Cerebroside Research Articles

Detergent-Resistant Membranes in Chloroplasts and Mitochondria of the Halophyte Salicornia perennans under Salt Stress

Halophytes represent important models for studying the key mechanisms of salt tolerance. One approach to the development of new knowledge of salt tolerance is to study the properties of detergent-resistant membranes (DRMs). In this work, the lipid profiles of DRMs of chloroplasts and mitochondria of euhalophyte Salicornia perennans Willd, before and after their exposure to shock concentrations of NaCl, have been investigated. We found that DRMs of chloroplasts are enriched in cerebrosides (CERs) and that sterols (STs) dominate the mass of mitochondrial DRMs. Also, it has been proven that (i) the impact of salinity provokes obvious growth in the content of CERs in DRMs of chloroplasts; (ii) the content of STs in DRMs of chloroplasts does not change under the influence of NaCl; (iii) salinity also causes some elevation in the content of monounsaturated and saturated fatty acids (FAs). Considering the fact that DRMs represent integral parts of both chloroplast and mitochondrial membranes, the authors have come to the conclusion that the cells of euhalophyte S. perennans, under the impact of salinity, presumes the choice (by the cell) of some specific composition of lipids and FAs in the membrane. This may be considered as a specific protection reaction of the plant cell against salinity.

Selective Capture-Based Single-Cell Mass Spectrometry for Enhancing Sphingolipid Profiling of Neurons with Differentiation of Cell Body from Synapse.

Currently, single-cell lipidomic mass spectrometry (MS) techniques are mostly limited to detection of high-abundance phosphatidylcholines (PCs). Herein, for enhancing the coverage to low-abundance sphingolipids in single-cell analysis, in-tube solid-phase microextraction (SPME) was combined with a single-probe MS system for selective enrichment of sphingolipids during singe-cell sampling. From the results, a lab-made single probe with a 30 μm tip size proved to be able to resolve the axon from the cell body of neuron HT22 in ambient conditions. TiO2 was immobilized onto the inner wall of the transfer capillary of the single-probe device for online selective capture of sphingolipids in ammonia-acetonitrile and rapid desorption in formic acid-methanol. The results showed that the breakthrough volume of the capillary with sample loading flow rate at 500 nL/min was >14 μL. Standard experiments showed that the signals of cerebroside (CB), ceramide (Cer), and sphingomyelin (SM) were largely enhanced after selective capture in the coated capillary, while PCs were totally removed. The reusability (>10 times) and stability of the lab-made TiO2-coated capillary was verified. By introducing the coated capillary into the single-probe MS system, the new system proved to have low detection limits of SM, Cer, and CB (0.007-0.027 μg/mm2) and acceptable linearity (r > 0.98) and repeatability (RSD < 30%). Lipid coverage of the new method to SMs and CBs proved to be largely improved (SM, 21 vs 2; CB, 10 vs 0) with the new method in comparison to conventional single-probe MS without selective capture by ambient analysis of a single spot of rat cerebellum. Finally, the new system was used to perform single-neuron analysis of sphingolipids in the control and lipopolysaccharide (LPS)-treated HT22 with differentiation of the cell body from the axonal synapse. Results showed that 5 sphingolipids had significantly higher concentrations in the synapse than in the cell body, while 3 oxidized sphingolipids had significantly higher levels in the cell body than in the synapse. After LPS treatment, most of the sphingolipids largely decreased and became more accumulated in the synapse, providing new information on LPS-induced neuroinflammation.

