Chunguang Han

A food‐derived synergist of NGF signaling: identification of protein tyrosine phosphatase 1B as a key regulator of NGF receptor‐initiated signal transduction

Neurotrophins, such as the nerve growth factor (NGF), play an essential role in the growth, development, survival and functional maintenance of neurons in the central and peripheral systems. They also prevent neuronal cell death under various stressful conditions, such as ischemia and neurodegenerative disorders. NGF induces cell differentiation and neurite outgrowth by binding with and activating the NGF receptor tyrosine kinase followed by activation of a variety of signaling cascades. We have investigated the NGF‐dependent neuritogenesis enhancer potential of a food‐derived small molecule contained in Brassica vegetables and identified the protein tyrosine phosphatase (PTP) 1B as a key regulator of the NGF receptor‐initiated signal transduction. Based on an extensive screening of Brassica vegetable extracts for the neuritogenic‐promoting activity in the rat pheochromocytoma cell line PC12, we found the Japanese horseradish, wasabi (Wasabia japonica, syn. Eutrema wasabi), as the richest source and identified 6‐methylsulfinylhexyl isothiocyanate (6‐HITC), an analogue of sulforaphane isolated from broccoli, as one of the major neuritogenic enhancers in the wasabi. 6‐HITC strongly enhanced the neurite outgrowth and neurofilament expression elicited by a low‐concentration of NGF that alone was insufficient to induce neuronal differentiation. 6‐HITC also facilitated the sustained‐phosphorylation of the extracellular signal‐regulated kinase and the autophosphorylation of the NGF receptor TrkA. It was found that PTP1B act as a phosphatase capable of dephosphorylating Tyr‐490 of TrkA and was inactivated by 6‐HITC in a redox‐dependent manner. The identification of PTP1B as a regulator of NGF signaling may provide new clues about the chemoprotective potential of food components, such as isothiocyanates.

Bioactive secondary metabolites from symbiotic marine dinoflagellates: symbiodinolide and durinskiols.

Symbiotic relationships play critical roles in marine ecosystems. Among symbionts, marine dinoflagellates have attracted the attention of natural products chemists, biologists, and ecologists, since they are rich sources of unique bioactive secondary metabolites. The polyol compound symbiodinolide, which was isolated from the symbiotic dinoflagellate Symbiodinium sp., exhibits significant voltage-dependent N-type Ca(2+) channel-opening activity and may serve as a defense substance to prevent digestion of the host animals. Durinskiols are also unique long carbon-chain polyol compounds that were isolated from the dinoflagellate Durinskia sp. We found a selective cleavage reaction of allylic 1,2-diol using an olefin metathesis catalyst, and developed a fluorescent-labeling method for MS/MS analysis to achieve the structural elucidation of huge polyol compounds. This review highlights recent advances in structural and biological studies on symbiodinolide, durinskiols, and related polyol compounds.

Stereoselective synthesis and absolute configuration of the C33–C42 fragment of symbiodinolide

Abstract Cross-metathesis of methyl ester which was prepared from symbiodinolide with ethylene was performed to give the C33–C42 degraded fragment. This fragment was estimated to be (36 S ,40 S )-diol by the modified Mosher method. Stereoselective synthesis of the (36 S ,40 S )-diol and its diastereomer (36 R ,40 S )-diol was achieved from l -aspartic acid. Synthetic bis-( S )- and ( R )-MTPA esters which were derivatized from the (36 S ,40 S )-diol exhibited spectroscopic data identical with those of bis-( S )- and ( R )-MTPA esters derived from the degraded product. Thus, the absolute stereochemistry of the C33–C42 fragment was elucidated to be (36 S ,40 S ).

