Xylaria insolita and X. subescharoidea: two newly described species collected from a termite nesting site in Hua-lien, Taiwan.

BACKGROUND: A number of Xylaria species are exclusively associated with nests of macrotermitine termites. A nesting site of Odontotermes formosanus in eastern Taiwan, which is the only macrotermitine termite known on the island, had been inundated during the raining season of 2010, and hundreds of Xylaria stromata emerged from it thereafter. A thorough examination of these stromata showed that they represent a mixture of different species. RESULTS: Five Xylaria species were identified from the stromata collected from the nesting site, including two undescribed species, which are newly described as X. insolita and X. subescharoidea herein, and three known species X. brunneovinosa, X. escharoidea, and X. furcata. CONCLUSION: Totally, there are 28 Xylaria species growing on termite nests or ground in the world. Although O. formosanus is the only macrotermitine species known in Taiwan, the Xylaria diversity associated with its nests is fairly high; the species number has reached 12 with X. furcata, X. insolita, and X. subescharoidea added to the Taiwan mycobiota.

Optimization of Protoplast Preparation and Regeneration of a Medicinal Fungus Antrodia cinnamomea.

Antrodia cinnamomea is a unique medicinal fungus in Taiwan. It has been found rich in some pharmacologically active compounds for anti-cancer, hangover, and immune regulation etc. With the in-depth study of these components, it would be interesting and important to establish a molecular system for basic studies of A. cinnamomea. Thus, we would like to set up a foundation for this purpose by studying the A. cinnamomea protoplast preparation and regeneration. Firstly, we studied the optimization method of protoplast preparation of A. cinnamomea, and found various factors that may affect the yield during protoplast preparation, such as mycelial ages, pH values, and osmotic stabilizers. Secondly, in the regeneration of protoplasts, we explored the effects of various conditions on the regeneration of protoplasts, including different media and osmotic pressure. In addition, we found that citrate buffer with pH value around 3 dramatically increased the regeneration of protoplasts of A. cinnamomea, and provided a set of regeneration methodology for A. cinnamomea.

A termite symbiotic mushroom maximizing sexual activity at growing tips of vegetative hyphae.

BACKGROUND: Termitomyces mushrooms are mutualistically associated with fungus-growing termites, which are widely considered to cultivate a monogenotypic Termitomyces symbiont within a colony. Termitomyces cultures isolated directly from termite colonies are heterokaryotic, likely through mating between compatible homokaryons. RESULTS: After pairing homokaryons carrying different haplotypes at marker gene loci MIP and RCB from a Termitomyces fruiting body associated with Odontotermes formosanus, we observed nuclear fusion and division, which greatly resembled meiosis, during each hyphal cell division and conidial formation in the resulting heterokaryons. Surprisingly, nuclei in homokaryons also behaved similarly. To confirm if meiotic-like recombination occurred within mycelia, we constructed whole-genome sequencing libraries from mycelia of two homokaryons and a heterokaryon resulting from mating of the two homokaryons. Obtained reads were aligned to the reference genome of Termitomyces sp. J132 for haplotype reconstruction. After removal of the recombinant haplotypes shared between the heterokaryon and either homokaryons, we inferred that 5.04% of the haplotypes from the heterokaryon were the recombinants resulting from homologous recombination distributed genome-wide. With RNA transcripts of four meiosis-specific genes, including SPO11, DMC1, MSH4, and MLH1, detected from a mycelial sample by real-time quantitative PCR, the nuclear behavior in mycelia was reconfirmed meiotic-like. CONCLUSION: Unlike other basidiomycetes where sex is largely restricted to basidia, Termitomyces maximizes sexuality at somatic stage, resulting in an ever-changing genotype composed of a myriad of coexisting heterogeneous nuclei in a heterokaryon. Somatic meiotic-like recombination may endow Termitomyces with agility to cope with termite consumption by maximized genetic variability.

Litchi seed extract inhibits epidermal growth factor receptor signaling and growth of Two Non-small cell lung carcinoma cells.

BACKGROUND: Litchi seeds possess rich amounts of phenolics and have been shown to inhibit proliferation of several types of cancer cells. However, the suppression of EGFR signaling in non-small cell lung cancer (NSCLC) by litchi seed extract (LCSE) has not been fully understood. METHODS: In this study, the effects of LCSE on EGFR signaling, cell proliferation, the cell cycle and apoptosis in A549 adenocarcinoma cells and NCI- H661 large-cell carcinoma cells were examined. RESULTS: The results demonstrated that LCSE potently reduced the number of cancer cells and induced growth inhibition, cell-cycle arrest in the G1 or G2/M phase, and apoptotic death in the cellular experiment. Only low cytotoxicity effect was noted in normal lung MRC-5 cells. LCSE also suppressed cyclins and Bcl-2 and elevated Kip1/p27, Bax and caspase 8, 9 and 3 activities, which are closely associated with the downregulation of EGFR and its downstream Akt and Erk-1/-2 signaling. CONCLUSION: The results implied that LCSE suppressed EGFR signaling and inhibited NSCLC cell growth. This study provided in vitro evidence that LCSE could serve as a potential agent for the adjuvant treatment of NSCLC.

