RESUMO
Brown spider phospholipases D from Loxosceles venoms are among the most widely studied toxins since they induce dermonecrosis, triggering inflammatory responses, increase vascular permeability, cause hemolysis, and renal failure. The catalytic (H12 and H47) and metal-ion binding (E32 and D34) residues in Loxosceles intermedia phospholipase D (LiRecDT1) were mutated to understand their roles in the observed activities. All mutants were identified using whole venom serum antibodies and a specific antibody to wild-type LiRecDT1, they were also analyzed by circular dichroism (CD) and differential scanning calorimetry (DSC). The phospholipase D activities of H12A, H47A, H12A-H47A, E32, D34 and E32A-D34A, such as vascular permeability, dermonecrosis, and hemolytic effects were inhibited. The mutant Y228A was equally detrimental to biochemical and biological effects of phospholipase D, suggesting an essential role of this residue in substrate recognition and binding. On the other hand, the mutant C53A-C201A reduced the enzyme's ability to hydrolyze phospholipids and promote dermonecrosis, hemolytic, and vascular effects. These results provide the basis understanding the importance of specific residues in the observed activities and contribute to the design of synthetic and specific inhibitors for Brown spider venom phospholipases D.
Assuntos
Domínio Catalítico/genética , Fosfolipase D/química , Fosfolipídeos/química , Venenos de Aranha/enzimologia , Animais , Aranha Marrom Reclusa/química , Aranha Marrom Reclusa/enzimologia , Permeabilidade Capilar , Dicroísmo Circular , Hemólise , Mutação , Fosfolipase D/metabolismo , Fosfolipídeos/metabolismo , Diester Fosfórico Hidrolases/química , Venenos de Aranha/químicaRESUMO
This is the first study on the hemolymph from a spider of the Loxosceles genus. These animals are responsible for a great number of envenomation cases worldwide. Several studies on Loxosceles venoms have been published, and the knowledge about the venom and its toxins is considerable, not only regarding the biological and biochemical characterization, but also regarding structural, genetic and phylogenetic approaches. However, the literature on Loxosceles hemolymph is nonexistent. The main goal of the present study was to characterize biochemically the hemolymph content, and especially, to identify its different hemocytes. Moreover, many papers have already shown molecules whose source is the hemolymph and their very interesting activities and biomedical applications, for example, antifungal and antibacterial activities. A 2D-SDS-PAGE of brown spider hemolymph showed approximately 111 spots for pH 3-10 and 150 spots for pH 4-7. A lectin-blotting assay showed that hemolymph carbohydrate residues were similar to those found in venom. Several types of TAG and DAG phospholipids were found in the hemolymph and characterized by HPTLC and mass spectrometry. Four different hemocytes were characterized in Loxosceles intermedia hemolymph: prohemocyte, plasmatocyte, granulocyte and adipohemocyte. This paper opens new possibilities on toxinology, studying an unknown biological material, and it characterizes a source of molecules with putative biotechnological applications.
Assuntos
Aranha Marrom Reclusa , Hemolinfa/química , Diester Fosfórico Hidrolases/química , Venenos de Aranha/química , Animais , Mordeduras e Picadas/patologia , Cromatografia em Camada Fina , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , FilogeniaRESUMO
Multiple cell membrane alterations have been reported to be the cause of various forms of hypertension. The present study focuses on the lipid portion of the membranes, characterizing the microviscosity of membranes reconstituted with lipids extracted from the aorta and mesenteric arteries of spontaneously hypertensive (SHR) and normotensive control rat strains (WKY and NWR). Membrane-incorporated phospholipid spin labels were used to monitor the bilayer structure at different depths. The packing of lipids extracted from both aorta and mesenteric arteries of normotensive and hypertensive rats was similar. Lipid extract analysis showed similar phospholipid composition for all membranes. However, cholesterol content was lower in SHR arteries than in normotensive animal arteries. These findings contrast with the fact that the SHR aorta is hyporeactive while the SHR mesenteric artery is hyperreactive to vasopressor agents when compared to the vessels of normotensive animal strains. Hence, factors other than microviscosity of bulk lipids contribute to the vascular smooth muscle reactivity and hypertension of SHR. The excess cholesterol in the arteries of normotensive animal strains apparently is not dissolved in bulk lipids and is not directly related to vascular reactivity since it is present in both the aorta and mesenteric arteries. The lower cholesterol concentrations in SHR arteries may in fact result from metabolic differences due to the hypertensive state or to genes that co-segregate with those that determine hypertension during the process of strain selection.
