RESUMO
Tri(n-butyl)phosphate (TNBP) and sodium cholate (SC) mixtures have been used to inactivate lipid-enveloped viruses like HIV and hepatitis B. We exploited the use of this combination to purify fibroblast growth factor-2 (FGF-2) from human placenta. Human placentas were extracted in the presence of 0.3% TNBP/0.2% SC and the clarified homogenate was adsorbed to S-Sepharose. The active fractions were further loaded onto a heparin-Sepharose column and purified FGF-2 was eluted with 2.0 M NaCl. FGF-2 purified this way was indistinguishable from FGF-2 purified without TNBP/SC in the extraction step in terms of yield, specific activity and biological response. The lipid-enveloped vaccinia virus was used in a parallel experiment to evaluate the inactivation capacity of our protocol. Under the conditions described here, the combined use of TNBP/SC did not eliminate but reduced significantly the number of vaccinia virus PFUs by log 2-3.
Assuntos
Ácidos Cólicos , Fator 2 de Crescimento de Fibroblastos/isolamento & purificação , Organofosfatos , Placenta/química , Células 3T3 , Animais , Ácido Cólico , Ácidos Cólicos/farmacologia , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Indicadores e Reagentes , Camundongos , Mitógenos/farmacologia , Peso Molecular , Organofosfatos/farmacologia , Placenta/virologia , Vaccinia virus/efeitos dos fármacos , Ensaio de Placa ViralRESUMO
We determined the circulating level of bioactivity for skeletal muscle proteolysis-inducing factors (PIF) in the blood samples from cancer patients whose body weight loss was greater than 10%. The level of bioactivity was estimated by measurement of tyrosine release from isolated 1at diaphragm muscles incubated with an ultrafiltered fraction of plasma or serum proteins containing molecules from 0 to 25 kDa in molecular weight. Significant levels of bioactivity were detected in 25 of the 50 cancer samples. No activity was found in 18 of the samples from healthy human blood donors. The ability of 13 of the cancer samples to induce muscle proteolysis was significantly inhibited by incubation of muscles in presence of indomethacin (10 microM). The neutralisation of 12 of the cancer samples with the antibodies to recombinant human interleukin-1 (IL-1), alpha and beta forms, partially abrogated the activity in five samples. These results suggest that the accelerated breakdown of proteins induced by the cancer plasma factors is at least in part mediated by IL-1 in cooperation with other active factors not yet defined. Additionally, we have shown that the increased breakdown of proteins induced by PIF in the crude supernatant derived from activated mouse peritoneal macrophages is prevented by the treatment of muscles with either indomethacin or quin-2 (1 microM). These observations provide indirect evidence for a possible causal relationship between the production of PIF and the body-weight loss of cancer patients.
Assuntos
Proteínas Sanguíneas/farmacologia , Caquexia/sangue , Proteínas Musculares/metabolismo , Neoplasias/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Proteínas Sanguíneas/antagonistas & inibidores , Criança , Diafragma/metabolismo , Feminino , Humanos , Indometacina/farmacologia , Interleucina-1/análise , Interleucina-1/antagonistas & inibidores , Interleucina-1/farmacologia , Masculino , Camundongos , Pessoa de Meia-Idade , Peso Molecular , Ratos , Ratos Endogâmicos , Proteínas Recombinantes/análise , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/farmacocinética , Redução de PesoRESUMO
We determined the circulating level of bioactivity for skeletal muscle proteolysis-inducing factors (PIF) in the blood samples from cancer patients whose body weight loss was greater than 10%. The level of bioactivity was estimated by measurement of tyrosine release from isolated rat diagphragm muscles incubated with an ultrafiltered fraction of plasma or serum proteins containing molecules from 0 to 25 kDa in molecular weight. Significant levels of bioactivity were detected in 25 of the 50 cancer samples. No activity was found in 18 of the samples from healthy human blood donors. The ability of 13 of the cancer samples to induce muscle proteolysis was significantly inhibited by incubation of muscles in presence of indomethacin (10 gM). The neutralisation of 12 of the cancer samples with the antibodies to recombinant human interleukin-I (IL-1), a and P forms, partially abrogated the activity in five samples. These results suggest that the accelerated breakdown of proteins induced by the cancer plasma factors is at least in part mediated by IL-1 in cooperation with other active factors not yet defined. Additionally, we have shown that the increased breakdown of proteins induced by PIF in the crude supernatant derived from activated mouse peritoneal macrophages is prevented by the treatment of muscles with either indomethacin or quin-2 (1 juM). These observations provide indirect evidence for a possible causal relationship between the production of PIF and the body-weight loss of cancer patients.