Per-pixel absolute quantitation for mass spectrometry imaging of endogenous lipidomes by model prediction of mass transfer kinetics in single-probe-based ambient liquid extraction

With the development of mass spectrometry imaging (MSI), techniques providing quantitative information on the spatial distribution have attracted more attentions recent years. However, for MSI of endogenous compounds in bio-samples, the uncertainty of locally varied sampling efficiencies always hinders accurate absolute quantitation. Here single-probe was used for ambient liquid extraction MSI in rat cerebellum, and standards of phosphatidylcholines (PCs) and cerebrosides (CBs) were doped in extraction solvent. The extraction kinetic curves of endogenous lipids in the ambient liquid extraction during probe parking in single pixel of tissue were investigated. From the results, the extraction kinetic curves were varied between different lipid species in different brain regions, resulting in variations of extraction efficiencies between imaging pixels, and calibration with standards deposited in tissue could not compensate for the variations. In our approach, the theoretical kinetic model of ambient liquid extraction was established, and original concentrations of endogenous lipids in each pixel of tissue were predicted by fitting the experimental extraction kinetic curve in each imaging pixel to the model. The experimental data was demonstrated to be well fitted to the kinetic model with R2 > 0.86, and only with 18-s extraction in each pixel, the original lipid concentrations were predicted accurately with relative errors <23%. With the new method, totally 157 lipids and small metabolites were imaged, and per-pixel quantitation was achieved for 19 PCs and 4 CBs. Compared with conventional quantitative MSI (q-MSI) method, the new q-MSI method had better reproducibility and wider linear range, and produced better contrast in the quantitative images of lipids in brain tissue with less hot spots and noises. The absolute quantitation results by the new method were verified by quantitative LC–MS method with Pearson'r > 0.9 and the slope of the linear fitting line of the correlation plot near 1.

Triple-Knockout, Synuclein-Free Mice Display Compromised Lipid Pattern.

Recent studies have implicated synucleins in several reactions during the biosynthesis of lipids and fatty acids in addition to their recognised role in membrane lipid binding and synaptic functions. These are among aspects of decreased synuclein functions that are still poorly acknowledged especially in regard to pathogenesis in Parkinson’s disease. Here, we aimed to add to existing knowledge of synuclein deficiency (i.e., the lack of all three family members), with respect to changes in fatty acids and lipids in plasma, liver, and two brain regions in triple synuclein-knockout (TKO) mice. We describe changes of long-chain polyunsaturated fatty acids (LCPUFA) and palmitic acid in liver and plasma, reduced triacylglycerol (TAG) accumulation in liver and non-esterified fatty acids in plasma of synuclein free mice. In midbrain, we observed counterbalanced changes in the relative concentrations of phosphatidylcholine (PC) and cerebrosides (CER). We also recorded a notable reduction in ethanolamine plasmalogens in the midbrain of synuclein free mice, which is an important finding since the abnormal ether lipid metabolism usually associated with neurological disorders. In summary, our data demonstrates that synuclein deficiency results in alterations of the PUFA synthesis, storage lipid accumulation in the liver, and the reduction of plasmalogens and CER, those polar lipids which are principal compounds of lipid rafts in many tissues. An ablation of all three synuclein family members causes more profound changes in lipid metabolism than changes previously shown to be associated with γ-synuclein deficiency alone. Possible mechanisms by which synuclein deficiency may govern the reported modifications of lipid metabolism in TKO mice are proposed and discussed.

Diverse Biological Functions of Sphingolipids in the CNS: Ceramide and Sphingosine Regulate Myelination in Developing Brain but Stimulate Demyelination during Pathogenesis of Multiple Sclerosis.

