Fang-Hua Chu

Completion of the genome sequence of Watermelon silver mottle virus and utilization of degenerate primers for detecting tospoviruses in five serogroups.

ABSTRACT The nucleotide sequence of the L RNA of Watermelon silver mottle virus (WSMoV) was determined. Combined with the previous work on M and S RNAs, the whole genomic sequence of this member of the genus Tospovirus was completed. The L RNA is 8,917 nucleotides in length, with one large open reading frame encoding a translation product of 2,878 amino acids (331.8 kDa) on the viral complementary strand. The L protein shares amino acid identities of only 44.3 and 46.5% with Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus, respectively; but an amino acid identity of 91.3% with Peanut bud necrosis virus. Among the sequenced tospoviruses, L protein was the most conserved gene product, whereas the nonstructural S protein was generally the most variable. Comparison of the deduced L protein of WSMoV with those of other members of the family Bunyaviridae revealed that its amino acid sequence includes the reported conserved motifs of RNA-dependent RNA polymerases. To develop a method for detecting tospo-viruses by reverse transcription-polymerase chain reaction (RT-PCR), two pairs of degenerate primers were designed from conserved regions of the L genes and used to amplify the corresponding regions of the L genes from total RNAs extracted from plant tissues infected with five serologically distinct tospoviruses. The DNA fragments obtained were identified as those of tospoviruses by restriction enzyme digestion and DNA sequencing. For field samples, watermelon and wax gourd infected with WSMoV, and lisianthus infected with TSWV were also successfully detected by these two pairs of degenerate primers, with a sensitivity similar to N-gene-specific primers. The results indicated that the RT-PCR with the degenerate primers is a fast and reliable method for detecting tospoviruses in different serogroups.

Essential oil from leaves of Cinnamomum osmophloeum acts as a xanthine oxidase inhibitor and reduces the serum uric acid levels in oxonate-induced mice

The xanthine oxidase (XOD) inhibitory activity and anti-hyperuricemia effect in mice of Cinnamomum osmophloeum, which is an endemic tree in Taiwan, were evaluated in this study. The results demonstrated that the essential oil of C. osmophloeum leaves presented the strongest XOD inhibition activity (IC(50)=16.3 μg/ml); however, no significant XOD inhibition activities were found in ethanolic and hot water extracts. Furthermore, among the main compounds of essential oil, the cinnamaldehyde exhibited the potent XOD inhibition activity with an IC(50)=8.4 μg/ml. Besides, the reducing serum uric acid levels in oxonate-induced mice by cinnamaldehyde were further investigated. The hyperuricemic mice were oral administrated cinnamaldehyde at a dosage of 150 mg/kg, the uric acid value in serum was reduced from 5.25±0.63 to 2.10±0.04 mg/dl, the levels of serum uric acid in mice was lowered down by 84.48% as compared to the hyperuricemic control group. Based on the results obtained in this study, cinnamaldehyde may be a potential lead compound for developing the pharmaceutic for anti-hyperuricemia agent.

Antrocamphin A, an Anti-inflammatory Principal from the Fruiting Body of Taiwanofungus camphoratus, and Its Mechanisms

The fungus Taiwanofungus camphoratus is commonly used for medicinal purposes in Taiwan. It is used as a detoxicant for food poisoning and considered to be a precious folk medicine for hepatoprotection and anti-inflammation. In this study, a lipopolysaccaride (LPS)-challenged ICR mouse acute inflammation model and a LPS-induced macrophage model were used to evaluate the anti-inflammatory activity of T. camphoratus. Ethanol extract of T. camphoratus significantly inhibited expression of iNOS and COX-2 in the liver of LPS-challenged acute inflammatory mice. The ethyl acetate fraction and its isolated compound, antrocamphin A, significantly suppressed nitrite/nitrate concentration in LPS-challenged RAW 264.7 cells. Antrocamphin A showed potent anti-inflammatory activity by suppressing pro-inflammatory molecule release via the down-regulation of iNOS and COX-2 expression through the NF-kappaB pathway. This study, therefore, first demonstrates the bioactive compound of T. camphoratus and illustrates the mechanism by which it confers its anti-inflammatory activity.

