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1.
Toxicon ; 150: 235-250, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29902540

RESUMO

Preparations of palytoxin (PLTX, derived from Japanese Palythoa tuberculosa) and the congeners 42-OH-PLTX (from Hawaiian P. toxica) and ovatoxin-a (isolated from a Japanese strain of Ostreopsis ovata), as well as a 50:50 mixture of PLTX and 42-OH-PLTX derived from Hawaiian P. tuberculosa were characterized as to their concentration, composition, in-vitro potency and interaction with an anti-PLTX monoclonal antibody (mAb), after which they were evaluated for lethality and tissue histopathology after intraperitoneal (IP) and aerosol administration to rats. Once each preparation was characterized as to its toxin composition by LC-HRMS and normalized to a total PLTX/OVTX concentration using HPLC-UV, all four preparations showed similar potency towards mouse erythrocytes in the erythrocyte hemolysis assay and interactions with the anti-PLTX mAb. The IP LD50 values derived from these experiments (0.92, 1.93, 1.81 and 3.26 µg/kg, for the 50:50 mix, 42-OH-PLTX, PLTX, and ovatoxin-a, respectively) were consistent with published values, although some differences from the published literature were seen. The aerosol LD50 values (0.063, 0.045, 0.041, and 0.031 µg/kg for the 50:50 mix, 42-OH PLTX, PLTX, and ovatoxin-a, respectively) confirmed the exquisite potency of PLTX suggested by the literature. The tissue histopathology of the different toxin preparations by IP and aerosol administration were similar, albeit with some differences. Most commonly affected tissues were the lungs, liver, heart, salivary glands, and adrenal glands. Despite some differences, these results suggest commonalities in potency and mechanism of action among these PLTX congeners.


Assuntos
Acrilamidas/química , Acrilamidas/toxicidade , Acrilamidas/administração & dosagem , Acrilamidas/metabolismo , Aerossóis , Animais , Venenos de Cnidários , Dinoflagellida/metabolismo , Relação Dose-Resposta a Droga , Feminino , Injeções Intraperitoneais , Toxinas Marinhas/administração & dosagem , Toxinas Marinhas/química , Toxinas Marinhas/toxicidade , Estrutura Molecular , Ratos , Ratos Endogâmicos F344
2.
J Food Prot ; 66(6): 1055-62, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12801009

RESUMO

Two multiplex polymerase chain reactions were developed for the detection of enterotoxigenic strains of Staphylococcus aureus: one multiplex reaction for the simultaneous detection of enterotoxigenic strains type A (entA), type B (entB), and type E (entE) and another for the simultaneous detection of enterotoxigenic strains type C (entC) and type D (entD). Both reactions were standardized with the use of the reference enterotoxigenic strains of S. aureus: FRI 722, producer of staphylococcal enterotoxin (SE) type A (SEA); FRI 1007, producer of SEB; FRI 137, producer of SEC1; FRI 472, producer of SED; and FRI 326, producer of SEE. Optimized methods were used to determine the presence of enterotoxigenic types for 51 S. aureus strains isolated from meat (sausage, ham, and chorizo) and dairy (powdered milk and cheese) products by the Baird-Parker technique. The enterotoxigenic capacities of the strains were determined by the indirect enzyme-linked immunosorbent assay (ELISA) with the use of reference staphylococcal toxins and antitoxins. Fifty of the 51 strains isolated were enterotoxigenic and produced one to four enterotoxin types, with the most frequently produced types being SEA and SED. Levels of correlation between the presence of genes that code for the production of SE (as determined by polymerase chain reaction) and the expression of these genes (as determined by the indirect ELISA) were 100% for SEA and SEE, 86% for SEC, 89% for SED, and 47% for SEB.


Assuntos
Laticínios/microbiologia , Enterotoxinas/isolamento & purificação , Microbiologia de Alimentos , Carne/microbiologia , Reação em Cadeia da Polimerase/métodos , Staphylococcus aureus/genética , Animais , Sequência de Bases , DNA Bacteriano/análise , Enterotoxinas/classificação , Sensibilidade e Especificidade , Staphylococcus aureus/química , Suínos
3.
Immunol Cell Biol ; 87(5): 400-12, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19417771

RESUMO

Attenuated Salmonella Typhi vaccine strains hold great promise as live vectors for presentation of foreign antigens from unrelated bacterial, viral and parasitic pathogens to the immune system. Although this approach has proved quite successful in experimental animal models for eliciting antigen-specific mucosal, humoral and cellular responses, results have been disappointing for clinical trials carried out thus far. We hypothesize that the paucity of human responses to foreign antigens delivered by live vectors suggests that the strains and genetic approaches used to date have resulted in overattenuated vaccine strains with severely reduced immunogenicity. However, remarkable advances have now been made in the genetics of foreign antigen expression, understanding mechanisms of live vector immunity and refining immunization strategies. The time has now come for development of multivalent live vectors in which stable antigen expression and export is balanced with metabolic fitness to create highly immunogenic vaccines.


Assuntos
Antígenos/imunologia , Vetores Genéticos/genética , Salmonella typhi/genética , Vacinas Atenuadas/imunologia , Animais , Antígenos/genética , Ensaios Clínicos como Assunto , DNA Recombinante/genética , Vetores Genéticos/administração & dosagem , Humanos , Imunização/métodos , Vacinas Atenuadas/genética
4.
Plasmid ; 50(1): 12-27, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12826054

RESUMO

The Salmonella enterica MisL (protein of membrane insertion and secretion) is an autotransporter with high homology to AIDA-I (adhesin involved in diffuse adherence) of enteropathogenic Escherichia coli. Considering that it has been reported that the MisL beta translocator domain is able to display heterologous passenger peptides to the bacterial surface, we developed a system to display proteins and release them to the external environment by means of proteolytic cleavage. Plasmids were constructed encoding 8 or 53 repeats of the NANP (Asp-Ala-Asp-Pro) tetrapeptide, which is the main B cell epitope of the Plasmodium falciparum circumsporozoitic protein (CSP), fused to the the MisL beta-domain and including the recognition cleavage sequence from the E. coli OmpT surface protease. E. coli XL-10Gold and BL21(DE3) (OmpT positive and negative, respectively) and Salmonella enterica serovar Typhimurium SL3261 (Aro A(-)) were transformed with the plasmids and, both expression and localization of the fusion proteins were assessed by Western blot, indirect immunofluorescence, and flow cytometry, using a monoclonal antibody against (NANP)(3). Higher expression of the (NANP)(8) and (NANP)(53) fusion proteins was demonstrated on the bacterial surface of the OmpT negative E. coli strains and the (NANP)(53) in the culture supernatant of E. coli XL-10Gold indicating a protease mediated cleavage. The flow cytometry analysis suggested 71 and 98% cleavage efficiency for the (NANP)(8) and (NANP)(53), respectively, in E. coli XL-10Gold. Similar results were obtained in S. enterica serovar Typhimurium SL3261, suggesting the involvement of other proteases related to OmpT. These results demonstrate that MisL may be used for the autodisplay and release of passenger proteins in attenuated Salmonella or E. coli strains, which may have several applications in vaccine design.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Proteínas de Membrana Transportadoras , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Salmonella enterica/genética , Adesinas de Escherichia coli/genética , Sequência de Aminoácidos , Animais , Escherichia coli/genética , Vetores Genéticos , Dados de Sequência Molecular , Engenharia de Proteínas , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
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