ExoU, uma proteína do sistema de secreção do tipo III de Pseudomonas aeruginosa, induz a secreção de eicosanóides por células epiteliais respiratórias. Papel dos corpúsculos lipídicos neste processo

Para pesquisar o papel de ExoU no desencadeamento de resposta inflamatória nas vias aéreas, células epiteliais respiratórias humanas (CERs) da linhagem BEAS-2B foram tratadas com AA radiomarcado e infectadas com a cepa PA103 de P. aeruginosa, que secreta ExoU, e com os mutantes PA103exoU (com deleção do gene exoU), PA103ΔUT/exoU (com deleção de exoU e complementação com o gene funcional) e PA103UT/S142A (com deleção de exoU e complementação com gene com mutagênese sítio-específica no domínio catalítico da enzima). Após 1 hora, a liberação de AA pelas culturas infectadas com as cepas produtoras de ExoU foi significativamente superior à observada em culturas infectadas pelas cepas não-produtoras ou por células controle. O tratamento das bactérias com MAFP, um inibidor de PLA2, resultou em significativa redução na liberação de AA. Células infectadas pelas cepas PA103 e PA103ΔUT/exoU secretaram PGE2 e LTB4 em concentrações significativamente maiores que as secretadas por células infectadas pelas demais cepas ou não infectadas. O tratamento com o MAFP reduziu significativamente a secreção de PGE2. A análise, por citometria de fluxo, de células infectadas e não infectadas tratadas com anticorpo anti-COX-2 mostrou que o percentual de células infectadas por PA103 marcadas foi significativamente superior ao percentual encontrado em culturas controle. Nenhuma diferença foi observada quanto ao percentual de células marcadas em culturas infectadas por PA103ΔexoU. O tratamento das culturas com NS-398 (um inibidor seletivo de COX-2) resultou na diminuição significativa da concentração de PGE2, secretada por células infectadas com PA103, mas não por células infectadas com PA103ΔexoU ou por células controle. Corpúsculos lipídicos (CLs) são domínios citoplasmáticos ricos em COX-2 e outras enzimas responsáveis pelo metabolismo do AA, sede da produção de eicosanóides. Como células infectadas pelas cepas produtoras de ExoU liberam AA livre, formulamos a hipótese de que a maior produção de eicosanóides por estas células seria dependente da indução do aumento no número dos CLs. No entanto, a análise por citometria de fluxo de células tratadas com uma sonda lipofílica com afinidade com os CLs mostrou que o percentual de células marcadas em culturas infectadas pelas cepas produtoras de ExoU foi significativamente inferior ao percentual em culturas controle ou infectadas pelas outras 2 cepas bacterianas. O tratamento das células com MAFP inibiu significativamente a redução do percentual de células contendo CLs. A análise, por citometria de fluxo, de células controle ou infectadas tratadas simultaneamente com a sonda lipofílica e com o anticorpo anti-PGE2, mostrou, em células infectadas com PA103, a redução da mediana da intensidade de marcação com a sonda lipofílica e o aumento da mediana da intensidade de marcação com o anticorpo anti-PGE2. Nossa hipótese é que a presença de ExoU nas células infectadas com a cepa PA103 resulte no metabolismo de glicerofosfolipídios presente nos CLs levando à diminuição da afinidade dos CLs pela sonda lipofílica e à síntese local de PGE2.

Development, validation, and utilization of a competitive enzyme-linked immunosorbent assay for the detection of antibodies against Brucella species in marine mammals

A competitive enzyme-linked immunosorbent assay (cELISA) was developed by using a whole-cell antigen from a marine Brucella sp. isolated from a harbor seal (Phoca vitulina). The assay was designed to screen sera from multiple marine mammal species for the presence of antibodies against marine-origin Brucella. Based on comparisons with culture-confirmed cases, specificity and sensitivity for cetacean samples tested were 73% and 100%, respectively. For pinniped samples, specificity and sensitivity values were 77% and 67%, respectively. Hawaiian monk seal (Monachus schauinslandi; n = 28) and bottlenose dolphin (Tursiops truncatus; n = 48) serum samples were tested, and the results were compared with several other assays designed to detect Brucella abortus antibodies. The comparison testing revealed the marine-origin cELISA to be more sensitive than the B. abortus tests by the detection of additional positive serum samples. The newly developed cELISA is an effective serologic method for detection of the presence of antibodies against marine-origin Brucella sp. in marine mammals.

