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1.
Sci Rep ; 13(1): 22560, 2023 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-38110595

RESUMO

This study explores the sphingolipid class of oligohexosylceramides (OHCs), a rarely studied group, in barley (Hordeum vulgare L.) through a new lipidomics approach. Profiling identified 45 OHCs in barley (Hordeum vulgare L.), elucidating their fatty acid (FA), long-chain base (LCB) and sugar residue compositions; and was accomplished by monophasic extraction followed by reverse-phased high performance liquid chromatography electrospray ionisation quadrupole-time-of-flight tandem mass spectrometry (HPLC-ESI-QqTOF-MS/MS) employing parallel reaction monitoring (PRM). Results revealed unknown ceramide species and highlighted distinctive FA and LCB compositions when compared to other sphingolipid classes. Structurally, the OHCs featured predominantly trihydroxy LCBs associated with hydroxylated FAs and oligohexosyl residues consisting of two-five glucose units in a linear 1 → 4 linkage. A survey found OHCs in tissues of major cereal crops while noting their absence in conventional dicot model plants. This study found salinity stress had only minor effects on the OHC profile in barley roots, leaving questions about their precise functions in plant biology unanswered.


Assuntos
Glicoesfingolipídeos Neutros , Espectrometria de Massas em Tandem , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Grão Comestível , Esfingolipídeos , Ácidos Graxos , Espectrometria de Massas por Ionização por Electrospray/métodos
2.
Glycoconj J ; 40(5): 551-563, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37606864

RESUMO

Globo A is a neutral Globo-series glycosphingolipid (GSL) that shows natural properties of a cytotoxicity receptor NKp44 binding ligand. The highly complex heptasaccharide glycan structure of Globo A combined with its biological profile provides a unique target for the development of a synthetic method to facilitate its bioactivity studies. Here, a concise chemoenzymatic route to the synthesis of Globo A and its α1,3-galactose-linked congener Globo B is reported. The key to success was the use of a synthetic azido ß-Globo H sphingosine (Globo H-ßSph) as an acceptor substrate and two glycosyl transferases, an α1,3-N-acetylgalactosaminyltransferase from Helicobacter mustelae (BgtA) and a human blood group B α1,3-galactosyltransferase (h1,3GTB), for stereoselective construction of the terminal α1,3-GalNAc and α1,3-Gal linkages, respectively. The azido-Sph lipid sidechain is further elaborated by reduction and a chemoselective N-acylation to complete the total synthesis of the neutral Globo-series GSLs. In addition, the synthesis of Forssman and para-Forssman antigens were prepared. The strategy may be suitable for accessing other complex GSLs and related lipid-modified GSL derivatives.


Assuntos
Glicoesfingolipídeos , Glicoesfingolipídeos Neutros , Humanos , Glicoesfingolipídeos/metabolismo , Ligação Proteica
3.
Methods Mol Biol ; 2613: 127-144, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36587076

RESUMO

This chapter describes the protocols for mass spectrometry (MS) applied to the structural characterization of neutral glycosphingolipids (GSLs) and the determination of neutral GSL contents in biological materials. The structural characterization is performed by thin layer chromatography-matrix assisted laser desorption ionization/mass spectrometry (TLC-MALDI/MS) and liquid chromatography-electrospray ionization/mass spectrometry (LC-ESI/MS) with reversed phase separation. The content determination is carried out by LC-ESI/MS with multiple reaction monitoring (MRM). These protocols provide clues for the functions of neutral GSLs at the level of a single GSL molecular species.


