Simultaneous extraction of plant organelle (mitochondria and plastid) genes during the DNA extraction step is major limitation in investigating the community structures of plant-associated bacteria. Although locked nucleic acid (LNA) oligonucleotides was designed to selectively amplify the bacterial small subunit rRNA genes by applying the PCR clamping technique, those for plastids were applicable only for particular plants, while those for mitochondria were available throughout most plants. To widen the applicable range, new LNA oligonucleotides specific for plastids were designed, and the efficacy was investigated. PCR without LNA oligonucleotides predominantly amplified the organelle genes, while bacterial genes were predominantly observed in having applied the LNA oligonucleotides. Denaturing gradient gel electrophoresis (DGGE) analysis displayed additional bacterial DGGE bands, the amplicons of which were prepared using the LNA oligonucleotides. Thus, new designed LNA oligonucleotides specific for plastids were effective and have widened the scope in investigating the community structures of plant-associated bacteria.

Ricinoleic acid (RA) is the main fatty acid component of castor oil and was found to inhibit Ca2+‐signal transduction pathway‐mediated cell cycle regulation in a yeast‐based drug screening assay. RA is expected to have antidiabetic, antiallergy, and/or anticancer properties but its target molecule is unknown. To identify a novel pharmacological effect of RA, we investigated its target molecule in the Ca2+‐signal transduction pathway. RA inhibition of calcineurin (CN) was examined in a yeast‐based CN inhibitor screening assay using the rsp5A401E mutant and in a phosphatase assay using recombinant human CN. RA showed growth‐restoration activity at 5 μg/spot in the CN inhibitor screening assay with the rsp5A401E yeast strain. Furthermore, it directly inhibited CN without immunophilins at Ki = 33.7 μM in a substrate‐competitive manner. The effects of RA on CN in mammalian cells were further evaluated by measuring β‐hexosaminidase (β‐HEX) release in RBL‐2H3 cells. RA at 50 μM suppressed the release of β‐HEX from RBL‐2H3 cells. Moreover, this compound was found to inhibit glycogen synthase kinase‐3β (GSK‐3β), as determined by a kinase assay using recombinant human GSK‐3β. RA inhibited GSK‐3β at Ki = 1.43 μM in a peptide substrate‐competitive manner. The inhibition of GSK‐3β by this molecule was further assessed in mammalian cells by measuring the inhibition of glucose production in H4IIE rat hepatoma cells. RA at 25 μM suppressed glucose production in these cells. These findings indicate that RA and/or castor oil could be a useful functional fatty acid to treat allergy or type 2 diabetes.

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