Phenologic variation of major triterpenoids in regular and white Antrodia cinnamomea.

BACKGROUND: Antrodia cinnamomea and its host Cinnamomum kanehirae are both endemic species unique to Taiwan. Many studies have confirmed that A. cinnamomea is rich in polysaccharides and triterpenoids that may carry medicinal effects in anti-cancer, anti-inflammation, anti-hypertension, and anti-oxidation. Therefore it is of interest to study the chemical variation of regular orange-red strains and white strains, which included naturally occurring and blue-light induced white A. cinnamomea. RESULTS: The chemical profiles of A. cinnamomea extracts at different growth stages were compared using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The TLC and HPLC profiles indicated that specific triterpenoids varied between white and regular strains. Moreover, the compounds of blue-light induced white strain were similar to those of naturally occurring white strain but retained specific chemical characteristics in more polar region of the HPLC chromatogram of regular strain. CONCLUSIONS: Blue-light radiation could change color of the regular A. cinnamomea from orange-red to white by changing its secondary metabolism and growth condition. Naturally occurring white strain did not show a significantly different composition of triterpenoid profiles up to eight weeks old when compared with the triterpenoid profiles of the regular strain at the same age. The ergostane-type triterpenoids were found existing in both young mycelia and old mycelia with fruiting body in artificial agar-plate medium culture, suggesting a more diversified biosynthetic pathway in artificial agar-plate culture rather than wild or submerged culture.

Induction of apoptosis and cell cycle arrest in human colorectal carcinoma by Litchi seed extract.

The Litchi (Litchi chinensis) fruit products possess rich amounts of flavanoids and proanthocyanidins. Its pericarp has been shown to inhibit breast and liver cancer cell growth. However, the anticolorectal cancer effect of Litchi seed extract has not yet been reported. In this study, the effects of polyphenol-rich Litchi seed ethanol extract (LCSP) on the proliferation, cell cycle, and apoptosis of two colorectal cancer cell lines Colo320DM and SW480 were examined. The results demonstrated that LCSP significantly induced apoptotic cell death in a dose-dependent manner and arrested cell cycle in G2/M in colorectal carcinoma cells. LCSP also suppressed cyclins and elevated the Bax : Bcl-2 ratio and caspase 3 activity. This study provides in vitro evidence that LCSP serves as a potential chemopreventive agent for colorectal cancer.

Arginine-rich intracellular delivery peptides synchronously deliver covalently and noncovalently linked proteins into plant cells.

Protein transduction domains (PTDs) are small peptides with a high content of basic amino acids, and they are responsible for cellular uptake. Many PTDs, including arginine-rich intracellular delivery (AID) peptides, have been shown to transport macromolecules across membranes and into cells. In this study, we demonstrated for the first time that AID peptides could rapidly and efficiently deliver proteins into plant cells in both covalent and noncovalent protein transductions (CNPT) simultaneously. The optimal molecular ratio between an AID peptide carrier and cargo in CNPT was about 3:1. Fluorescence resonance energy transfer (FRET) analysis revealed protein-protein interactions between AID peptide carriers and cargos after CNPT in cells. The possible mechanisms of AID peptides-mediated cellular entry might involve a combination of multiple internalization pathways. Therefore, applications by AID peptide-mediated CNPT may provide a simple and direct transport strategy for delivering two proteins in agricultural systems.

Fructification of Antrodia cinnamomea was strain dependent in malt extract media and involved specific gene expression.

Antrodia cinnamomea is an expensive medicinal fungus that grows only inside the rotten trunk of Cinnamomum kanehirae . In vitro culture of A. cinnamomea fruiting body is difficult and, therefore, of value for further investigation. To study whether the fructification of A. cinnamomea is strain dependent in artificial media, we grew four different A. cinnamomea strains on malt extract agar (MEA) media. The standard MEA and a series of dilution of the MEA nutrient components were made to culture A. cinnamomea. The formation of fruiting body was determined by visual and microscopic observation on A. cinnamomea's porous morphogenesis and HPLC analysis. All A. cinnamomea strains cultured grew best in 50% MEA, but carried different capabilities of fructification. In addition, we studied four antioxidation- or senescence-related genes, including a cytochrome P450, a glutathione-S-transferase, a peroxiredoxin, and a manganese superoxide dismutase. We found both cytochrome P450 and glutathione-S-transferase were expressed 3.66- and 2.75-fold in fruiting body compared with mycelium, respectively, and perxoiredoxin and manganese superoxide dismutase were found with similar expressions in both fruiting body and mycelium.

Cell membrane diversity in noncovalent protein transduction.