Assuntos
Glicoesfingolipídeos Neutros , Glicoesfingolipídeos Neutros/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas por Ionização por Electrospray , Cromatografia Líquida , Cromatografia em Camada Delgada/métodos , Glicoesfingolipídeos/química
4.
Analyst ; 147(13): 3101-3108, 2022 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-35695136

RESUMO

LcGg4, a neutral glycosphingolipid (GSL) and cancer antigen, its epimers GalNAc-LcGg4 and GlcNAc-LcGg4, and three lipid forms of GalNAc-LcGg4 were studied by mass spectrometry (MS). It was found that different forms of GalNAc-LcGg4 carrying homologous (d16:1/18:0) and (d18:1/18:0) lipids were easily separated and identified using liquid chromatography (LC)-MS. In addition, like gangliosides, homologous lipid forms of GalNAc-LcGg4 showed the same fragmentation pattern, except for a uniform shift of their glycolipid product ions by a certain m/z number determined by the varied lipid structure. It was also disclosed that LcGg4 and its epimers GalNAc-LcGg4 and GlcNAc-LcGg4, which are different only in the C4-configuration of their non-reducing end sugar residues, gave the same MS/MS product ions in similar relative intensities, as well as the same LC retention time, suggesting the challenge to differentiate epimeric GSLs by LC-MS. However, ion mobility spectrometry (IMS)-MS was able to efficiently separate and distinguish these epimers. This study has demonstrated the promise of IMS-MS for isomeric GSL characterization and the IMS-MS and LC-MS/MS combination for natural GSL analysis.


Assuntos
Espectrometria de Mobilidade Iônica , Glicoesfingolipídeos Neutros , Cromatografia Líquida/métodos , Gangliosídeos , Espectrometria de Massas em Tandem
5.
Nutrients ; 12(4)2020 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-32252241

RESUMO

Sphingolipid metabolism plays a critical role in cell growth regulation, lipid regulation, neurodevelopment, type 2 diabetes, and cancer. Animal experiments suggest that vitamin D may be involved in sphingolipid metabolism regulation. In this study, we tested the hypothesis that vitamin D supplementation would alter circulating long-chain ceramides and related metabolites involved in sphingolipid metabolism in humans. We carried out a post-hoc analysis of a previously conducted randomized, placebo-controlled clinical trial in 70 overweight/obese African-Americans, who were randomly assigned into four groups of 600, 2000, 4000 IU/day of vitamin D3 supplements or placebo for 16 weeks. The metabolites were measured in 64 subjects (aged 26.0 ± 9.4 years, 17% male). Serum levels of N-stearoyl-sphingosine (d18:1/18:0) (C18Cer) and stearoyl sphingomyelin (d18:1/18:0) (C18SM) were significantly increased after vitamin D3 supplementation (ps < 0.05) in a dose-response fashion. The effects of 600, 2000, and 4000 IU/day vitamin D3 supplementation on C18Cer were 0.44 (p = 0.049), 0.52 (p = 0.016), and 0.58 (p = 0.008), respectively. The effects of three dosages on C18SM were 0.30 (p = 0.222), 0.61 (p = 0.009), and 0.68 (p = 0.004), respectively. This was accompanied by the significant correlations between serum 25-hydroxyvitamin D3 [25(OH)D] concentration and those two metabolites (ps < 0.05). Vitamin D3 supplementations increase serum levels of C18Cer and C18SM in a dose-response fashion among overweight/obese African Americans.


Assuntos
Negro ou Afro-Americano , Calcifediol/sangue , Colecalciferol/administração & dosagem , Glicoesfingolipídeos Neutros/metabolismo , Obesidade/metabolismo , Adulto , Negro ou Afro-Americano/etnologia , Colecalciferol/metabolismo , Suplementos Nutricionais , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Humanos , Masculino , Obesidade/etnologia , Sobrepeso/etnologia , Sobrepeso/metabolismo
6.
Anal Biochem ; 581: 113348, 2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31251925

