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1.
J Neurosci ; 23(10): 4127-33, 2003 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-12764100

RESUMO

Type 1 vanilloid receptors (VR1) have been identified recently in the brain, in which they serve as yet primarily undetermined purposes. The endocannabinoid anandamide (AEA) and some of its oxidative metabolites are ligands for VR1, and AEA has been shown to afford protection against ouabain-induced in vivo excitotoxicity, in a manner that is only in part dependent on the type 1 cannabinoid (CB1) receptor. In the present study, we assessed whether VR1 is involved in neuroprotection by AEA and by arvanil, a hydrolysis-stable AEA analog that is a ligand for both VR1 and CB1. Furthermore, we assessed the putative involvement of lipoxygenase metabolites of AEA in conveying neuroprotection. Using HPLC and gas chromatography/mass spectroscopy, we demonstrated that rat brain and blood cells converted AEA into 12-hydroxy-N-arachidoylethanolamine (12-HAEA) and 15-hydroxy-N-arachidonoylethanolamine (15-HAEA) and that this conversion was blocked by addition of the lipoxygenase inhibitor nordihydroguaiaretic acid. Using magnetic resonance imaging we show the following: (1) pretreatment with the reduced 12-lipoxygenase metabolite of AEA, 12-HAEA, attenuated cytotoxic edema formation in a CB1 receptor-independent manner in the acute phase after intracranial injection of the Na+/K+-ATPase inhibitor ouabain; (2) the reduced 15-lipoxygenase metabolite, 15-HAEA, enhanced the neuroprotective effect of AEA in the acute phase; (3) modulation of VR1, as tested using arvanil, the VR1 agonist capsaicin, and the antagonist capsazepine, leads to neuroprotective effects in this model, and arvanil is a potent neuroprotectant, acting at both CB1 and VR1; and (4) the in vivo neuroprotective effects of AEA are mediated by CB1 but not by lipoxygenase metabolites or VR1.


Assuntos
Ácidos Araquidônicos/fisiologia , Canabinoides/farmacologia , Capsaicina/análogos & derivados , Capsaicina/metabolismo , Ácidos Graxos Insaturados/fisiologia , Lipoxigenase/fisiologia , Degeneração Neural/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Receptores de Droga/fisiologia , Animais , Animais Recém-Nascidos , Células Sanguíneas/efeitos dos fármacos , Células Sanguíneas/enzimologia , Células Sanguíneas/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/metabolismo , Química Encefálica , Mapeamento Encefálico , Moduladores de Receptores de Canabinoides , Endocanabinoides , Etanolaminas/análise , Etanolaminas/metabolismo , Lipoxigenase/metabolismo , Masculino , Masoprocol/farmacologia , Degeneração Neural/induzido quimicamente , Degeneração Neural/enzimologia , Ouabaína/farmacologia , Alcamidas Poli-Insaturadas , Ratos , Ratos Wistar , Receptores de Droga/metabolismo
2.
Parasitol Res ; 90(4): 330-6, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12695908

RESUMO

Gut-associated glycoproteins constitute a major group of the circulating excretory antigens produced by human Schistosoma species. The O-glycans of the relatively abundant circulating anodic antigen (CAA) from S. mansoni carry long stretches of unique -->6(GlcA beta 1-->3)GalNAc beta 1--> repeats. Specific anti-carbohydrate monoclonal antibodies (mAbs) are essential tools for the immunodiagnostic detection of CAA in the serum or urine of Schistosoma-infected subjects. In order to define the epitopes recognised by these anti-CAA mAbs, we screened a series of protein-coupled synthetic di- to pentasaccharide building blocks of the CAA polysaccharide for immunoreactivity, using ELISA and surface plasmon resonance spectroscopy. It was shown that anti-CAA IgM mAbs preferentially recognise -->6(GlcA beta 1-->3)GalNAc beta 1--> disaccharide units. Interestingly, no mouse anti-CAA mAbs of the IgG class were found that bind to the synthetic epitopes, although many of the IgG mAbs tested do recognise native CAA in a carbohydrate-dependent manner. In addition, both IgM and IgG class antibodies could be detected in human infection sera using the synthetic CAA fragments. These synthetic schistosome glycan epitopes and their matching set of specific mAbs are useful tools that further the development of diagnostic methods and are helpful in defining the immunological responses of the mammalian hosts to schistosome glycoconjugates.


