RESUMO
BACKGROUND: Mycobacterium ulcerans disease (Buruli ulcer) is a neglected tropical disease common amongst children in rural West Africa. Animal experiments have shown that tissue destruction is caused by a toxin called mycolactone. METHODOLOGY/PRINCIPAL FINDINGS: A molecule was identified among acetone-soluble lipid extracts from M. ulcerans (Mu)-infected human lesions with chemical and biological properties of mycolactone A/B. On thin layer chromatography this molecule had a retention factor value of 0.23, MS analyses showed it had an m/z of 765.6 [M+Na(+)] and on MS:MS fragmented to produce the core lactone ring with m/z of 429.4 and the polyketide side chain of mycolactone A/B with m/z of 359.2. Acetone-soluble lipids from lesions demonstrated significant cytotoxic, pro-apoptotic and anti-inflammatory activities on cultured fibroblast and macrophage cell lines. Mycolactone A/B was detected in all of 10 tissue samples from patients with ulcerative and pre-ulcerative Mu disease. CONCLUSIONS/SIGNIFICANCE: Mycolactone can be detected in human tissue infected with Mu. This could have important implications for successful management of Mu infection by antibiotic treatment but further studies are needed to measure its concentration.
Assuntos
Toxinas Bacterianas/química , Úlcera de Buruli/metabolismo , Úlcera de Buruli/microbiologia , Mycobacterium ulcerans/metabolismo , Adolescente , Adulto , Animais , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/farmacologia , Linhagem Celular , Criança , Cromatografia em Camada Fina , Feminino , Humanos , Macrolídeos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Mycobacterium ulcerans/fisiologia , Pele/metabolismo , Pele/microbiologia , Pele/patologia , Espectrometria de Massas por Ionização por Electrospray , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
Glucose in airway surface liquid (ASL) is maintained at low concentrations compared to blood glucose. Using radiolabelled [(3)H]-D: -glucose and [(14)C]-L: -glucose, detection of D: - and L: -glucose by high-performance liquid chromatography and metabolites by nuclear magnetic resonance, we found that glucose applied to the basolateral side of H441 human airway epithelial cell monolayers at a physiological concentration (5 mM) crossed to the apical side by paracellular diffusion. Transepithelial resistance of the monolayer was inversely correlated with paracellular diffusion. Appearance of glucose in the apical compartment was reduced by uptake of glucose into the cell by basolateral and apical phloretin-sensitive GLUT transporters. Glucose taken up into the cell was metabolised to lactate which was then released, at least in part, across the apical membrane. We suggest that glucose transport through GLUT transporters and its subsequent metabolism in lung epithelial cells help to maintain low glucose concentrations in human ASL which is important for protecting the lung against infection.
Assuntos
Células Epiteliais/metabolismo , Glucose/metabolismo , Acetatos/metabolismo , Transporte Biológico , Células Cultivadas , Difusão , Células Epiteliais/efeitos dos fármacos , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Homeostase , Humanos , Ácido Láctico/metabolismo , Pulmão/citologia , Floretina/farmacologia , EstereoisomerismoRESUMO
BACKGROUND: Glioblastoma, the most common primary brain tumor, has variable prognosis. We aimed to identify serum biomarkers that predict survival of patients with glioblastoma. METHODS: In phase 1 (biomarker discovery), SELDI-TOF mass spectra were studied in 200 serum samples from 58 control subjects and 36 patients with grade II astrocytoma, 15 with anaplastic astrocytoma, and 91 with glioblastoma. To identify potential biomarkers, we searched for peptide peaks that changed progressively in size with increasing malignancy. One peak, identified as the B-chain of alpha 2-Heremans-Schmid glycoprotein (AHSG), was less prominent with increasing tumor grade. We therefore investigated AHSG as a survival predictor in glioblastoma. We measured serum AHSG by turbidimetry and determined indices of malignancy, including tumor proliferation (Ki67 immunolabel) and necrosis (tumor lipids on magnetic resonance spectroscopy). In phase 2 (biomarker validation), the prognostic power of AHSG was validated in an independent group of 72 glioblastoma patients. RESULTS: Median survival was longer (51 vs 29 weeks) in glioblastoma patients with normal vs low serum AHSG concentrations (hazard ratio 2.7, 95% CI 1.5-5.0, P <0.001), independent of age and Karnofsky score. Serum AHSG inversely correlated with Ki-67 immunolabeling and tumor lipids. A prognostic index combining serum AHSG with patient age and Karnofsky score separated glioblastoma patients with short (<3 months) and long (>2 years) median survival. The prognostic value of serum AHSG was validated in a different cohort of glioblastoma patients. CONCLUSIONS: We conclude that serum AHSG concentration, measured before starting treatment, predicts survival in patients with glioblastoma.
