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1.
Adv Immunol ; 102: 245-76, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19477323

RESUMO

To better understand the immune basis for chronic inflammatory lung disease, we analyzed a mouse model of lung disease that develops after respiratory viral infection. The disease that develops in this model is similar to asthma and chronic obstructive pulmonary disease (COPD) in humans and is manifested after the inciting virus has been cleared to trace levels. The model thereby mimics the relationship of paramyxoviral infection to the development of childhood asthma in humans. When the acute lung disease appears in this model (at 3 weeks after viral inoculation), it depends on an immune axis that is initiated by expression and activation of the high-affinity IgE receptor (FcvarepsilonRI) on conventional lung dendritic cells (cDCs) to recruit interleukin (IL)-13-producing CD4(+) T cells to the lower airways. However, when the chronic lung disease develops fully (at 7 weeks after inoculation), it is driven instead by an innate immune axis that relies on invariant natural killer T (iNKT) cells that are programmed to activate macrophages to produce IL-13. The interaction between iNKT cells and macrophages depends on contact between the semi-invariant Valpha14Jalpha18-TCR on lung iNKT cells and the oligomorphic MHC-like protein CD1d on macrophages as well as NKT cell production of IL-13 that binds to the IL-13 receptor (IL-13R) on the macrophage. This innate immune axis is also activated in the lungs of humans with severe asthma or COPD based on detection of increased numbers of iNKT cells and alternatively activated IL-13-producing macrophages in the lung. Together, the findings identify an adaptive immune response that mediates acute disease and an innate immune response that drives chronic inflammatory lung disease in experimental and clinical settings.


Assuntos
Pneumopatias/etiologia , Viroses/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Quimiocinas CC/biossíntese , Doença Crônica , Via Alternativa do Complemento , Células Dendríticas/imunologia , Humanos , Interleucina-13/fisiologia , Macrófagos/fisiologia , Células T Matadoras Naturais/imunologia , Receptores de IgE/análise , Viroses/complicações
2.
J Lipid Res ; 48(8): 1801-24, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17488996

RESUMO

Acidic glycosphingolipid components were extracted from the opportunistic mycopathogen Aspergillus fumigatus and identified as inositol phosphorylceramide and glycosylinositol phosphorylceramides (GIPCs). Using nuclear magnetic resonance sppectroscopy, mass spectrometry, and other techniques, the structures of six major components were elucidated as Ins-P-Cer (Af-0), Manp(alpha1-->3)Manp(alpha1-->2)Ins-P-Cer (Af-2), Manp(alpha1-->2)Manp(alpha1-->3)Manp(alpha1-->2)Ins-P-Cer (Af-3a), Manp(alpha1-->3)[Galf(beta1-->6)]Manp(alpha1-->2)-Ins-P-Cer (Af-3b), Manp(alpha1-->2)-Manp(alpha1-->3)[Galf(beta1-->6)]Manp(alpha1-->2)Ins-P-Cer (Af-4), and Manp(alpha1-->3)Manp(alpha1-->6)GlcpN(alpha1-->2)Ins-P-Cer (Af-3c) (where Ins = myo-inositol and P = phosphodiester). A minor A. fumigatus GIPC was also identified as the N-acetylated version of Af-3c (Af-3c*), which suggests that formation of the GlcNalpha1-->2Ins linkage may proceed by a two-step process, similar to the GlcNalpha1-->6Ins linkage in glycosylphosphatidylinositol (GPI) anchors (transfer of GlcNAc, followed by enzymatic de-N-acetylation). The glycosylinositol of Af-3b, which bears a distinctive branching Galf(beta1-->6) residue, is identical to that of a GIPC isolated previously from the dimorphic mycopathogen Paracoccidioides brasiliensis (designated Pb-3), but components Af-3a and Af-4 have novel structures. Overlay immunostaining of A. fumigatus GIPCs separated on thin-layer chromatograms was used to assess their reactivity against sera from a patient with aspergillosis and against a murine monoclonal antibody (MEST-1) shown previously to react with the Galf(beta1-->6) residue in Pb-3. These results are discussed in relation to pathogenicity and potential approaches to the immunodiagnosis of A. fumigatus.