Assuntos
Anticorpos Monoclonais , Antígenos de Helmintos/imunologia , Mapeamento de Epitopos , Glicoproteínas/imunologia , Proteínas de Helminto/imunologia , Oligossacarídeos/metabolismo , Schistosoma mansoni/imunologia , Animais , Anticorpos Anti-Helmínticos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Helmintos/sangue , Antígenos de Helmintos/urina , Sequência de Carboidratos , Ensaio de Imunoadsorção Enzimática , Glicoconjugados/síntese química , Glicoconjugados/química , Glicoconjugados/imunologia , Glicoproteínas/sangue , Glicoproteínas/urina , Proteínas de Helminto/sangue , Proteínas de Helminto/urina , Humanos , Hibridomas , Camundongos , Dados de Sequência Molecular , Oligossacarídeos/síntese química , Oligossacarídeos/química , Oligossacarídeos/imunologia , Esquistossomose mansoni/diagnóstico , Esquistossomose mansoni/parasitologia , Ressonância de Plasmônio de Superfície
3.
Exp Parasitol ; 105(3-4): 219-25, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14990315

RESUMO

The development of the humoral anti-glycan immune response of chimpanzees, either or not vaccinated with radiation-attenuated Schistosoma mansoni cercariae, was followed during 1 year after infection with S. mansoni. During the acute phase of infection both the vaccinated and the control chimpanzees produce high levels of immunoglobulin G (IgG) antibodies against carbohydrate structures that are characteristic for schistosomes carrying the Fucalpha1-3GalNAc and Fucalpha1-2Fucalpha1-3GlcNAc motifs, but not to the more widespread occurring structures GalNAcbeta1-4GlcNAc, GalNAcbeta1-4(Fucalpha1-3)GlcNAc, and Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis(x)). In addition, high levels of IgM antibodies were found against the trimeric Lewis(x) epitope. Apparently, the schistosome-characteristic carbohydrate structures are dominant epitopes in the anti-glycan humoral immune response of the chimpanzees. All chimpanzees showed an increase in the level of antibodies against most of the carbohydrate structures tested directly after vaccination, peaking at challenge time and during the acute phase of infection. With the exception of anti-F-LDN antibody responses, the anti-carbohydrate antibody responses upon schistosome infection of the vaccinated animals were muted in comparison to the control animals.


Assuntos
Anticorpos Anti-Helmínticos/biossíntese , Dissacarídeos/imunologia , Epitopos/imunologia , Schistosoma mansoni/imunologia , Trissacarídeos/imunologia , Animais , Sequência de Carboidratos , Dissacarídeos/síntese química , Dissacarídeos/química , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Estudos Longitudinais , Masculino , Dados de Sequência Molecular , Pan troglodytes , Polissacarídeos/síntese química , Polissacarídeos/química , Polissacarídeos/imunologia , Esquistossomose mansoni/imunologia , Análise Espectral/métodos , Trissacarídeos/síntese química , Trissacarídeos/química , Vacinação
4.
J Neurosci ; 21(22): 8765-71, 2001 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-11698588

RESUMO

The endocannabinoid anandamide [N-arachidonoylethanolamine (AEA)] is thought to function as an endogenous protective factor of the brain against acute neuronal damage. However, this has never been tested in an in vivo model of acute brain injury. Here, we show in a longitudinal pharmacological magnetic resonance imaging study that exogenously administered AEA dose-dependently reduced neuronal damage in neonatal rats injected intracerebrally with the Na(+)/K(+)-ATPase inhibitor ouabain. At 15 min after injury, AEA (10 mg/kg) administered 30 min before ouabain injection reduced the volume of cytotoxic edema by 43 +/- 15% in a manner insensitive to the cannabinoid CB(1) receptor antagonist SR141716A. At 7 d after ouabain treatment, 64 +/- 24% less neuronal damage was observed in AEA-treated (10 mg/kg) rats compared with control animals. Coadministration of SR141716A prevented the neuroprotective actions of AEA at this end point. In addition, (1) no increase in AEA and 2-arachidonoylglycerol levels was detected at 2, 8, or 24 hr after ouabain injection; (2) application of SR141716A alone did not increase the lesion volume at days 0 and 7; and (3) the AEA-uptake inhibitor, VDM11, did not affect the lesion volume. These data indicate that there was no endogenous endocannabinoid tone controlling the acute neuronal damage induced by ouabain. Although our data seem to question a possible role of the endogenous cannabinoid system in establishing a brain defense system in our model, AEA may be used as a structural template to develop neuroprotective agents.