Assuntos
Biomarcadores Tumorais/sangue , Proteínas Sanguíneas/análise , Neoplasias Encefálicas/diagnóstico , Glioblastoma/diagnóstico , Adulto , Astrocitoma/diagnóstico , Astrocitoma/mortalidade , Astrocitoma/patologia , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Feminino , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Taxa de Sobrevida , alfa-2-Glicoproteína-HSRESUMO
Nearly 50 million people worldwide suffer from age-related macular degeneration (AMD), which causes severe loss of central vision. A single-nucleotide polymorphism in the gene for the complement regulator factor H (FH), which causes a Tyr-to-His substitution at position 402, is linked to approximately 50% of attributable risks for AMD. We present the crystal structure of the region of FH containing the polymorphic amino acid His402 in complex with an analogue of the glycosaminoglycans (GAGs) that localize the complement regulator on the cell surface. The structure demonstrates direct coordination of ligand by the disease-associated polymorphic residue, providing a molecular explanation of the genetic observation. This glycan-binding site occupies the center of an extended interaction groove on the regulator's surface, implying multivalent binding of sulfated GAGs. This finding is confirmed by structure-based site-directed mutagenesis, nuclear magnetic resonance-monitored binding experiments performed for both H402 and Y402 variants with this and another model GAG, and analysis of an extended GAG-FH complex.
Assuntos
Envelhecimento/fisiologia , Fator H do Complemento/química , Fator H do Complemento/metabolismo , Sítios de Ligação , Fator H do Complemento/genética , Cristalografia por Raios X , Produtos do Gene gag/química , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Ligantes , Modelos Moleculares , Mutação/genética , Polissacarídeos/química , Polissacarídeos/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Sacarose/análogos & derivados , Sacarose/química , Sacarose/metabolismo , Propriedades de SuperfícieRESUMO
Cultivated vascular smooth muscle cells (SMCs) were surface-labeled with CyDyes followed by biotinylation. After enrichment on avidin columns, proteins were separated on large format gradient gels by SDS-PAGE. A comparison between CyDye-tagged and non-tagged gel bands revealed a substantial increase of protein identifications from membrane, membrane-associated, and extracellular matrix proteins with a corresponding reduction in co-purified intracellular proteins. Notably the majority of identified proteins were involved in cellular adhesion processes. To demonstrate the quantitative potential of this platform, we performed a comparison between mature and embryonic stem cell-derived smooth muscle cells (esSMCs) and identified the membrane proteins E-cadherin, integrin alpha6, and CD98 (4F2) to be significantly up-regulated in esSMCs suggesting that SMCs derived from embryonic stem cells maintain characteristics of their embryonic stem cell origin. This was subsequently confirmed by RT-PCR: despite expressing a panel of smooth muscle markers (calponin, Sm22, and aortic smooth muscle actin), esSMCs remained positive for markers of stem cell pluripotency (Oct4, Nanog, and Rex1). In summary, we describe a novel strategy for the profiling of cell membrane proteins. The procedure combines DIGE technology with biotin/avidin labeling to discriminate membrane and membrane-associated proteins from intracellular contaminants by fluorescence tagging and permits semiquantitative differential expression analysis of membrane proteins.
Assuntos
Biotina/metabolismo , Corantes/metabolismo , Células-Tronco Embrionárias/química , Proteínas de Membrana/análise , Miócitos de Músculo Liso/química , Miócitos de Músculo Liso/citologia , Coloração e Rotulagem/métodos , Sequência de Aminoácidos , Animais , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Células-Tronco Embrionárias/citologia , Espectrometria de Massas , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismoRESUMO
Pools of O-glycopeptides (and their deglycosylated analogues) derived from trypsin-digested normal human serum IgA1 have been treated with ammonia under conditions reported to result in complete liberation of O-glycans linked to serine and threonine residues in glycopeptides and glycoproteins. MALDI-TOF MS analysis has revealed that only one of the six glycosylated sites is susceptible to beta-elimination under these conditions. It is likely that resistance to beta-elimination is due to very close proximity of proline to the glycosylated serine or threonine residues. Preliminary results using 0.1M NaOH (instead of ammonia) to perform beta-elimination indicated that there was also selective de-O-glycosylation with this reagent, however, these results were complicated by the concomitant hydrolysis of the peptide bonds. These findings may have implications for similarly O-glycosylated peptides and proteins and possibly for other chemical methods that are used to carry out beta-eliminations of O-glycans.
Assuntos
Amônia/química , Química/métodos , Glicopeptídeos/química , Glicosilação , Imunoglobulina A/química , Sequência de Aminoácidos , Animais , Humanos , Modelos Químicos , Dados de Sequência Molecular , Neuraminidase/química , Peptídeos/química , Serina/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Treonina/químicaRESUMO
Henoch-Schönlein purpura is characterized by immunoglobulin A1 (IgA1) depositions in blood vessels of the skin or in glomeruli, resulting from altered hinge region O-glycosylation. Henoch-Schönlein purpura is seldom reported as a complication of IgA1 myeloma, even when the circulating IgA concentration is very high. We report two patients with IgA1 myeloma presenting with Henoch-Schönlein purpura. The O-glycosylation of these patients' IgA1 was studied. Both patients showed increased binding to peanut agglutinin lectin, suggesting a low degree of sialylation of the hinge region of IgA1 that was confirmed by mass spectrometry. IgA multiple myeloma, secreting IgA1 molecules with decreased sialylation, presenting with a Henoch-Schönlein purpura-like syndrome was diagnosed.