RESUMO
BACKGROUND: Extracellular matrix degradation, mediated by the urokinase plasminogen activation (uPA) system, is a critical step in tumor invasion and metastasis. High tumor levels of uPA and its inhibitor PAI-1 have been correlated with poor cancer prognosis. We examined four single nucleotide polymorphisms (SNPs) with a potential effect on expression of genes in the uPA system for their role in colorectal cancer susceptibility and prognosis. PATIENTS AND METHODS: We genotyped the SNPs in 308 Swedish incident colorectal cancer patients with up to 16 years of follow-up and in 585 age- and sex-matched controls. We evaluated the associations between genotypes and colorectal cancer and Dukes' stage. Survival probabilities were compared between different subgroups. RESULTS: Patients with PAI-1 -675 5G/5G genotype had better survival than patients with 4G/4G or 4G/5G genotypes when they had Dukes' stage A or B tumors (P = 0.023 and P = 0.015, respectively). No statistically significant association was observed between the SNPs and the risk of colorectal cancer or Dukes' stage. CONCLUSIONS: Our results suggest a role for the PAI-1 genotype in colorectal cancer prognosis, but further studies are needed to evaluate the impact of our finding in the clinic.
Assuntos
Neoplasias Colorretais/genética , Polimorfismo de Nucleotídeo Único , Ativador de Plasminogênio Tipo Uroquinase/genética , Estudos de Casos e Controles , Estudos de Coortes , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Análise de Sobrevida , Suécia , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Specific serine and threonine residues of recombinant human beta-casein produced in Escherichia coli were shown to be phosphorylated in vivo when human casein kinase II was coexpressed in the same plasmid. All of the phosphorylated forms found in the native protein were also detected in the recombinant protein. The phosphorylation of recombinant human beta-casein was confirmed by immunoblots, fast protein liquid chromatography, urea-polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis, and liquid chromatography-mass spectrometry. The results indicate that the substrate specificity of casein kinase II in vivo was unaffected in its recombinant form. This is the first demonstration of in vivo phosphorylation of specific residues of a multiphosphorylated protein produced in E. coli with a single plasmid.
Assuntos
Caseínas/biossíntese , Caseínas/química , Caseína Quinase II , Caseínas/genética , Caseínas/isolamento & purificação , Coenzimas/análise , Coenzimas/genética , Humanos , Espectrometria de Massas , Mutagênese Insercional , Fosforilação , Plasmídeos/genética , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/químicaRESUMO
The adherence of pyelonephritic Escherichia coli isolates to mammalian host cells is mediated by the P-pili structures on the bacterial surface. The protein constituting the distal part of the pili structure, papG, interacts with glycan receptors on the host cell. Variation in specificity for different glycoconjugates between the isolates, that may reflect variation in host tropism, has been correlated to three different classes of papG. Truncated variants of the class I, II and III papG adhesins were produced as fusion protein in E. coli and analysed for carbohydrate binding. The results showed that both carbohydrate binding and specificity of the papG adhesin resided in a linear part of the N-terminus of the protein.
Assuntos
Adesinas de Escherichia coli/química , Adesinas de Escherichia coli/metabolismo , Metabolismo dos Carboidratos , Proteínas de Fímbrias , Fímbrias Bacterianas/química , Fragmentos de Peptídeos/química , Adesinas de Escherichia coli/genética , Adulto , Sequência de Bases , Sítios de Ligação , Cromatografia de Afinidade , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Hemaglutinação , Humanos , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Relação Estrutura-AtividadeRESUMO
Human plasminogen activator inhibitor type 1, PAI-1, was expressed in Chinese hamster ovary cells. A production level of 10-15 mg latent PAI-1 per liter of media was achieved after methotrexate amplification. Latent recombinant PAI-1 was purified by two chromatographic steps, cation exchange chromatography on CM-Sepharose and affinity chromatography on heparin-Sepharose. The obtained latent PAI-1 was approximately 90-95% pure showing one homogenous peak upon size-exclusion chromatography. However, four different isoforms due to different degrees of sialylation could be seen upon isoelectric focusing. Purified latent PAI-1 was activated by incubation in 6 M guanidine-HCl. By this method, 40-60% of PAI-1 was converted to an active form after removing the denaturant. The active fraction of PAI-1 was separated from inactive material by size exclusion chromatography on Superdex 200. Active PAI-1 migrated as expected for a 43-kDa large protein, while inactive PAI-1 migrated as larger protein complexes, suggesting that the remaining inactive PAI-1 was in the form of aggregates. This method for the separation of active and inactive PAI-1 could also be used for activated native PAI-1 prepared from human endothelial cells. Active recombinant PAI-1 was remarkably stable at pH 5.5, both when stored on ice and when stored at room temperature.
