RESUMO
A transcriptome analysis of the venom glands of the spider Loxosceles laeta, performed by our group, in a previous study (Fernandes-Pedrosa et al., 2008), revealed a transcript with a sequence similar to the human complement component C3. Here we present the analysis of this transcript. cDNA fragments encoding the C3 homologue (Lox-C3) were amplified from total RNA isolated from the venom glands of L. laeta by RACE-PCR. Lox-C3 is a 5178 bps cDNA sequence encoding a 190kDa protein, with a domain configuration similar to human C3. Multiple alignments of C3-like proteins revealed two processing sites, suggesting that Lox-C3 is composed of three chains. Furthermore, the amino acids consensus sequences for the thioester was found, in addition to putative sequences responsible for FB binding. The phylogenetic analysis showed that Lox-C3 belongs to the same group as two C3 isoforms from the spider Hasarius adansoni (Family Salcitidae), showing 53% homology with these. This is the first characterization of a Loxosceles cDNA sequence encoding a human C3 homologue, and this finding, together with our previous finding of the expression of a FB-like molecule, suggests that this spider species also has a complement system. This work will help to improve our understanding of the innate immune system in these spiders and the ancestral structure of C3.
Assuntos
Proteínas de Artrópodes/genética , Complemento C3/genética , Aranhas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Glândulas Exócrinas/imunologia , Feminino , Diester Fosfórico Hidrolases , Filogenia , Análise de Sequência de DNA , Venenos de Aranha , Aranhas/imunologiaRESUMO
Envenomation by Loxosceles species (brown spider) can lead to local dermonecrosis and to serious systemic effects. The main toxic component in the venom of these spiders is sphingomyelinase D (SMase D) and various isoforms of this toxin are present in Loxosceles venoms. We have produced a new anti-loxoscelic serum by immunizing horses with recombinant SMase D. In the present study, we compared the neutralization efficacy of the new anti-loxoscelic serum and anti-arachnidic serum (the latter serum is used for therapy for loxoscelism in Brazil) against the toxic effects of venoms from spiders of the genus Loxosceles. Neutralization tests showed that anti-SMase D serum has a higher activity against toxic effects of L. intermedia and L. laeta venoms and similar or slightly weaker activity against toxic effects of L. gaucho than that of Arachnidic serum. These results demonstrate that recombinant SMase D can replace venom for anti-venom production and therapy.
Assuntos
Animais , Venenos de Aranha/intoxicação , Intoxicação/terapia , Soros Imunes , Testes de Neutralização/métodosRESUMO
BACKGROUND AND AIMS: This study sought genetic evidence of long-term isolation in populations of Monstera adansonii var. klotzschiana (Araceae), a herbaceous, probably outbreeding, humid forest hemi-epiphyte, in the brejo forests of Ceará (north-east Brazil), and clarification of their relationships with populations in Amazonia and the Atlantic forest of Brazil. METHODS: Within-population genetic diversity and between-population dissimilarity were estimated using AFLP molecular markers in 75 individuals from eight populations located in Ceará, the Brazilian Atlantic Forest and Amazonia. KEY RESULTS: The populations showed a clinal pattern of weak genetic differentiation over a large geographical region (F(ST) = 0.1896). A strong correlation between genetic and geographical distance (Mantel test: r = 0.6903, P = 0.002) suggests a historical pattern of isolation by distance. Genetic structure analysis revealed at least two distinct gene pools in the data. The two isolated Ceará populations are significantly different from each other (pairwise Phi(PT) = 0.137, P = 0.003) and as diverse (Nei's gene diversity, average H(e) = 0.1832, 0.1706) as those in the Atlantic and Amazon forest regions. The population in southern Brazil is less diverse (Nei's gene diversity, average H(e) = 0.127) than the rest. The Ceará populations are related to those of the Atlantic forest rather than those from Amazonia (AMOVA, among-groups variation = 11.95 %, P = 0.037). CONCLUSIONS: The gene pools detected within an overall pattern of clinal variation suggest distinct episodes of gene flow, possibly correlated with past humid forest expansions. The Ceará populations show no evidence of erosion of genetic diversity, although this was expected because of their isolation. Their genetic differentiation and relatively high diversity reinforce the importance of conserving the endangered brejo forests.
