The Competition of Yin and Yang: Exploring the Role of Wild-Type and Mutant p53 in Tumor Progression.

The protein p53 is a well-known tumor suppressor that plays a crucial role in preventing cancer development [...].

Identification and Isolation of an Intermediate Metabolite with Dual Antioxidant and Anti-Proliferative Activity Present in the Fungus Antrodia cinnamomea Cultured on an Alternative Medium with Cinnamomum kanehirai Leaf Extract.

The fungus Antrodia cinnamomea has been used as a folk medicine for various diseases, especially cancer. When A. cinnamomea is cultured on the original host, an endangered woody plant Cinnamomum kanehirai Hayata, the fungus produces more active ingredients, but its growth is slow. Here, C. kanehirai leaf ethanol extract (KLEE) was used as a substitute for C. kanehirai wood to culture A. cinnamomea on solid medium to shorten the culture period and produce active metabolites en masse. The antioxidant activities of methanol extracts from A. cinnamomea cultured on KLEE (MEAC-KLEE) were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging effect, reducing power, and ferrous ion-chelating effect, and the effective concentration (EC50) values were 0.27, 0.74, and 0.37 mg mL-1, respectively. MEAC-KLEE exhibited specific anti-proliferative activity against a non-small-cell lung cancer cell line (A549) by Annexin V assay. A secondary metabolite (2,4-dimethoxy-6-methylbenzene-1,3-diol, DMMB) present in the extract (MEAC-KLEE) was purified by high-performance liquid chromatography (HPLC) and identified by nuclear magnetic resonance (NMR) spectra. DMMB exhibited moderate antioxidant activity against DPPH radicals and reducing power, with EC50 values of 12.97 and 25.59 µg mL-1, respectively, and also induced apoptosis in A549 cells. Our results provide valuable insight into the development of DMMB for nutraceutical biotechnology.

Trends in the Immunomodulatory Effects of Cordyceps militaris: Total Extracts, Polysaccharides and Cordycepin.

Cordyceps militaris (C. militaris) is a fungus with a long history of widespread use in folk medicine, and its biological and medicinal functions are well studied. A crucial pharmacological effect of C. militaris is immunomodulation. In this review, we catalog the immunomodulatory effects of different extracts of C. militaris, namely total extracts, polysaccharides and cordycepin. Total extracts obtained using water or 50% ethyl alcohol and polysaccharides from C. militaris were discovered to tend to promote type 1 immunity, whereas total extracts obtained using 70-80% ethyl alcohol and cordycepin from C. militaris were more likely to promote type 2 immunity. This article is the first to classify the immunomodulatory effects of different extracts of C. militaris. In addition, we discovered a relationship between different segments or extracts and differing types of immunity. This review can provide the readers a comprehensive understanding on the immunomodulatory effects of the precious folk medicine and guidance on its use for both health people and those with an immunodeficiency.

Cytotoxic Effects of Chlorophyllides in Ethanol Crude Extracts from Plant Leaves.

Chlorophyllide (chlide) is a natural catabolic product of chlorophyll (Chl), produced through the activity of chlorophyllase (chlase). The growth inhibitory and antioxidant effects of chlide from different plant leaf extracts have not been reported. The aim of this study is to demonstrate that chlide in crude extracts from leaves has the potential to exert cytotoxic effects on cancer cell lines. The potential inhibitory and antioxidant effects of chlide in crude extracts from 10 plant leaves on breast cancer cells (MCF7 and MDA-MB-231), hepatocellular carcinoma cells (Hep G2), colorectal adenocarcinoma cells (Caco2), and glioblastoma cells (U-118 MG) were studied using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and DPPH (1,1-diphenyl-2-picrylhydrazyl) assays. The results of the MTT assay showed that chlide in crude extracts from sweet potato were the most effective against all cancer cell lines tested. U-118 MG cells were the most sensitive, while Caco2 cells were the most resistant to the tested crude extracts. The cytotoxic effects of chlide and Chl in crude extracts from sweet potato and of commercial chlorophyllin (Cu-chlin), in descending order, were as follows: chlide > Chl > Cu-chlin. Notably, the IC50 of sweet potato in U-118 MG cells was 45.65 µg/mL while those of Chl and Cu-chlin exceeded 200 µg/mL. In the DPPH assay, low concentrations (100 µg/mL) of chlide and Cu-chlin from crude extracts of sweet potato presented very similar radical scavenging activity to vitamin B2. The concentration of chlide was negatively correlated with DPPH activity. The current study was the first to demonstrate that chlide in crude extracts from leaves have potential cytotoxicity in cancer cell lines. Synergism between chlide and other compounds from leaf crude extracts may contribute to its cytotoxicity.