Assuntos
Animais , Masculino , Ratos , Aorta/química , Membrana Celular/química , Colesterol/análise , Hipertensão/metabolismo , Artérias Mesentéricas/química , Fosfolipídeos/análise , Colesterol/química , Espectroscopia de Ressonância de Spin Eletrônica , Cromatografia Gasosa-Espectrometria de Massas , Hipertensão/etiologia , Músculo Liso Vascular/química , Músculo Liso Vascular/citologia , Fosfolipídeos/química , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
Multiple cell membrane alterations have been reported to be the cause of various forms of hypertension. The present study focuses on the lipid portion of the membranes, characterizing the microviscosity of membranes reconstituted with lipids extracted from the aorta and mesenteric arteries of spontaneously hypertensive (SHR) and normotensive control rat strains (WKY and NWR). Membrane-incorporated phospholipid spin labels were used to monitor the bilayer structure at different depths. The packing of lipids extracted from both aorta and mesenteric arteries of normotensive and hypertensive rats was similar. Lipid extract analysis showed similar phospholipid composition for all membranes. However, cholesterol content was lower in SHR arteries than in normotensive animal arteries. These findings contrast with the fact that the SHR aorta is hyporeactive while the SHR mesenteric artery is hyperreactive to vasopressor agents when compared to the vessels of normotensive animal strains. Hence, factors other than microviscosity of bulk lipids contribute to the vascular smooth muscle reactivity and hypertension of SHR. The excess cholesterol in the arteries of normotensive animal strains apparently is not dissolved in bulk lipids and is not directly related to vascular reactivity since it is present in both the aorta and mesenteric arteries. The lower cholesterol concentrations in SHR arteries may in fact result from metabolic differences due to the hypertensive state or to genes that co-segregate with those that determine hypertension during the process of strain selection.
Assuntos
Aorta/química , Membrana Celular/química , Colesterol/análise , Hipertensão/metabolismo , Artérias Mesentéricas/química , Fosfolipídeos/análise , Animais , Colesterol/química , Espectroscopia de Ressonância de Spin Eletrônica , Cromatografia Gasosa-Espectrometria de Massas , Hipertensão/etiologia , Masculino , Músculo Liso Vascular/química , Músculo Liso Vascular/citologia , Fosfolipídeos/química , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
An increasing number of studies support a potential role for coccoid forms in Helicobacter pylori infection. Evidence for this was obtained through scanning microscopy, genetic analysis for virulence traits, examination of the presence and activity of key enzymes, and other methods. We studied the serum immunoglobulin G responses to coccoid H. pylori forms by enzyme-linked immunosorbent assay (ELISA) and immunoblotting and compared them with those of bacillary cells. Sera from a total of 295 infected individuals were studied; these included sera from 100 patients with duodenal ulcers, 98 patients with nonulcer dyspepsia, 11 patients with gastroduodenal cancer, and 86 asymptomatic individuals. Initially, we characterized and selected coccoid and bacillary antigenic preparations by one-dimensional (1-D) and 2-D gel electrophoresis and immunoblotting. Data showed that coccoid and bacillary preparations with comparable protein contents have similar patterns in 1-D and 2-D electrophoresis gels and antigenic recognition at blotting. These results revealed that coccoid and spiral antigens in ELISA can equally recognize specific antibodies to H. pylori in sera from infected individuals. The analysis of the spiral and coccoid preparations by Western blotting showed no major differences in antigen recognition. No specific bands or profiles associated with a single gastric condition were identified.