Prostaglandins from a Zoanthid: Paclitaxel-Like Neurite-Degenerating and Microtubule-Stabilizating Activities

Two prostaglandins, PGA2 and PGB2, were isolated from the Okinawan zoanthid, Palythoa kochii, during a search for paclitaxel-like neurite-degenerating compounds from natural sources using a cell-based assay method. In the presence of PGA2 at 30 μM, the neuronal processes induced in PC12 cells by the nerve growth factor (NGF) degenerated over 24 h, whereas PGB2 had no effect on the neuronal processes of PC12 cells. This activity of PGA2 was similar to that of the microtubule-stabilizing agents, paclitaxel (Taxol®) and epothilone A, unlike the microtubule-depolymerizing agent, colchicine, which brought about quick neurite degeneration within 3 h. PGA2 stimulated tubulin polymerization, although less potently than paclitaxel. An examination of structure-activity relationships across several PGs suggests that the cyclopentenone ring structure and the orientation of its dipolar moment played an important role in the paclitaxel-like neurite-degenerating activity. These results suggest that the cyclopentenone-type PGs can interact with microtubules to inhibit their function like paclitaxel.

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.

Recent insights into natural venoms

Toxic substances that occur in nature have various structures and functions. In fact, the very novelty of their structures and functions sometimes extends far beyond the realm of human imagination, and the capabilities of these compounds are still largely untapped despite the major advances of modern science. In this report we focus on the most recent developments in this field, with a particular emphasis on natural venoms, marine sunscreen, and marine huge molecules.

Bioactive Maleic Anhydrides and Related Diacids from the Aquatic Hyphomycete Tricladium castaneicola.

Four maleic anhydride derivatives, tricladolides A-D (1-4), and three alkylidene succinic acid derivatives, tricladic acids A-C (5-7), were isolated from the aquatic hyphomycete Tricladium castaneicola. The structures of these compounds were determined by spectroscopic analysis, and all were found to be novel. The compounds exhibited inhibitory activity against fungi, particularly Phytophthora sp., a plant pathogen of oomycetes. The inhibitory activity of these metabolites revealed the importance of the cyclic anhydride structure and the lipophilicity of the alkyl side chain. On the other hand, the cytotoxicity of the compounds against B16 melanoma cells indicated that the cyclic anhydride structure was not essential.

Lycosides, Unusual Carotenoid-Derived Terpenoid Glycosides from a Vegetable Juice, Inhibit Asexual Reproduction of the Plant Pathogen Phytophthora

Vegetable juices, typical culture media for the plant pathogen Phytophthora, effectively induce its asexual reproduction (zoosporangia formation). However, some chromatographic fractions from a vegetable juice were found to inhibit asexual reproduction. Bioassay-guided chromatographic steps led to the isolation of four novel compounds, named lycosides A-D, 1-4, that could be metabolic products from a carotenoid. They showed 50% inhibitory activity against the asexual reproduction of P. capsici at 2.1-7.6 μM. The structure-activity relationship and the universality of the inhibitory activity within the Phytophthora genus were also investigated. In addition, the quantitative analysis of lycosides in fresh vegetables and vegetable juices revealed that tomato is the source of these active substances. These food-derived chemicals could help provide safe agents to control the outbreak of the agricultural pest Phytophthora in fields.

Linckosides enhance proliferation and induce morphological changes in human olfactory ensheathing cells

Linckosides are members of the steroid glycoside family isolated from the starfish Linckia laevigata. These natural compounds have notable neuritogenic activity and synergistic effects on NGF-induced neuronal differentiation of PC12 cells. Neurogenic factors or molecules that are able to mimic their activities are known to be involved in the survival, proliferation and migration of neurons and glial cells; however how glial cells respond to specific neurogenic molecules such as linckosides has not been investigated. This study aimed to examine the effect of three different linckosides (linckoside A, B and granulatoside A) on the morphological properties, proliferation and migration of human olfactory ensheathing cells (hOECs). The proliferation rate after all the treatments was higher than control as detected by MTS assay. Additionally, hOECs displayed dramatic morphological changes characterized by a higher number of processes after linckoside treatment. Interestingly changes in microtubule organization and expression levels of some early neuronal markers (GAP43 and βIII-tubulin) were also observed. An increase in the phosphorylation of ERK 1/2 after addition of the compounds suggests that this pathway may be involved in the linckoside-mediated effects particularly those related to morphological changes. These results are the first description of the stimulating effects of linckosides on hOECs and raise the potential for this natural compound or its derivatives to be used to regulate and enhance the therapeutic properties of OECs, particularly for cell transplantation therapies.