Immunomodulatory Effect of Cordyceps militaris Polysaccharide on RAW 264.7 Macrophages by Regulating MAPK Signaling Pathways.

Polysaccharide is one of the principal bioactive components found in medicinal mushrooms and has been proven to enhance host immunity. However, the possible mechanism of immunomodulatory activity of Cordyceps militaris polysaccharide is not fully understood. Hot water extraction and alcohol precipitation, DEAE-Sephadex A-25 chromatography, and Sephadex G-100 chromatography were used to isolate polysaccharide from C. militaris. A high-molecular-weight polysaccharide isolated from C. militaris was designated as HCMP, which had an Mw of 6.18 × 105 Da and was composed of arabinose, galactose, glucose, mannose, and xylose in a mole ratio of 2.00:8.01:72.54:15.98:1.02. The polysaccharide content of HCMP was 91.2% ± 0.16. The test in vitro showed that HCMP activated mouse macrophage RAW 264.7 cells by enhancing phagocytosis and NO production, and by regulating mRNA expressions of inflammation-related molecules in RAW 264.7 cells. Western blotting revealed that HCMP induced the phosphorylation of mitogen-activated protein kinases (MAPKs). Moreover, using inhibitors of MAPKs decreased the mRNA levels of inflammation-related molecules induced by HCMP. These data evidenced that the immunomodulatory effect of HCMP on RAW 264.7 macrophages was mediated via the MAPK signaling pathway. These findings suggested that HCMP could be developed as a potent immunomodulatory agent for use in functional foods and dietary supplements.

RNA-seq analysis reveals an immunomodulatory peptide from highland barley activating RAW264.7 macrophages via TNF/NF-κB signaling pathway.

Highland barley (HB) is an important cereal crop distributed in the plateau region. Bioactive peptides (BAPs) derived from cereal proteins have shown biological functions. However, the knowledge of highland barley peptide (HBP) is limited. This study aims to explore the immunomodulatory activity of HBP and the relationship between immunomodulatory activity and related gene expression through RNA-seq. Firstly, HBP is isolated from protease hydrolysates of HB protein, yielding 12.04% of crude HB protein. The molecular weight of HBP is about 1702 Da analyzed by gel filtration chromatography, and HBP has a specific amino acid sequence as Gln-Pro-Gln-Gln-Pro-Phe-Pro-Gln (QPQPFPQ) analyzed by LC-MS. Besides, HBP contains 42.20% hydrophobic amino acids and 10.86% basic amino acids. Next, the immunomodulatory activity of HBP in vitro shows that HBP enhances the phagocytosis of RAW264.7 macrophages, promotes nitric oxide (NO) production and the mRNA expression of pro-inflammatory genes including tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and inducible nitric oxide synthase (iNOS), and decreases the mRNA expression of anti-inflammatory gene, transforming growth factor ß1 (TGF-ß1). RNA-seq analysis reveals TNF and nuclear factor kappa B (NF-κB) pathways are upregulated, and RT-qPCR is performed to verify RNA-seq analysis. In conclusion, HBP activates RAW264.7 macrophages via TNF/NF-κB signaling pathway. HBP, as a significant immunomodulatory peptide, might be a promising resource for future functional foods.

Biochar-extracted liquor stimulates nitrogen related gene expression on improving nitrogen utilization in rice seedling.

Introduction: Biochar has been shown to be an effective soil amendment for promoting plant growth and improving nitrogen (N) utilization. However, the physiological and molecular mechanisms behind such stimulation remain unclear. Methods: In this study, we investigated whether biochar-extracted liquor including 21 organic molecules enhance the nitrogen use efficiency (NUE) of rice plants using two N forms (NH4 +-N and NO3 --N). A hydroponic experiment was conducted, and biochar-extracted liquor (between 1 and 3% by weight) was applied to rice seedlings. Results: The results showed that biochar-extracted liquor significantly improved phenotypic and physiological traits of rice seedlings. Biochar-extracted liquor dramatically upregulated the expression of rice N metabolism-related genes such as OsAMT1.1, OsGS1.1, and OsGS2. Rice seedlings preferentially absorbed NH4 +-N than NO3 --N (p < 0.05), and the uptake of NH4 +-N by rice seedlings was significantly increased by 33.60% under the treatment of biochar-extracted liquor. The results from molecular docking showed that OsAMT1.1protein can theoretically interact with 2-Acetyl-5-methylfuran, trans-2,4-Dimethylthiane, S, S-dioxide, 2,2-Diethylacetamide, and 1,2-Dimethylaziridine in the biochar-extracted liquor. These four organic compounds have similar biological function as the OsAMT1.1 protein ligand in driving NH4 +-N uptakes by rice plants. Discussion: This study highlights the importance of biochar-extracted liquor in promoting plant growth and NUE. The use of low doses of biochar-extracted liquor could be an important way to reduce N input in order to achieve the purpose of reducing fertilizer use and increasing efficiency in agricultural production.

