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1.
Genet Mol Res ; 14(2): 6929-42, 2015 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-26125901

RESUMEN

Isoflurane can induce widespread cytotoxicity. We hypothesized that isoflurane induces apoptosis partly by causing excessive calcium release from the endoplasmic reticulum (ER) via direct activation of inositol 1,4,5-trisphosphate receptors (IP3R). Rat pheochromocytoma cells cultured for seven days with nerve growth factor were divided into four groups: control group (C), IP3R antagonist group (X), isoflurane group (I) and isoflurane + IP3R antagonist group (I+X). Groups I and I+X were treated with 1 MAC isoflurane for 12 h. Groups X and I+X were pretreated with IP3R antagonist. Annexin V/PI apoptosis and TUNEL assays were performed to evaluate cell apoptosis. TEM was used to observe changes in cell ultrastructure. Changes in calcium concentration ([Ca(2+)]i) in the cytoplasm were measured by flow cytometry. RT-PCR was performed to evaluate IP3R mRNA expression. TEM showed that isoflurane treatment altered cell ultrastructure. Compared to group C, cell apoptosis rate and [Ca(2+)]i increased in groups I and I+X (P < 0.05). Compared to group C, IP3R mRNA expression was lower in group X and higher in group I (P < 0.05). Compared to group X, cell apoptosis rate, [Ca(2+)]i and IP3R mRNA expression increased in groups I and I+X (P < 0.05). Compared to group I, cell apoptosis rate, [Ca(2+)]i and IP3R mRNA expression decreased in group I+X (P < 0.05). These results suggest that exposure to 1 MAC isoflurane for 12 h causes excessive calcium release partly by direct activation of IP3R on the ER membrane and triggers cell apoptosis.


Asunto(s)
Anestésicos por Inhalación/toxicidad , Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Isoflurano/toxicidad , ARN Mensajero/metabolismo , Animales , Anexina A5/metabolismo , Apoptosis/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Expresión Génica , Etiquetado Corte-Fin in Situ , Receptores de Inositol 1,4,5-Trifosfato/agonistas , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Receptores de Inositol 1,4,5-Trifosfato/genética , Transporte Iónico , Compuestos Macrocíclicos/farmacología , Factor de Crecimiento Nervioso/farmacología , Oxazoles/farmacología , Células PC12 , ARN Mensajero/agonistas , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/genética , Ratas
2.
Genet Mol Res ; 9(2): 935-40, 2010 May 18.
Artículo en Inglés | MEDLINE | ID: mdl-20486089

RESUMEN

We developed a straightforward, rapid, and inexpensive method to determine transgene copy number in tobacco. The plasmid (pSSRCopy) used for tobacco transformation contains a simple sequence repeat (SSR) locus, PT1199, which was partially deleted in the middle, a homogenous SSR locus in tobacco K326. A 168-bp segment of the cloned PT1199 was shortened to 95 bp by deleting a 73-bp internal fragment. Using a pair of SSR primers, competitive PCR was amplified from genomic DNA from transgenic tobacco harboring pSSRCopy, and the two expected bands were found. The 168-bp band (SSR-168) corresponds to endogenous PT1199 and the 95-bp band (SSR-95) comes from the integrated pSSRCopy. A single copy of a transgene can be easily distinguished from multiple copies by comparing band densities.


Asunto(s)
Cartilla de ADN/genética , Nicotiana/genética , Reacción en Cadena de la Polimerasa/métodos , Transgenes , Southern Blotting , Dosificación de Gen , Genes de Plantas , Técnicas Genéticas , Modelos Genéticos , Hojas de la Planta/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética
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