The high throughput screening of neuropeptide FF2 receptor ligands from Korean herbal plant extracts.

We have screened 356 libraries of Korean herbal plant extracts to find potential anti-obesity drugs. We employed the recently developed fluorescence polarization high throughput screening (FP HTS) assays of human neuropeptide FF (NPFF) receptors in 384-well microtiter plates. The primary hits were cherry-picked from the libraries and further analyzed by secondary displacement curve assays, in vitro GTPgammaS binding assays and cell-based CRE luciferase reporter assays.

Point mutation of (+)-germacrene A synthase from Ixeris dentata.

Sesquiterpene cyclases catalyze the conversion of common precursor, farnesyl pyrophosphate, into various terpene backbones. X-ray crystallography of tobacco epi-aristolochene synthase has previously proposed a cyclization mechanism wherein the allylic carbocation intermediate is stabilized by the main chain carbonyl oxygens of three consecutive threonine residues. Alignment of amino acid sequences of plant terpene cyclases shows that the first position of the triad is almost invariably threonine or serine.

Reverse transcription quantitative-PCR of three genes with high homology encoding 3-hydroxy-methylglutaryl-CoA reductase in rice.

Reverse transcription followed by RT Q-PCR is useful for the systematic measurement of changes in gene expression. RT Q-PCR with two pairs of primers for each gene was used for relative expression of three genes with high homology encoding 3-hydroxy-methylglutaryl-CoA reductase (HMGR) in rice. At various growth stages of etiolated seedling and various times after UV-irradiation treatment, RT Q-PCR of each HMGR gene showed a consistent pattern of relative expression with the RT Q-PCR data, using two pairs of primers, giving a high degree of accuracy.

Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice.

Reverse transcription followed by real-time quantitative polymerase chain reaction (RT Q-PCR) is useful for the systematic measurement of plant physiological changes in gene expression. The validity of using 18S rRNA and three housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase, actin, and tubulin, was tested as a reference of RT Q-PCR. Under various growth stages of etiolated seedlings, different cultivars, and various times after UV-irradiation treatment, expression level of 18S rRNA correlated with total RNA suggesting the uniformity of RT Q-PCR efficiencies among samples.