Assuntos
Ácidos Araquidônicos/farmacologia , Lesões Encefálicas/prevenção & controle , Neurônios/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Western Blotting , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Edema Encefálico/induzido quimicamente , Edema Encefálico/patologia , Edema Encefálico/prevenção & controle , Lesões Encefálicas/induzido quimicamente , Lesões Encefálicas/patologia , Moduladores de Receptores de Canabinoides , Canabinoides/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Endocanabinoides , Inibidores Enzimáticos , Glicerídeos/metabolismo , Estudos Longitudinais , Imageamento por Ressonância Magnética , Microinjeções , Neurônios/metabolismo , Neurônios/patologia , Ouabaína , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas , Pirazóis/farmacologia , Ratos , Ratos Wistar , Receptores de Canabinoides , Receptores de Droga/antagonistas & inibidores , Rimonabanto
5.
Chemistry ; 7(20): 4411-21, 2001 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-11695675

RESUMO

The preparation is described of a range of neoglycoproteins containing synthesised fragments of the capsular polysaccharide of Streptococcus pneumoniae type 3, that is beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->O)-(CH2)3NH2 (1), beta-D-Glcp-(1-->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->O)-(CH2)3NH2 (2), and beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->O)-(CH4)NH2 (3). A blockwise approach was developed for the synthesis of the protected carbohydrate chains, in which the carboxylic groups were introduced prior to deprotection by selective oxidation of HO-6 in the presence of HO-4 by using TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy radical). After deprotection, the 3-aminopropyl spacer of the fragments was elongated with diethyl squarate (3,4-diethoxy-3-cyclobutene-1,2-dione) and the elongated oligosaccharides were conjugated to CRM197 (cross-reacting material of diphtheria toxin), KLH (keyhole limpet hemocyanin) or TT (tetanus toxoid). The resulting neoglycoconjugates varied in oligosaccharide chain length, oligosaccharide loading and protein carrier. These well-defined conjugates are ideal probes for evaluating the influence of the different structural parameters in immunological tests.


Assuntos
Glicoproteínas/síntese química , Vacinas Pneumocócicas/síntese química , Polissacarídeos Bacterianos/química , Streptococcus pneumoniae/química , Adjuvantes Imunológicos/química , Cápsulas Bacterianas , Proteínas de Bactérias/química , Proteínas de Transporte/química , Hemocianinas/química , Oligossacarídeos/síntese química , Oligossacarídeos/química , Polissacarídeos Bacterianos/imunologia , Toxoide Tetânico/química
6.
J Ind Microbiol Biotechnol ; 26(1-2): 90-3, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11548755

RESUMO

The interaction of crystalline and amorphous amylopectin with the plasticisers glycerol and ethylene glycol in the absence of water was studied with differential scanning calorimetry (DSC) and solid state NMR. At room temperature glycerol interacts mainly with the amorphous regions, while for ethylene glycol the amylopectin crystallinity does not effect the interaction. After heating the mixtures, an additional immobilisation of the plasticiser occurs.


Assuntos
Amilopectina/química , Etilenoglicol/química , Glicerol/química , Espectroscopia de Ressonância Magnética/métodos , Plastificantes/química , Varredura Diferencial de Calorimetria/métodos
7.
J Neurosci ; 21(17): 6475-9, 2001 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-11517236

RESUMO

Excitotoxicity is a paradigm used to explain the biochemical events in both acute neuronal damage and in slowly progressive, neurodegenerative diseases. Here, we show in a longitudinal magnetic resonance imaging study that Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the main active compound in marijuana, reduces neuronal injury in neonatal rats injected intracerebrally with the Na(+)/K(+)-ATPase inhibitor ouabain to elicit excitotoxicity. In the acute phase Delta(9)-THC reduced the volume of cytotoxic edema by 22%. After 7 d, 36% less neuronal damage was observed in treated rats compared with control animals. Coadministration of the CB(1) cannabinoid receptor antagonist SR141716 prevented the neuroprotective actions of Delta(9)-THC, indicating that Delta(9)-THC afforded protection to neurons via the CB(1) receptor. In Delta(9)-THC-treated rats the volume of astrogliotic tissue was 36% smaller. The CB(1) receptor antagonist did not block this effect. These results provide evidence that the cannabinoid system can serve to protect the brain against neurodegeneration.