Evaluating the Application Potential of a Recombinant Ganoderma Protein as Bioactive Ingredients in Cosmetics.

The aim of this study was to evaluate the application potential of a recombinant fungal immunomodulatory protein from Ganoderma lucidum (rFIP-glu). First, a recombinant plasmid pPIC9K::FIP-glu-His was transferred into Pichia pastoris for the production of protein. The protein was then to assess its free radical scavenging abilities and the effect on the viability of both human immortalized keratinocytes (HaCaT cells) and mouse B16-F10 melanoma cells (B16 cells) in vitro, followed by the effect on the melanin synthesis of B16 cells. The results of SDS-PAGE and western blot showed that rFIP-glu was successfully expressed. Furtherly, a bioactivity assay in vitro indicated that the scavenging rate of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals reached 84.5% at 6.0 mg/mL (p ≤ 0.0001) of rFIP-glu, showing strong antioxidant activity. Subsequently, a safety evaluation demonstrated that rFIP-glu promoted the proliferation of HaCaT cells, with the cell viability reaching 124.3% at 48 µg/mL (p ≤ 0.01), regarding the cell viability of B16 cells after exposure to rFIP-glu (48 µg/mL) significantly inhibited, to 80.7% (p ≤ 0.01). Besides, rFIP-glu inhibited the melanin synthesis of B16 cells in a dose-dependent manner from 100-1000 µg/mL, and rFIP-glu at 500 µg/mL (p ≤ 0.01) exhibited the highest intracellular melanin amount reduction of 16.8%. Furthermore, a mechanism analysis showed that rFIP-glu inhibited tyrosinase (TYR) activity by up-regulating the expression of the microphthalmia-associated transcription factor (MITF) and down-regulating the gene expression of TYR and tyrosinase-related protein-1 (TYRP-1), thus inhibiting melanin synthesis. The data implied that rFIP-glu had significant antioxidant activity and whitening potency. It should be used as raw materials for cosmeceutical applications.

Recent advances of bioactive proteins/polypeptides in the treatment of breast cancer.

Proteins do not only serve as nutrients to fulfill the demand for food, but also are used as a source of bioactive proteins/polypeptides for regulating physical functions and promoting physical health. Female breast cancer has the highest incidence in the world and is a serious threat to women's health. Bioactive proteins/polypeptides exert strong anti-tumor effects and exhibit inhibition of multiple breast cancer cells. This review discussed the suppressing effects of bioactive proteins/polypeptides on breast cancer in vitro and in vivo, and their mechanisms of migration and invasion inhibition, apoptosis induction, and cell cycle arrest. This may contribute to providing a basis for the development of bioactive proteins/polypeptides for the treatment of breast cancer.

Integrated transcriptome and metabolome analysis reveals molecular responses of spider to single and combined high temperature and drought stress.

High temperature and drought are abiotic stresses restricting many arthropods' survival and growth. Wolf spiders are poikilothermic arthropods that are vital in managing insects and pests. Nonetheless, investigating changes in spiders under temperature and drought stress are limited, especially at the molecular and gene expression levels. The study found that the combined effects of high temperature and drought stress significantly reduced survival rates and raised superoxide dismutase and malondialdehyde levels in the wolf spider Pardosa pseudoannulata. An integrated transcriptome and metabolome analysis showed that differentially expressed genes and metabolites were highly enriched in pathways involved in the proteolysis and oxidation-reduction process. The gene expression profiles displayed that heat shock protein (HSP) families (i.e., small heat shock protein, HSP70, HSP90, and HSP beta protein) were up-regulated under temperature and/or drought stresses. Additionally, a conjoint analysis revealed that under the combined stress, several important enzymes, including maltase-glucoamylase, glycerol-6-phosphate transporter, alanine-glyoxylate transaminase, and prostaglandin-H2 D-isomerase, were altered, affecting the metabolism of starch, sucrose, amino acids, and arachidonic acid. The protein interaction network further confirmed that under the combined stress, metabolic processes, peptide metabolic processes, and ATP generation from ADP were up-regulated, indicating that spiders could accelerate the metabolism of carbohydrates and proteins to combat stress and maintain homeostasis. Overall, this work showed that exposure to a combination of pressures might cause distinct defensive reactions in spiders and offered novel perspectives to research the molecular underpinnings of spider adaptation to a changing climate.