Sphingolipids are enriched in the Central Nervous System (CNS) and display multiple biological functions. They participate in tissue development, cell recognition and adhesion, and act as receptors for toxins. During myelination, a variety of interactive molecules such as myelin basic protein, myelin associated glycoprotein, phospholipids, cholesterol, sphingolipids, etc., participate in a complex fashion. Precise roles of some sphingolipids in myelination still remain unexplored. Our investigation delineated participation of several sphingolipids in myelination during rat brain development as well as in human brain demyelination during pathogenesis of Multiple Sclerosis (MS). These sphingolipids included Ceramide (Cer)/dihydroceramide (dhCer), Sphingosine (Sph)/dihydrosphingosine (dhSph), and glucosyl/galactosylceramide (glc/galCer) as we detected these by column chromatography, high performance thin-layer chromatography, gas chromatography-mass spectrometry, and high-performance liquid chromatography. Cer/dhCer level rises during rat brain development starting at Embryonic stage (E) until postnatal day (P21), then gradually falls until the maturity (P30 and onwards), and remains steady maintaining a constant ratio (4–4.5:1) throughout the brain development. GlcCer is the initial Monoglycosylceramide (MGC) that appears at early Postnatal stage (P8) and then GalCer appears at P10 with an increasing trend until P21 and its concentration remains unaltered. Sph and dhSph profiles show a similar trend with an initial peak at P10 and then a comparatively smaller peak at P21 maintaining a ratio of (2–2.5:1) of Sph:dhSph. The profiles of all these sphingolipids, specifically at P21, clearly indicate their importance during rat brain development but somewhat unspecified roles in myelination. While Cer has been reported to involve in neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease, Sph being a potent inhibitor of protein kinase C has recently been implicated in CNS demyelination due to MS. Inflammatory cytokines stimulate Sph elevation in MS brains and lead to demyelination due to oligodendrocyte death as we examined by using human oligodendroglioma culture. In conclusions, sphingolipids are essential for brain development but they have deleterious effects in demyelinating diseases such as MS.

Effects of dietary glucocerebrosides from sea cucumber on the brain sphingolipid profiles of mouse models of Alzheimer's disease.

Herein, we investigated the potential relationship between sphingolipids and Alzheimer's disease (AD) with special attention to the relationship between dietary sea cucumber glucocerebrosides (SCGs) and sphingolipid metabolism. We assessed animal behavior using the Morris water maze test, determined Aβ1-42 concentration in the hippocampus using ELISA, and assessed the sphingolipid profile of the hippocampus and the cortex in normal mice (SAMR1), AD mice (SAMP8), and AD mice (SAMP8) fed with SCG using liquid chromatography-triple quadrupole mass spectrometry. We found that the SAMP8 mice had impaired memory and an SCG diet significantly rescued spatial memory deficits in these mice. As expected, we found that the profiles of sphingolipid species and the levels of total cerebrosides (CBS), ceramides (Cer), and sulfatides (ST) were significantly different in both the hippocampus and the cortex between the three groups; moreover, there were significantly lower ST levels and higher Cer and CBS levels in these regions in the SAMP8 mice. In the AD-SCG group, Cer and ST levels were altered only in the hippocampus, in contrast to the AD group. The major molecular species ST (d18:1-C24:1) and Cer (d18:1/18:0) were especially different between those of the two groups. Unexpectedly, sphingolipid profiles, including the nonhydroxylated fatty acid-ST/hydroxylated fatty acid-ST, very long fatty acid-galactocerebroside/long fatty acid-galactocerebroside, nonhydroxylated fatty acid-galactocerebroside/hydroxylated fatty acid-galactocerebroside and galactocerebroside/glucocerebroside ratios, were affected by AD. Thus, the ST and Cer levels and the profiles of sphingolipid species in the AD-SCG group were significantly different compared to those of the AD model group. Therefore, SCG has potential ameliorative effects in AD, and exogenous sphingolipid intake may potentially influence sphingolipid metabolism in vivo.

Normobaric Hyperoxia Preconditioning Induces Changes in the Brain Lipidome.

Recent investigations have demonstrated that normobaric hyperoxia induces neuroprotection against ischemic injury. The aim of study was to determine the survey of HO (hyperoxia) preconditioning on brain lipidome.The animals were assigned into three groups, the first experimental group was exposed to 95% inspired HO for 4 h /day for six consecutive days. The second experimental group considered as the control group and was exposed to 21% oxygen as room air (RA) in the same chamber. The third group acted as sham, which was under the stress of surgery condition without ischemia. The first two groups were divided into 2 subgroups, intact (without any surgery) and middle cerebral artery occlusion- operated (MCAO). Twenty-four hours after exposure to hyperoxia, MCAO subgroups were subjected to 60 min of right middle cerebral artery occlussion. After 24 h reperfusion, infarct volume (IV) and neurological deficit score (NDS) were assessed in MCAO subgroup. Brain lipidomics were measured in the intact subgroup. Preconditioning with HO significantly reduced NDS and IV and elevated the level of phosphatidylethanolamine (PE), sphingomyelin (SM), cholesterol ester (CE), cholesterol (Chol), phosphatidylcholine (PC), triglyceride (TG) and cerebroside (CB) in the brain as compared with the control (sham and RA). HO preconditioning, significantly decreased the brain ceramide (Cer) and lyso- phosphatidylcholine (Lyso-PC or LPC) levels. Preconditioning with HO decreases brain ischemia injury via changes in brain lipidomics and significantly decreases the brain ceramides (CER).Although more studies are required to explain the mechanisms of time course of neuroprotection, HO preconditioning partly decreases brain ischemia injury via changes in brain lipidome.