Conversion of crude Jatropha curcas seed oil into biodiesel using liquid recombinant Candida rugosa lipase isozymes.

The versatile Candida rugosa lipase (CRL) has been widely used in biotechnological applications. However, there have not been feasibility reports on the transesterification of non-edible oils to produce biodiesel using the commercial CRL preparations, mixtures of isozymes. In the present study, four liquid recombinant CRL isozymes (CRL1-CRL4) were investigated to convert various non-edible oils into biodiesel. The results showed that recombinant CRL2 and CRL4 exhibited superior catalytic efficiencies for producing fatty acid methyl ester (FAME) from Jatropha curcas seed oil. A maximum 95.3% FAME yield was achieved using CRL2 under the optimal conditions (50 wt% water, an initial 1 equivalent of methanol feeding, and an additional 0.5 equivalents of methanol feeding at 24h for a total reaction time of 48 h at 37 °C). We concluded that specific recombinant CRL isozymes could be excellent biocatalysts for the biodiesel production from low-cost crude Jatropha oil.

MicroRNA-like small RNAs prediction in the development of Antrodia cinnamomea.

Antrodia cinnamomea, a precious, host-specific brown-rot fungus that has been used as a folk medicine in Taiwan for centuries is known to have diverse bioactive compounds with potent pharmaceutical activity. In this study, different fermentation states of A. cinnamomea (wild-type fruiting bodies and liquid cultured mycelium) were sequenced using the next-generation sequencing (NGS) technique. A 45.58 Mb genome encoding 6,522 predicted genes was obtained. High quality reads were assembled into a total of 13,109 unigenes. Using a previously constructed pipeline to search for microRNAs (miRNAs), we then identified 4 predicted conserved miRNA and 63 novel predicted miRNA-like small RNA (milRNA) candidates. Target prediction revealed several interesting proteins involved in tri-terpenoid synthesis, mating type recognition, chemical or physical sensory protein and transporters predicted to be regulated by the miRNAs and milRNAs.

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

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.

Antroquinonol from ethanolic extract of mycelium of Antrodia cinnamomea protects hepatic cells from ethanol-induced oxidative stress through Nrf-2 activation.

AIM OF THE STUDY: In recent years, the medicinal mushroom Antrodia cinnamomea, known as "niu-chang chih" has received much attention with regard to its possible health benefits; especially its hepatoprotective effects against various drugs, toxins, and alcohol induced liver diseases. However, the molecular mechanism underlying this protective effect of Antrodia cinnamomea and its active compound antroquinonol was poorly understood. In the present study we evaluated to understand the hepatoprotective efficacy of antroquinonol and ethanolic extracts of mycelia of Antrodia cinnamomea (EMAC) in vitro and in vivo. MATERIALS AND METHODS: The protective mechanism of antroquinonol and EMAC against ethanol-induced oxidative stress was investigated in cultured human hepatoma HepG2 cells and ICR mice model, respectively. HepG2 cells were pretreated with antroquinonol (1-20µM) and oxidative stress was induced by ethanol (100mM). Meanwhile, male ICR mice were pretreated with EMAC for 10 days and hepatotoxicity was generated by the addition of ethanol (5g/kg). Hepatic enzymes, cytokines and chemokines were determined using commercially available assay kits. Western blotting and real-time PCR were subjected to analyze HO-1 and Nr-2 expression. EMSA was performed to monitor Nrf-2 ARE binding activity. Possible changes in hepatic lesion were observed using histopathological analysis. RESULTS: Antroquinonol pretreatment significantly inhibited ethanol-induced AST, ALT, ROS, NO, MDA production and GSH depletion in HepG2 cells. Western blot and RT-PCR analysis showed that antroquinonol enhanced Nrf-2 activation and its downstream antioxidant gene HO-1 via MAPK pathway. This mechanism was then confirmed in vivo in an acute ethanol intoxicated mouse model: serum ALT and AST production, hepatocellular lipid peroxidation and GSH depletion was prevented by EMAC in a dose-dependent manner. EMAC significantly enhanced HO-1 and Nrf-2 activation via MAPKs consistent with in vitro studies. Ethanol-induced hepatic swelling and hydropic degeneration of hepatocytes was significantly inhibited by EMAC in a dose-dependent manner. CONCLUSIONS: These results provide a scientific basis for the hepatoprotective effects of Antrodia cinnamomea. Data also imply that antroquinonol, a potent bioactive compound may be responsible for the hepatoprotective activity of Antrodia cinnamomea. Moreover, the present study highly supported our traditional knowledge that Antrodia cinnamomea as a potential candidate for the treatment of alcoholic liver diseases.

