Sesamin and sesamolin reduce amyloid-ß toxicity in a transgenic Caenorhabditis elegans.

Alzheimer's disease (AD) is a devastating neurodegenerative disease characterized by ß-amyloid (Aß) plaques in the brain. At the present, there is no approved drug with a proven disease-modifying effect. Sesame seed (Sesame indicum) has long been known as a healthy food in Southeast Asian countries. Sesame lignans obtained from sesame seed possess antioxidant property that exhibit a variety of beneficial effects in various models. The objective of this study was to investigate the protective effects of sesame lignans including sesamin, sesamolin, and sesamol against Aß toxicity in Caenorhabditis elegans (C. elegans) model of Aß toxicity and to address whether these sesame lignans have a positive effect on lifespan extension. A transgenic C. elegans expressing human Aß was used to investigate protective effects of sesame lignans against Aß toxicity. Sesamin and sesamolin significantly alleviated Aß-induced paralysis. The real-time PCR revealed that both sesamin and sesamolin did not affect the expression of Aß transgene. However, we found that only sesamin inhibited Aß oligomerization. These findings demonstrated that, among three sesame lignans tested, sesamin protected against Aß toxicity by reducing toxic Aß oligomers. Sesamin and sesamolin also significantly improved Aß-induced defect in chemotaxis behavior and reversed the defect to normal. Moreover, sesamin prolonged median and mean lifespan of the wild type worm. On the other hand, sesamolin and sesamol failed to extend lifespan. These results offer valuable evidence for the future use of sesamin in the development of agents for the treatment of AD. It is also worth investigating the structure-activity relationship of lignan-related structures and their anti-Aß toxicity activities in the future.

Functional expression of a putative geraniol 8-hydroxylase by reconstitution of bacterially expressed plant CYP76F45 and NADPH-cytochrome P450 reductase CPR I from Croton stellatopilosus Ohba.

While attempting to isolate the enzyme geranylgeraniol 18-hydroxylase, which is involved in plaunotol biosynthesis in Croton stellatopilosus (Cs), the cDNAs for a cytochrome P450 monooxygenase(designated as CYP76F45) and an NADPH-cytochrome P450 reductase (designated as CPR I based on its classification) were isolated from the leaf. The CYP76F45 and CsCPR I genes have open reading frames (ORFs) encoding 507- and 711-amino acid proteins with predicted relative molecular weights of 56.7 and 79.0 kDa,respectively. Amino acid sequence comparison showed that both CYP76F45 (63­73%) and CsCPR I (79­83%) share relatively high sequence identities with homologous proteins in other plant species.Phylogenetic tree analysis confirmed that CYP76F45 belongs to the CYP76 family and that CsCPR I belongs to Class I of dicotyledonous CPRs, with both being closely related to Ricinus communis genes. Functional characterization of both enzymes, each expressed separately in Escherichia coli as recombinant proteins,showed that only simultaneous incubation of the membrane bound proteins with the substrate geraniol (GOH) and the coenzyme NADPH could form 8-hydroxygeraniol. The enzyme mixture could also utilize acyclic sesquiterpene farnesol (FOH) with a comparable substrate preference ratio (GOH:FOH) of 54:46. The levelsof the CYP76F45 and CsCPR I transcripts in the shoots, leaves and twigs of C. stellatopilosus were correlated with the levels of a major monoterpenoid indole alkaloid, identified tentatively as 19-Evallesamine,that accumulated in these plant parts. These results suggested that CYP76F45 and CPR I function as the enzyme geraniol-8-hydroxylase (G8H), which is likely to be involved in the biosynthesis of the indole alkaloid in C. stellatopilosus [corrected].

Molecular cloning, bacterial expression and functional characterisation of cytochrome P450 monooxygenase, CYP97C27, and NADPH-cytochrome P450 reductase, CPR I, from Croton stellatopilosus Ohba.