RESUMO

Skin fibroblasts are recognized as a valuable model of primary human cells able of mirroring the chronological and biological aging. Here, a lipidomic study of glycosphingolipids (GSL) occurring in the easily accessible human dermal fibroblasts (HDF) is presented. Reversed-phase liquid chromatography with negative electrospray ionization (RPLC-ESI) coupled to either orbitrap or linear ion-trap multiple-stage mass spectrometry was applied to characterize GSL in commercially adult and neonatal primary human fibroblast cells and in skin samples taken from an adult volunteer. Collision-induced dissociation in negative ion mode allowed us to get information on the monosaccharide number and ceramide composition, whereas tandem mass spectra on the ceramide anion was useful to identify the sphingoid base. Nearly sixty endogenous GSL species were successfully recognized, namely 33 hexosyl-ceramides (i.e., HexCer, Hex2Cer and Hex3Cer) and 24 gangliosides as monosialic acid GM1, GM2 and GM3, along with 5 globosides Gb4. An average content of GSLs was attained and the most representative GSL in skin fibroblasts were Hex3Cer, also known as Gb3Cer, followed by Gb4, HexCer and Hex2Cer , while gangliosides were barely quantifiable. The most abundant GSLs in the examined cell lines share the same ceramide base (i.e. d18:1) and the relative content was d18:1/24:1 > d18:1/24:0 > d18:1/16:0 > d18:1/22:0.


Assuntos
Glicoesfingolipídeos Acídicos , Derme/metabolismo , Fibroblastos/metabolismo , Glicoesfingolipídeos Neutros , Glicoesfingolipídeos Acídicos/análise , Glicoesfingolipídeos Acídicos/metabolismo , Adulto , Células Cultivadas , Derme/citologia , Fibroblastos/citologia , Humanos , Glicoesfingolipídeos Neutros/análise , Glicoesfingolipídeos Neutros/metabolismo
7.
J Biochem ; 163(3): 201-214, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29069405

RESUMO

Glycosphingolipids (GSLs) from the silkworm Bombyx mori were identified and GSL expression patterns between larvae and pupae were compared. The structural analysis of neutral GSLs from dried pupae revealed the following predominant species: Glcß1Cer, Manß4Glcß1Cer, GlcNAcß3Manß4Glcß1Cer, Galß3Manß4Glcß1Cer, GalNAcα4Galß3Manß4Glcß1Cer, GlcNAcß3Galß3Manß4Glcß1Cer, Galα4Galß3Manß4Glcß1Cer and (GalNAcα4)1-4 GalNAcα4Galß3Manß4Glcß1Cer. Lin-ear elongation of α4-GalNAc was observed at the non-reducing end of Galß3Manß4Glcß1Cer with up to five GalNAc repeats. The arthro-series GSL GlcNAcß3Manß4Glcß1Cer, a characteristic GSL-glycan sequence of other Arthropoda, was detected in silkworms. The main ceramide species in each purified GSL fraction were h20:0-d14:1 and h22:0-d14:1. GSL expression patterns in larvae and pupae were compared using thin-layer chromatography, which demonstrated differences among acidic, polar and neutral GSL fractions, while the zwitterionic fraction showed no difference. Neutral GSLs such as ceramides di-, tri- and tetrasaccharides in larvae showed less abundant than those in pupae. MALDI-TOF MS analysis revealed that larval GSLs contained four types of ceramide species, whereas pupal GSLs contained only two types. The structural analysis of neutral GSLs from silkworms revealed a novel series of GSLs. The comparison of GSL expression patterns between larvae and pupae demonstrated differences in several fractions. Alterations in GSL ceramide composition between larvae and pupae were observed by MALDI-TOF MS analysis.


Assuntos
Bombyx/química , Ceramidas/química , Larva/química , Glicoesfingolipídeos Neutros/química , Pupa/química , Animais , Configuração de Carboidratos
8.
Glycoconj J ; 34(1): 127-138, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27796613