Assuntos
Edema Encefálico/prevenção & controle , Cannabis , Dronabinol/farmacologia , Fármacos Neuroprotetores/farmacologia , Ouabaína/toxicidade , Doença Aguda , Animais , Animais Recém-Nascidos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Edema Encefálico/induzido quimicamente , Edema Encefálico/diagnóstico , Edema Encefálico/metabolismo , Doença Crônica , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Estudos Longitudinais , Imageamento por Ressonância Magnética , Microinjeções , Ouabaína/administração & dosagem , Ratos , Ratos Wistar , Receptores de Canabinoides , Receptores de Droga/antagonistas & inibidores , Receptores de Droga/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Água/metabolismo
8.
Biochimie ; 83(7): 653-8, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11522394

RESUMO

The binding properties of a spacer-linked synthetic Sd(a) tetrasaccharide beta-D-GalpNAc-(1-->4)-alpha-Neu5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (1), two tetrasaccharide mimics beta-D-Galp-(1-->4)-alpha-Neu5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (2) and beta-D-GlcpNAc-(1-->4)-alpha-Neu5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (3), and two trisaccharide mimics beta-D-GalpNAc-(1-->4)-3-O-(SO(3)H)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (4) and beta-D-GalpNAc-(1-->4)-3-O-(CH(2)COOH)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (5) with lectins from Dolichos biflorus (DBL), Maackia amurensis (MAL), Phaseolus limensis (PLL), Ptilota plumosa (PPL), Ricinus communis 120 (RCL120) and Triticum vulgaris (wheat germ agglutinin, WGA) have been investigated by surface plasmon resonance (SPR) detection. MAL, PPL, RCL120 and WGA did not display any binding activity with compounds 1-5. However, DBL and PLL, both exhibiting GalNAc-specificity, showed strong binding activity with compounds 1, 4 and 5, and 1, 3, 4 and 5, respectively. The results demonstrate that SPR is a very useful analysis system for identifying biologically relevant oligosaccharide mimics of the Sd(a) determinant.


Assuntos
Lectinas/química , Oligossacarídeos/química , Ressonância de Plasmônio de Superfície/métodos , Trissacarídeos/síntese química , Sequência de Carboidratos , Antígenos de Histocompatibilidade/química , Cinética , Modelos Biológicos , Lectinas de Plantas , Plantas , Trissacarídeos/química
9.
Proc Natl Acad Sci U S A ; 98(16): 9419-24, 2001 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-11459930

RESUMO

Sponges (Porifera), the simplest and earliest multicellular organisms, are thought to have evolved from their unicellular ancestors about 1 billion years ago by developing cell-recognition and adhesion mechanisms to discriminate against "non-self." Consequently, they are used as models for investigating recognition phenomena. Cellular adhesion of marine sponges is an event involving adherence of extracellular proteoglycan-like molecules, otherwise known as aggregation factors (AFs). In a calcium-independent process the AFs adhere to the cell surface, and in a calcium-dependent process they exhibit AF self-association. A mechanism which has been implied but not definitely proven to play a role in the calcium-dependent event is self-recognition of defined carbohydrate epitopes. For the red beard sponge, Microciona prolifera, two carbohydrate epitopes, a sulfated disaccharide and a pyruvylated trisaccharide, have been implicated in cellular adhesion. To investigate this phenomenon a system has been designed, by using surface plasmon resonance detection, to mimic the role of carbohydrates in cellular adhesion of M. prolifera. The results show self-recognition of the sulfated disaccharide to be a major force behind the calcium-dependent event. The interaction is not simply based on electrostatic interactions, as other sulfated carbohydrates analyzed by using this procedure did not self-associate. Furthermore, the interaction is completely eradicated on substitution of Ca(2+) ions by either Mg(2+) or Mn(2+) ions. This physiologically relevant recognition mechanism confirms the existence of true carbohydrate self-recognition, and may have significant implications for the role of carbohydrates in cellular recognition of higher organisms.