Detection of the galactose cerebroside enzyme activity by dry blood spot specimens on filter paper

目的 通过分别检测球形细胞脑白质营养不良(Krabbe病)滤纸片干血斑和外周血白细胞中半乳糖脑苷脂－β－半乳糖苷酶(galactocerebrosidase，GALC)的活性，判断利用滤纸片干血斑检测GALC活性的可行性。 方法 利用GALC特异性水解荧光底物6－十六酰胺－4－甲基伞形酮－β－D－半乳糖苷检测3例Krabbe病患儿滤纸片干血斑中该酶活性，再检测外周血白细胞中该酶活性，以验证两种检测方法结果是否一致。 结果 3例临床诊断为Krabbe病患儿滤纸片干血斑中GALC酶活性分别为0、0.13、0.12 μmol/(h·μl)，外周血白细胞GALC酶活性分别为1.22、0.92、0.8 nmol/(17 h·mg protein)，两组均明显低于正常参考值。 结论 两种方法检测GALC酶活性结果一致，利用滤纸片干血斑行酶活性检测方法简便快速，可作为临床Krabbe病筛查时检测酶活性的新方法。

Ultra-high-performance liquid chromatography electrospray ionization tandem mass spectrometry for accurate analysis of glycerophospholipids and sphingolipids in drug resistance tumor cells

Glycerophospholipids and sphingolipids are important signaling molecules which are involved in many physiological and pathological processes. Here we reported an effective method for accurate analysis of these lipids by liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS). The methanol method was adopted for extraction of lipids due to its simplicity and high efficiency. It was found that two subclasses of sphingolipids, sulfatide (ST) and cerebroside (CB), were heat labile, so a decreased temperature in the ion source of MS might be necessary for these compounds analysis. In addition, it was found that the isobaric interferences were commonly existent, for example, the m/z of 16:0/18:1 PC containing two 13C isotope being identical to that of 16:0/18:0 PC determined by a unit mass resolution mass spectrometer; therefore, a baseline separation of interferential species was required to maintain selectivity and accuracy of analysis. In this work, an ultra-high-performance liquid chromatography (UHPLC)-based method was developed for separation of interferential species. Moreover, in order to deal with the characteristics of different polarity and wide dynamic range of glycerophospholipids and sphingolipids in biological systems, three detecting conditions were combined together for comprehensive and rational analysis of glycerophospholipids and sphingolipids. The method was utilized to profile glycerophospholipids and sphingolipids in drug resistant tumor cells. Our results showed that many lipids were significantly changed in drug resistant tumor cells compared to paired drug sensitive tumor cells. This is a systematic report about the isobaric interferences and heat labile compounds interferences when analyzing glycerophospholipids and sphingolipids by ESI-MS/MS, which aids in ruling out one potential source of systematic error to ensure the accuracy of analysis.