Crossing of the plasma membrane for all macromolecules without energy, receptors or any artificial methods was thought to be difficult. Our previous studies demonstrated that arginine-rich intracellular delivery (AID) peptides are able to deliver macromolecules, such as proteins, RNAs and DNAs, into either animal or plant cells. Cellular internalization could be mediated by effective and nontoxic AID peptides in either a covalent or noncovalent protein transduction (NPT) manner. AID peptides were so versatile that the procedure seemed to replace the current artificial transfection methods. However, the utilization of AID peptides has been limited to animal or plant systems so far. None has proposed that AID peptides could work in other species. Here, we select some representative organisms to screen whether NPT mediated by AID peptides works in them. They include cyanobacteria, bacteria, archaea, algae, fungi and yeasts. The results reveal that not all living beings possess this capability of protein transduction. Interestingly, all species of prokaryotes tested, which were thought to be highly diverse from the animal and plant systems, appear to be capable of NPT. The mechanism of AID-mediated NPT in cyanobacteria is in a classical endocytosis- and energy-independent pathway and may involve macropinocytosis. In contrast, green algae and multicellular fungi of the eukaryotes are impermeable to protein passage. Our results bring an interesting clue to the reexamination of the phylogeny of both algae and fungi.

Transfection and expression of plasmid DNA in plant cells by an arginine-rich intracellular delivery peptide without protoplast preparation.

The delivery and expression of exogenous genes in plant cells have been of particular interest for plant research and biotechnology. Here, we present results demonstrating a simple DNA transfection system in plants. Short arginine-rich intracellular delivery peptide, a protein transduction domain, was capable of delivering plasmid DNA into living plant cells non-covalently. This peptide-mediated DNA delivery conferred several advantages, such as nuclear targeting, non-toxic effect, and ease of preparation without protoplast formulation. Thus, this novel technology shall provide a powerful tool to investigate gene function in vivo, and lay the foundation for the production of transgenic plants in future.

Noncovalent protein transduction in plant cells by macropinocytosis.

* Protein delivery across cellular membranes or compartments is primarily limited by low biomembrane permeability. * Many protein transduction domains (PTDs) have previously been generated, and covalently cross-linked with cargoes for cellular internalization. * An arginine-rich intracellular delivery (AID) peptide could rapidly deliver fluorescent proteins or beta-galactosidase enzyme into plant and animal cells in a noncovalent fashion. The possible mechanism of this noncovalent protein transduction (NPT) may involve macropinocytosis. * The NPT via a nontoxic AID peptide provides a powerful tool characterized by its simplicity and quickness to have active proteins function in living cells in vivo. This should be of broad utility for functional enzyme assays and protein therapies in both plant biology research as well as biomedical applications.

Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells.

The protein delivery across cellular membranes or compartments is limited by low biomembrane permeability because of the hydrophobic characteristics of cell membranes. Usually the delivery processes utilize passive protein channels or active transporters to overcome the membrane impediment. In this report, we demonstrate that arginine-rich intracellular delivery (AID) peptide is capable of efficiently delivering fused fluorescent proteins unpreferentially into different plant tissues of both tomato (a dicot plant) and onion (a monocot plant) in a fully bioactive form. Thus, cellular internalization via AID peptide can be a powerful tool characterized by its simplicity, non-invasion and high efficiency to express those bioactive proteins in planta or in plant cells in vivo. This novel method may alternatively provide broader applications of AID chimera in plants without the time-consuming transgenic approaches.

His-404 and His-405 are essential for enzyme catalytic activities of a bacterial indole-3-acetyl-L-aspartic acid hydrolase.

Bacterial indole-3-acetyl-l-aspartic acid (IAA-Asp) hydrolase has shown very high substrate specificity compared with similar IAA-amino acid hydrolase enzymes found in Arabidopsis thaliana. The IAA-Asp hydrolase also exhibits, relative to the Arabidopsis thaliana-derived enzymes, a very high Vmax (fast reaction rate) and a higher Km (lower substrate affinity). These two characteristics indicate that there are fundamental differences in the catalytic activity between this bacterial enzyme and the Arabidopsis enzymes. By employing a computer simulation approach, a catalytic residue, His-385, from a non-sequence-related zinc-dependent exopeptidase of Pseudomonas was found to structurally match His-405 of IAA-Asp hydrolase. The His-405 residue is conserved in all related sequences of bacteria and Arabidopsis. Point mutation experiments of this His-405 to seven different amino acids resulted in complete elimination of enzyme activity. However, point mutation on the neighboring His-404 to eight other residues resulted in reduction, to various degrees, of enzyme activity. Amino acid substitutions for His-404 also showed that this residue influenced the minor activity of the IAA-Asp hydrolase for the substrates IAA-Gly, IAA-Ala, IAA-Ser, IAA-Glu and IAA-Asn. These results show the value and potential of structural modeling for predicting target residues for further study and for directing bioengineering of enzyme structure and function.