Assuntos
Adesão Celular , Glicoconjugados/metabolismo , Poríferos/citologia , Animais , Cálcio/metabolismo , Configuração de Carboidratos , Sequência de Carboidratos , Glicoconjugados/química , Antígenos CD15/metabolismo , Biologia Marinha , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Poríferos/metabolismo , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Ressonância de Plasmônio de Superfície
10.
Biochemistry ; 40(23): 6819-27, 2001 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-11389595

RESUMO

Lipoxygenases are key enzymes in the metabolism of unsaturated fatty acids. Soybean lipoxygenase-1 (LOX-1), a paradigm for lipoxygenases isolated from different sources, is composed of two domains: a approximately 30 kDa N-terminal domain and a approximately 60 kDa C-terminal domain. We used limited proteolysis and gel-filtration chromatography to generate and isolate a approximately 60 kDa fragment of LOX-1 ("mini-LOX"), produced by trypsin cleavage between lysine 277 and serine 278. Mini-LOX was subjected to N-terminal sequencing and to electrophoretic, chromatographic, and spectroscopic analysis. Mini-LOX was found to be more acidic and more hydrophobic than LOX-1, and with a higher content of alpha-helix. Kinetic analysis showed that mini-LOX dioxygenates linoleic acid with a catalytic efficiency approximately 3-fold higher than that of LOX-1 (33.3 x 10(6) and 10.9 x 10(6) M(-1) x s(-1), respectively), the activation energy of the reaction being 4.5 +/- 0.5 and 8.3 +/- 0.9 kJ x mol(-1) for mini-LOX and LOX-1, respectively. Substrate preference, tested with linoleic, alpha-linolenic, and arachidonic acids, and with linoleate methyl ester, was the same for LOX-1 and mini-LOX, and also identical was the regio- and stereospecificity of the products generated thereof, analyzed by reversed-phase and chiral high-performance liquid chromatography, and by gas chromatography/mass spectrometry. Mini-LOX was able to bind artificial vesicles with higher affinity than LOX-1, but the binding was less affected by calcium ions than was that of LOX-1. Taken together, these results suggest that the N-terminal domain of soybean lipoxygenase-1 might be a built-in inhibitor of catalytic activity and membrane binding ability of the enzyme, with a possible role in physio(patho)logical conditions.


Assuntos
Glycine max/enzimologia , Lipoxigenase/metabolismo , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/metabolismo , Tripsina/metabolismo , Sítios de Ligação , Dicroísmo Circular , Ativação Enzimática , Hidrólise , Cinética , Lipossomos/metabolismo , Lipoxigenase/química , Proteínas de Membrana/química , Peso Molecular , Fragmentos de Peptídeos/química , Ligação Proteica , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Especificidade por Substrato
11.
Infect Immun ; 69(7): 4698-701, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11402020

RESUMO

Di-, tri-, and tetrasaccharides, synthesized according to the chemical structure of pneumococcal polysaccharide type 3 (PS3), were coupled to the cross-reactive material (CRM(197)) of modified diphtheria toxin in different molar carbohydrate/protein ratios using the squarate coupling method. To study protective immunity, female BALB/c mice were subcutaneously immunized twice (with a 3-week interval) using the amount of conjugates corresponding to 2.5 microg of oligosaccharide per mouse. The conjugates evoked PS3 binding immunoglobulin G antibodies that lasted for at least 7 weeks after the booster. Immunogenicity was not influenced by the carbohydrate/protein ratio. All mice with PS3-specific antibodies survived the intraperitoneal challenge with Streptococcus pneumoniae type 3. Therefore, synthetic oligosaccharide-protein conjugates might have potential as vaccines.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Toxina Diftérica/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Polissacarídeos Bacterianos/imunologia , Animais , Sequência de Carboidratos , Reações Cruzadas , Dissacarídeos/imunologia , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Streptococcus pneumoniae/imunologia , Trissacarídeos/imunologia
12.
J Biol Chem ; 276(33): 30834-44, 2001 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-11410585