Composition and Antiradical Power of Syzygium aromaticum Lipids

2) determine the total phenolic content and in vitro radical scavenging activity of CO. The results from the present study will be used to develop novel edible oil products rich in bioactive molecules with a desirable shelf life. The proportion of lipid classes present in CO as well as the R f values of the subclasses are shown in Table 1. The level of NL was the highest (ca. 94.7%), followed by GL (0.76%) and PL (0.39%). The data are in agreement with Suzuki et al. [13] who analyzed the lipid classes of clove lipidic extract. Subclasses of NL contained triacylglycerol (TG), free fatty acids (FFA), diacylglycerol (DG), esterified sterols (STE), and monoacylglycerol (MG) in decreasing order. A significant amount of TG was found (ca. 96.6% of total NL) followed by a relatively low level of FFA (ca. 1.4% of total NL), while DG and STE were recovered in lower levels. The subclasses of GL found in CO were sulfoquinovosyl diacylglycerol (SQD), digalactosyl diglycerides (DGD), cerebrosides (CER), steryl glycosides (SG), monogalactosyl diglycerides (MGD), and esterified steryl glycosides (ESG) as presented in Table 1. The proportion of each component was estimated by lipid-carbohydrate determination. CER, SG, and ESG were the prevalent components and made up about 92% of the total GL. The PL subclasses in CO were separated into four major fractions via TLC. The TLC fractions of PL revealed that the predominant PL subclasses were PC followed by PE, PI, and PS (Table 1). About half of the total PL was in PC and a quarter was in PE, while PI and PS were isolated in smaller quantities. The fatty acid profiles of CO and lipid classes (NL, GL, and PL) are presented in Table 2. According to the results, nine fatty acids were identified in CO. Linoleic and oleic acids were the main fatty acids in CO. Both fatty acids comprise together about 80% of the total FAME. The cold-pressed oil contained significant levels of monounsaturated fatty acids (MUFA, 39.7 g/100 g total fatty acids), which is comparable to cold pressed hemp, cranberry, blueberry, onion, and milk thistle seed oils but is much lower than the 81 and 82% in carrot and parsley cold pressed seed oils 14, 15.

OXIDATION AND POLYMORPHISM OF FATTY ACIDS AND NUTRITIONAL ASPECTS CONJUGATED OF LINOLEIC ACIDS

Fats and oils are triglycerides containing one unit of glycerol with three units of fatty acids. It also contained mono- and diglycerides, phosphatides, cerebrosides, sterols, terpenes, fatty alcohols, free fatty acids, fat-soluble vitamins, and other substances. Fats and oils are recognized as essential nutrients in both human and animal diets. They provide the most concentrated source of energy and also provide essential fatty acids which are precursors for important hormones. Conjugated linoleic acid (CLA) is a group of geometrical and positional isomers of linoleic acid. In contrast to linoleic acid, double bonds in CLA are usually located at positions 9 and 11 or 10 and 12 and each double bond can be either in the cis or trans configuration. Meat and dairy products from ruminant animals are the principal natural sources of CLA in the human diet. Dietary CLA has been shown to have potent anti-carcinogenic, antiatherogenic, immune modulating and also have other biological activities. The CLA was also reported to reduce body fat content. Key words: anti-atherogenic, anticarcinogenic, conjugated linoleic acid, Fats and oils, immune modulator

Direct and Potent Regulation of γ-Secretase by Its Lipid Microenvironment

gamma-Secretase is an unusual and ubiquitous aspartyl protease with an intramembrane catalytic site that cleaves many type-I integral membrane proteins, most notably APP and Notch. Several reports suggest that cleavage of APP to produce the Abeta peptide is regulated in part by lipids. As gamma-secretase is a multipass protein complex with 19 transmembrane domains, it is likely that the local lipid composition of the membrane can regulate gamma-activity. To determine the direct contribution of the lipid microenvironment to gamma-secretase activity, we purified the human protease from overexpressing mammalian cells, reconstituted it in vesicles of varying lipid composition, and examined the effects of individual phospholipids, sphingolipids, cholesterol, and complex lipid mixtures on substrate cleavage. A conventional gamma-activity assay was modified to include a detergent-removal step to facilitate proteoliposome formation, and this increased baseline activity over 2-fold. Proteoliposomes containing sphingolipids significantly increased gamma-secretase activity over a phosphatidylcholine-only baseline, whereas the addition of phosphatidylinositol significantly decreased activity. Addition of soluble cholesterol in the presence of phospholipids and sphingolipids robustly increased the cleavage of APP- and Notch-like substrates in a dose-dependent manner. Reconstitution of gamma-secretase in complex lipid mixtures revealed that a lipid raft-like composition supported the highest level of activity compared with other membrane compositions. Taken together, these results demonstrate that membrane lipid composition is a direct and potent modulator of gamma-secretase and that cholesterol, in particular, plays a major regulatory role.

