylceramide. The ceramide moiety of both cerebrosides consisted mainly of 2-hydroxystearic acid and either 9-methyl-octadeca-4, 8-sphingadienine or octadeca-4, 8-sphingadienine. To our knowledge, this is the first study to provide evidence for the presence of ß-galactosylceramide in A. oryzae.

Biomarker-guided screening of Juzen-taiho-to, an oriental herbal formulation for immunostimulation.

Juzen-taiho-to is an immunostimulatory herbal formulation that is clinically used in East Asia for cancer patients undergoing chemotherapy and radiation. The formulation stimulates various leukocytes, including T, B, and NK cells and macrophages. Although Juzen-taiho-to is known to contain numerous compounds with various pharmacological activities, it is not clear which compounds are responsible for the stimulation of individual cell types. Here, we conducted what we call "biomarker-guided screening" to purify compounds responsible for the macrophages stimulatory activity. To this end, gene expression was analyzed by a DNA array for macrophages treated with Juzen-taiho-to and DMSO (vehicle control), which identified intercellular adhesion molecule 1 as a biomarker of macrophage stimulation by Juzen-taiho-to. A quantitative reverse transcription polymerase chain reaction assay of intercellular adhesion molecule 1 was then used to guide the purification of active compounds. The screening resulted in the purification of a glycolipid mixture, containing ß-glucosylceramides. The glycolipid mixture potently stimulated intercellular adhesion molecule 1 expression in primary dendritic cells as well as in primary CD14+ (macrophages) cells. The identification of this glycolipid mixture opens up an opportunity for further studies to understand how plant-derived glycolipids stimulate macrophages and dendritic cells in a safe and effective manner as demonstrated by Juzen-taiho-to.

Structural elucidation of the neutral glycosphingolipids, mono-, di-, tri- and tetraglycosylceramides from the marine crab Erimacrus isenbeckii.

The neutral glycosphingolipids, mono-, di-, tri- and tetraglycosylceramides (GL-1, GL-2, GL-3, GL-4a and GL-4b), were identified from whole tissues of the marine crab Erimacrus isenbeckii by successive column chromatography with ion exchange Sephadex (QAE-Sephadex), magnesium silicate (Florisil) and silicic acid (Iatrobeads) resins. Through component analysis, sugar analysis, methylation studies, exoglycosidase cleavage, and various chromatographic and spectrometric techniques, their structures were proposed to be as follows: GL-1, Glcß1-1Cer; GL-2, Manß1-4Glcß1-1Cer; GL-3, Galß1-3Manß1-4Glcß1-1Cer; and GL-4a and GL-4b, Gal3Meα1-4Galß1-3Manß1-4Glcß1-1Cer. The main molecular species of the aliphatic moiety in each purified glycolipid were 18:0, 22:0, 22:1-d14:1 (fatty acid-sphingoid) and 18:0-d16:1 for GL-1; 18:0-d16:1 and 22:1-d14:1, d16:1 for GL-2; 22:1, 24:1-d16:1 for GL-3; 22:1, 24:1-d16:1 for GL-4a; and h22:1, h24:1-d16:1 for GL-4b, respectively. By immunological detection, an arthro-series glycosphingolipid (At3Cer; GlcNAcß1-3Manß1-4Glcß1-1Cer) was also detected as a minor component. The characteristic arthro-series glycosphingolipid has been observed in most animals belonging to the phylum Arthropoda.

Quantitation and structural determination of glucosylceramides contained in sake lees.

Sake lees are solid parts filtered from the mash of sake, the traditional rice wine of Japan, which is brewed with Aspergillus oryzae and Saccharomyces cerevisiae. The moisture-holding activity of sake lees has long been recognized in Japan. However, the constituent responsible for this activity has not been elucidated. In this study, we first determined the structure of the glucosylceramides contained in sake lees. The glucosylceramides contained in sake lees were N-2'-hydroxyoctadecanoyl-l-O-ß-D-glucopyranosyl-9-methyl-4,8-sphingadienine (d19:2/C18:0h), N-2'-hydroxyoctadecanoyl-l-O-ß-D-glucopyranosyl-4,8-sphingadienine (d18:2/C18:0h), N-2'-hydroxyicosanoyl-l-O-ß-D-glucopyranosyl-4,8-sphingadienine (d18:2/C20:0h) and N-2'-hydroxyicosanoyl-l-O-ß-D-glucopyranosyl-4,8-sphingadienine (d18:2/C22:0h), which corresponded to those of A. oryzae and rice. The glucosylceramide produced by A. oryzae constituted the most abundant species (43% of the total glucosylceramide) in the sake lees. These results will be of value in the utilization of sake lees for cosmetics and functional foods.

Isolation of cytotoxic glucoerebrosides and long-chain bases from sea cucumber Cucumaria frondosa using high speed counter-current chromatography.