Essential oil from the leaves of Cryptomeria japonica acts as a silverfish (Lepisma saccharina) repellent and insecticide

This is the first article to report the evaluation of a natural product used as an antisilverfish agent. Silverfish (Lepisma saccharina), primitive wingless insects, feed on a variety of materials, including paper, cotton, starch, and cereals. They can be a problem in libraries and other places where books, documents, and papers are stored. In this pilot study, the essential oil from leaves of Cryptomeria japonica was investigated to test its properties as a silverfish repellent and insecticide. The results from a repellency bioassay show that the essential oil significantly repelled silverfish. The repellent activity was 80% at a dosage of 0.01 mg/cm3. When silverfish were exposed to a concentration of 0.16 mg/cm3 of essential oil, they were killed within 10h. The chemical composition of essential oil, the emissions from a test chamber, and the residue left on filter papers previously soaked with the essential oil in a chamber were analyzed by gas chromatography-mass spectrometry. The components of the essential oil were found to be: elemol (18.22%), 16-kaurene (11.63%), 3-carene (9.66%), sabinene (9.37%), 4-terpineol (9.06%), β-eudesmol (5.70%), α-pinene (5.62%), and limonene (5.26%). Only some constituents of the essential oil compounds collected by solid-phase microextraction were found to be emitted in the test chamber. The main constituents were: 3-carene (21.03%), p-cymene (10.95%), limonene (9.49%), β-myrcene (9.39%), γ-terpinene (9.10%), α-terpinene (8.57%), and 4-terpineol (7.97%).

Genomic and transcriptomic analyses of the medicinal fungus Antrodia cinnamomea for its metabolite biosynthesis and sexual development.

Significance Antrodia cinnamomea, a mushroom, has long been used as a remedy for cancer, hypertension, and hangover. However, the molecular basis of its medicinal effects is unclear and its genome has not been studied. We obtained a genome draft and conducted gene annotation. Genome ontology enrichment and pathway analyses shed light on sexual development and metabolite biosynthesis. We identified genes differentially expressed between mycelium and fruiting body and also proteins in the mevalonate pathway, terpenoid pathways, cytochrome P450s, and polyketide synthases, which may contribute to production of medicinal metabolites. Genes of metabolite biosynthesis pathways showed expression enrichment for tissue-specific compounds in mycelium and in fruiting body. Our data will be useful for developing a strategy to increase the production of valuable metabolites. Antrodia cinnamomea, a polyporus mushroom of Taiwan, has long been used as a remedy for cancer, hypertension, and hangover, with an annual market of over

Metabolite profiles for Antrodia cinnamomea fruiting bodies harvested at different culture ages and from different wood substrates.

100 million (US) in Taiwan. We obtained a 32.15-Mb genome draft containing 9,254 genes. Genome ontology enrichment and pathway analyses shed light on sexual development and the biosynthesis of sesquiterpenoids, triterpenoids, ergostanes, antroquinonol, and antrocamphin. We identified genes differentially expressed between mycelium and fruiting body and 242 proteins in the mevalonate pathway, terpenoid pathways, cytochrome P450s, and polyketide synthases, which may contribute to the production of medicinal secondary metabolites. Genes of secondary metabolite biosynthetic pathways showed expression enrichment for tissue-specific compounds, including 14-α-demethylase (CYP51F1) in fruiting body for converting lanostane to ergostane triterpenoids, coenzymes Q (COQ) for antroquinonol biosynthesis in mycelium, and polyketide synthase for antrocamphin biosynthesis in fruiting body. Our data will be useful for developing a strategy to increase the production of useful metabolites.

Bioactivity Investigation of Lauraceae Trees Grown in Taiwan

Antrodia cinnamomea is a precious edible fungus endemic to Taiwan that has long been used as a folk remedy for health promotion and for treating various diseases. In this study, an index of 13 representative metabolites from the ethanol extract of A. cinnamomea fruiting body was established for use in quality evaluation. Most of the index compounds selected, particularly the ergostane-type triterpenoids and polyacetylenes, possess good anti-inflammation activity. A comparison of the metabolite profiles of different ethanol extracts from A. cinnamomea strains showed silmilar metabolites when the strains were grown on the original host wood (Cinnamomum kanehirai) and harvested after the same culture time period (9 months). Furthermore, the amounts of typical ergostane-type triterpenoids in A. cinnamomea increased with culture age. Culture substrates also influenced metabolite synthesis; with the same culture age, A. cinnamomea grown on the original host wood produced a richer array of metabolites than A. cinnamomea cultured on other wood species. We conclude that analysis of a fixed group of compounds including triterpenoids, benzolics, and polyacetylenes constitutes a suitable, reliable system to evaluate the quality of ethanol extract from A. cinnamomea fruiting bodies. The evaluation system established in this study may provide a platform for analysis of the products of A. cinnamomea.

