Jianhua Qi

Termitomycesphins A-D, Novel Neuritogenic Cerebrosides from the Edible Chinese Mushroom Termitomyces albuminosus

Abstract Four novel cerebrosides, termitomycesphins A–D, were isolated from the edible Chinese mushroom Termitomyces albuminosus (Berk.) Heim. (‘Jizong’ in Chinese), shown to induce neuronal differentiation in rat PC12 cells. The absolute stereostructures were elucidated by spectroscopic methods and chemical derivatization. These new cerebrosides have a unique C19 hydroxylated sphingosine base with branching around the middle. Termitomycesphins A and C possessing a C16 α-hydroxy fatty acid showed a higher neuritogenic activity than did termitomycesphins B and D possessing a C18 α-hydroxy fatty acid.

Gentisides C-K: nine new neuritogenic compounds from the traditional Chinese medicine Gentiana rigescens Franch.

Two new alkyl 2,3-dihydroxybenzoates, gentisides A and B, were isolated from the traditional Chinese medicine Gentiana rigescens Franch. Their structures and stereochemistry were elucidated by spectroscopic methods and chemical derivatization. These compounds showed a significant neuritogenic activity at 30 microM against PC12 cells that was comparable to that seen for the best nerve growth factor (NGF) concentration of 40 ng/mL. Gentisides A and B showed parallel activity, indicating that the observed structural difference at the end of their alkyl chain did not affect neuritogenic activity.

Ganodermasides A and B, two novel anti-aging ergosterols from spores of a medicinal mushroom Ganoderma lucidum on yeast via UTH1 gene

Two novel ergosterol derivatives, ganodermasides A and B, hydroxylated at C-15 were isolated from the methanol extract of spores of a medicinal mushroom, Ganoderma lucidum, showed to extend the replicative life span of Saccharomyces cerevisiae, a yeast of K6001 strain. The stereostructures of ganodermasides A and B were determined based on the spectroscopic analysis and comparison of spectroscopic data. These new sterols have a 4, 6, 8(14), 22-tetraene-3-one unit with a unique hydroxylation at C-15. The anti-aging activity of these compounds on yeast is comparable to a well-known substance, resveratrol. Based on results of the investigation of the mechanism of biological activity, ganodermasides A and B regulated UTH1 expression in order to extend the replicative life span of yeast.

Synthesis and absolute configuration of hormone α1

An important biological event in phytopathogens of the genus Phytophthora is sexual reproduction, which is conducted by two mating types, A1 and A2. A factor known as hormone alpha1 is secreted by the A1 mating type and induces the formation of sexual spores (oospores) in the A2 mating type. Here we describe the asymmetric synthesis and assignment of the absolute configuration of hormone alpha1 by oospore-inducing assays of the synthesized isomers.

The second Phytophthora mating hormone defines interspecies biosynthetic crosstalk

The heterothallic species of the agricultural pest Phytophthora use mating hormones α1 and α2 to regulate their sexual reproduction. Here we describe the absolute stereostructure of the second mating hormone α2 as defined by spectroscopic analysis and total synthesis. We have uncovered not only the interspecies universality of α hormones but also the pathway by which α2 is biosynthesized from phytol by A2-mating type strains and metabolized to α1 by A1 strains.

Leptin gene transfer regulates fibromuscular development and lipid deposition in muscles via SIRT1, FOXO3a and PGC-1α in mice in vivo.

Leptin gene transfer in the liver by hydrodynamic-based gene delivery instead of peptide administration was used to investigate the effects of leptin on muscle mass accretion and lipid accumulation in muscles of wild-type mice. Food intake (P<0.01), body weight (P<0.01) and white adipose tissue (P<0.01) were significantly reduced in the leptin gene-treated group compared with the control group. Moreover, plasma leptin concentration was significantly increased after administration of the mouse leptin gene at a dose of 15 µg per mouse for 1 day (P<0.01) or 1 week (P<0.05). Furthermore, the mRNA abundance of myosin heavy chain type I (MyHC-I), myosin heavy chain type II (MyHC-IIa, MyHC-IIx), adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and lipoprotein lipase (LPL) genes in gastrocnemius muscle and extensor digitorum longus after administration of leptin for 1 week were significantly increased compared with the control group. Finally, we investigated the mechanism by which leptin gene transfer affects fibromuscular and fat deposition in muscle. Gene expression and protein levels of SIRT1, and proliferator-activated receptor-γ coactivator-1α (PGC-1α) were remarkably increased in extensor digitorum longus. On the other hand, PGC-1α and FOXO3a gene expression was observed to have significantly increased in gastrocnemius muscle. However, only changes in the protein levels of PGC-1α were observed (P<0.05). These results suggest that leptin may affect the growth and development of muscle, and fat deposition in wild-type mice via SIRT1 and FOXO3a and their downstream targets, including PGC-1α.

