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Allyl Isothiocyanate, a Pungent Ingredient of Wasabi and Mustard Oil, Impairs Gastric Paracellular Barrier in Primary Cultures from the Rat Stomach via TRPA1-Independent Pathway

K. Tashima,M. Kabashima,Kenjiro Matsumoto,ShingoYano,S. Hagen,S. Horie

Published 2014 in Unknown venue

ABSTRACT

of primary cultured rat gastric epithelial cells, which were grown for 4 days after platting, the confluent monolayer of RGM-1 cells, or freshly isolated rat dorsal root ganglia was immediately submerged in Buffer RLT (Qiagen), which inhibited RNase activation, and was homogenized by using a Multi-beads shocker (Yasui Kikai, Osaka, Japan). Reverse transcription-polymerase chain reaction (RT-PCR) was performed by using a one-step RT-PCR Kit (Qiagen) and a thermal cycler GeneAmp PCR System 9700 (Applied Biosystems, CA, USA) for 35 cycles (TRPA1, TRPV1, and GAPDH) under the following conditions: reverse transcription at 50°C for 30 min, initial denaturation; 15 min at 95°C and then 30 sec at 94°C, followed by a 30 sec annealing step at 56°C for TRPA1, TRPV1, and GAPDH and 1 min elongation at 72°C. The primers sequences were 5’-CCC CAC TAC ATT GGG CTG CA-3’ and 5’-CCG CTG TCC AGG CAC ATC TT-3’ for rat TRPA1, 5’-TCG TCT ACC TCG TGT TCT TGT TTG-3’ and 5’-CCA GAT GTT CTT GCT CTC TTG TGC-3’ for rat TRPV1, and 5’-TCC CTC AAG ATT GTC AGC AA-3’ and 5’-AGA TCC ACA ACG GAT ACA TT-3’ for rat GAPDH. The PCR products were separated on 3% (wt/vol) agarose gel in Tris-acetate EDTA buffer, stained with ethidium bromide, and analyzed by LAS 3000 (FUJI‐ FILM, Tokyo, Japan). The sequence of the PCR product was analyzed using the BLAST program (NCBI).

PUBLICATION RECORD

  • Publication year

    2014

  • Venue

    Unknown venue

  • Publication date

    2014-07-16

  • Fields of study

    Medicine, Chemistry

  • Identifiers
    DOI 10.5772/58603
  • External record

    Open on Semantic Scholar

  • Source metadata

    Semantic Scholar

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