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.

The sweet potato sporamin promoter confers high-level phytase expression and improves organic phosphorus acquisition and tuber yield of transgenic potato.

The sweet potato sporamin promoter was used to control the expression in transgenic potato of the E. coli appA gene, which encodes a bifunctional enzyme exhibiting both acid phosphatase and phytase activities. The sporamin promoter was highly active in leaves, stems and different size tubers of transgenic potato, with levels of phytase expression ranging from 3.8 to 7.4% of total soluble proteins. Phytase expression levels in transgenic potato tubers were stable over several cycles of propagation. Field tests showed that tuber size, number and yield increased in transgenic potato. Improved phosphorus (P) acquisition when phytate was provided as a sole P source and enhanced microtuber formation in cultured transgenic potato seedlings when phytate was provided as an additional P source were observed, which may account for the increase in leaf chloroplast accumulation (important for photosynthesis) and tuber yield of field-grown transgenic potato supplemented with organic fertilizers. Animal feeding tests indicated that the potato-produced phytase supplement was as effective as a commercially available microbial phytase in increasing the availability of phytate-P to weanling pigs. This study demonstrates that the sporamin promoter can effectively direct high-level recombinant protein expression in potato tubers. Moreover, overexpression of phytase in transgenic potato not only offers an ideal feed additive for improving phytate-P digestibility in monogastric animals but also improves tuber yield, enhances P acquisition from organic fertilizers, and has a potential for phytoremediation.

Promoter analysis and differential expression of the Candida rugosa lipase gene family in response to culture conditions.

Five lipase genes have been identified and sequenced from Candida rugosa. However, as the sequences of LIP multigene family are extremely closely related, it is difficult to characterize the expression spectrum of LIP genes. In the present work we have cloned, sequenced, and analyzed the promoters of these five LIP isoform genes, and several putative transcriptional elements including oleate response element (ORE) and upstream activation sequence 1 (UAS1) were identified. A quantitative real-time RT-PCR method was developed for determining the differential expression of C. rugosa lipase family genes in response to various environmental and nutritional factors. While all five LIP genes display significant changes in mRNA expression under oleic acid and/or olive oil culture conditions, LIP2 showed the strongest induction (456-fold) in response to oleic acid. LIP transcription and promoter regulation were studied by assaying the beta-galactosidase activities of promoter-lacZ fusions in Saccharomyces cerevisiae. Three of the LIP genes, LIP3, LIP4, and LIP5, showed significant induction by oleic acid, and their ORE and UAS1 elements are essential for induction by oleic acid. Together, this suggests that the multiple lipase expression profiles may be due to differential transcriptional regulation of the LIP genes in response to environment or nutritional factors.

Synthesis and characterization of canola oil-stearic acid-based trans-free structured lipids for possible margarine application.

