Identification and Characterization of Genes in the Curcuminoid Pathway of Curcuma zedoaria Roscoe.

BACKGROUND: Curcuminoid genes have an important role in the biosynthesis of curcumin, a valuable bioactive compound, in Curcuma species. However, there have not been any reports of these genes in Curcuma zedoaria. OBJECTIVE: The present work reports on the isolation of genes encoding enzymes in curcuminoid metabolic pathway and their expression in C. zedoaria. METHOD: The primers were designed from untranslation regions of DCS, CURS1, CURS2 and CURS3 genes which are involved in curcuminoid biosynthesis in C. longa to isolate the corresponding fulllength genes in C. zedoaria. RT-PCR amplification and HPLC analysis are used to estimate the expression of genes and biosynthesis of curcumin in both rhizome and callus. RESULTS: The results showed that all four genes from C. zedoaria (named CzDCS, CzCURS1, CzCURS2 and CzCURS3) and C. longa have a high identity (approximately 99%) and lengths of genes from C. zedoaria are 1382, 1240, 1288 and 1265 nu, respectively. CzCURS1, 2 and 3 genes have one intron while CzDCS has two introns. RT-PCR amplification indicated that curcuminoid genes expressed mRNA in rhizome and callus of C. zedoaria. Curcumin, a major component of curcuminoids, was also found in callus by HPLC analysis. CONCLUSION: The sequence information of DCS and CURS1-3 genes in C. zedoaria will be very valuable for a subsequent study on the effects of elicitors on the transcription of genes involved in curcuminoid biosynthesis pathway.

Discovery and characterization of potent small molecule inhibitors of the high affinity proline transporter.

The mammalian proline transporter (PROT) is a high affinity Na(+)/Cl(-)-dependent transporter expressed in specific regions of the brain. It is homologous to other neurotransmitter transporters such as glycine, norepinephrine, serotonin, and dopamine transporters. PROT is enriched in glutamatergic synaptic terminals and may play an important role in the regulation of excitatory neurotransmission. No non-peptide small molecule inhibitors have been described for this transporter. To study its physiological role in the central nervous system and evaluate its potential as a therapeutic target, we established cell lines that stably express recombinant hPROT and characterized its kinetic properties for proline uptake. We then screened for inhibitors and identified a series of compounds that inhibit hPROT-mediated proline uptake. A known compound, benztropine, was found to inhibit hPROT with an IC(50) of 0.75microM. A series of novel compounds were also found, one of which, LP-403812, showed an IC(50) of approximately 0.1microM on both recombinant human and mouse PROT without significant inhibition of glycine and dopamine transporters at concentrations up to 10microM. This compound also inhibited proline transporter activity of mouse brain synaptosomes with the same potency. These inhibitors provide important tools for the understanding of PROT functions in the brain and may lead to the development of therapeutic agents for certain neurological disorders.