Yoshihisa Nakazawa

High throughput and exhaustive analysis of diverse lipids by using supercritical fluid chromatography-mass spectrometry for metabolomics.

We have developed an analytical system that enables the simultaneous rapid analysis of lipids with varied structures and polarities through the use of supercritical fluid chromatography-mass spectrometry (SFC-MS). The separation conditions for SFC (column, modifier, back pressure, etc.) and the detection conditions for mass spectrometry (ionization method, parameters, etc.) were investigated to develop a simultaneous analytical method for lipid mixtures that included phospholipids, glycolipids, neutral lipids, and sphingolipids. When cyanopropylated silica gel-packed column was used for the separation, all lipids were successfully detected and the analysis time was less than 15 min. The use of an octadecylsilylated column resulted in separation, which was dependent on the differences in the unsaturation of the fatty acid side chains and isomer separation. This system is a powerful tool for studies on lipid metabolomics because it is useful not only as a fingerprinting method for the screening of diverse lipids but also for the detailed profiling of individual components.

Histochemical study of detailed laticifer structure and rubber biosynthesis-related protein localization in Hevea brasiliensis using spectral confocal laser scanning microscopy

In Hevea brasiliensis, laticifers produce and accumulate rubber particles. Despite observation using histochemical methods, development stage structure and structures with ceasing functions have rarely been described. Spectral confocal laser scanning microscopy with Nile red staining simplifies laticifer structure observation in tangential sections while enhancing the resolution. Laticifer and ray images were extracted from unmixed images and used to monitor changes during growth. A laticifer network structure developed from increased anastomoses between adjoining laticifers outside of the conducting phloem, but because of increased radial division and growth of rays, the network structure ruptured and disintegrated. We also investigated immunohistochemical localization of two rubber particle-associated proteins in the laticifers: small rubber particle protein (SRPP) and rubber elongation factor (REF). Mature bark test results show that SRPP is localized only in the laticifer layers in the conducting phloem; REF is localized in all laticifer layers. Because SRPP plays a positive role in rubber biosynthesis, results show that the rubber biosynthesis capability of laticifers is concentrated where rays and the sieve tube actively transport metabolites.

Two Arabidopsis thaliana Golgi α-mannosidase I enzymes are responsible for plant N-glycan maturation

N-Glycosylation is an important post-translational modification that occurs in many secreted and membrane proteins in eukaryotic cells. Golgi alpha-mannosidase I hydrolases (MANI) are key enzymes that play a role in the early N-glycan modification pathway in the Golgi apparatus. In Arabidopsis thaliana, two putative MANI genes, AtMANIa (At3g21160) and AtMANIb (At1g51590), were identified. Biochemical analysis using bacterially produced recombinant AtMANI isoforms revealed that both AtMANI isoforms encode 1-deoxymannojirimycin-sensitive alpha-mannosidase I and act on Man(8)GlcNAc(2) and Man(9)GlcNAc(2) structures to yield Man(5)GlcNAc(2). Structures of hydrolytic intermediates accumulated in the AtMANI reactions indicate that AtMANIs employ hydrolytic pathways distinct from those of mammalian MANIs. In planta, AtMANI-GFP/DsRed fusion proteins were detected in the Golgi stacks. Arabidopsis mutant lines manIa-1, manIa-2, manIb-1, and manIb-2 showed N-glycan profiles similar to that of wild type. On the other hand, the manIa manIb double mutant lines produced Man(8)GlcNAc(2) as the predominant N-glycan and lacked plant-specific complex and hybrid N-glycans. These data indicate that either AtMANIa or AtMANIb can function as the Golgi alpha-mannosidase I that produces the Man(5)GlcNAc(2) N-glycan structure necessary for complex N-glycan synthesis.