The cDNAs for cytochrome P450 monooxygenase (designated as CYP97C27 by D. Nelson's group) and NADPH-cytochrome P450 reductase (designated as CPR I based on its classification) were isolated from Croton stellatopilosus leaves, which actively biosynthesise plaunotol (18-OH geranylgeraniol). CYP97C27 and CPR I contain open reading frames encoding proteins of 471 and 711 amino acids with predicted molecular masses of 53 and 79kDa, respectively. By aligning the deduced sequences of CYP97C27 and CPR I with other plant species, all functional domains of CYP97C27 (heme and oxygen binding) and CPR I (CYP- and FMN, FAD, and NADPH cofactor binding) were identified. Amino acid sequence comparison indicated that both CYP97C27 (85-93%) and CPR I (79-83%) share high sequence identities with homologous proteins in other plant species, suggesting that CYP97C27 belongs to the CYP97C subfamily and that CPR I belongs to class I of the dicotyledonous CPR. Functional characterisation of both enzymes, produced in Escherichia coli (pET32a/BL21(DE3)) as recombinant proteins, showed that simultaneous incubation of CYP97C27 and CPR I with the substrate geranylgeraniol (GGOH) and coenzyme NADPH led to formation of the product plaunotol. In C. stellatopilosus, the levels of the CYP97C27 and CPR I transcripts were highly correlated with those of several mRNAs involved in the plaunotol biosynthetic pathway, suggesting that CYP97C27 and CPR I are the enzymes that catalyse the last hydroxylation step of the pathway.

Cloning and expression of 1-deoxy-d-xylulose 5-phosphate synthase cDNA from Croton stellatopilosus and expression of 2C-methyl-d-erythritol 4-phosphate synthase and geranylgeranyl diphosphate synthase, key enzymes of plaunotol biosynthesis.

1-Deoxy-d-xylulose 5-phosphate synthase (DXS, EC: 4.1.3.37), the first enzyme in the 2C-methyl-d-erythritol 4-phosphate (MEP) pathway, is known to be responsible for the rate-limiting step of isoprenoid biosynthesis in Escherichia coli and Arabidopsis thaliana. In this study, the dxs gene from Croton stellatopilosus, designated csdxs, was cloned from leaf tissue using the rapid amplification of cDNA ends (RACE) technique. Leaves of C. stellatopilosus contain plaunotol, an acyclic diterpene alcohol. The csdxs cDNA containing the open reading frame of 2163 base pairs appeared to encode a polypeptide of 720 amino acids. Analysis of the deduced amino acid sequence revealed that the NH(2)-terminus of CSDXS carried a chloroplast transit peptide, a thiamine diphosphate binding site, and a transketolase motif, which are the important characteristics of DXS enzymes in higher plants. Multiple alignments of CSDXS with other plant DXSs have indicated that CSDXS has identity ranging between 68% and 89%. Expression levels of csdxs and genes encoding key enzymes in the plaunotol biosynthetic pathway, namely 2C-methyl-d-erythritol 4-phosphate synthase (meps) and geranylgeranyl diphosphate synthase (ggpps), were analysed by measuring transcript levels in leaves of different developmental stages. The results showed that dxs, meps, and ggpps are all active in young leaves prior to full expansion when plaunotol is synthesised from the DXP precursor in chloroplasts. The dense presence of chloroplasts and oil globules in the palisade cells of these leaves support the view that these genes are involved in plaunotol biosynthesis in chloroplast-containing tissues.

Productivity and quality of volatile oil extracted from Mentha spicata and M. arvensis var. piperascens grown by a hydroponic system using the deep flow technique.

The purpose of this study was to determine the differences between spearmint (Mentha spicata L.) and Japanese mint (M. arvensis L. var. piperascens Malinv.) cultivated in either soil or nutrient solution using the deep flow technique (DFT). The differences were measured in terms of harvest period (full bloom period) and quantity and chemical components of volatile oils. The spearmint and Japanese mint were cultivated in four different nutrient formulas: plant standard nutrient, plant standard nutrient with an amino acid mixture, plant standard nutrient with a sulphur compound, and a combination of plant standard nutrient with an amino acid mixture and a sulphur compound. We observed that cultivation of spearmint and Japanese mint in nutrient solution using DFT is an effective method to provide high production of volatile oil, since it results in an earlier harvest period and higher quantity of volatile oil. We determined that for spearmint an amino acid mixture is an appropriate nutrient supplement to enhance production of volatile oil with optimum carvone content. Finally, we observed high menthol content in Japanese mint grown in all four nutrient formulas; however, supplementation with a combination of sulphur fertilisation and amino acid mixture yields the highest quantity of volatile oil.