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1.
Genet Mol Res ; 10(4): 3747-59, 2011 Nov 22.
Artículo en Inglés | MEDLINE | ID: mdl-22179998

RESUMEN

An electrochemical biosensor, using a disposable electrochemical printed chip aggregation by the bisbenzimide dye (Hoechst 33258), was used for detecting the expression of ß-actin and RAGE genes. Using linear sweep voltammetry, the expression of these two genes in HeLa and HepG2 cell lines was determined based on anodic peak current, and the results were compared with conventional agarose gel electrophoresis. Total cellular RNA was reverse transcribed to complementary DNA, and amplification by PCR was carried out. Subsequently, the PCR products were subjected to detection by either electrophoresis or electrochemical biosensor. Precision of the electrochemical biosensor technique was acceptable (ß-actin: CV = 1.875% for 10(4) copies and 4.684% for 10(9) copies; RAGE: CV = 2.253% for 10(9) copies, and 3.743% for 10 copies). In the electrochemical biosensor technique, the PCR products were measured in the same run with various concentrations of standards, and copy numbers of ß-actin gene were interpolated from a standard curve. Copy numbers of the ß-actin gene were then compared between the two techniques. At the 95% confidence limit, the two methods had no significant differences and were significantly correlated (y = -40383.0623 + 1.0233x; P > 0.10). The electrochemical biosensor method was more sensitive than the conventional electrophoresis method because it could detect as low as 10 copies of the RAGE gene. The conventional electrophoresis method detected the RAGE gene at concentrations of at least 10(4) copies, and the linearity for semi-quantitative measurement was in the range of 10(6)-10(9) copies. When the electrochemical biosensor was applied to detect the RAGE gene expression in both cell types, we found that HeLa cells expressed the RAGE gene about 2-fold higher than in HepG2 cells (relative value of 0.000905 vs 0.0004670). Therefore, this study suggests the potential modification of the electrochemical biosensor with the use of bisbenzimide dye (Hoechst 33258) for detecting gene expression.


Asunto(s)
Bisbenzimidazol/metabolismo , Colorantes/metabolismo , Técnicas Electroquímicas/métodos , Regulación Neoplásica de la Expresión Génica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Actinas/genética , Técnicas Biosensibles , Electrodos , Electroforesis en Gel de Agar , Dosificación de Gen/genética , Células HeLa , Células Hep G2 , Humanos , Reacción en Cadena de la Polimerasa , Receptor para Productos Finales de Glicación Avanzada/genética , Estándares de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas
2.
Parasitology ; 137(12): 1791-7, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20561394

RESUMEN

The use of chemotherapy on a mass scale in endemic areas may lead to the appearance of resistant isolates through the mechanism of selective drug pressure. Studies have demonstrated that praziquantel (PZQ) is able to inhibit the excretory activity and to cause tegumental damage in Schistosoma mansoni adult worms. The use of the probe resorufin to evaluate excretory activity, as well as the probe Hoechst 33258 to detect tegumental damage in adult worms, may represent a method to identify resistant (or less susceptible) isolates. The purpose of the present work was to compare the changes caused by PZQ in the function of the excretory system and in the integrity of the tegument of adult worms from the LE isolate (susceptible to PZQ) and the LE-PZQ isolate (less susceptible to PZQ). Worms from the isolate LE-PZQ showed less severe tegumental lesions, in both in vitro and in vivo experiments, detected by labelling with Hoechst 33258 and continued to have a functional excretory system as shown by labelling with resorufin in vitro.


