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1.
J Immunol Methods ; 307(1-2): 135-43, 2005 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-16277988

RESUMO

The systems for display of foreign peptides and polypeptides on filamentous bacteriophage have exploited genetic fusion to all of the five coat proteins. Multivalent display systems allowing selection of low affinity antibody fragments have been devised for fusions to gene III. However, since pIII has to interact with the bacterial receptors during the infection process, reduced infectivity can be observed. Alternative display systems utilizing other coat protein have been examined. These, however, take advantage of phagemid systems, in which a mixture of fusion and non-fusion coat proteins becomes displayed, thus preventing multivalent display. In this paper, we describe genetically stable fusion of scFv fragments to gene VII directly in the phage genome, thus giving rise to a multivalent display system where infectivity is not comprised. A hundred-fold enrichments factor can be obtained in model selection. Our results demonstrate that the small size of pVII (33 amino acids) is not structurally compromised by fusion of scFv antibody fragments at their N-terminus, thus demonstrating the feasibility of utilizing pVII as a fusion partner.


Assuntos
Bacteriófago M13/genética , Proteínas do Capsídeo/genética , Fragmentos de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Biblioteca de Peptídeos , Animais , Western Blotting , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Bovinos , Ensaio de Imunoadsorção Enzimática , Escherichia coli/virologia , Humanos , Fragmentos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Soroalbumina Bovina/imunologia , Estreptavidina/metabolismo , Ubiquitina/imunologia
2.
Kardiol Pol ; 63(2): 107-13; discussion114, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16136407

RESUMO

INTRODUCTION: The glycoprotein complex Ia/IIa (GP Ia/IIa) is a major collagen receptor on platelets and other cell types. Recently, linked polymorphisms within the coding region of the GP Ia gene (C807T and G873A) related to GP Ia/IIa surface expression have been identified. The 807T/873A allele is associated with high expression, whereas the 807C/873G allele is associated with low surface expression of GP Ia/IIa. Subsequently, the 807T allele was found to be associated with coronary artery disease (CAD) and cerebral infarction in younger patients. Moreover, platelet thrombus formation is significantly influenced by genetic variations of the GPIb alpha and GPIa receptors and is dependent on the blood flow rate. AIM: 1. To determine the frequency of C807T polymorphism of the GPIa gene in young survivors of myocardial infarction (MI) and 2. to evaluate the relationship between the intensity of CAD in the coronary angiography examination and the 807C/T genetic status of the patients. METHODS: 102 young male survivors of MI (YSMI) -- mean age 43, range 29-49 years, mean age at the time of the first episode 37+/-3 years -- were studied. Obesity was found in 15%, diabetes in 14%, hyperlipidemia in 87%, hypertension in 22% and smoking history in 90% of cases. Familial CAD and/or MI were confirmed in 50% of patients. The control group consisted of 106 healthy volunteers with a negative family history of CAD, both medical staff members and blood donors (mean age 40, range 18-42 years). The genetic study was performed using genomic DNA obtained from peripheral blood leukocytes. The C807T polymorphism of platelet glycoprotein Ia (GPIa) was investigated using the PCR method introduced by Santoso et al. RESULTS: Coronary angiography (Siemens Bicor system) revealed single-artery disease in 34%, two-artery disease in 36% and three-artery disease in 26% of patients. In two patients there were no signs of CAD. The C807T polymorphism of GPIa was found in 73.5% of investigated patients (heterozygotes CT 59.8%, homozygotes TT 13.7%). The CC genotype was confirmed in 26.5% of patients. A similar analysis performed in the group of healthy men showed C807T polymorphism of the GPIa gene in 73.6% (CT in 58.5% and TT in 15.1% of persons, ns). CC genotype was found in 26.4% of persons. Interestingly, the T genotype frequency was similar in patients with three- or two-artery disease in comparison with patients with single-vessel or without CAD (49.3% vs. 50.7%, respectively, ns). In 75 YSMI carrying C807T polymorphism of the GPIa gene additional genetic abnormalities were confirmed in 21 patients - BclI polymorphism of b-chain fibrinogen gene, G4070A and G1691A (FV Leiden) mutation of factor V gene and C677T polymorphism of methylenetetrahydrofolate reductase gene. Partial occurrence of combined polymorphisms was found. This was confirmed independently of the number of coronary arteries involved. CONCLUSIONS: Our results may question the potential role of C807T the GPIa anomaly as a single genetic abnormality predisposing young men to coronary artery disease.


Assuntos
Angiografia Coronária , Integrina alfa2/genética , Mutação , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/genética , Polimorfismo Genético , Adulto , Alanina , Estudos de Casos e Controles , Cisteína , Expressão Gênica , Frequência do Gene , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Treonina
3.
Kardiol Pol ; 59(9): 205-12, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-14618197

RESUMO

BACKGROUND: Several polymorphisms in the genes encoding for three separate chains of fibrinogen have been described. Some of them (Hae III and B854) are associated with elevated fibrinogen plasma level. AIM: To determine the frequency of BclI polymorphism in the fibrinogen beta-chain gene (BclI betaFb) in young survivors of myocardial infraction (MI) and to assess the relationship between allele status, plasma fibrinogen concentration and the number of affected coronary arteries. METHODS: The study group consisted of 99 male patients (mean age 43.5, range 29-49 years) with premature coronary artery disease (CAD) diagnosed by coronary angiography who had MI in the mean age of 37.4+/-3.2 years. The control group involved 78 age- and gender-matched healthy volunteers. DNA was extracted from peripheral blood leukocytes using standard methods. Fibrinogen blood concentration was determined using biuretic method. The BclI polymorphism in the fibrinogen beta-chain gene was investigated using polymerase chain reaction (PCR). RESULTS: Obesity was found in 15%, smoking - in 89%, hypertension - in 21%, diabetes - in 14% and hyperlipidemia - in 86% of MI patients. A family history of MI was present in 50% of patients. Coronary angiography revealed single-vessel disease in 34%, two-vessel disease in 36%, and three-vessel disease in 16% of patients. In two patients coronary angiography was normal. The frequency of BclI polymorphism of the beta-fibrinogen gene was significantly higher in MI patients than in controls (40.4% vs 29.5%, p<0.01). Moreover, in MI patients carrying the mutant allele a higher concentration of blood fibrinogen was found in comparison to patients without this anomaly (3.87 vs 3.55 g/L, p=0.05). There was no evidence of an association between the number of affected coronary arteries and polymorphism of BclI betaFbg gene status. However, all patients carrying BclI polymorphism of betaFbg gene had abnormal coronary angiography, contrary to patients without any defect. CONCLUSIONS: 1. Polymorphism of BclI betaFbg gene is associated with an increased fibrinogen plasma level. 2. There is no association between BclI polymorphism of betaFbg gene and the number of affected coronary arteries. This may confirm the hypothesis of multi-factorial aetiology of CAD in young patients suffering MI.


Assuntos
Fibrinogênio/genética , Fibrinogênio/metabolismo , Genes bcl-1 , Infarto do Miocárdio/sangue , Infarto do Miocárdio/genética , Polimorfismo Genético , Adulto , Estudos de Casos e Controles , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase
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