Total glucocerebrosides of the sea cucumber Cucumaria frondosa (CFC) have been isolated from the less polar lipid fraction of the chloroform-methanol extract using high speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of petroleum ether-methanol-water (5:4:1, v/v). Three glucocerebroside molecular species (CFC-1, CFC-2 and CFC-3) were isolated from crude total cerebrosides with repeated column chromatography. The structures of these three glucocerebroside molecular species were determined on the basis of chemical and spectroscopic evidence: fatty acids were mainly saturated (C22:0 and C18:0), monounsaturated (C24:1 and C20:1) and α-hydroxyl fatty acids (C24:1h, C23:0h, C23:1h and C22:0h), the structures of long-chain bases were dihydroxy (d17:1, d18:2 and d18:1) and trihydroxy (t17:0 and t16:0), and the glycosylation was glucose. High purity long-chain bases of sea cucumber Cucumaria frondosa (CF-LCB) were prepared from total lipids by HSCCC with a two-phase solvent system composed of n-hexane-methyl tert butyl ether-methanol-water (1:1:2:1, v/v). Compare with traditional preparative methods, the method of HSCCC is short cycle, high yield and less solvent consumption. The composition analysis of CF-LCB showed that the ratio of d18:2 and d17:1 was approximately 2:1. The four glucocerebrosides and long-chain bases from sea cucumber Cucumaria frondosa were evaluated for activity in vitro assays for the cytotoxic activities against Caco-2 colon cancer cells. The results indicated that both glucocerebrosides and long-chain bases exhibited an inhibitory effect on cell proliferation. Moreover, CFC-3 was most effective in four glucocerebrosides to Caco-2 cell viability. The inhibition effect of CF-LCB was much stronger than glucocerebrosides.

Novel cerebroside, termitomycesphin I, from the mushroom, Termitomyces titanicus.

The novel cerebroside, termitomycesphin I (1), and two known cerebrosides (2 and 3) were isolated from the edible mushroom, Termitomyces titanicus. The structures of 1-3 were determined and identified by interpreting the spectroscopic data.

Termitomycesphins G and H, additional cerebrosides from the edible Chinese mushroom Termitomyces albuminosus.

Two new cerebrosides, termitomycesphins G and H, were isolated from the edible Chinese mushroom, Termitomyces albuminosus (Berk.) Herm., and exhibited neuritogenic activity against PC12 cells. Their structures and absolute stereochemistry were elucidated by spectroscopic methods and by a comparison of the specific rotation of the hydrogenated products from termitomycesphins H and C. These cerebrosides possessed a unique modification by a hydroxyl group at the middle of the long-chain base, like earlier congeners termitomycesphins A-F. Termitomycesphin G with a 16-carbon-chain fatty acid showed higher neuritogenic activity than that of termitomycesphin H with an 18-carbon-chain fatty acid. This effect was observed within the termitomycesphins, suggesting that the chain length of the fatty acyl moiety played a key role in the neuritogenic activity.

Characterization of glucosylceramides in the Polygonaceae, Rumex obtusifolius L. injurious weed.

Rumex obtusifolius L., a member of Polygonaceae, is one of the world's worst weeds. We characterized the glucosylceramide molecular species in leaves of R. obtusifolius by liquid chromatography/tandem mass spectrometry. 4,8-Sphingadienines were principally paired with 2-hydroxy palmitic acids. In contrast, 4-hydroxy-8-sphingenines were chiefly attached to 2-hydroxy fatty acids with 22 to 26 carbon-chain length. A unique characteristic of the 2-hydroxy fatty acid composition of R. obtusifolius was the high content of n-9 monoenoic 2-hydroxy fatty acids with 22 and 24 carbon-chain length. The levels of the Z and E stereoisomers of the 8-unsaturated long-chain bases were reliably distinguished from those in other plant families in ten species of Polygonaceae.

Analysis of glucosylceramides from various sources by liquid chromatography-ion trap mass spectrometry.

Liquid chromatography-mass spectrometry is one of the most powerful methods for the identification and detection of chemical structures of lipids. In this study, we attempted to identify the chemical structures of glucosylceramides from maize, rice, mushroom (maitake) and sea cucumber by liquid chromatography-ion trap mass spectrometry. For structural analysis of glucosylceramides, [M+H]+, [M+H-18]+ or [M+H-162]+ in the positive scan mode was used for MS/MS analysis to obtain product ion spectra. The typical signals which are characteristic for the sphingoid base moieties were observed while the isomers could not be distinguished. This method should be useful for the structural determination of diverse glucosylceramide molecular species.

Two new cerebrosides from the pollen of Typha angustifolia.