Functional Analysis of Two Solanesyl Diphosphate Synthases from Arabidopsis thaliana

Solanesyl diphosphate (SPP) is regarded as the precursor of the side-chains of both plastoquinone and ubiquinone in Arabidopsis thaliana. We previously analyzed A. thaliana SPP synthase (At-SPS1) (Hirooka et al., Biochem. J., 370, 679–686 (2003)). In this study, we cloned a second SPP synthase (At-SPS2) gene from A. thaliana and characterized the recombinant protein. Kinetic analysis indicated that At-SPS2 prefers geranylgeranyl diphosphate to farnesyl diphosphate as the allylic substrate. Several of its features, including the substrate preference, were similar to those of At-SPS1. These data indicate that At-SPS1 and At-SPS2 share their basic catalytic machinery. Moreover, analysis of the subcellular localization by the transient expression of green fluorescent protein-fusion proteins showed that At-SPS2 is transported into chloroplasts, whereas At-SPS1 is likely to be localized in the endoplasmic reticulum in the A. thaliana cells. It is known that the ubiquinone side-chain originates from isopentenyl diphosphate derived from the cytosolic mevalonate pathway, while the plastoquinone side-chain is synthesized from isopentenyl diphosphate derived from the plastidial methylerythritol phosphate pathway. Based on this information, we propose that At-SPS1 contributes to the biosynthesis of the ubiquinone side-chain and that At-SPS2 supplies the precursor of the plastoquinone side-chain in A. thaliana.

Quantification of trans-1,4-polyisoprene in Eucommia ulmoides by fourier transform infrared spectroscopy and pyrolysis-gas chromatography/mass spectrometry

Commercial development of trans-1,4-polyisoprene from Eucommia ulmoides Oliver (EU-rubber) requires specific knowledge on selection of high-rubber-content lines and establishment of agronomic cultivation methods for achieving maximum EU-rubber yield. The development can be facilitated by high-throughput and highly sensitive analytical techniques for EU-rubber extraction and quantification. In this paper, we described an efficient EU-rubber extraction method, and validated that the accuracy was equivalent to that of the conventional Soxhlet extraction method. We also described a highly sensitive quantification method for EU-rubber by Fourier transform infrared spectroscopy (FT-IR) and pyrolysis-gas chromatography/mass spectrometry (PyGC/MS). We successfully applied the extraction/quantification method for study of seasonal changes in EU-rubber content and molecular weight distribution.

Analysis of long-chain polyprenols using supercritical fluid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

The separation of long-chain polyprenols was successfully achieved using supercritical fluid chromatography (SFC). Each 100-mer greater component was separated using tetrahydrofuran as a mobile phase modifier. The molecular mass distributions derived from SFC analyses agreed with the results of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses. The number-average molecular mass calculated by MALDI-TOF-MS data were also in accord with the results of quantitative 1H-NMR analysis of terminal groups. A combination of SFC and MALDI-TOF-MS analyses is a powerful tool for the elucidation of the complicated structures of natural polyprenols.

羅布麻（紅麻:Apocynum venetum L.）葉エキスの降圧作用に関する研究(3)

To clarify the mechanisms underlying the antihypertensive effect of Luobuma (Apocynum venetum L. (Apocynaceae)) leaf extract (LLE), we investigated the vasodilator effect of LLE in the rat mesenteric vascular bed, which plays an important role in changes in peripheral resistance and thus the regulation of blood pressure. In the perfused mesenteric vascular bed with active tone and intact endothelium, perfusion of LLE (0.1 ng to 100 mg/ml for 15 min) caused dose-dependent vasodilation, which was abolished by chemical removal of the endothelial layer with perfusion of sodium deoxycholate, but not by N(G)-nitro-L-arginine-methyl ester (L-NAME), a competitive inhibitor of nitric oxide (NO), which instead increased the effect. The LLE-induced vasodilation was partially inhibited by high K(+)-containing Krebs solution and tetraethylammonium (a K(+) channel blocker) and completely by the combination of L-NAME and high K(+)-Krebs solution. However, atropine (a muscarinic acetylcholine receptor antagonist) did not affect the vasodilation. These results suggest that the vasodilation induced by LLE is endothelium-dependent and mediated by endothelium-derived hyperpolarizing factor, which involves the activation of K(+)-channels. The higher concentrations of LLE may enhance NO production/release to cause vasodilation.