RESUMO

Silurus asotus egg lectin (SAL), an α-galactoside-binding protein isolated from the eggs of catfish, is a member of the rhamnose-binding lectin family that binds to Gb3 glycan (Galα1-4Galß1-4Glc). We have previously demonstrated that SAL reduces the proliferation of Gb3-expressing Burkitt's lymphoma Raji cells and confirm here that it does not reduce their viability, indicating that unlike other lectins, it is not cytotoxic. The aim of this study was to determine the signal transduction mechanism(s) underlying this novel SAL/Gb3 binding-mediated effect profile. SAL/Gb3 interaction arrested the cell cycle through increasing the G0/1 phase population of Raji cells. SAL suppressed the transcription of cell cycle-related factors such as c-MYC, cyclin D3, and cyclin-dependent protein kinase (CDK)-4. Conversely, the CDK inhibitors p21 and p27 were elevated by treatment with SAL. In particular, the production of p27 in response to SAL treatment increased steadily, whereas p21 production was maximal at 12 h and lower at 24 h. Activation of Ras-MEK-ERK pathway led to an increase in expression of p21. Notably, treatment of Raji cells with anti-Gb3 mAb alone did not produce the above effects. Taken together, our findings suggest that Gb3 on the Raji cell surface interacts with SAL to trigger sequential GDP-Ras phosphorylation, Ras-MEK-ERK pathway activation, p21 production, and cell cycle arrest at the G0/1 phase.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Peixes/farmacologia , Lectinas/farmacologia , Glicoesfingolipídeos Neutros/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/toxicidade , Linfoma de Burkitt/metabolismo , Peixes-Gato , Linhagem Celular Tumoral , Ciclina D3/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Proteínas de Peixes/química , Proteínas de Peixes/toxicidade , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Lectinas/química , Lectinas/toxicidade , Sistema de Sinalização das MAP Quinases , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ramnose/metabolismo
9.
Sci Rep ; 6: 30336, 2016 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-27458147

RESUMO

Shiga toxin (Stx), an AB5 toxin, binds specifically to the neutral glycosphingolipid Gb3 at the cell surface before being transported into cells. We here demonstrate that addition of conical lysophospholipids (LPLs) with large head groups inhibit Stx binding to cells whereas LPLs with small head groups do not. Lysophosphatidylinositol (LPI 18:0), the most efficient LPL with the largest head group, was selected for in-depth investigations to study how the binding of Stx is regulated. We show that the inhibition of Stx binding by LPI is reversible and possibly regulated by cholesterol since addition of methyl-ß-cyclodextrin (mßCD) reversed the ability of LPI to inhibit binding. LPI-induced inhibition of Stx binding is independent of signalling and membrane turnover as it occurs in fixed cells as well as after depletion of cellular ATP. Furthermore, data obtained with fluorescent membrane dyes suggest that LPI treatment has a direct effect on plasma membrane lipid packing with shift towards a liquid disordered phase in the outer leaflet, while lysophosphoethanolamine (LPE), which has a small head group, does not. In conclusion, our data show that cellular treatment with conical LPLs with large head groups changes intrinsic properties of the plasma membrane and modulates Stx binding to Gb3.


Assuntos
Lisofosfolipídeos/farmacologia , Glicoesfingolipídeos Neutros/metabolismo , Toxina Shiga/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células HeLa , Humanos , Lisofosfolipídeos/química , Ligação Proteica , beta-Ciclodextrinas/farmacologia
10.
Anal Chem ; 88(11): 5595-9, 2016 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-27212679

RESUMO

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to simultaneously visualize the lateral distribution of different lipid classes in tissue sections, but the applicability of the method to real-life samples is often limited by ion suppression effects. In particular, the presence of abundant phosphatidylcholines (PCs) can reduce the ion yields for all other lipid species in positive ion mode measurements. Here, we used on-tissue treatment with buffer-free phospholipase C (PLC) to near-quantitatively degrade PCs in fresh-frozen tissue sections. The ion signal intensities of mono-, di-, and oligohexosylceramides were enhanced by up to 10-fold. In addition, visualization of Shiga toxin receptor globotriaosylceramide (Gb3Cer) in the kidneys of wild-type and α-galactosidase A-knockout (Fabry) mice was possible at about ten micrometer resolution. Importantly, the PLC treatment did not decrease the high lateral resolution of the MS imaging analysis.


Assuntos
Encéfalo/enzimologia , Rim/enzimologia , Glicoesfingolipídeos Neutros/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fosfolipases Tipo C/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Glicoesfingolipídeos Neutros/metabolismo , Fosfolipases Tipo C/química
11.
Lipids ; 50(9): 913-7, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26017029

RESUMO

In recent years, obesity has been considered a pathological stage of early lifestyle-related diseases, and adipose tissue and adipocyte research has been active. Glycosphingolipids are involved in the pathogenesis of type 2 diabetes induced by insulin resistance, but the details of the glycosphingolipid molecular species composition of adipocytes have yet to be elucidated. We used 3T3-L1 adipocytes and the 1,2-dichloroethane-wash method to remove triacylglycerols, which are abundant in adipocytes, and analyzed the structures of glycosphingolipids, particularly neutral glycosphingolipids, using liquid chromatography-mass spectrometry.