Myelin Lipid Abnormalities in the Aspartoacylase-Deficient Tremor Rat

The high concentration of N-acetylaspartate (NAA) in neurons of the central nervous system and its growing clinical use as an indicator of neuronal viability has intensified interest in the biological function of this amino acid derivative. The biomedical relevance of such inquiries is highlighted by the myelin-associated pathology of Canavan disease, an inherited childhood disorder resulting from mutation of aspartoacylase (ASPA), the NAA-hydrolyzing enzyme. This enzyme is known to be localized in oligodendrocytes with bimodal distribution in cytosol and the myelin sheath, and to produce acetyl groups utilized in myelin lipid synthesis. Loss of this acetyl source in Canavan disease and rodent models such as the tremor rat are thought to account for the observed myelin deficit. This study was undertaken to further define and quantify the specific lipid abnormalities that occur as a result of ASPA deficit in the tremor rat. Employing mass spectrometry together with high performance thin-layer chromatography, we found that myelin from 28-day-old animals showed major reduction in cerebrosides (CB) and sulfatides (Sulf) with unsubstituted fatty acids, and equal if not greater changes in myelin from 7-month-old tremors. Cerebrosides with 2-hydroxyfatty acids showed little if any change at either age; Sulf with 2-hydroxyfatty acids showed no significant change at 28 days, but surprisingly a major increase at 7 months. Two species of phosphatidylcholine, 32:0 and 34:1, also showed significant increase, but only at 28 days. One form of phosphatidylethanolamine, PE36:1, was reduced a modest amount at both ages, whereas the plasmalogen form did not change. The dysmyelination that results from inactivation of ASPA is thus characterized by selective decreases as well as some increases in specific lipids.

Cerebrosides of baifuzi, a novel potential blocker of calcium‐activated chloride channels in rat pulmonary artery smooth muscle cells

Calcium-activated chloride channels (CaCCs) are crucial regulators of vascular tone by promoting a depolarizing influence on the resting membrane potential of vascular smooth muscle cells. However, the lack of a special blocker of CaCCs has limited the investigation of its functions for long time. Here, we report that CB is a novel potential blocker of I(Cl(Ca)) in rat pulmonary artery smooth muscle cells (PASMC). Cerebrosides (CB) were isolated from Baifuzi which is dried root tuber of the herb Typhonium giganteum Engl used for treatment of stroke in traditional medicine. Using the voltage-clamp technique, sustained Ca(2+)-activated Cl(-) current (I(Cl(Ca))) was evoked by a K(+)-free pipette solution containing 500nM Ca(2+) which exhibited typical outwardly rectifying and voltage-/time-dependence characterization. Data showed that CB played a distinct inhibitory role in modulating the CaCCs. Moreover, we investigated the kinetic effect of CB on I(Cl(Ca)) and found that it could slow the activation dynamics of the outward current, accelerate the decay of the inward tail current and change the time-dependence characterization. We conclude that CB is a novel potent blocker of CaCCs. The interaction between CB and CaCCs is discussed.