Incorporation of stearic acid into canola oil to produce trans-free structured lipid (SL) as a healthy alternative to partially hydrogenated fats for margarine formulation was investigated. Response surface methodology was used to study the effects of lipozyme RM IM from Rhizomucor miehei and Candida rugosa lipase isoform 1 (LIP1) and two acyl donors, stearic acid and ethyl stearate, on the incorporation. Lipozyme RM IM and ethyl stearate gave the best result. Gram quantities of SLs were synthesized using lipozyme RM IM, and the products were compared to SL made by chemical catalysis and fat from commercial margarines. After short-path distillation, the products were characterized by GC and RPHPLC-MS to obtain fatty acid and triacylglycerol profiles, 13C NMR spectrometry for regiospecific analysis, X-ray diffraction for crystal forms, and DSC for melting profile. Stearic acid was incorporated into canola oil, mainly at the sn-1,3 positions, for the lipase reaction, and no new trans fatty acids formed. Most SL products did not have adequate solid fat content or beta' crystal forms for tub margarine, although these may be suitable for light margarine formulation.

Enzymatic approach to biodiesel production.

The need for alternative energy sources that combine environmental friendliness with biodegradability, low toxicity, renewability, and less dependence on petroleum products has never been greater. One such energy source is referred to as biodiesel. This can be produced from vegetable oils, animal fats, microalgal oils, waste products of vegetable oil refinery or animal rendering, and used frying oils. Chemically, they are known as monoalkyl esters of fatty acids. The conventional method for producing biodiesel involves acid and base catalysts to form fatty acid alkyl esters. Downstream processing costs and environmental problems associated with biodiesel production and byproducts recovery have led to the search for alternative production methods and alternative substrates. Enzymatic reactions involving lipases can be an excellent alternative to produce biodiesel through a process commonly referred to alcoholysis, a form of transesterification reaction, or through an interesterification (ester interchange) reaction. Protein engineering can be useful in improving the catalytic efficiency of lipases as biocatalysts for biodiesel production. The use of recombinant DNA technology to produce large quantities of lipases, and the use of immobilized lipases and immobilized whole cells, may lower the overall cost, while presenting less downstream processing problems, to biodiesel production. In addition, the enzymatic approach is environmentally friendly, considered a "green reaction", and needs to be explored for industrial production of biodiesel.

A highly stable cambialistic-superoxide dismutase from Antrodia camphorata: expression in yeast and enzyme properties.

A cDNA encoding a putative superoxide dismutase (SOD) was identified in expressed sequence tags of Antrodia camphorata, a medicinal mushroom found only in Taiwan. The deduced protein was aligned with Mn-SODs and Fe-SODs from other organisms, this SOD showed greater homology to Mn-SOD. Functional A. camphorata SOD protein was overexpressed in yeast and purified. The purified enzyme showed two active forms on a 12.5% native PAGE, a dimer and a monomer. The dimeric protein's half-life of deactivation at 80 degrees C was 7 min, and its thermal inactivation rate constant K(d) was 9.87 x 10(-2)min(-1). The enzyme was stable in a broad pH range from 5-11; in the presence of 0.4M imidazole and 2% SDS. The atomic absorption spectrometric assay showed that 1.0 atom of manganese/iron (9:1) was present in each SOD subunit. The high stability of the enzyme make it better suited than other cambialistic-SODs for use in cosmetics. The SOD also documents its future utility in developing anti-inflammatory agent and in the treatment of chronic diseases.

Characterization of fish Cu/Zn-superoxide dismutase and its protection from oxidative stress.

Copper/zinc superoxide dismutase was cloned from the zebrafish ( Danio rerio). The full coding region of the zebrafish superoxide dismutase (ZSOD) complementary DNA was ligated with pET-20b(+) and successfully expressed in Escherichia coli strain AD494(DE3)pLysS. The active enzyme was purified by His tagging. The ZSOD yield was 6 mg from 0.2 L of E. coli culture, and the specific activity was 2000 U/mg as assayed using a RANSOD kit. The enzyme stability was characterized by reaction to temperature, pH, and detergent treatment. The results showed enzyme activity was still active after heat treatment at 70 degrees C for 10 minutes, resistant to pH treatment from 2.3 to 12, and resistant to treatment with sodium dodecyl sulfate (SDS) under 4%. In addition, the recombinant ZSOD was used to protect fish from 100 ppm of paraquat-induced oxidative injury by soaking fish larva in 55 micro g/ml SOD enzyme. The results were significant.