Assuntos
Aspergillus fumigatus/metabolismo , Glicoesfingolipídeos/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Sequência de Carboidratos , Glicoesfingolipídeos/metabolismo , Dados de Sequência Molecular , Monossacarídeos/química
3.
J Biol Chem ; 280(44): 37204-16, 2005 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-16115860

RESUMO

In humans and rodents, the lysosomal catabolism of core Man(3)GlcNAc(2) N-glycan structures is catalyzed by the concerted action of several exoglycosidases, including a broad specificity lysosomal alpha-mannosidase (LysMan), core-specific alpha1,6-mannosidase, beta-mannosidase, and cleavage at the reducing terminus by a di-N-acetylchitobiase. We describe here the first cloning, expression, purification, and characterization of a novel human glycosylhydrolase family 38 alpha-mannosidase with catalytic characteristics similar to those established previously for the core-specific alpha1,6-mannosidase (acidic pH optimum, inhibition by swainsonine and 1,4-dideoxy-1,4-imino-d-mannitol, and stimulation by Co(2+) and Zn(2+)). Substrate specificity studies comparing the novel human alpha-mannosidase with human LysMan revealed that the former enzyme efficiently cleaved only the alpha1-6mannose residue from Man(3)GlcNAc but not Man(3)GlcNAc(2) or other larger high mannose oligosaccharides, indicating a requirement for chitobiase action before alpha1,6-mannosidase activity. In contrast, LysMan cleaved all of the alpha-linked mannose residues from high mannose oligosaccharides except the core alpha1-6mannose residue. alpha1,6-Mannosidase transcripts were ubiquitously expressed in human tissues, and expressed sequence tag searches identified homologous sequences in murine, porcine, and canine databases. No expressed sequence tags were identified for bovine alpha1,6-mannosidase, despite the identification of two sequence homologs in the bovine genome. The lack of conservation in 5'-flanking sequences for the bovine alpha1,6-mannosidase genes may lead to defective transcription similar to transcription defects in the bovine chitobiase gene. These results suggest that the chitobiase and alpha1,6-mannosidase function in tandem for mammalian lysosomal N-glycan catabolism.


Assuntos
Lisossomos/enzimologia , Manose/análogos & derivados , Manosidases/metabolismo , Oligossacarídeos/metabolismo , Acetilglucosaminidase/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Clonagem Molecular , Cobalto/farmacologia , Cães , Inibidores Enzimáticos/farmacologia , Etiquetas de Sequências Expressas , Humanos , Imino Furanoses/farmacologia , Manitol/análogos & derivados , Manitol/farmacologia , Manose/metabolismo , Manosidases/antagonistas & inibidores , Manosidases/genética , Dados de Sequência Molecular , Oligossacarídeos/isolamento & purificação , Pirrolidinas/farmacologia , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Swainsonina/farmacologia , Suínos , Zinco/farmacologia
4.
J Lipid Res ; 46(4): 759-68, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15654124

RESUMO

To defend themselves against fungal pathogens, plants produce numerous antifungal proteins and peptides, including defensins, some of which have been proposed to interact with fungal cell surface glycosphingolipid components. Although not known as a phytopathogen, the filamentous fungus Neurospora crassa possesses numerous genes similar to those required for plant pathogenesis identified in fungal pathogens (Galagan, J. E., et al. 2003. Nature 422: 859-868), and it has been used as a model for studying plant-phytopathogen interactions targeting fungal membrane components (Thevissen, K., et al. 2003. Peptides. 24: 1705-1712). For this study, neutral glycolipid components were extracted from wild-type and plant defensin-resistant mutant strains of N. crassa. The structures of purified components were elucidated by NMR spectroscopy and mass spectrometry. Neutral glycosphingolipids of both wild-type and mutant strains were characterized as beta-glucopyranosylceramides, but those of the mutants were found with structurally altered ceramides. Although the wild type expressed a preponderance of N-2'-hydroxy-(E)-Delta3-octadecenoate as the fatty-N-acyl component attached to the long-chain base (4E,8E)-9-methyl-4,8-sphingadienine, the mutant ceramides were found with mainly N-2'-hydroxyhexadecanoate instead. In addition, the mutant strains expressed highly increased levels of a sterol glucoside identified as ergosterol-beta-glucoside. The potential implications of these findings with respect to defensin resistance in the N. crassa mutants are discussed.


Assuntos
Defensinas/farmacologia , Farmacorresistência Fúngica/genética , Glicolipídeos/química , Glicolipídeos/metabolismo , Mutação/genética , Neurospora crassa/genética , Neurospora crassa/metabolismo , Proteínas de Plantas/farmacologia , Cromatografia em Camada Fina , Defensinas/metabolismo , Farmacorresistência Fúngica/efeitos dos fármacos , Glicolipídeos/isolamento & purificação , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular , Neurospora crassa/química , Neurospora crassa/efeitos dos fármacos , Proteínas de Plantas/metabolismo
5.
Fungal Genet Biol ; 40(2): 176-85, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14516770

RESUMO

Twenty-five Neurospora crassa mutants obtained by chemical mutagenesis were screened for increased resistance to various antifungal plant defensins. Plant defensin-resistant N. crassa mutants were further tested for their cross-resistance towards other families of structurally different antimicrobial peptides. Two N. crassa mutants, termed MUT16 and MUT24, displaying resistance towards all plant defensins tested but not to structurally different antimicrobial peptides were selected for further characterization. MUT16 and MUT24 were more resistant towards plant defensin-induced membrane permeabilization as compared to the N. crassa wild-type. Based on the previously demonstrated key role of fungal sphingolipids in the mechanism of growth inhibition by plant defensins, membrane sphingolipids of MUT16 and MUT24 were analysed. Membranes of these mutants contained structurally different glucosylceramides, novel glycosylinositolphosphorylceramides, and an altered level of steryl glucosides. Evidence is provided to link these clear differences in sphingolipid profiles of N. crassa mutants with their resistance towards different plant defensins.