RESUMO

The carbohydrate structures present on the glycoproteins in the central and peripheral nerve systems are essential in many cell adhesion processes. The P0 glycoprotein, expressed by myelinating Schwann cells, plays an important role during the formation and maintenance of myelin, and it is the most abundant constituent of myelin. Using monoclonal antibodies, the homophilic binding of the P0 glycoprotein was shown to be mediated via the human natural keller cell (HNK)-1 epitope (3-O-SO(3)H-GlcUA(beta1-3)Gal(beta1-4)GlcNAc) present on the N-glycans. We recently described the structure of the N-glycan carrying the HNK-1 epitope, present on bovine peripheral myelin P0 (Voshol, H., van Zuylen, C. W. E. M., Orberger, G., Vliegenthart, J. F. G., and Schachner, M. (1996) J. Biol. Chem. 271, 22957-22960). In this study, we report on the structural characterization of the detectable glycoforms, present on the single N-glycosylation site, using state-of-the-art NMR and mass spectrometry techniques. Even though all structures belong to the hybrid- or biantennary complex-type structures, the variety of epitopes is remarkable. In addition to the 3-O-sulfate present on the HNK-1-carrying structures, most of the glycans contain a 6-O-sulfated N-acetylglucosamine residue. This indicates the activity of a 6-O-sulfo-GlcNAc-transferase, which has not been described before in peripheral nervous tissue. The presence of the disialo-, galactosyl-, and 6-O-sulfosialyl-Lewis X epitopes provides evidence for glycosyltransferase activities not detected until now. The finding of such an epitope diversity triggers questions related to their function and whether events, previously attributed merely to the HNK-1 epitope, could be mediated by the structures described here.


Assuntos
Epitopos , Proteína P0 da Mielina/química , Polissacarídeos/química , Animais , Bovinos , Células Matadoras Naturais/imunologia , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Polissacarídeos/imunologia
13.
J Ind Microbiol Biotechnol ; 26(1/2): 90-93, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11360176

RESUMO

The interaction of crystalline and amorphous amylopectin with the plasticisers glycerol and ethylene glycol in the absence of water was studied with differential scanning calorimetry (DSC) and solid state NMR. At room temperature glycerol interacts mainly with the amorphous regions, while for ethylene glycol the amylopectin crystallinity does not effect the interaction. After heating the mixtures, an additional immobilisation of the plasticiser occurs.

14.
Carbohydr Res ; 331(2): 173-82, 2001 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-11322731

RESUMO

The exopolysaccharide of Streptococcus thermophilus S3, produced in skimmed milk, is composed of D-galactose and L-rhamnose in a molar ratio of 2:1. The polysaccharide contains 0.4 equiv of O-acetyl groups per repeating unit. Linkage analysis and 1D/2D NMR (1H and 13C) studies on native and O-deacetylated EPS together with nanoES-CID tandem mass spectrometry studies on oligosaccharides generated by a periodate oxidation protocol, show the polysaccharide to have the following structure: [structure: see text].


Assuntos
Leite/microbiologia , Polissacarídeos Bacterianos/química , Streptococcus/metabolismo , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Espectroscopia de Ressonância Magnética , Metilação , Dados de Sequência Molecular , Estrutura Molecular , Polissacarídeos Bacterianos/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray
15.
Carbohydr Res ; 331(2): 183-94, 2001 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-11322732

RESUMO

The lactic acid bacterium Lactobacillus delbrueckii subsp. bulgaricus 291, when grown in skimmed milk, produced 80 mg/L exopolysaccharide with an average molecular mass of 1.4 x 10(3) kDa. Monosaccharide analysis, methylation analysis, MS, and 1D/2D NMR (1H and 13C) studies performed on the native polysaccharide, and on oligosaccharides obtained from a mild acid hydrolysate of the native polysaccharide, showed the polysaccharide to consist of branched pentasaccharide repeating units with the following structure: [structure: see text].