A Steroidal Saponin from Ophiopogon japonicus Extends the Lifespan of Yeast via the Pathway Involved in SOD and UTH1

Nolinospiroside F is a steroidal saponin isolated from Ophiopogon japonicus (O. japonicus). In this study, we found that nolinospiroside F significantly extends the replicative lifespan of K6001 yeast at doses of 1, 3 and 10 μM, indicating that it has an anti-aging effect. This may be attributed to its anti-oxidative effect, as nolinospiroside F could increase yeast survival under oxidative stress conditions and decrease the level of malondialdehyde (MDA), an oxidative stress biomarker. It could also increase anti-oxidative stress genes, SOD1 and SOD2, expression, and the activity of superoxide dismutase (SOD). It increase the activity of SIRT1, an upstream inducer of SOD2 expression. In sod1 and sod2 mutant yeast strains, nolinospiroside F failed to extend their replicative lifespan. These results indicate that SOD participates in the anti-aging effect of nolinospiroside F. Furthermore, nolinospiroside F inhibited the expression of UTH1, a yeast-aging gene that is involved in the oxidative stress of yeast, and failed to extend the replicative lifespan of uth1 or skn7 mutant yeast cells. SKN7 is the transcriptional activator of UTH1. We also demonstrate that SOD and UTH1 regulate each other’s expression. Together, these results suggest that SOD and UTH1 genes are required for and play interactive roles in nolinospiroside F-mediated yeast lifespan extension.

Structure–Activity Relationships of Neuritogenic Gentiside Derivatives

Neurotrophic factors are target-derived peptides that play an important role in the development and survival of responsive neuronal populations. Nerve growth factor (NGF), one of the most important neurotrophic factors, is essential for neuronal differentiation, growth, survival, function maintenance, and prevention of aging in the central and peripheral systems. 2] However, because of its large molecular size and hydrophilic properties, NGF cannot pass through the blood–brain barrier, limiting its use as a therapeutic agent. Therefore, synthetic low-molecular weight compounds that possess equivalent or better neuritogenic activity compared with NGF are promising agents for the treatment of neurodegenerative diseases, such as Alzheimer’s disease. The PC12 cell line, cloned from rat pheochromocytoma, is widely used as a model system to evaluate the biological activity of neuritogenic substances. Recently, screening for neuritogenic substances from traditional Chinese medicine resulted in the isolation of 11 novel alkyl benzoates (gentisides A–K) from Gentiana rigescens (Franch.). 9] These compounds are structurally different from one another through varying alkyl chain lengths and the presence or absence of an isobutyl or isopropyl group at the end of the alkyl chain. The structure–activity relationships within gentisides reveal that the alkyl chain length is important for activity, but structural diversity at the end of the alkyl chain is not. Gentisides D, E, and F have similar alkyl chain lengths. In spite of the different structures at the end of the alkyl chain, these compounds exhibit similar neuritogenic activities at the optimum concentration of 3 mm. Gentiside E, which has a straight alkyl chain of 18 carbon atoms, exhibits higher activity (74 % at 3 mm) than gentiside K, which has 24 carbon atoms in the alkyl chain (48 % at 30 mm). To study the structure–activity relationships and discover lead compounds for drug development, a series of gentiside derivatives were designed and synthesized. First, 2,3-dihydroxybenzoates 1 a–j (Table 1) with different alkyl chain lengths were synthesized to find the optimum length of the alkyl chain. Second, tetradecylbenzoates 1 k–u (Table 1) were pre-

Sterols from Mytilidae show anti-aging and neuroprotective effects via anti-oxidative activity.