Characterization of novel myelin components 3‐O‐acetyl‐sphingosine galactosylceramides by electrospray ionization Q‐TOF MS and MS/CID‐MS of Li+ adducts

Glycosphingolipids with R(f) values higher than those of monoglycosylceramides (MGCs) in normal phase HPTLC appear to be normal components of myelin. A series of such low polarity components, referred to as 'fast moving cerebrosides' (FMCs), have been isolated from rat brain, and two of these fractions (FMC-1 and FMC-2) were found to be novel derivatives of galactosylceramide (GalCer) exhibiting O-acetylation at the 3-hydroxy group of the sphingoid moiety, and incorporating either non-hydroxy or 2-hydroxy fatty-N-acylation (Dasgupta S, Levery SB, Hogan EL. J. Lipid Res. 2002; 43: 751-761). Similar to the parent compounds, the 3-O-acetyl-sphingoid derivatives exhibit considerable diversity with respect to fatty-N-acyl chain length, manifested by heterogeneous molecular ion (Li(+) adduct) profiles. However, a detailed analysis of the individual molecular variants ('lipoforms'), e.g. by tandem MS/CID-MS analysis, was not carried out. In addition, several other FMCs distinguished by even lower polarity (higher HPTLC R(f) values) were isolated but have remained uncharacterized. For this study, analysis of both the known and unknown FMC components was carried out by positive ion ESI-MS and MS/CID-MS of their Li(+) adducts on a Q-TOF mass spectrometer. Since a Q-TOF instrument has not yet been applied to MS of lithiated cerebrosides and FMCs, MS/CID-MS spectra of bovine brain GalCer (both types) and the previously characterized rat brain FMCs (FMC-1 and FMC-2), having 3-O-acetylation of the sphingoid, were systematically acquired and their fragmentation behavior compared. This was followed by systematic analysis of previously uncharacterized FMC fractions (FMC-3 through FMC-5/6/7). The GalCer and FMC components proved to be amenable to analysis by this technique, and the data confirm that the latter are all related 3-O-acetyl-sphingoid derivatives, with the higher R(f) components carrying additional O-acetyl modifications on the galactosyl residue, which further reduce their polarity. The utility of the technique, the structures of unknown FMCs, and their characteristic fragmentation patterns are described.

Cerebrosides from the Roots of Serratula chinensis

A new cerebroside, 1-O-beta-D-glucopyranosyl-(2S,3R,8E)-2-[(2'R)-2-hydroxy- palmitoylamino]-8-octadecene-1,3-diol, along with aralia cerebroside and 1-O-beta-D- glucopyranosyl-(2S,3S,4R,8E)-2-[(2'R)-2-hydroxybehenoylamino]-8-octadecene-1,3,4-triol were isolated from the roots of Serratula chinensis S. Moore. The structure of the new cerebroside was established by spectroscopic and chemical means. Occurrence of cerebrosides in Serratula is reported here for the first time.

Synthetic α-Mannosyl Ceramide as a Potent Stimulant for an NKT Cell Repertoire Bearing the Invariant Vα19-Jα26 TCR α Chain

A NKT cell repertoire is characterized by the expression of the Valpha19-Jalpha26 invariant TCR alpha chain (Valpha19 NKT cell). This repertoire, as well as a well-established Valpha14-Jalpha281 invariant TCR alpha(+) NKT cell subset (Valpha14 NKT cell), has been suggested to have important roles in the regulation of the immune system and, thus, is a major therapeutic target. Here, we attempted to find specific antigens for Valpha19 NKT cells. Valpha19 as well as Valpha14 NKT cells exhibited reactivity to alpha-galactosyl ceramide (alpha-GalCer). Thus, a series of monoglycosyl ceramides with an axially oriented glycosidic linkage between the sugar and ceramide moiety were synthesized and their antigenicity to Valpha19 NKT cells was determined by measuring their immune responses in culture with glycolipids. Comprehensive examinations revealed substantial antigenic activity for Valpha19 NKT cells by alpha-mannosyl ceramide.