Assuntos
Lactobacillus/metabolismo , Polissacarídeos Bacterianos/química , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia , Lactobacillus/química , Espectroscopia de Ressonância Magnética , Metilação , Leite/microbiologia , Dados de Sequência Molecular , Polissacarídeos Bacterianos/isolamento & purificação , Polissacarídeos Bacterianos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
16.
Biol Chem ; 382(2): 299-311, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11308028

RESUMO

The enzymatic access to nucleotide-activated oligosaccharides by a glycosidase-catalyzed transglycosylation reaction was explored. The nucleotide sugars UDP-GlcNAc and UDP-Glc were tested as acceptor substrates for beta-galactosidase from Bacillus circulans using lactose as donor substrate. The UDP-disaccharides Gal(beta1-4)GlcNAc(alpha1-UDP) (UDP-LacNAc) and Gal(beta1-4)Glc(alpha1-UDP) (UDP-Lac) and the UDP-trisaccharides Gal(beta1-4)Gal(beta1-4)GlcNAc(alpha1-UDP and Gal(beta1-4)Gal(beta1-4)Glc(alpha1-UDP) were formed stereo- and regioselectively. Their chemical structures were characterized by 1H and 13C NMR spectroscopy and fast atom bombardment mass spectrometry. The synthesis in frozen solution at -5 degrees C instead of 30 degrees C gave significantly higher product yields with respect to the acceptor substrates. This was due to a remarkably higher product stability in the small liquid phase of the frozen reaction mixture. Under optimized conditions, at -5 degrees C and pH 4.5 with 500 mM lactose and 100 mM UDP-GlcNAc, an overall yield of 8.2% (81.8 micromol, 62.8 mg with 100% purity) for Gal(beta1-4)GlcNAc(alpha1-UDP) and 3.6% (36.1 micromol, 35 mg with 96% purity) for Gal(beta1-4)Gal(beta1-4)GlcNAc(alpha1-UDP) was obtained. UDP-Glc as acceptor gave an overall yield of 5.0% (41.3 micromol, 32.3 mg with 93% purity) for Gal(beta1-4)Glc(alpha1-UDP) and 1.6% (13.0 micromol, 12.2 mg with 95% purity) for Gal(beta1-4)Gal(beta1-4)Glc(alpha1-UDP). The analysis of other nucleotide sugars revealed UDP-Gal, UDP-GalNAc, UDP-Xyl and dTDP-, CDP-, ADP- and GDP-Glc as further acceptor substrates for beta-galactosidase from Bacillus circulans.


Assuntos
Bacillus/enzimologia , Nucleotídeos/metabolismo , Oligossacarídeos/síntese química , Açúcares de Uridina Difosfato/química , beta-Galactosidase/metabolismo , Bioquímica/métodos , Sequência de Carboidratos , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Nucleotídeos/química , Oligossacarídeos/metabolismo , Estereoisomerismo , Especificidade por Substrato , Temperatura , Açúcares de Uridina Difosfato/síntese química , Açúcares de Uridina Difosfato/metabolismo , beta-Galactosidase/química
17.
Infect Immun ; 69(2): 787-93, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11159969

RESUMO

The immunogenicity and protective capacity of Streptococcus pneumoniae 6B capsular polysaccharide (PS)-derived synthetic phosphate-containing disaccharide (Rha-ribitol-P-), trisaccharide (ribitol-P-Gal-Glc-), and tetrasaccharide (Rha-ribitol-P-Gal-Glc-)-protein conjugates in rabbits and mice were studied. In rabbits, all saccharides conjugated to keyhole limpet hemocyanin (KLH) evoked high levels of pneumococcal (Pn) type 6B antibodies that facilitated type-specific phagocytosis. Unlike the disaccharide rabbit antisera, tri- and tetrasaccharide rabbit antisera also reacted with 6A PS in an enzyme-linked immunosorbent assay (ELISA) and promoted phagocytosis of 6A pneumococci. All these rabbit antisera passively protected mice against a Pn 6B challenge. The disaccharide conjugate-induced antiserum, however, failed to protect mice against a 6A challenge. In mice, phagocytic and protective anti-Pn 6B antibodies were only induced by the tetrasaccharide conjugate and not by PS 6B or PS 6B-protein conjugates. These antibodies did not cross-react with 6A PS in ELISA and were unable to phagocytize 6A pneumococci. In conclusion, the disaccharide and tetrasaccharide conjugates already contain epitopes capable of inducing 6B-specific, fully protective antibodies in rabbits and mice, respectively.