Assuntos
Células 3T3-L1/química , Adipócitos/química , Glicoesfingolipídeos Neutros/química , Animais , Linhagem Celular , Cromatografia Líquida , Fibroblastos/química , Espectrometria de Massas , Camundongos
12.
Glycoconj J ; 31(3): 209-19, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24310545

RESUMO

We previously performed a systematic analysis of free ceramide (Cers) species, the constituent ceramide species of sphingomyelins and neutral glycosphingolipids (NGSLs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with high-energy collision-induced dissociation. As a result, distinct species differences were found among Cers, sphingomyelins and NGSLs in the kidneys. Using this method, we investigated various sphingolipid species from human colon cancer Caco-2 cells as well as the influence of environmental oxygen on these species in detail. Unexpectedly, even in normoxia, all Cers species were composed of dihydrosphingosine (d18:0) and non-hydroxy fatty acid (NFA), and 34% of sphingomyelins were composed of dihydrosphingomyelins with NFA. In contrast, major constituent ceramide species of NGSLs were composed of the usual long-chain base of sphingosine (d18:1) and hydroxy fatty acid (HFA). When the cells were cultured under hypoxic condition for 3 days, all the Cers and nearly 80% of the sphingomyelins were dihydrosphingolipids composed of d18:0-NFAs, but a significant proportion of d18:1-HFAs still remained in the NGSLs. When the cells were transferred from conditions of hypoxia to normoxia again (reoxygenation), Cer species composed of d18:1-NFAs, which were not found in Cers under the original normoxic conditions, appeared. Such Cers were probably synthesized as precursors for the constituent ceramides of sphingomyelins and NGSLs.


Assuntos
Ceramidas/análise , Glicoesfingolipídeos Neutros/química , Oxigênio/metabolismo , Esfingomielinas/química , Células CACO-2 , Hipóxia Celular , Ceramidas/química , Ceramidas/metabolismo , Ácidos Graxos/análise , Ácidos Graxos/química , Humanos , Glicoesfingolipídeos Neutros/análise , Glicoesfingolipídeos Neutros/metabolismo , Esfingomielinas/análise , Esfingomielinas/metabolismo , Esfingosina/análogos & derivados , Esfingosina/análise , Espectrometria de Massas em Tandem
13.
Glycoconj J ; 30(3): 257-68, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22890904

RESUMO

Brine shrimp are primitive crustacean arthropodal model organisms, second to daphnia, which can survive in high-salinity environments. Their oviposited cysts, cuticle-covered diapausing eggs, are highly resistant to dryness. To elucidate specialties of brine shrimp, this study characterized glycosphingolipids, which are signal transduction-associated material. A group of novel and complex fucosyl glycosphingolipids were separated and identified from cysts of the brine shrimp Artemia franciscana by repeated lipid extraction, alkaline methanolysis, acid treatment, successive column chromatography, and post-source decay measurements by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Structures of the glycosphingolipids were elucidated by conventional structural characterization and mass spectrometry, and the compounds were identified as GlcNAcß1-3GalNAcß1-4(GlcNAcα1-2Fucα1-3)GlcNAcß1-3Manß1-4Glcß1-Cer, GalNAcß1-4(Fucα1-3)GlcNAcß1-3GalNAcß1-4(GlcNAcα1-2Fucα1-3)GlcNAcß1-3Manß1-4Glcß1-Cer, and GalNAcß1-4(GlcNAcα1-2Fucα1-3)GlcNAcß1-3GalNAcß1-4(GlcNAcα1-2Fucα1-3)GlcNAcß1-3Manß1-4Glcß1-Cer. These compounds also contained a branching, non-arthro-series disaccharide with an α-GlcNAc terminus, similar to that found in a previously reported ceramide hexasaccharide (III(3)(GlcNAcα2Fucα)-At4Cer). The glycans within these complex GSLs are longer than reported glycans of the animal kingdom containing α-GlcNAc terminus. These complex GSLs as well as the longest GSL with ten sugar residues, ceramide decasaccharide (CDeS), contain the fucosylated LacdiNAc sequence reported to associate with parasitism/immunosuppression and the α-GlcNAc terminus reported to show a certain antibacterial effect in other reports. CDeS, the longest GSL of this species, was found in the highest amount, which indicates that CDeS may be functionally important.