Assuntos
Defensinas/farmacologia , Farmacorresistência Fúngica/genética , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/genética , Proteínas de Plantas/farmacologia , Anti-Infecciosos/farmacologia , Membrana Celular/química , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/genética , Metanossulfonato de Etila/farmacologia , Corantes Fluorescentes/metabolismo , Mutagênicos/farmacologia , Mutação , Neurospora crassa/crescimento & desenvolvimento , Neurospora crassa/metabolismo , Compostos Orgânicos , Esfingolipídeos/análise
6.
J Lipid Res ; 44(11): 2073-88, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12923229

RESUMO

Aspergillus nidulans is a well-established nonpathogenic laboratory model for the opportunistic mycopathogen, A. fumigatus. Some recent studies have focused on possible functional roles of glycosphingolipids (GSLs) in these fungi. It has been demonstrated that biosynthesis of glycosylinositol phosphorylceramides (GIPCs) is required for normal cell cycle progression and polarized growth in A. nidulans (Cheng, J., T.-S. Park, A. S. Fischl, and X. S. Ye. 2001. Mol. Cell Biol. 21: 6198-6209); however, the structures of A. nidulans GIPCs were not addressed in that study, nor were the functional significance of individual structural variants and the downstream steps in their biosynthesis. To initiate such studies, acidic GSL components (designated An-2, -3, and -5) were isolated from A. nidulans and subjected to structural characterization by a combination of one-dimensional (1-D) and 2-D NMR spectroscopy, electrospray ionization-mass spectrometry (ESI-MS), ESI-MS/collision-induced decomposition-MS (MS/CID-MS), ESI-pseudo-[CID-MS]2, and gas chromatography-MS methods. All three were determined to be GIPCs, with mannose as the only monosaccharide present in the headgroup glycans; An-2 and An-3 were identified as di- and trimannosyl inositol phosphorylceramides (IPCs) with the structures Man alpha 1-->3Man alpha 1-->2Ins1-P-1Cer and Man alpha 1-->3(Man alpha 1-->6)Man alpha 1-->2Ins1-P-1Cer, respectively (where Ins = myo-inositol, P = phosphodiester, and Cer = ceramide). An-5 was partially characterized, and is proposed to be a pentamannosyl IPC, based on the trimannosyl core structure of An-3.


Assuntos
Aspergillus nidulans/química , Ceramidas/análise , Ceramidas/química , Glicoesfingolipídeos/análise , Glicoesfingolipídeos/química , Manose/análise , Manose/química , Cromatografia em Camada Fina , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Fosforilação , Espectrometria de Massas por Ionização por Electrospray
7.
J Biol Chem ; 278(3): 1411-4, 2003 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-12454022

RESUMO

The neurogenic Drosophila genes brainiac and egghead are essential for epithelial development in the embryo and in oogenesis. Analysis of egghead and brainiac mutants has led to the suggestion that the two genes function in a common signaling pathway. Recently, brainiac was shown to encode a UDP-N-acetylglucosamine:beta Man beta 1,3-N-acetylglucosaminyltransferase (beta 3GlcNAc-transferase) tentatively assigned a key role in biosynthesis of arthroseries glycosphingolipids and forming the trihexosylceramide, GlcNAc beta 1-3Man beta 1-4Glc beta 1-1Cer. In the present study we demonstrate that egghead encodes a Golgi-located GDP-mannose:beta Glc beta 1,4-mannosyltransferase tentatively assigned a biosynthetic role to form the precursor arthroseries glycosphingolipid substrate for Brainiac, Man beta 1-4Glc beta 1-1Cer. Egghead is unique among eukaryotic glycosyltransferase genes in that homologous genes are limited to invertebrates, which correlates with the exclusive existence of arthroseries glycolipids in invertebrates. We propose that brainiac and egghead function in a common biosynthetic pathway and that inactivating mutations in either lead to sufficiently early termination of glycolipid biosynthesis to inactivate essential functions mediated by glycosphingolipids.


Assuntos
Proteínas de Drosophila , Glicoesfingolipídeos/metabolismo , Proteínas de Insetos/genética , Manosiltransferases/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Animais , Sequência de Bases , Células CHO , Cricetinae , Primers do DNA , Drosophila melanogaster , Proteínas de Insetos/metabolismo , Manosiltransferases/metabolismo , Ressonância Magnética Nuclear Biomolecular , Especificidade por Substrato
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