Norikazu Sakakibara

Metabolic analysis of the cinnamate/monolignol pathway in Carthamus tinctorius seeds by a stable-isotope-dilution method

The present study established a system for comprehensive metabolic analysis of the cinnamate/monolignol and lignan pathways by the use of a stable-isotope-dilution method. The system was successfully applied to characterization of the pathways in Carthamus tinctorius cv. Round-leaved White maturing seeds in combination with administration of stable-isotope-labelled precursors. Experimental results obtained using this technique strongly suggested the intermediacy of ferulic acid in lignan biosynthesis in the plant.

Biosynthesis of yatein in Anthriscus sylvestris

Little is known about the biosynthesis of yatein, in spite of its importance as a typical heartwood lignan and a key biosynthetic intermediate of the antitumor lignan podophyllotoxin. The present study, based on individual administration of [13C]phenylalanine and deuterium labelled lignans and simultaneous administration of two distinct lignans labelled with deuterium atoms to Anthriscus sylvestris, established the two independent branch pathways from matairesinol, one to afford yatein via thujaplicatin, 5-methylthujaplicatin, and 4,5-dimethylthujaplicatin and the other to bursehernin via pluviatolide. The latter pathway did not lead to yatein, eliminating the presence of a metabolic grid from matairesinol to yatein.

At5g54160 gene encodes Arabidopsis thaliana 5-hydroxyconiferaldehyde O-methyltransferase

The function of an Arabidopsis thaliana gene, At5g54160 annotated as a caffeic acid O-methyltransferase CAOMT gene was characterized. The recombinant enzyme of this gene (AtOMT1) catalyzed the O-methylation of phenylpropanoid and flavonoid substrates. The specificity constants (kcat/Km) for 5-hydroxyconiferaldehyde (5-HCAld) and quercetin were both 0.11 μM−1·min−1. On the other hand, lignins of At5g54160-knockout Arabidopsis mutants lacked syringyl units. In addition, we showed that the gene silencing also resulted in significant accumulation of caffeyl alcohol (CaAlc). These results strongly suggested that At5g54160 gene is involved in syringyl lignin synthesis for the methylation of both 5-hydroxyconiferaldehyde and 3,4-dihydroxyphenyl compound(s).

Survey and enzymatic formation of lignans of Anthriscus sylvestris

Gas chromatography — mass spectrometry analysis of the β-glucosidase-treated MeOH extracts ofAnthriscus sylvestris showed, based on comparison of the mass spectra and retention times with those of authentic samples, the presence of lignans, yatein, secoisolariciresinol, lariciresinol, matairesinol, hinokinin, and pluviatolide. The existence of small amounts of bursehernin was suggested by mass chromatography. In addition, nemerosin and deoxypodophyllotoxin were tentatively identified by comparing the mass spectra with those reported in the literature. Enzyme preparations fromA. sylvestris catalyzed the formation of secoisolariciresinol and lariciresinol from coniferyl alcohol. Furthermore, the enzyme preparation catalyzed the formation of lariciresinol from (±)-pinoresinols and the formation of secoisolariciresinol from (±)-lariciresinols. Thus, pinoresinol/lariciresinol reductase (PLR) activity was detected. Chiral high-performance liquid chromatography analysis showed selective formation of (+)-lariciresinol and (−)-secoisolariciresinol from (±)pinoresinols with theA. sylvestris PLR preparation, indicating that the stereochemical property ofA. sylvestris PLR-catalyzed reduction was similar to those ofForsythia PLR andArctium lappa ripening seed PLR.

Stereochemistry of matairesinol formation by Daphne secoisolariciresinol dehydrogenase

Secoisolariciresinol dehydrogenase activity was detected for the first time from Daphne odora and Daphne genkwa (Thymelaeaceae), which are known to produce optically pure (+)-matairesinol. In sharp contrast, (−)-matairesinol was formed selectively over the (+)-antipode by the secoisolariciresinol dehydrogenase preparation from both D. odora callus and D. genkwa shoots.

A novel nucleic acid analogue shows strong angiogenic activity

A novel nucleic acid analogue (2Cl-C.OXT-A) significantly stimulated tube formation of human umbilical endothelial cells (HUVEC). Its maximum potency at 100muM was stronger than that of vascular endothelial growth factor (VEGF), a positive control. At this concentration, 2Cl-C.OXT-A moderately stimulated proliferation as well as migration of HUVEC. To gain mechanistic insights how 2Cl-C.OXT-A promotes angiogenic responses in HUVEC, we performed immunoblot analyses using phospho-specific antibodies as probes. 2Cl-C.OXT-A induced robust phosphorylation/activation of MAP kinase ERK1/2 and an upstream MAP kinase kinase MEK. Conversely, a MEK inhibitor PD98059 abolished ERK1/2 activation and tube formation both enhanced by 2Cl-C.OXT-A. In contrast, MAP kinase responses elicited by 2Cl-C.OXT-A were not inhibited by SU5416, a specific inhibitor of VEGF receptor tyrosine kinase. Collectively these results suggest that 2Cl-C.OXT-A-induces angiogenic responses in HUVEC mediated by a MAP kinase cascade comprising MEK and ERK1/2, but independently of VEGF receptor tyrosine kinase. In vivo assay using chicken chorioallantoic membrane (CAM) and rabbit cornea also suggested the angiogenic potency of 2Cl-C.OXT-A.