Cerebrosides alter the lyotropic and thermotropic phase transitions of DOPE:DOPC and DOPE:DOPC:sterol mixtures

Freezing injury in rye and oat is a consequence of the formation of the inverted hexagonal (H II) phase in regions where the plasma membrane is brought into close proximity with cytoplasmic membranes during freeze-induced dehydration. Susceptibility to plasma membrane destabilization and H II phase formation during freezing is associated with alterations in plasma membrane lipid composition. This paper examines the influence of lipid composition and hydration on the propensity of lipid mixtures of DOPE:DOPC and DOPE:DOPC:sterols with added cerebrosides (CER) to form the H II phase during dehydration. The addition of CER to DOPE:DOPC: β-sitosterol mixtures decreased the water content of the dispersions in a manner suggesting that most or all of the water in the dehydrated mixtures was associated with the phospholipids. The addition of CER significantly decreased the osmotic pressure at which the L α →H II phase transition occurred from an osmotic pressure of 76.1 MPa for DOPE:DOPC (50:50) to 20 MPa in DOPE:DOPC: β-sitosterol:CER (22.5:22.5:50:5) and 8 MPa in DOPE:DOPC: β-sitosterol:CER (15:15:50:20). Experiments examining the effects of CER on the thermally-induced formation of the H II phase in fully hydrated mixtures and examining the influence of CER on the formation of the H II phase in DOPE:DOPC mixtures lacking β-sitosterol suggested that CER facilitated the L α →H II phase transition by effecting a decrease in bilayer hydration and by increased lateral packing pressures within the acyl domain of the bilayer. Taken in sum, these data indicate that the differential propensity of the rye and oat plasma membranes to undergo freeze-induced formation of the L α →H II phase cannot be attributed to one lipid species. Rather, the propensity towards freeze-induced membrane destabilization is a consequence of the summation of physical characteristics of the membrane lipid components that included bilayer hydration, packing pressures within the hydrophobic domain of the membrane, the propensity of the lipid components to demix, and the relative proportions of the various lipid components.

Neutral monoglycosylceramides in rat brain: occurrence, molecular expression and developmental variation.

Developmental expression of neutral monoglycosylceramides (MGCs) has been examined in rat brain from embryonic day 15 (E15) to postnatal day 30 (P30) and adulthood. To this point, glucosylceramide (GlcCer) is the only MGC that has been characterized in embryonic brain. Galactosylceramide (GalCer) appears at P1, increases with age until P25 and remains constant thereafter. The developmental occurrence of GlcCer and GalCer agrees well with their respective glucosyl- and galactosyltransferase activities. Cerebroside fatty-acid and base compositions, examined by gas chromatography, also change during development. Several alkali-labile fast-migrating cerebrosides (FMCs) with a higher thin-layer chromatography RF than GalCer/ GlcCer are expressed early at P10, increase in concentration with age (P25-P30) and are unchanged until maturity. They are derivatives of GalCer. By employing a newly developed neutral methylation procedure, we have confirmed the structure of one of the FMCs as 6-acylGalCer. A reduction in brain FMC concentrations along with GalCer in murine genetic dysmyelinating disorders (jimpy and quaking) further supports the conclusion that they are myelin constituents.

Cerebrosides in mature-green and red-ripe bell pepper and tomato fruits

Cerebrosides (CBs) isolated from pericarp lipids of green and red bell pepper and tomato fruits were separated by a new HPLC method into ⩾ six components. HPLC profiles were similar for the two fruits and changed little with ripening. CB 1 was the major peak, CB 2 was found only in bell pepper, and CBs 4, 5 and 6 were more abundant in pepper than in tomato. Total CBs and HPLC CB fractions were cleaved for analysis of fatty acid (FA), sphingoid (SP) and sugar moieties. FAs of total CBs were >95% saturated 2-hydroxyl. In CBs 1 and 2, 2-hydroxyl 16:0 was ⩾98% of the total FAs, whereas 2-hydroxyl 22:0, 23:0 and 24:0 predominated in CBs 4–6. The SPs of CBs 1 and 2 were tentatively identified as the 8- cis and 8- trans isomers of 4,8-sphingadienine and 8-sphingenine, respectively, whereas the SPs of CBs 4–6 were identified as isomers of 4-hydroxy-8-sphingenine. Glucose was the only sugar in bell pepper and tomato CBs. The major CB in both fruits was deduced to be 1- O- β-glucosyl- N-(2′hydroxypalmitoyl)-4- trans-8- cis-sphingadienine.