Assuntos
Artemia/química , Glicoesfingolipídeos Neutros/química , Animais , Sequência de Carboidratos , Polissacarídeos/química
14.
Biochim Biophys Acta ; 1811(11): 861-74, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21740982

RESUMO

This mini-review summarizes the protocol we have developed for the analysis of neutral glycosphingolipids (GSLs) by high-performance thin layer chromatography (HPTLC)-mass spectrometry (MS). We also present results obtained using this glycolipidomic approach to study neutral GSLs from mouse kidney, spleen, and small intestine. Finally, we discuss what is required for further development of this method, as well as what is expected for the future of glycolipid biology.


Assuntos
Cromatografia em Camada Delgada/métodos , Espectrometria de Massas/métodos , Glicoesfingolipídeos Neutros/análise , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Glicoesfingolipídeos Neutros/química , Especificidade de Órgãos , Soluções
15.
Ideggyogy Sz ; 64(1-2): 29-35, 2011 Jan 30.
Artigo em Húngaro | MEDLINE | ID: mdl-21428036

RESUMO

BACKGROUND: Fabry-disease (FD) is a rare X-linked lysosomal storage disease. Deficiency of alpha-galactosidase A activity leads to the accumulation of neutral glycosphingolipids, primarily globotriaosylceramide (GL-3) in various tissues, particularly blood vessels, kidneys, myocardium and in ganglions of the peripheral and autonomic nervous system and causes diverse symptoms. The classical phenotype is seen in most males and rarely in females. In women, symptoms start later and the severity is milder. Both peripheral and central nervous system can be both affected. OBJECTIVES: Fabry-patiens and gene-carriers in the central region of Hungary are treated in the 2nd Pediatric Department, Semmelweis University. These patients are consulted by an interdisciplinary team. At present, four hemizygous male, four heterozygous female Fabry-patients and three asymptomatic heterozygous gene carriers are followed. RESULTS: After reviewing the neurological complications of FD, we present clinical and neuroimaging data of our patients. CONCLUSION: We emphasize that neurologists should suspect the rare monogenic FD in the case of acroparaesthesia and heat-cold intolerance in childhood or adolescence; clinical signs of TIA/stroke or unexplained MRI alterations suggesting small vessel disease in young adults. Early diagnosis and introduction of enzyme replacement therapy (ERT) can halt or reverse progression.


Assuntos
Doença de Fabry/complicações , Doença de Fabry/diagnóstico , Doenças do Sistema Nervoso/enzimologia , Fatores Etários , Doenças do Sistema Nervoso Central/enzimologia , Progressão da Doença , Terapia de Reposição de Enzimas , Doença de Fabry/enzimologia , Doença de Fabry/genética , Doença de Fabry/metabolismo , Feminino , Heterozigoto , Humanos , Masculino , Doenças do Sistema Nervoso/metabolismo , Glicoesfingolipídeos Neutros/metabolismo , Doenças do Sistema Nervoso Periférico/enzimologia , Fenótipo , Transtornos das Sensações/enzimologia , Índice de Gravidade de Doença , Fatores Sexuais , alfa-Galactosidase/uso terapêutico
16.
Glycoconj J ; 28(2): 67-87, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21400001

