RESUMO
The aim of this work was to obtain the recombinant single chain variable fragments of antibodies (scFv) against human protein C, the key component of blood anticoagulation system. For this purpose a peptide that mimics a Pro144-Leu155 sequence of protein C was synthesized and the murine immune scFv library against this peptide was constructed. The protein C specific scFv 9E were selected from the constructed library by the phage-display method. The scFv 9E dissociation constant was found to be 2â10(-9) M. It was shown that scFv 9E were suitable for protein C detection by ELISA and Western blotting. Selected scFv could be further used for protein C investigation and for the development of quantitative methods for protein C detection in human blood.
Assuntos
Especificidade de Anticorpos , Oligopeptídeos/química , Proteína C/análise , Anticorpos de Cadeia Única/isolamento & purificação , Animais , Bovinos , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virologia , Feminino , Hemocianinas/química , Hemocianinas/imunologia , Humanos , Imunoconjugados/química , Imunoconjugados/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Moluscos , Oligopeptídeos/imunologia , Biblioteca de Peptídeos , Ligação Proteica , Proteína C/química , Proteína C/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Soroalbumina Bovina/química , Soroalbumina Bovina/imunologia , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/químicaRESUMO
The values of endoplasmic reticulum Ca(2+)-pool and store-operated Ca2+ entry (SOCE) were estimated in rat thymocytes and Jurkat cells loaded with indo-1 and treated with thapsigargin. It was shown that the relative value of SOCE in thymocytes was substantially lower than in Jurkat cells. Significant increase of SOCE in Jurkat cells preincubated with 10(-5) M C60 and exposed to uv/visible light irradiation was detected at 1-3 h after exposure. At this time FCCP-induced Ca(2+)-release from mitochondria was shown to be reduced, while cytochrome c level into the cytoplasm of Jurkat cells, detected by Western blot analysis, to be increased. It is supposed that Ca2+ flux remodulation induced by photoexcited fullerene C60 in Jurkat cells might be involved in the initiation of signalling events leading to cell apoptosis.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Citocromos c/metabolismo , Fulerenos/farmacologia , Mitocôndrias/efeitos dos fármacos , Fármacos Fotossensibilizantes/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/efeitos da radiação , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes , Humanos , Indóis , Células Jurkat , Luz , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Especificidade de Órgãos , Ionóforos de Próton/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Tapsigargina/farmacologia , Timócitos/citologia , Timócitos/efeitos dos fármacos , Timócitos/metabolismo , Timócitos/efeitos da radiaçãoRESUMO
AIM: To estimate the viability of normal and transformed T-lymphocytes after UV/Vis irradiation in the presence of pristine fullerenes C(60). METHODS: Thymocytes were isolated from Wistar rats' thymus. Murine leukemia L1210 and human lymphoma Jurkat cells were used in this study. Mercury-vapor lamp was used for fullerenes C(60) photoexcitation. Cytotoxicity was etermined by MTT assay. Changes in cell morphology were monitored by phase-contrast light microscopy. RESULTS: fullerenes C(60) exhibit cytotoxic effect against transformed T-lymphocytes when combined with UV/Vis irradiation (320-600 nm). Photoinduced effect was enhanced with the increasing of irradiation time period and C(60) concentration, cell death was registered after 24 hours incubation. Fullerenes C(60) photocytotoxicity against normal T-lymphocytes (thymocytes) was not observed. CONCLUSION: The present study suggests that pristine fullerenes C(60) have the potential to be an effective photosensitizer and exhibit cytotoxic effect on transformed T-cells in vitro .
Assuntos
Fulerenos/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/efeitos da radiação , Raios Ultravioleta , Animais , Linhagem Celular Transformada , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Fotoquímica , Ratos , Ratos Wistar , Linfócitos T/patologia , Células Tumorais CultivadasRESUMO
The viability of normal (Wistar rat thymocytes) and transformed (human leukemia Jurkat cells) T cells after UV/Vis irradiation in the presence of pristine C60 fullerene was studied. The data obtained have shown that C60 fullerene exhibits cytotoxic effect against transformed T lymphocytes when combined with UV/Vis irradiation using mercury-vapor lamp (320-600 nm). C60 fullerene photocytotoxicity was not detected in thymocytes. C60-dependent photoinduced apoptosis of Jurkat cells was confirmed by DNA fragmentation and caspase-3 activation. No substantial increase of caspase-3 activation was observed in thymocytes treated with C60 fullerene plus irradiation, while antileukemic agent cytosine arabinoside was shown to induce caspase-3 activation both in Jurkat cells and thymocytes. The data obtained may be useful for development of photosensitizers for photodynamic therapy with selective action on leukemia cells.