Metabolome analysis reveals the toxic effects of cadmium exposure on the egg sac of spider Pardosa pseudoannulata.

The investigation of the toxic effects of cadmium (Cd) on rice field invertebrates has attracted accumulating attention. Spider grants a novel insight into the impacts of Cd stress on invertebrates, but the effects of Cd-induced toxicity and molecular response mechanism of related metabolites in spider's egg sacs remain elusive. This investigation found that Cd stress distinctively decreased vitellogenin (Vg) content and hatched spiderlings numbers in the egg sac of Pardosa pseudoannulata. In addition, Cd stress exerted oxidative stress in the egg sac, manifested as the increase of superoxide dismutase and malondialdehyde levels. Further results showed that Cd exposure could affect egg sacs' energy metabolism, including protein and lipid contents. Metabolome analysis generated 73 up-regulated and 63 down-regulated differentially expressed metabolites (DEMs), mainly affecting phenylalanine metabolism, alpha-linolenic acid metabolism, pentose phosphate pathway, and biosynthesis of amino acids. Specifically, pathway analysis showed that Cd exposure down-regulated several key factors, including tyrosine, L-phenylalanine, O-phospho-L-serine, and L-cystathionine, and inhibited the metabolism of amino acids in the egg sacs. The subsequent correlation analysis found that three metabolite indicators, 9-Oxo-ODE, PG (17:0/18:2), and PE (17:0/20:5), were the dominant contributors to the egg sec's properties (i.e., Vg content and gained spiderlings). Collectively, this study hopes to provide valuable data for the protection of rice field spiders and offer novel perspectives for Cd pollution assessment and management.

Chinese Cordyceps: Bioactive Components, Antitumor Effects and Underlying Mechanism-A Review.

Chinese Cordyceps is a valuable source of natural products with various therapeutic effects. It is rich in various active components, of which adenosine, cordycepin and polysaccharides have been confirmed with significant immunomodulatory and antitumor functions. However, the underlying antitumor mechanism remains poorly understood. In this review, we summarized and analyzed the chemical characteristics of the main components and their pharmacological effects and mechanism on immunomodulatory and antitumor functions. The analysis revealed that Chinese Cordyceps promotes immune cells' antitumor function by via upregulating immune responses and downregulating immunosuppression in the tumor microenvironment and resetting the immune cells' phenotype. Moreover, Chinese Cordyceps can inhibit the growth and metastasis of tumor cells by death (including apoptosis and autophagy) induction, cell-cycle arrest, and angiogenesis inhibition. Recent evidence has revealed that the signal pathways of mitogen-activated protein kinases (MAPKs), nuclear factor kappaB (NF-κB), cysteine-aspartic proteases (caspases) and serine/threonine kinase Akt were involved in the antitumor mechanisms. In conclusion, Chinese Cordyceps, one type of magic mushroom, can be potentially developed as immunomodulator and anticancer therapeutic agents.

Transcriptomic analysis of cadmium toxicity and molecular response in the spiderling of Pirata subpiraticus.

Cadmium (Cd) is a kind of toxic heavy metal widely distributed in the environment, posing life-threatening challenges to organisms. The paddy field spider is a natural enemy of pests and an essential component of rice biodiversity. Nonetheless, the effects of Cd stress on the postembryonic development of spiders and its detailed mechanism remain to be investigated. In the present study, we found that Cd stress posed adverse impacts on the growth indicators (e.g., carapace length, development duration, and survival rate) and increased the levels of three antioxidants (i.e., superoxide dismutase, glutathione S-transferase, and glutathione peroxidase) in the spiderlings of Pirata subpiraticus. An in-depth transcriptome analysis was employed in the study, and the results displayed that differentially expressed genes (DEGs) involved in postembryonic morphogenesis, development involved in symbiotic interaction, postembryonic development, and growth were distinctively altered under Cd stress. Further enrichment analysis showed that Cd exposure could activate the apoptosis pathway in the spider via the up-regulation of several key factors, including caspase-10, α-tubulin, actin, etc. In addition, we demonstrated that the increased level of glutathione-related enzymes in spiderlings was caused by the activation of glutathione metabolic pathway. The altered hedgehog signaling pathway might affect cell proliferation, tissue patterning, and development of spiderlings. Further protein interaction network displayed that Cd stress could affect multiple biological processes in spiderlings, particularly cellular response to stimulus and system development. To sum up, this study can provide multi-level perspectives to understand the toxicity of Cd on the growth and development of spiders.