A High-Throughput and Solvent-free Method for Measurement of Natural Polyisoprene Content in Leaves by Fourier Transform Near Infrared Spectroscopy

Commercial development of natural polyisoprene from polyisoprene-producing plants requires detailed knowledge on how to select high-polyisoprene-content lines and establish agronomic cultivation methods for achieving maximum polyisoprene yield. This development can be facilitated by a high-throughput quantification method for natural polyisoprene. In this paper, we describe the Fourier transform near infrared spectroscopy (FT-NIR) technique coupled with a partial least squares (PLS) regression model to quantify natural polyisoprene in Eucommia ulmoides leaves. PLS regression models are discussed with respect to linearity, root-mean-square error of estimation (RMSEE), and root-mean-square error of prediction (RMSEP). The best PLS regression model was obtained with second derivative NIR spectra in the region between 4000-6000 cm(-1) (R2Y, 0.95; RMSEE, 0.25; RMSEP, 0.37). This is the first report to employ FT-NIR analysis for high throughput and solvent-free quantification of natural polyisoprene in leaves.

Construction and analysis of EST libraries of the trans-polyisoprene producing plant, Eucommia ulmoides Oliver

Eucommia ulmoides Oliver is one of a few woody plants capable of producing abundant quantities of trans-polyisoprene rubber in their leaves, barks, and seed coats. One cDNA library each was constructed from its outer stem tissue and inner stem tissue. They comprised a total of 27,752 expressed sequence tags (ESTs) representing 10,520 unigenes made up of 4,302 contigs and 6,218 singletons. Homologues of genes coding for rubber particle membrane proteins that participate in the synthesis of high-molecular poly-isoprene in latex were isolated, as well as those encoding known major latex proteins (MLPs). MLPs extensively shared ESTs, indicating their abundant expression during trans-polyisoprene rubber biosynthesis. The six mevalonate pathway genes which are implicated in the synthesis of isopentenyl diphosphate (IPP), a starting material of poly-isoprene biosynthesis, were isolated, and their role in IPP biosynthesis was confirmed by functional complementation of suitable yeast mutants. Genes encoding five full-length trans-isoprenyl diphosphate synthases were also isolated, and two among those synthesized farnesyl diphosphate from IPP and dimethylallyl diphosphate, an assumed intermediate of rubber biosynthesis. This study should provide a valuable resource for further studies of rubber synthesis in E. ulmoides.

Overexpression of an isopentenyl diphosphate isomerase gene to enhance trans-polyisoprene production in Eucommia ulmoides Oliver.

BackgroundNatural rubber produced by plants, known as polyisoprene, is the most widely used isoprenoid polymer. Plant polyisoprenes can be classified into two types; cis-polyisoprene and trans-polyisoprene, depending on the type of polymerization of the isoprene unit. More than 2000 species of higher plants produce latex consisting of cis-polyisoprene. Hevea brasiliensis (rubber tree) produces cis-polyisoprene, and is the key source of commercial rubber. In contrast, relatively few plant species produce trans-polyisoprene. Currently, trans-polyisoprene is mainly produced synthetically, and no plant species is used for its commercial production.ResultsTo develop a plant-based system suitable for large-scale production of trans-polyisoprene, we selected a trans-polyisoprene-producing plant, Eucommia ulmoides Oliver, as the target for genetic transformation. A full-length cDNA (designated as EuIPI, Accession No. AB041629) encoding isopentenyl diphosphate isomerase (IPI) was isolated from E. ulmoides. EuIPI consisted of 1028 bp with a 675-bp open reading frame encoding a protein with 224 amino acid residues. EuIPI shared high identity with other plant IPIs, and the recombinant protein expressed in Escherichia coli showed IPI enzymatic activity in vitro. EuIPI was introduced into E. ulmoides via Agrobacterium-mediated transformation. Transgenic lines of E. ulmoides overexpressing EuIPI showed increased EuIPI expression (up to 19-fold that of the wild-type) and a 3- to 4-fold increase in the total content of trans-polyisoprenes, compared with the wild-type (non-transgenic root line) control.ConclusionsIncreasing the expression level of EuIPI by overexpression increased accumulation of trans-polyisoprenes in transgenic E. ulmoides. IPI catalyzes the conversion of isopentenyl diphosphate to its highly electrophilic isomer, dimethylallyl diphosphate, which is the first step in the biosynthesis of all isoprenoids, including polyisoprene. Our results demonstrated that regulation of IPI expression is a key target for efficient production of trans-polyisoprene in E. ulmoides.