RESUMO

Free ceramides and glycosphingolipids (GSLs) are important components of the membrane microdomain and play significant roles in cell survival. Recent studies have revealed that both fatty acids and long-chain bases (LCBs) are more diverse than expected, in terms of i) alkyl chain length, ii) hydroxylation and iii) the presence or absence of double bonds. Electrospray ionization mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been well utilized to characterize sphingolipids with high throughput, but reports to date have not fully characterized various types of ceramide species such as hydroxyl fatty acids and/or trihydroxy-LCBs of both free ceramides and the constituent ceramides in neutral GSLs. We performed a systematic analysis of both ceramide species, including LCBs with nona-octadeca lengths using MALDI-TOF MS with high-energy collision-induced dissociation (CID) at 20 keV. Using both protonated and sodiated ions, this technique enabled us to propose general rules to discriminate between isomeric and isobaric ceramide species, unrelated to the presence or absence of sugar chains. In addition, this high-energy CID generated (3,5)A ions, indicating Hex 1-4 Hex linkage in the sugar chains. Using this method, we demonstrated distinct differences among ceramide species, including free ceramides, sphingomyelins, and neutral GSLs of glucosylceramides, galactosylceramides, lactosylceramides, globotriaosylceramides and Forssman glycolipids in the equine kidneys.


Assuntos
Ceramidas/química , Glicoesfingolipídeos Neutros/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Galactosilceramidas/química , Glucosilceramidas/química , Cavalos , Rim/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
17.
Microbiol Immunol ; 55(2): 135-40, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21265875

RESUMO

Dengue viruses infect cells by attaching to a surface receptor which remains unknown. The putative receptor molecules of dengue virus type 2 on the surface of mosquito (AP-61) and mammalian (LLC-MK2) cell lines were investigated. The immunochemical detection and structural analysis of carbohydrates demonstrated that the neutral glycosphingolipids, L-3 (GlcNAcß1-3Manß1-4Glcß1-1'Cer) in AP-61 cells, and nLc(4) Cer (Galß1-4GlcNAcß1-3Galß1-4Glcß1-1'Cer) in LLC-MK2 cells were recognized by the virus. These findings strongly suggest that neutral glycosphingolipids share the key determinant for virus binding and that the ß-GlcNAc residue may play an important role in dengue virus binding to the host cell surface.


Assuntos
Culicidae/metabolismo , Vírus da Dengue/metabolismo , Dengue/metabolismo , Insetos Vetores/metabolismo , Mamíferos/metabolismo , Glicoesfingolipídeos Neutros/metabolismo , Animais , Sequência de Carboidratos , Linhagem Celular , Culicidae/virologia , Dengue/virologia , Humanos , Insetos Vetores/virologia , Células K562 , Macaca mulatta , Mamíferos/virologia , Dados de Sequência Molecular , Glicoesfingolipídeos Neutros/química
18.
J Lipid Res ; 52(2): 308-17, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21062954

RESUMO

Neutral glycosphingolipids containing one to six sugars in their oligosaccharide chains have been isolated from cysts of the brine shrimp Artemia franciscana. The structures of these glycolipids were identified by methylation analysis, partial acid hydrolysis, gas-liquid chromatography, combined gas-liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and proton nuclear magnetic resonance spectroscopy to be Glcß1-Cer, Manß1-4Glcß1-Cer, Fucα1-3Manß1-4Glcß1-Cer, GlcNAcß1-3Manß1-4Glcß1-Cer, GlcNAcα1-2Fucα1-3Manß1-4Glcß1-Cer, GalNAcß1-4GlcNAcß1-3Manß1-4Glcß1-Cer, GalNAcß1-4(Fucα1-3)GlcNAcß1-3Manß1-4Glcß1-Cer (CPS), and GalNAcß1-4(GlcNAcα1-2Fucα1-3)GlcNAcß1-3Manß1-4Glcß1-Cer (CHS). Two glycosphingolipids, CPS and CHS, were characterized as novel structures. Because Artemia contains a certain series of glycosphingolipids (-Fucα3Manß4GlcßCer), which differ from the core sugar sequences reported thus far, we tentatively designated the glycosphingolipids characterized as nonarthro-series ones. Furthermore, CHS exhibited a hybrid structure of arthro-series and nonarthro-series sugar chain. Two novel glycosphingolipids were characterized from the brine shrimp Artemia franciscana; one was composed of arthrotetraose and a branching fucose attached to N-acetylglucosamine residue, and the other was composed of CPS with an additional N-acetylglucosamine residue attached to the branching fucose.