Assuntos
Araceae/genética , Variação Genética , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Brasil , Fluxo Gênico/genética , Marcadores Genéticos/genética , Genética Populacional , Polimorfismo GenéticoRESUMO
Bites by Loxosceles spiders can induce severe clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, haemolysis and persistent inflammation. The causative toxin is a sphingomyelinase D (SMase D) that cleaves sphingomyelin into choline and ceramide-1-phosphate. A similar enzyme, showing comparable bioactivity, is secreted by certain pathogenic corynebacteria and acts as a potent virulence factor. We have previously found that SMase D toxins led to an increased susceptibility of human erythrocytes (E) to activation of complement (C) via the classical pathway (CP) in the absence of antibodies. In the present study we have investigated the CP initiating components involved in the haemolysis induced by SMases from Corynebacterium pseudotuberculosis (PLD) and from Loxosceles intermedia venom (P1). When P1 or PLD treated E were incubated with C8-depleted human serum, an increase in C1q, serum amyloid protein (SAP) and C-reactive protein (CRP) binding was observed. While purified C1q, SAP and CRP were found to bind to P1 or PLD treated E, depletion of SAP or CRP from human serum did not prevent C-mediated lysis, suggesting that pentraxins are not involved in the initiation of C-activation. However depletion of C1 lead to a greatly reduced haemolysis, demonstrating that the activation of the CP is caused by direct binding of C1q to the SMase treated cells. Binding of fluid phase C-regulators C4b-binding protein and factor H was also observed, however these C-regulators in conjunction with the membrane bound C-regulators were unable to prevent haemolysis, demonstrating the potency of SMase D facilitated binding of C1 and activation of C.
Assuntos
Complemento C1q/imunologia , Membrana Eritrocítica/imunologia , Hemólise , Diester Fosfórico Hidrolases/imunologia , Animais , Ativação do Complemento/efeitos dos fármacos , Corynebacterium pseudotuberculosis/enzimologia , Membrana Eritrocítica/metabolismo , Eritrócitos/imunologia , Eritrócitos/metabolismo , Hemólise/efeitos dos fármacos , Humanos , Diester Fosfórico Hidrolases/metabolismo , Diester Fosfórico Hidrolases/toxicidade , Ligação Proteica , Coelhos , Venenos de Aranha/enzimologiaRESUMO
Envenomation by Loxosceles spiders causes two main clinical manifestations: cutaneous and systemic loxoscelism. The factors contributing to the severity of loxoscelism are not fully understood. We have analysed biochemical and toxicity variations in venom of L. laeta and L. intermedia, with the aim to find a correlation with the seriousness of loxoscelism. Differences in expression of proteins, glycoproteins and sphingomyelinase activity were observed between venom from male and female spiders and between venom from the two species. These differences were reflected in the toxicity of the venoms including the capacity to induce complement-dependent haemolysis, dermonecrosis and lethality. Comparative analysis of gender and species, showed that these biological activities were more prominent in venom from female spiders, especially from L. laeta. Antiserum raised against venom from females L. laeta spiders had the highest efficacy in neutralizing venoms of males and females of both species. These results indicate that the severity of loxoscelism depends, at least partially, on the species and sex of the spider and suggest that for accidents involving L. laeta an specific serum therapy is necessary. Furthermore, it emphasizes the efficacy of the antiserum produced against L. laeta female venom in neutralizing Loxosceles venoms from different species and gender.
Assuntos
Antivenenos/metabolismo , Pele/patologia , Picada de Aranha/metabolismo , Venenos de Aranha/química , Aranhas/química , Análise de Variância , Animais , Antivenenos/uso terapêutico , Western Blotting , Eletroforese em Gel de Poliacrilamida , Eritrócitos/efeitos dos fármacos , Feminino , Citometria de Fluxo , Hemólise/efeitos dos fármacos , Humanos , Dose Letal Mediana , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Necrose/induzido quimicamente , Fatores Sexuais , Especificidade da Espécie , Esfingomielina Fosfodiesterase/metabolismo , Picada de Aranha/induzido quimicamente , Picada de Aranha/tratamento farmacológico , Venenos de Aranha/toxicidadeRESUMO
