Species identification of animal cells by nested PCR targeted to mitochondrial DNA.

We developed a highly sensitive and convenient method of nested polymerase chain reaction (PCR) targeted to mitochondrial deoxyribonucleic acid (DNA) to identify animal species quickly in cultured cells. Fourteen vertebrate species, including human, cynomolgus monkey, African green monkey, mouse, rat, Syrian hamster, Chinese hamster, guinea pig, rabbit, dog, cat, cow, pig, and chicken, could be distinguished from each other by nested PCR. The first PCR amplifies mitochondrial DNA fragments with a universal primer pair complementary to the conserved regions of 14 species, and the second PCR amplifies the DNA fragments with species-specific primer pairs from the first products. The species-specific primer pairs were designed to easily distinguish 14 species from each other under standard agarose gel electrophoresis. We further developed the multiplex PCR using a mixture of seven species-specific primer pairs for two groups of animals. One was comprised of human, mouse, rat, cat, pig, cow, and rabbit, and the other was comprised of African green monkey, cynomolgus monkey, Syrian hamster, Chinese hamster, guinea pig, dog, and chicken. The sensitivity of the PCR assay was at least 100 pg DNA/reaction, which was sufficient for the detection of each species of DNA. Furthermore, the nested PCR method was able to identify the species in the interspecies mixture of DNA. Thus, the method developed in this study will provide a useful tool for the authentication of animal species.

A novel oxyiminoalkanoic acid derivative, TAK-559, activates human peroxisome proliferator-activated receptor subtypes.

A novel oxyiminoalkanoic acid derivative, TAK-559, (E)-4-[4-[(5-methyl-2-phenyl-1, 3-oxazol-4-yl)methoxy]benzyloxyimino]-4-phenylbutyric acid, was synthesized as a candidate of a new type of insulin-sensitizing agent. We report here activation of human peroxisome proliferator-activated receptor (hPPAR) subtypes by TAK-559. In a transient transactivation assay, TAK-559 was a potent hPPARgamma1 and hPPARalpha agonist with EC50 values of 31 and 67 nM, respectively. Furthermore, TAK-559 was a partial agonist for hPPARgamma1 with about 68% of maximal activation obtained with rosiglitazone (5-(4-(2-(methyl(2-pyridinyl)amino)ethoxy) benzyl)-1,3-thiazolidine-2,4-dione), a thiazolidinedione derivative, which is known as a PPARgamma agonist. PPARdelta was significantly activated at a high concentration (10 microM) of TAK-559. Competition-binding assays using radiolabeled ligand indicated that the transactivation of all hPPAR subtypes by TAK-559 was due to direct binding of TAK-559 to each subtype. We also demonstrated that TAK-559 acts to recruit the coactivator SRC-1 to each of hPPARgamma1 and hPPARalpha, and to dissociate the corepressor NCoR from each of hPPARgamma1 and hPPARalpha. Taken together, we conclude that TAK-559 is a dual agonist for hPPARgamma1 and hPPARalpha with nearly equal EC50 values, a partial agonist for hPPARgamma1, and has a rather slight agonist activity for hPPARdelta.