Assuntos
Artemia/química , Glicoesfingolipídeos Neutros/química , Animais , Sequência de Carboidratos , Metilação , Dados de Sequência Molecular
19.
J Lipid Res ; 51(8): 2282-94, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20444989

RESUMO

Shiga toxin (Stx)-producing Escherichia coli are the leading cause of hemorrhagic colitis and life-threatening extraintestinal complications in humans. Stx1 and Stx2 are transferred by yet to be delineated mechanisms from the intestine to the circulation where they injure microvascular endothelial cells. The resulting vascular lesions cause renal failure and brain damage. Because lipoproteins are potential carriers of Stx through the circulation, we investigated human lipoprotein-associated neutral glycosphingolipids (GSLs) with emphasis on high (globotriaosylceramide) and low (globotetraosylceramide) affinity Stx-receptors. TLC overlay employing Stx1, Stx2, and anti-GSL antibodies demonstrated preferential distribution of globo-series GSLs to very low- and low-density lipoproteins compared with minor association with high-density lipoproteins. Electrospray ionization quadrupole time-of-flight mass spectrometry portrayed C24:0/C24:1 and C16:0 as the major fatty acid of the ceramide moieties of Stx-receptors carrying nonvarying d18:1 sphingosine. This structural heterogeneity was also found in precursor lactosylceramide, glucosylceramide, and galactosylceramide, the last showing an exceptionally high degree of hydroxylated C24 fatty acids. Our findings provide the basis for exploring the functional role of lipoprotein-associated Stx-receptors in human blood.


Assuntos
Análise Química do Sangue/métodos , Análise Química do Sangue/normas , Lipoproteínas/química , Espectrometria de Massas/métodos , Espectrometria de Massas/normas , Glicoesfingolipídeos Neutros/sangue , Triexosilceramidas/sangue , Anticorpos/imunologia , Cerebrosídeos/sangue , Cromatografia em Camada Delgada , Gangliosídeos/sangue , Humanos , Imunoensaio , Glicoesfingolipídeos Neutros/química , Glicoesfingolipídeos Neutros/imunologia , Padrões de Referência , Triexosilceramidas/química
20.
J Dairy Sci ; 93(1): 19-26, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20059899

RESUMO

Milk glycosphingolipids (GSL) have been reported to participate in the newborn's defense against pathogens. Taking this into account, in this study we determined the neutral GSL content of ovine milk, including its fatty acid profile. Its role in bacterial adhesion was also addressed by immunodetection of separate GSL in a high-performance thin-layer chromatography overlay assay. Ovine milk has a neutral GSL pattern similar to human milk and includes lactosylceramide (LacCer; 45.7%), monohexosylceramide (glucosylceramide and galactosylceramide, 31.2%), globotriaosylceramide (Gb3; 19.1%), and globotetraosylceramide (Gb4; 3.5%). Globotriaosylceramide and Gb4 are present in human but not bovine milk. Neutral GSL contained C23:0 and C24:0 as the most abundant fatty acids, a finding consistent with its high content of very long chain fatty acids (longer than C20). Most fatty acids were saturated and had a low content of polyunsaturated fatty acids. Bovine enterotoxigenic Escherichia coli strains bound strongly to LacCer and showed a weak binding to monohexosylceramide. The K99 strain also bound strongly to Gb3, and F41 to Gb4. Lactosylceramide, monohexosylceramide, and Gb3 were also observed to bind to human uropathogenic E. coli strains. The results reported here show the ability of neutral GSL in ovine milk to bind to E. coli strains. These compounds could be used as an alternative and available source to supplement infant or bovine formulas with a view to preventing bacterial infections.


Assuntos
Leite/química , Glicoesfingolipídeos Neutros/análise , Glicoesfingolipídeos Neutros/química , Animais , Bovinos , Escherichia coli/metabolismo , Ácidos Graxos/análise , Humanos , Leite/microbiologia , Ovinos
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