Asunto(s)
Antihelmínticos/farmacología , Resistencia a Medicamentos , Colorantes Fluorescentes , Praziquantel/farmacología , Schistosoma mansoni/efectos de los fármacos , Animales , Bisbenzimidazol/metabolismo , Sistema Digestivo/metabolismo , Sistema Digestivo/patología , Colorantes Fluorescentes/metabolismo , Oxazinas/metabolismo , Pruebas de Sensibilidad Parasitaria/métodos , Esquistosomiasis mansoni/tratamiento farmacológico , Esquistosomiasis mansoni/parasitología , Piel/metabolismo , Piel/patología
3.
Behav Brain Res ; 193(1): 17-27, 2008 Nov 03.
Artículo en Inglés | MEDLINE | ID: mdl-18571743

RESUMEN

We have previously shown that intranigral transplants of immortalized GABAergic cells decrease the number of kainic acid-induced seizures [Castillo CG, Mendoza S, Freed WJ, Giordano M. Intranigral transplants of immortalized GABAergic cells decrease the expression of kainic acid-induced seizures in the rat. Behav Brain Res 2006;171:109-15] in an animal model. In the present study, recurrent spontaneous behavioral seizures were established by repeated systemic injections of this excitotoxin into male Sprague-Dawley rats. After the seizures had been established, cells were transplanted into the substantia nigra. Animals with transplants of control cells (without hGAD67 expression) or with sham transplants showed a death rate of more than 40% over the 12 weeks of observation, whereas in animals with M213-2O CL-4 transplants, the death rate was reduced to less than 20%. The M213-2O CL-4 transplants significantly reduced the percentage of animals showing behavioral seizures; animals with these transplants also showed a lower occurrence of stage V seizures than animals in the other groups. In vivo and in vitro analyses provided evidence that the GABAergic cells show sustained expression of both GAD67 and hGAD67 cDNA, as well as increased gamma-aminobutyric acid (GABA) levels in the ventral mesencephalon of transplanted animals. Therefore, transplantation of GABA-producing cells can produce long-term alleviation of behavioral seizures in an animal model.


Asunto(s)
Glutamato Descarboxilasa/metabolismo , Neuronas/trasplante , Convulsiones/cirugía , Sustancia Negra/cirugía , Ácido gamma-Aminobutírico/biosíntesis , Animales , Conducta Animal/efectos de los fármacos , Bisbenzimidazol/metabolismo , Línea Celular Transformada , Cromatografía Líquida de Alta Presión , Técnica del Anticuerpo Fluorescente Indirecta , Glutamato Descarboxilasa/genética , Inyecciones Intraperitoneales , Ácido Kaínico/administración & dosificación , Ácido Kaínico/toxicidad , Masculino , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Neuronas/citología , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Convulsiones/inducido químicamente , Sustancia Negra/citología , Sustancia Negra/metabolismo , Ácido gamma-Aminobutírico/administración & dosificación , Ácido gamma-Aminobutírico/farmacología
4.
Artículo en Inglés | MEDLINE | ID: mdl-10102382

RESUMEN

Eicosatrienoic acid (ETA 5,8,11, n-9) is abnormally increased by essential fatty acid deficiency (EFAD), a condition associated with alterations of cell proliferation and differentiation. In comparison to certain EFAs, addition of ETA at a low concentration resulted in a reduction in the expression of the cell-cell adhesion molecule, E-cadherin, and to a lesser degree, of desmoglein, along with increased invasion of Matrigel by human squamous cell carcinoma (SCC) cells in vitro. At higher concentrations, ETA stimulated the growth of SCC cells. As previously shown, n-6 EFAs (mainly 18:3 n-6, GLA), up-regulated the expression of E-cadherin and desmoglein. This is the first report showing that the abnormal 20:3 n-9 (Mead's acid) is a down regulator of antimetastatic E-cadherin and desmoglein expression.


Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Cadherinas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Ácido 8,11,14-Eicosatrienoico/farmacología , Bisbenzimidazol/metabolismo , Western Blotting , Cadherinas/inmunología , Moléculas de Adhesión Celular , División Celular , Desmogleínas , Desmoplaquinas , Relación Dosis-Respuesta a Droga , Humanos , Inmunohistoquímica , Células Tumorales Cultivadas , Ácido gammalinolénico/metabolismo
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