Assuntos
Anticorpos Antibacterianos/biossíntese , Oligossacarídeos/imunologia , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Epitopos , Feminino , Hemocianinas/imunologia , Humanos , Soros Imunes/imunologia , Imunização Passiva , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose , Coelhos , Vacinas Conjugadas/imunologia
18.
Biopolymers ; 58(3): 279-94, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11169388

RESUMO

Structural characteristics of pectic substances extracted from soybean meal cell walls (water unextractable solids) with a chelating agent-containing buffer (0.05M 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) and 0.05M NH(4)-oxalate in 0.05M NaOAc buffer) were studied. The arabinogalactans present as side chains to the rhamnogalacturonan backbone were largely removed by enzymatic hydrolysis using endo-galactanase, exo-galactanase, endo-arabinanase, and arabinofuranosidase B. The remaining pectic backbone appeared to be resistant to enzymatic degradation by pectolytic enzymes. After partial acid hydrolysis of the isolated pectic backbone, one oligomeric and two polymeric populations were obtained by size-exclusion chromatography. Monosaccharide and linkage analyses, enzymatic degradation, and NMR spectroscopy of these populations showed that the pectic substances in the original extract contain both rhamnogalacturonan and xylogalacturonan regions, while homogalacturonan is absent.


Assuntos
Ácido Edético/análogos & derivados , Glycine max/química , Pectinas/química , Ácidos/química , Ácido Edético/química , Enzimas/química , Hidrólise , Espectroscopia de Ressonância Magnética , Conformação Molecular
19.
FEBS Lett ; 489(2-3): 229-32, 2001 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-11165255

RESUMO

Expression of high quantities of alfalfa hydroperoxide lyase in Escherichia coli made it possible to study its active site and structure in more detail. Circular dichroism (CD) spectra showed that hydroperoxide lyase consists for about 75% of alpha-helices. Electron paramagnetic resonance (EPR) spectra confirmed its classification as a cytochrome P450 enzyme. The positive influence of detergents on the enzyme activity is paralleled by a spin state transition of the heme Fe(III) from low to high spin. EPR and CD spectra showed that detergents induce a subtle conformational change, which might result in improved substrate binding. Because hydroperoxide lyase is thought to be a membrane bound protein and detergents mimic a membrane environment, the more active, high spin form likely represents the in vivo conformation. Furthermore, the spin state appeared to be temperature-dependent, with the low spin state favored at low temperature. Point mutants of the highly conserved cysteine in domain D indicated that this residue might be involved in heme binding.


Assuntos
Aldeído Liases/química , Sistema Enzimático do Citocromo P-450/química , Aldeído Liases/genética , Aldeído Liases/metabolismo , Sítios de Ligação/genética , Western Blotting , Dicroísmo Circular , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Detergentes/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Medicago sativa/enzimologia , Mutagênese Sítio-Dirigida , Conformação Proteica/efeitos dos fármacos
20.
Glycoconj J ; 18(7): 511-8, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12151712

RESUMO

Gp273, a glycoprotein of the egg extracellular coats of the mollusc bivalve Unio elongatulus, is the ligand molecule for sperm-egg interaction during fertilization. In this study we have analyzed the N-glycans from gp273. N-glycans were enzymatically released by PNGase F digestion and their structures were elucidated by normal phase HPLC profiling of the 2-aminobenzamide-labeled N-glycans, MALDI-TOF mass spectrometry and 1H NMR spectroscopy. The combined data revealed that the N-glycans of gp273 consist of Glc1Man9GlcNAc2 and Man9GlcNAc2. In Unio, the presence of noncomplex-type N-glycans parallels the inefficacy of these glycans in the ligand function. Their role in the protection of the polypeptide chain from proteolytic attack is suggested by the electrophoretic patterns obtained after enzymatic digestion of the native and the N-deglycosylated protein. These results are discussed in the light of the evolution of the recognition and adhesion properties of oligosaccharide chains in the fertilization process.


Assuntos
Glicoproteínas/metabolismo , Polissacarídeos/química , Interações Espermatozoide-Óvulo , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Glicoproteínas/química , Ligantes , Moluscos , Ressonância Magnética Nuclear Biomolecular , Mapeamento de Peptídeos , Polissacarídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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