Two new cerebrosides, 1-O-(beta-d-glucopyranosyloxy)-(2S,3S,4R,8Z)-2-[(2'R)-2'-hydroxytricosanoylamino]-8-nonadecene-3,4-diol (1) and 1-O-(beta-D-glucopyranosyloxy)-(2S,3R,4E,8Z)-2-[(2'R)-2'-hydroxynonadecanoylamino]-4,13-nonadecene-3-diol (2), were isolated from the pollen of Typha angustifolia. Their structures were elucidated by chemical and spectral means. This is the first report on the occurrence of cerebroside in Typha (Typhaceae). Compounds 1 and 2 exhibited effect on the proliferation of cultured vascular smooth muscle cell (VSMCs) induced by fatal bovine serum (FBS).

Structural determination of glucosylceramides isolated from marine sponge by fast atom bombardment collision-induced dissociation linked scan at constant B/E.

Five glucosylceramides (GlcCers) were isolated by reversed phase high-performance liquid chromatography from the MeOH extracts of a marine sponge, Haliclona (Reniera) sp., collected from the coast of Ulleung Island, Korea, and analyzed by fast atom bombardment mass spectrometry (FAB-MS) in positive-ion mode. FAB-mass spectra of these compounds included protonated molecules [M + H](+) and abundant sodiated molecules [M + Na](+) from a mixture of m-NBA and NaI. The structures of these GlcCers, which were similar, were elucidated by FAB-linked scan at constant B/E. To find diagnostic ions for their characterization, the GlcCers were analyzed by collision-induced dissociation (CID) linked scan at constant B/E. The CID-linked scan at constant B/E of [M + H](+) and [M + Na](+) precursor ions resulted in the formation of numerous characteristic product ions via a series of dissociative processes. The product ions formed by charge-remote fragmentation provided important information for the characterization of the fatty N-acyl chain moiety and the sphingoid base, commonly referred to as the long-chain base. The product ions at m/z 203 and 502 were diagnostic for the presence of a sodiated sugar ring and beta-D-glucosylsphinganine, respectively. For further confirmation of the structure of the fatty N-acyl chain moiety in each GlcCer, fatty acid methyl esters were obtained from the five GlcCers by methanolysis and analyzed by FAB-MS in positive-ion mode. On the basis of these dissociation patterns, the structures of the five GlcCers from marine sponge were elucidated. In addition, the accurate mass measurement was performed to obtain the elemental composition of the GlcCers isolated from marine sponge.

Glycosylceramides obtain from the starfish Asterias amurensis Lütken.

Complex lipids in the starfish Asterias amurensis were characterized and the influence of sphingoid bases on human colon carcinoma Caco-2 cells was also investigated. Lipid content of gonad and viscera were 3.3% and 6.8%, respectively, in wet basis. The main lipid class in gonad was ceramide monohexoside (CMH) while triglyceride (TG) was predominant in the viscera. The most abundant fatty acid in the polar lipid was eicosapentaenoic acid (EPA, C20:5n-3), with the gonad and viscera samples having the highest proportion of 41.5% and 32.7%, respectively, of total fatty acids. Starfish internal organ contained enormous amount (0.7% in wet base) of glycosylceramide. Sphingoid bases of the glycosylceramide were mainly consisted of d22:2, d22:1 and d18:3. This sphingoid base exerted an apoptotic activity on Caco-2 cells. Thus, starfish could be used as a potential source of precious and useful complex lipids.

Two new protease-inhibiting glycosphingolipids from Buddleja crispa.

Crispins A (1) and B (2), two new glycosphingolipids, were isolated from the whole plant Buddleja crispa, along with three known compounds: alpha-amyrin, linoleic acid, and stigmasterol. Their structures were elucidated by chemical and spectroscopic techniques. Both 1 and 2 showed significant inhibitory activity against alpha-chymotrypsin in a concentration-dependent manner.

Tumor specific cytotoxicity of glucosylceramide.

To develop a new taxon of anti-cancer agent with lower side effect, this study described a tumor selective cytotoxicity of glucosylceramide extracted from malt feed of beer brewing waste. Interpretation of (13)C- and (1)H-NMR spectra identified the chemical structure of major component of glucosylceramide as 1-O-beta-D: -glucopyranosyl-2(2'-hydroxyeicosanoylamino)-4,11-octadecadiene-1,3-diol. Selective cytotoxicity was studied with three pairs of normal and cancer cells: liver, skin and lung. The glucosylceramide selectively lowered the relative viability of cancer cells. Of the pairs, the selectivity was most pronounced with the liver cells, and, for this reason, further experiment was conducted with this pair of normal (CS-HC) and cancer cells (HepG2) to get more insight into the selective toxicity. The glucosylceramide significantly increased the cell population at G(2)/M phase in HepG2 cells, and also increased the numbers of apoptotic (sub-G(0)/G(1)) cells, but to much lesser extent compared with the increase in G(2)/M phase. Treatment of HepG2 cells with this agent selectively disrupted the mitochondrial membrane integrity without activation of caspase pathway to induce apoptosis. These findings suggested that the glucosylceramide specifically suppressed the growth of cancer cells by inhibiting cell renewal capacity rather than induction of apoptosis. The underlying mechanism for the selectivity remains to be answered in the forthcoming study.