For screening anti-aging samples from marine natural products, K6001 yeast strain was employed as a bioassay system. The active mussel extract was separated to give an active sterol fraction (SF). SF was further purified, and four sterol compounds were obtained. Their structures were determined to be cholesterol (CHOL), brassicasterol, crinosterol, and 24-methylenecholesterol. All compounds showed similar anti-aging activity. To understand the action mechanism involved, anti-oxidative experiments, reactive oxygen species (ROS) assays, and malondialdehyde (MDA) tests were performed on the most abundant compound, CHOL. Results indicated that treatment with CHOL increases the survival rate of yeast under oxidative stress and decreases ROS and MDA levels. In addition, mutations of uth1, skn7, sod1, and sod2, which feature a K6001 background, were employed and the lifespans of the mutations were not affected by CHOL. These results demonstrate that CHOL exerts anti-aging effects via anti-oxidative stress. Based on the connection between neuroprotection and anti-aging, neuroprotective experiments were performed in PC12 cells. Paraquat was used to induce oxidative stress and the results showed that the CHOL and SF protect the PC12 cells from the injury induced by paraquat. In addition, these substance exhibited nerve growth factor (NGF) mimic activities again confirmed their neuroprotective function.

Granulatoside A, a starfish steroid glycoside, enhances PC12 cell neuritogenesis induced by nerve growth factor through an activation of MAP kinase.

Nerve growth factor (NGF) is the first and best characterized member of the neurotrophin family and an important factor for growth, differentiation, survival, and function maintenance of neurons. Although NGF is expected to have therapeutic potential, it is also unstable and cannot readily pass through the blood-brain barrier. Thus, attempts have been made to identify the exogenous substances that mimic and/or enhance its physiological action. Since the PC12 cell line derived from rat pheochromocytoma cells expresses neuronal differentiation in response to NGF, this is a useful model not only for screening studies but also for investigating the signal transduction pathways of PC12 cell neuritogenesis. Our previous search for compounds that mimic the neuritogenic activity of NGF using the PC12 cell line system resulted in the isolation of a series of steroid glycosides named linckosides (for example, linckoside B (4)) from the Okinawan blue starfish Linckia laevigata. A further examination of the related compounds led to identification of a known metabolite, granulatoside A (1), which was originally isolated from the starfish Choriaster granulatus. Although granulatoside A (1) did not induce any neurite outgrowth in PC12 cells, it potently enhanced the neuritogenic activity of NGF. Here we report the novel NGF-enhancing activity of granulatoside A (1), a brief description of the structure–activity relationship, and the cellular mechanism involving mitogen-activated protein (MAP) kinase. Granulatoside A (1) was obtained in 0.002% yield (based on the wet weight of the starfish L. laevigata) using the HPLC process that previously yielded linckoside F. This metabolite was originally isolated from a different starfish and identified by comparing its spectral properties with the reported data. Granulatoside A (1) did not induce the NGF-like neuronal differentiation in PC12 cells up to a concentration of 40 mm (Figure 1, 2a), despite its structural similarity to some active linckosides. However, in the presence of a trace amount of NGF (1.5 ngmL ) that scarcely induced neurite outgrowth (Figure 1, 2b), granulatoside A (1) significantly induced neurite outgrowth in a dose-dependent manner (Figure 1). In the presence of 1 at 40 mm, the neuritogenesis reached the maximum of 95%, which was comparable to the activity of 40 ngmL 1 of NGF (Figure 1, 2c). The cells cultured without the agents did not exhibit any neurite outgrowth (Figure 2d). At a concentration higher than 80 mm, the compound showed a little cytotoxicity. The IC50 value of 1 against PC12 cells was estimated to be higher than 200 mm by MTT assay (data not shown), suggesting that 1 is a low-toxicity agent with interesting biological activity. The synergistic effect exerted on the PC12 cell neuritogenesis by granulatoside A (1) and NGF was examined by varying their concentrations (Figure 3). The effect significantly increased as the concentration of 1 became higher and the concentration of NGF became lower, indicating that 1 potently enhanced NGF but not vice versa. For example, the peak activity was achieved using the combination of 1 ngmL 1 of NGF and 40 mm of 1, and was comparable to the activity by 40 ngmL 1 of NGF alone, meaning an approximately 40-fold enhancement of the activity of NGF. [a] Dr. J. Qi, C. Han, Y. Sasayama, H. Nakahara, Dr. T. Shibata, Prof. K. Uchida, Prof. M. Ojika Graduate School of Bioagricultural Sciences Nagoya University, Chikusa-ku, Nagoya 464-8601 (Japan) Fax: (+81)52-789-4284 E-mail : ojika@agr.nagoya-u.ac.jp Figure 1. Dose-dependent enhancement of NGF-induced neuritogenesis in PC12 cells by granulatoside A (1) 3 days after treatment.