Profiling of phenylpropanoid monomers in developing xylem tissue of transgenic aspen (Populus tremuloides)

Here we describe alterations in the cinnamate/monolignol pathway in three transgenic aspen lines: one with downregulated expression of 4-coumarate:CoA ligase (4CL), one with upregulated expression of coniferaldehyde 5-hydroxylase (CAld5H), and a 4CL downregulated/CAld5H upregulated line. Compared with the wild type, the 4CL downregulated line showed significantly increased levels of p-hydroxycinnamic acids such as p-coumaric, ferulic, and sinapic acids. In contrast, the CAld5H upregulated line had increased content of p-coumaryl and 5-hydroxyconiferyl alcohols. In the 4CL downregulated line, it was likely that most hydroxycinnamic acids were glycosylated. These results strongly suggest that the downregulation of 4CL and upregulation of CAld5H disrupt the metabolic flow through the cinnamate/monolignol pathway and thus alter the amount and structure of its final product, lignin.

Synthesis of 1-benzyl-3-(3,5-dimethylbenzyl)uracil derivatives with potential anti-HIV activity

Background: Nine novel uracil analogues were synthesized and evaluated as inhibitors of HIV-1. Methods: Key structural modifications included replacement of the 6-chloro group of 1-benzyl-6-chloro-3-(3,5-dimethylbenzyl)uracil by other functional groups or N1-alkylation of 3-(3,5-dimethylbenzyl)-5-fluorouracil. Results: These compounds showed only micromolar potency against HIV-1 in MT-4, though two of them; 6-azido-1-benzyl-3-(3,5-dimethylbenzyl) uracil and 6-amino-1-benzyl-3-(3,5-dimethylbenzyl) uracil were highly potent (half maximal effective concentration =0.067 and 0.069 μM) and selective (selectivity index =685 and 661), respectively. Structure–activity relationships among the newly synthesized uracil analogues suggest the importance of the H-bond formed between 6-amino group of 6-amino-1-benzyl-3-(3,5-dimethylbenzyl) uracil and amide group of HIV-1 reverse transcriptase. Conclusions: We discovered two 6-substituted 1-benzyl-3-(3,5-dimethylbenzyl) uracils, (6-azido-1-benzyl-3-(3,5-dimethylbenzyl) uracil and 6-amino-1-benzyl-3-(3,5-dimethylbenzyl) uracil) as novel anti-HIV agents. These compounds should be further pursued for their toxicity and pharmacokinetics in vivo as well as antiviral activity against non-nucleoside reverse transcriptase inhibitor-resistant strains.

Lignan production in Daphne odora cell cultures

Lignan production in callus and cell suspension cultures ofDaphne odora is reported for the first time. The cell suspension culture produced pinoresinol, lariciresinol, secoisolariciresinol, matairesinol, and wikstromol. The production of matairesinol in the cell suspension culture was much higher than that inDaphne odora stem tissues.

Delayed administration of the nucleic acid analog 2Cl-C.OXT-A attenuates brain damage and enhances functional recovery after ischemic stroke

2Cl-C.OXT-A (COA-Cl) is a novel nucleic acid analog that enhances angiogenesis through extracellular signal-regulated kinase 1 or 2 (ERK1/2) activation. ERK1/2 is a well-known kinase that regulates cell survival, proliferation and differentiation in the central nervous system. We performed in vitro and in vivo experiments to investigate whether COA-Cl can attenuate neuronal damage and enhance recovery after brain ischemia. In primary cortical neuron cultures, COA-Cl prevented neuronal injury after 2h of oxygen-glucose deprivation. COA-Cl increased phospho-ERK levels in a dose-dependent manner and COA-Cl-induced neuroprotection and ERK1/2 activation was inhibited by suramin or PD98059. The effect of COA-Cl was evaluated in vivo with 60min of middle cerebral artery occlusion combined with bilateral common carotid artery occlusion. COA-Cl or saline was injected intracerebroventricularly 5min after reperfusion. COA-Cl significantly reduced infarct volume and improved neurological deficits upon injection of 15 or 30μg/kg COA-Cl. Moreover, COA-Cl reduced the number of TUNEL positive cells in ischemic boundary, while rCBF was not significantly changed by COA-Cl administration. We also evaluated the effect of delayed COA-Cl administration on recovery from brain ischemia by continuous administration of COA-Cl from 1 to 8 days after reperfusion. Delayed continuous COA-Cl administration also reduced infarct volume. Furthermore, COA-Cl enhanced peri-infarct angiogenesis and synaptogenesis, resulting in improved motor function recovery. Our findings demonstrate that COA-Cl exerts both neuroprotective and neurorestorative effects over a broad therapeutic time window, suggesting COA-Cl might be a novel and potent therapeutic agent for ischemic stroke.