A rational protocol for the successful crystallization of L-amino-acid oxidase from Bothrops atrox.

Despite the valuable contributions of robotics and high-throughput approaches to protein crystallization, the role of an experienced crystallographer in the evaluation and rationalization of a crystallization process is still crucial to obtaining crystals suitable for X-ray diffraction measurements. In this work, the difficult task of crystallizing the flavoenzyme L-amino-acid oxidase purified from Bothrops atrox snake venom was overcome by the development of a protocol that first required the identification of a non-amorphous precipitate as a promising crystallization condition followed by the implementation of a methodology that combined crystallization in the presence of oil and seeding techniques. Crystals were obtained and a complete data set was collected to 2.3 Šresolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 73.64, b = 123.92, c = 105.08 Å, ß = 96.03°. There were four protein subunits in the asymmetric unit, which gave a Matthews coefficient V(M) of 2.12 Å(3) Da(-1), corresponding to 42% solvent content. The structure has been solved by molecular-replacement techniques.

Evidence of caspase-mediated apoptosis induced by l-amino acid oxidase isolated from Bothrops atrox snake venom.

The aim of this work was to investigate the involvement of caspases in apoptosis induced by l-amino acid oxidase isolated from Bothrops atrox snake venom. The isolation of LAAO involved three chromatographic steps: molecular exclusion on a G-75 column; ion exchange column by HPLC and affinity chromatography on a Lentil Lectin column. SDS-PAGE was used to confirm the expected high purity level of BatroxLAAO. It is a glycoprotein with 12% sugar and an acidic character, as confirmed by its amino acid composition, rich in "Asp and Glu" residues. It displays high specificity toward hydrophobic l-amino acids. The N-terminal amino acid sequence and internal peptide sequences showed close structural homology to other snake venom LAAOs. This enzyme induces in vitro platelet aggregation, which may be due to H2O2 production by LAAOs, since the addition of catalase completely inhibited the aggregation effect. It also showed cytotoxicity towards several cancer cell lines: HL60, Jurkat, B16F10 and PC12. The cytotoxicity activity was abolished by catalase. A fluorescence microscopy evaluation revealed a significant increase in the apoptotic index of these cells after BatroxLAAO treatment. This observation was confirmed by phosphatidyl serine exposure and activation of caspases. BatroxLAAO is a protein with various biological functions that can be involved in envenomation. Further investigations of its function will contribute to toxicology advances.

Alteraçöes cefalométricas ocorridas na dentadura mista após o uso de um expansor fixo tipo quadrihélice / Cephalometrics changes that occur in the mixed dentition after the use of the quad-helix fixed appliance

Este estudo foi conduzido com o intento de avaliar as alteraçöes cefalométricas suscitadas pelo expansor fixo tipo quadrihélice aplicado em pacientes no estágio da dentadura mista. A amostra utilizada constituiu de 33 crianças, sendo 9 do sexo masculino e 24 do sexo feminino. A idade dessas crianças variou de 5 anos a 11 anos e 5 meses, perfazendo um valor médio de 8 anos e 3 meses com um desvio padräo de 1 ano e 9 meses. Em todos esses casos o expansor fixo tipo quadrihélice foi empregado padronizadamente para descruzar uma mordida cruzada posterior. O aparelho tipo quadrihélice, embora tenha corrigido eficientemente a mordida cruzada posterior em todos os casos tratados, näo promoveu alteraçöes significantes nas grandezas cefalométricas angulares estudadas, quais sejam: NAP, SNA, ANB, SND, SN.GoGn, SN.Gn, SNPO. Já as alteraçöes significativas ocorridas nas grandezas lineares apreciadas (S-A, FMO-A, S-A', S-M') foram atribuídas ao crescimento normal das estruturas faciais. Assim sendo, concluímos que o aparelho expansor tipo quadrihélice constitui um dispositivo eficaz para a correçäo da mordida posterior; no entanto, näo incita alteraçöes cefalométricas similares àquelas provocadas pela expansäo rápida da maxila