Assuntos
Anticorpos Antibacterianos/sangue , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Adulto , Antígenos de Bactérias/análise , Western Blotting , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Infecções por Helicobacter/microbiologia , Helicobacter pylori/patogenicidade , Helicobacter pylori/ultraestrutura , Humanos , Imunoglobulina G/sangue , VirulênciaRESUMO
The objective of this study was to evaluate the prevalence of antibodies to Helicobacter pylori CagA and VacA proteins and correlate this prevalence with gastric diseases in colonised Chileans. The study was performed in 418 adults colonised with H. pylori: 316 with gastroduodenal pathology (152 duodenal ulcer, 14 gastric cancer and 150 gastritis patients) and 102 asymptomatic subjects. Serum IgG antibodies to H. pylori were determined by enzyme immunoassay (EIA). Antibodies to VacA and CagA proteins were detected by Western blotting. In a subgroup of the patients, the vacuolating activity was determined by HeLa cell assay and the CagA product was confirmed by PCR assay. IgG antibodies to both VacA and CagA proteins of H. pylori were found in 270 (85%) of 316 colonised gastric patients and in 72 (71%) of 102 asymptomatic subjects. Colonisation with virulent strains was significantly higher among duodenal ulcer and gastric cancer patients than in gastritis patients or asymptomatic subjects. Infections with VacA+/ CagA+ H. pylori strains is common in Chile but, in contrast to some Asian countries, this phenotype was more prevalent in isolates from patients with more severe gastric pathologies.
Assuntos
Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/imunologia , Infecções por Helicobacter/epidemiologia , Helicobacter pylori/imunologia , Gastropatias/epidemiologia , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Western Blotting , Chile/epidemiologia , Citotoxinas/imunologia , Infecções por Helicobacter/imunologia , Helicobacter pylori/patogenicidade , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Imunoglobulina G/sangue , Estudos Soroepidemiológicos , Gastropatias/imunologia , Gastropatias/microbiologiaRESUMO
The purpose of this study was to analyze the profile of glycosphingolipids (GSLs) in periradicular lesions refractory to endodontic treatment. Sixteen periapical lesions were removed surgically from patients (experimental group) and compared with 10 samples of periodontal ligament removed from extracted intact third molars (control group). After the GSLs extraction and purification procedures were performed the neutral and acidic GSL fractions were analyzed by high-performance thin-layer chromatography and quantified by densitometry. Data reported herein show that: (i) tissues in the experimental group presented about twice as much GSLs as the control group; (ii) lesion tissues express lactoneotetraosylceramide, and lactofucopentaosyl (IV) ceramide, whereas these neutral GSLs are absent in normal tissues; and (iii) normal tissues express GT1b, whereas lesions cells do not express this ganglioside. In contrast lesion tissues express GM3, which is conspicuously absent in normal tissues.
Assuntos
Gangliosídeo G(M3)/análise , Doenças Periapicais/terapia , Tratamento do Canal Radicular , Glicoesfingolipídeos Acídicos/análise , Biomarcadores/análise , Cromatografia em Camada Fina , Densitometria , Gangliosídeo G(M1)/análise , Gangliosídeos/análise , Globosídeos/análise , Humanos , Lactosilceramidas/análise , Glicoesfingolipídeos Neutros/análise , Doenças Periapicais/metabolismo , Granuloma Periapical/metabolismo , Granuloma Periapical/terapia , Ligamento Periodontal/metabolismo , Cisto Radicular/metabolismo , Cisto Radicular/terapiaRESUMO
An IgG2a monoclonal antibody anti-glucosylceramide was established and termed MEST-2. High performance thin layer chromatography immunostaining, and solid-phase radioimmunoassay showed that MEST-2 reacts with glucosylceramide from yeast and mycelium forms of Paracoccidioides brasiliensis, Histoplasma capsulatum, and Sporothrix schenckii; from hyphae of Aspergillus fumigatus; and from yeast forms of Candida albicans, Cryptococcus neoformans, Cryptococcus laurentii, and Cryptococcus albidus. Studies on the fine specificity of MEST-2 showed that it recognizes the beta-D-glucose residue, and that the 2-hydroxy group present in the fatty acid is an important auxiliary feature for the antibody binding. It was also demonstrated that phosphatidylcholine and ergosterol modulate MEST-2 reactivity to glucosylceramide, by solid-phase radioimmunoassay. Indirect immunofluorescence showed that MEST-2 reacts with the surface of yeast forms of P. brasiliensis, H. capsulatum and S. schenckii. Weak staining of mycelial forms of P. brasiliensis and hyphae of A. fumigatus was also observed. The availability of a monoclonal antibody specific to fungal glucosylceramide, and its potential use in analyzing biological roles attributed to glucosylceramide in fungi are discussed.