Serological and Molecular Characterization of a High Temperature-recovered Virus Belonging to Tospovirus Serogroup IV

Abstract This research collected 27 Lauraceae tree species in Taiwan, and the extracts prepared from leaves and branches were selected to evaluate and characterize their putative bioactivities and potential medicinal applications. Several bioactivity assays, including antifungal tests, antioxidant evaluation, anti-inflammation activity, and cytotoxicity were preformed in this study.The results showed no significant antifungal activity by Lauraceae extracts. Neolitsea parvigemma. (Hay.) Kanehira et Sasaki expresses the best antioxidant activity (IC50 = 5.73 µg/mL) in the DPPH assay.The extracts of Litsea akoensis. Hay. and Cryptocarya concinna. Hance had significant anti-inflammation activity, and they can inhibit the nitric oxide (NO) production in the LPS-induced microphage assay at the dose of 25 µg/mL. According to the cytotoxicity assay, Lindera aggregate. (Sims) Kosterm and Cryptocarya concinna. Hance extracts showed in vitro. cytotoxicity against human umbilical vein endothelial cell line (HUVEC) with IC50 values of 43.15 µg/mL and 49.36 µg/mL, respectively, and Phoebe formosana. (Matsum. et Hay.) Hay. extract exhibited marked cytotoxicity (IC50 = 42.87 µg/mL) against a human leukemia cell line (HL-60). Results from this preliminary investigation suggest that these Lauraceae tree species may have a great potential for further development as cancer chemoprevention agents or food supplements for promoting human health.

Neuropharmacological activities of phytoncide released from Cryptomeria japonica

A serologically and cytologically distinct gloxinia tospovirus (HT-1) previously isolated from a gloxinia plant infected with Impatiens necrotic spot virus (INSV) when propagated in a high-temperature environment was characterized. Rabbit antisera produced for INSV and Tomato spotted wilt virus (TSWV) nucleocapsids (N) failed to react with HT-1 proteins in western blot analysis. The HT-1 antibodies reacted strongly with homologous antigen but failed to react with INSV and TSWV. However, the HT-1 antiserum reacted in ELISA with Watermelon silver mottle virus (WSMV) from Taiwan and in western blot analysis with the WSMV N protein. A reciprocal test showed that the antiserum prepared against the N protein of WSMV also reacted with the HT-1 N protein in both ELISA and western blot analysis. DNA probes derived from the N gene of HT-1 or WSMV hybridized to RNAs prepared from plants infected with either virus. Stronger signals were obtained with homologous than with heterologous reactions. Neither probe detected INSV or TSWV. The M and S RNAs of HT-1 were sequenced. The M RNA contains two open reading frames (ORF) ; one in the sense orientation encoding a nonstructural (NSm) protein of 308-amino-acids (aa) and the other in the ambisense orientation, a 1122-aa precursor of Gl and G2 glycoproteins. The S RNA also contains two ORFs ; one in the sense orientation encoding a nonstructural (NSs) protein of 439 aa and the other in the ambisense orientation, an N protein of 277 aa. HT-1 is distantly related to INSV and TSWV as shown by low nucleotide (40–52%) and amino acid (28–48%) similarities in the four ORF sequences. The HT-1 virus shares high nucleotide (76–81%) and amino acid (85–92%) similarities with WSMV and peanut bud necrosis virus (PBNV). Based on the serological properties and sequence data, we propose that HT-1 is a distinct species of serogroup IV in the genus Tospovirus. This is the first time that a tospovirus similar to those found in the Far East and in Southeast Asia has been identified in the US.

Molecular cloning and characterization of monoterpene synthases from Litsea cubeba (Lour.) Persoon

Forest-air bathing and walking (shinrin-yoku) is beneficial to human heath. In this study the phytoncide (volatile compounds) released from Cryptomeria japonica plantation forest was characterized by using solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS). The main volatile compounds were α-pinene (19.35%), β-myrcene (16.98%), d-limonene (15.21%), and γ-muurolene (7.42%). Furthermore, the neuropharmacological activity of the essential oils from leaves of C. japonica (ECJ) was evaluated by several animal behavior tests. ECJ could prolong the sleeping phase of ICR (imprinting control region) mice in the pentobarbital-induced sleeping time model. Furthermore, both EJC and one of its monoterpenes, d-limonene, possessed potent anxiolytic and analgesic activities based on the results obtained from elevated plus maze and writhing tests. The volatile compounds released from C. japonica provide relaxing and stress-relieving effects on mice, and further study on the effect of phytoncide on humans is worthwhile.