RESUMO
A very sensitive and convenient fluorescence nanobiosensor for rapid detection of DNA methylation based on Fe3O4/Au core/shell nanoparticles has been developed. Specific site of CpG islands of adenomatous polyposis coli (APC), a well studied tumor suppressor gene, was used as the detection target DNA sequence. The characteristics of nanoparticles were determined by scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive spectroscopy (EDS), UV-visible spectroscopy and X-ray diffraction (XRD) spectroscopy. Fe@Au nanoparticles functionalized by bounding of single stranded DNA (ssDNA) probe through sulfhydryl group at the 5' phosphate end. Then unmethylated and methylated complementary target ssDNA were hybridized with the immobilized ssDNA probe. Dipyridamole, a pharmaceutical agent used for the first time as a fluorescence probe which significantly interacted with hybridized unmethylated and methylated DNA. Upon the addition of the target unmethylated and methylated ssDNA, the fluorescence intensity increased in linear range by concentration of unmethylated ssDNA from 1.6 × 10(-15) to 6.6 × 10(-13)M with detection limit of 1.2 × 10(-16)M and on the other hand, fluorescence intensity declined linearly with concentration of 3.2 × 10(-15)-8.0 × 10(-13)M methylated DNA and detection limit was 3.1 × 10(-16)M. We have also shown that nanobiosensor could distinguish ratio of methylation in series of partially methylated DNA targets with identical sequences. A density functional theory (DFT) calculation was also performed to investigate the interaction between Dipyridamole with unmethylated and methylated cytosine. Finally real sample analysis suggested that nanobiosensor could have practical application for methylation detection in human plasma sample.
Assuntos
Técnicas Biossensoriais/instrumentação , Metilação de DNA/genética , DNA de Neoplasias/genética , Detecção Precoce de Câncer/instrumentação , Fluorometria/instrumentação , Nanotecnologia/instrumentação , Neoplasias/diagnóstico , Animais , DNA de Neoplasias/análise , DNA de Neoplasias/química , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Neoplasias/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA/instrumentaçãoAssuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA/genética , DNA de Neoplasias/genética , Neoplasias Pulmonares/genética , Regiões Promotoras Genéticas/genética , Carcinoma de Pequenas Células do Pulmão/genética , Adulto , Idoso , Biomarcadores Tumorais/genética , Feminino , Genes Supressores de Tumor , Humanos , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
A phospholipase C which hydrolyzes [14C]phosphatidylcholine has been purified 1782-fold from 70% ammonium sulfate extract of bull seminal plasma. Purification steps included acid precipitation, chromatography on DEAE-Sephacel, concanavalin A, octyl-Sepharose 4B and Ultrogel AcA 34. The final step provided homogeneous phospholipase C as determined by polyacrylamide gel electrophoresis. The enzyme comprised two subunits, Mr 69,000 and Mr 55,000, respectively. The enzyme had an optimum at pH 7.2 and pI 5.0. EDTA, Cd2+, Pb2+, Ni2+, Fe2+, and Zn2+ inhibited phospholipase C activity. Km and Vmax on p-nitrophenyl phosphorylcholine and phosphatidylcholine substrates were 20 mM and 17 mumol/min/mg of the purified enzyme and 100 microM and 18 mumol/min/mg of the purified enzyme, respectively. The enzyme appeared to be localized in the acrosome as judged by the binding of anti-phospholipase C to the acrosome. This phospholipase C, unlike other known phospholipases (C), did not hydrolyze [1-14C]phosphatidylinositol. The testicular extract of the guinea pig contained inactive phospholipase C which was activated on incubation with acrosin and trypsin but not chymotrypsin.
Assuntos
Fosfatidilcolinas/metabolismo , Sêmen/enzimologia , Fosfolipases Tipo C/isolamento & purificação , Acrosina/farmacologia , Animais , Radioisótopos de Carbono , Bovinos , Ácido Edético/farmacologia , Hidrólise , Focalização Isoelétrica , Cinética , Masculino , Peso Molecular , Fosfatidiletanolaminas/metabolismo , Fosfatidilinositóis/metabolismo , Tripsina/farmacologia , Fosfolipases Tipo C/análiseRESUMO
The antibody to the rabbit sperm inner acrosomal membrane, raised in guinea pig, completely inhibited the fertilization of rabbit ova in vitro. The F(ab')2 of the antibody was equally effective in inhibiting fertilization. The antibody appeared to exert its inhibitory effect by binding to the inner acrosomal membrane of acrosome-reacted sperm. The antibody-treated sperm did not attach to or penetrate the zona pellucida. Thus, anti-IAM offers a great potential as a contraceptive agent.