Assuntos
Anticorpos Monoclonais/imunologia , Fungos/metabolismo , Glucosilceramidas/imunologia , Plantas/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Glucosilceramidas/metabolismo , Metabolismo dos Lipídeos , TemperaturaRESUMO
Cerebroside (monohexosylceramide) components were identified in neutral lipids extracted from both the yeast and mycelial forms of the thermally dimorphic mycopathogen Histoplasma capsulatum. The components were purified from both forms and their structures elucidated by 1- and 2-dimensional nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and low energy tandem collision-induced dissociation mass spectrometry (ESI-MS/CID-MS). Both components were characterized as beta-glucopyranosylceramides (GlcCers) containing (4E,8E)-9-methyl-4,8-sphingadienine as the long-chain base, attached to 18-carbon 2-hydroxy fatty N-acyl components. However, while the fatty acid of the yeast form GlcCer was virtually all N-2'-hydroxyoctadecanoate, the mycelium form GlcCer was characterized by almost exclusive expression of N-2'-hydroxy-(E)-delta(3)-octadecenoate. These results suggest that the yeast-mycelium transition is accompanied by up-regulation of an as yet uncharacterized ceramide or cerebroside 2-hydroxy fatty N-acyl (E)-delta(3)-desaturase activity. They also constitute further evidence for the existence of two distinct pathways for ceramide biosynthesis in fungi, since glycosylinositol phosphorylceramides (GIPCs), the other major class of fungal glycosphingolipids, are found with ceramides consisting of 4-hydroxysphinganine (phytosphingosine) and longer chain 2-hydroxy fatty acids. In addition to identification of the major glucocerebroside components, minor components (< 5%) detectable by molecular weight differences in the ESI-MS profiles were also characterized by tandem ESI-MS/CID-MS analysis. These minor components were identified as variants differing in fatty acyl chain length, or the absence of the sphingoid 9-methyl group or (E)-delta(8)-unsaturation, and are hypothesized to be either biosynthetic intermediates or the result of imperfect chemical transformation by the enzymes responsible for these features. Possible implications of these findings with respect to chemotaxonomy, compartmentalization of fungal glycosphingolipid biosynthetic pathways, and regulation of morphological transitions in H.capsulatum and other dimorphic fungi are discussed.
Assuntos
Cerebrosídeos/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Histoplasma/metabolismo , Configuração de Carboidratos , Cerebrosídeos/química , Cromatografia em Camada Fina , Ressonância Magnética Nuclear Biomolecular , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Acidic glycosphingolipid components were extracted from the yeast form of the dimorphic mycopathogen Sporothrix schenckii. Two minor and the major fraction from the yeast form (Ss-Y1, -Y2, and -Y6, respectively) have been isolated. By a combination of 1- and 2-D 1H-nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and gas chromatography/mass spectrometry (GC/MS), Ss-Y6 was determined to be triglycosylinositol phosphorylceramide with a novel glycan structure, Manalpha1-->3Manalpha1-->6GlcNH(2)alpha1-->2Ins1-P-1Cer (where Ins=myo-inositol, P=phosphodiester). While the GlcNH(2)alpha1-->6Ins1-P- motif is found widely distributed in eukaryotic GPI anchors, the linkage GlcNH(2)alpha1-->2Ins1-P- has not been previously observed in any glycolipid. Ss-Y1 and Ss-Y2 were both found to have the known glycan structure Manalpha1-->3Manalpha1-->2Ins1-P-1Cer. Together with the results of a prior study [Toledo et al. (2001) Biochem. Biophys. Res. Commun. 280, 19-24] which showed that the mycelium form expresses GIPCs with the structures Manalpha1-->6Ins1-P-1Cer and Manalpha1-->3Manalpha1-->6Ins1-P-1Cer, these results demonstrate that S. schenckii can synthesize glycosylinositol phosphorylceramides with at least three different core linkages.