Technique for wavelength converting a 2.488 Gb/s signal whilst simultaneously monitoring and replacing a wavelength identifying pilot tone

A wavelength conversion device was demonstrated at the bit rate of 2.488 Gb/s with 2R (reamplification and reshaping) regenerative properties. A low frequency pilot tone was removed during the conversion process and a new one added. The wavelength converter is shown to operate well at 10 Gb/s, and tone identification/replacement should also be possible at this data rate.

ISOLATION AND PROPERTIES OF A BLOOD-COAGULATION FACTOR-X ACTIVATOR FROM THE VENOM OF KING COBRA (OPHIOPHAGUS HANNAH)

A specific blood coagulation factor X activator was purified from the venom of Ophiophagus hannah by gel filtration and two steps of FPLC Mono-Q column ion-exchange chromatography. It showed a single protein band both in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline polyacrylamide gel electrophoresis. The mol. wt was estimated to be 62,000 in non-reducing conditions and 64,500 in reducing conditions by SDS-PAGE. The isoelectric point was found to be pH 5.6. The enzyme had weak amidolytic activities toward CBS 65-25, but it showed no activities on S-2266, S-2302, thrombin substrate S-2238, plasmin substrate S-2251 or factor Xa substrate S-2222. It had no arginine esterase activity toward substrate benzoylarginine ethylester (BAEE). The enzyme activated factor X in vitro and the effect was absolutely Ca2+ dependent, with a Hill coefficient of 6.83. It could not activate prothrombin nor had any effect on fibrinogen and thus appeared to act specifically on factor X. The procoagulant activity of the enzyme was almost completely inhibited by serine protease inhibitors like PMSF, TPCK and soybean trypsin inhibitor; partially inhibited by L-cysteine. Metal chelator EDTA did not inhibit its procoagulant activity. These results suggest that the factor X activator from O. hannah venom is a serine protease.

AN ACTIVATOR OF BLOOD-COAGULATION FACTOR-X FROM THE VENOM OF BUNGARUS-FASCIATUS

A specific activator of blood coagulation factor X was purified from the venom of Bungarus fasciatus by gel filtration and by ion-exchange chromatography on a Mono-Q column (FPLC). It consisted of a single polypeptide chain, with a mel. wt of 70,000 in reducing and non-reducing conditions. The enzyme had an amidolytic activity towards the chromogenic substrates S-2266 and S-2302 but it did not hydrolyse S-2238, S2251 or S-2222, which are specific substrates for thrombin, plasmin and factor Xa, respectively. The enzyme activated factor X in vitro and the effect was Ca2+ dependent with a Hill coefficient of 7.9. As with physiological activators, the venom activator cleaves the heavy chain of factor X, producing the activated factor Xa alpha. The purified factor X activator from B. fasciatus venom did not activate prothrombin, nor did it cleave or clot purified fibrinogen. The amidolytic activity and the factor X activation activity of the factor X activator from B. fasciatus venom were readily inhibited by serine protease inhibitors such as diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), benzamidine and by soybean trypsin inhibitor but not by EDTA. These observations suggest that the factor X activator from B. fasciatus venom is a serine protease. It therefore differs from those of activators obtained from Vipera russelli and Bothrops atrox venoms, which are metalloproteinases.

Identification and cloning of a trypsin inhibitor from skin secretions of Chinese red-belly toad Bombina maxima

A novel trypsin inhibitor was identified and purified from skin secretions of Chinese red-belly toad Bombina maxima. The partial N-terminal 29 amino acid residues of the peptide, named BMTI, were determined by automated Edman degradation. This allowed the cloning of a full-length cDNA encoding BMTI from a cDNA library prepared from the toad skin. The deduced complete amino acid sequence of BMTI indicates that mature BMTI is composed of 60 amino acids. A FASTA search in the databanks revealed that BMTI exhibits 81.7% sequence identity with BSTI, a trypsin/thrombin inhibitor from European toad Bombina bombina skin secretions. Sequence differences between BMTI and BSTI were due to 11 substitutions at positions 2, 9, 25, 27, 36-37, 39, 41-42, 50 and 56. BMTI potently inhibited trypsin with a K-i value of 0.06 muM, similar to that of BSTI. However, unlike BSTI, which also inhibited thrombin with a K-i value of 1 muM, no inhibitory effect of BMTI on thrombin was observed under the assay conditions. (C) 2002 Elsevier Science Inc. All rights reserved.