Loxosceles is the most venomous spider in Brazil, and envenomation causes dermonecrosis and complement (C)-dependent intravascular hemolysis. The authors studied the mechanism of induction of C-induced hemolysis. Purified Loxosceles toxins rendered human erythrocytes susceptible to lysis by human C but did not have an effect on the E-bound C-regulators DAF, CR1, or CD59. However, incubation with venom toxins caused cleavage of glycophorin from the erythrocyte (E) surface, facilitating C activation and hemolysis. The results suggest that glycophorin is an important factor in the protection of E against homologous C. Cleavage of glycophorin (GP) A, GPB, and GPC occurred at sites close to the membrane but could not be accomplished using purified GPA and purified toxins, demonstrating that cleavage was not an effect of a direct proteolytic action of the Loxosceles toxins on the glycophorins. Inhibition of the cleavage of glycophorins induced by Loxosceles venom was achieved with 1,10-phenanthroline. The authors propose that the sphingomyelinase activity of the toxins induces activation of an endogenous metalloproteinase, which then cleaves glycophorins. They observed the transfer of C-dependent hemolysis to other cells, suggesting that the Loxosceles toxins can act on multiple cells. This observation can explain the extent of hemolysis observed in patients after envenomation. Identification of the mechanism of induction of susceptibility to C-mediated lysis after Loxosceles envenomation opens up the possibility of the development of an effective therapeutic strategy. (Blood. 2000;95:683-691)
Assuntos
Proteínas do Sistema Complemento/fisiologia , Membrana Eritrocítica/fisiologia , Eritrócitos/fisiologia , Glicoforinas/efeitos dos fármacos , Hemólise , Metaloendopeptidases/sangue , Diester Fosfórico Hidrolases/farmacologia , Venenos de Aranha/farmacologia , Animais , Ativação Enzimática , Membrana Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Glicoforinas/metabolismo , Hemólise/efeitos dos fármacos , Humanos , Técnicas In Vitro , Células Jurkat , Células K562 , Metaloendopeptidases/efeitos dos fármacos , Neuraminidase/farmacologia , Diester Fosfórico Hidrolases/isolamento & purificação , Inibidores de Proteases/farmacologia , Venenos de Aranha/isolamento & purificação , Aranhas , Células U937RESUMO
The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement (C) dependent haemolysis. The aim of this study was to characterise the toxins in the venom responsible for the different biological effects. We have previously shown that a 35 kDa protein, named F35, purified from Loxosceles intermedia venom, incorporates into the membranes of human erythrocytes and renders them susceptible to the alternative pathway of autologous C. Here we have further purified the F35 protein which was resolved by reversed phase chromatography into three tightly contiguous peaks termed P1, P2, and P3. P1 and P2 were shown to be homogeneous by SDS-PAGE and N-terminal aminoacid analysis, while P3 consisted of two highly homologous proteins. N-terminal sequencing of all four proteins showed a high degree of homology, which was confirmed by cross-reactivity of antisera raised against the individual purified proteins. Functional characterisation of P1 and P2 indicated the presence of sphingomyelinase activity and either protein in isolation was capable of inducing all the in vivo effects seen with whole spider venom, including C-dependent haemolysis and dermonecrosis. In all assays, P2 was more active than P1, while P3 was completely inactive. These data show that different biological effects of L. intermedia venom can be assigned to the sphingomyelinase activity of two highly homologous proteins, P1 and P2. Identification of these proteins as inducers of the principal pathological effects induced by whole venom will aid studies of the mechanism of action of the venom and the development of a effective therapy.
Assuntos
Dermotoxinas/farmacologia , Hemólise/efeitos dos fármacos , Esfingomielina Fosfodiesterase/farmacologia , Venenos de Aranha/farmacologia , Sequência de Aminoácidos , Animais , Ensaio de Atividade Hemolítica de Complemento , Reações Cruzadas , Dermotoxinas/sangue , Ensaio de Imunoadsorção Enzimática , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Necrose , Fragmentos de Peptídeos/isolamento & purificação , Coelhos , Alinhamento de Sequência , Esfingomielina Fosfodiesterase/sangue , Esfingomielina Fosfodiesterase/química , Venenos de Aranha/sangue , Venenos de Aranha/enzimologiaRESUMO
Two additional patients with alpha-N-acetylgalactosaminidase (alpha-NAGA) deficiency are described. An 11-month-old girl with nonconsanguineous parents had generalized seizures and no angiokeratoma. Biochemical investigation showed persistent slight oligosacchariduria; enzymatic analysis of plasma, leukocytes, and fibroblasts revealed profound alpha-NAGA deficiency. Heterozygote enzyme levels were found in both parents. The mother has epilepsy, and epilepsy is present in the father's family. A younger, clinically healthy brother also had the enzyme deficiency. Electron microscopy of lymphocytes from the index patient showed no vacuolization. Incubation of cultured fibroblasts with Helix pomatia lectin showed the presence of intracellular N-acetylgalactosamine-containing storage material, not present in a series of 12 normal fibroblast lines. Our cases cannot be classified definitely as infantile cases. Biochemically the diagnosis could easily have been missed. Urinary oligosaccharide pattern after resorcinol staining was identical to those previously described, but excretion was significantly lower than in the reported infantile cases and the bands disappeared after the urine was desalted. The enzyme defect in leukocytes would have been missed with one of the commercial substrates used. For this mild variant of alpha-NAGA deficiency, the clinical pattern is not yet clear; a longer follow-up period is needed.