Recent advances in bioactive peptides from cereal-derived Foodstuffs.

Cereal-derived proteins account for a major part of human dietary protein consumption. Natural bioactive peptides (NBPs) from these proteins involve a variety of physiological activities and play an important role in the promotion of human health. This review focuses on the characteristics of NBPs obtained from cereals, and the commonly used methods for preparation, separation, purification, and identification. We also discussed the biological functions of cereal-derived NBPs (CNBPs), including the activities of antioxidant, immunomodulatory, antimicrobial, and regulation of hyperglycaemia and hypertension. The paper summarised the latest progress in the research and application of CNBPs and analysed the prospects for the development and application of several protein by-products, providing an important way to improve the added value of protein by-products in cereal processing.

N-Glycosylated Ganoderma lucidum immunomodulatory protein improved anti-inflammatory activity via inhibition of the p38 MAPK pathway.

The global health emergency generated by coronavirus disease-2019 has prompted the search for immunomodulatory agents. There are many potential natural products for drug discovery and development to tackle this disease. One of these candidates is the Ganoderma lucidum fungal immunomodulatory protein (FIP-glu). In the present study, we clarify the influences of N-linked glycans on the improvement of anti-inflammatory activity and the potential mechanisms of action. Four proteins, including FIP-glu (WT) and its mutants N31S, T36N and N31S/T36N, were successfully expressed in P. pastoris, of which T36N and N31S/T36N were glycoproteins. After treatment with peptide-N-glycosidase F, the results of SDS-PAGE and Western blot showed that the glycan moiety was removed completely, indicating that the glycan moiety was N-linked. This was also demonstrated by UPLC-qTOF-MS. The cytotoxicity assay showed that N-linked glycans decreased the cytotoxicity of WT; while, the RT-qPCR assay showed that N-glycosylated WT regulated the mRNA expression of IL-6 and TGF-ß1. The Western blot results showed that N-glycosylated WT reduced the phosphorylation level of p38 MAPK. In conclusion, our findings revealed a novel mechanism by which N-glycosylation of FIP-glu improved its anti-inflammatory activity through the regulation of the expression of inflammatory cytokines in RAW264.7 via inhibition of p38 MAPK phosphorylation. It was proved that N-glycosylation significantly improved the functional properties of FIP-glu, providing theoretical and technical support for expanding the application of FIPs in the food and pharmaceutical industries.

Immunostimulatory effects of the intracellular polysaccharides isolated from liquid culture of Ophiocordyceps sinensis (Ascomycetes) on RAW264.7 cells via the MAPK and PI3K/Akt signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Chinese Cordyceps (DongChong XiaCao), a parasitic complex of a fungus Ophiocordyceps sinensis and a caterpillar, is a traditional Chinese medicine. Polysaccharides extracted from O. sinensis have immunomodulatory effects on macrophages. However, the mechanism of polysaccharides on macrophage and the composition of polysaccharides are not known. AIM OF STUDY: We aimed to investigate composition and structure of the intracellular polysaccharides from O. sinensis mycelia (designed as OSP), and evaluate its the immunomodulatory effect on macrophages and its underlying mechanism. MATERIALS AND METHODS: We performed a liquid-state fermentation of O. sinensis to produce mycelia. The DEAE-Sephadex-A25 cellulose column and Sephadex-G100 gel column chromatography were employed to purify and character the intracellular OSP. Macrophages RAW264.7 cells were employed to evaluate OSP's immunomodulatory activity and the possible mechanism responsible for the activation of macrophages in vitro. RESULTS: The average molecular weight of OSP was distributed at 27,972 Da, OSP was composed of xylose, mannose, glucose, and galactose with the ratio of 2.9 : 6.6 : 166 : 2.6, with a trace amount of fucose, arabinose and rhamnose. The phagocytosis of RAW264.7 cells was improved significantly and remarkable changes were observed in the morphology with OSP-treated cells. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis demonstrated that OSP had an ability to regulate the mRNA expression of pro-inflammatory and anti-inflammatory cytokines, and to induce the mRNA expression level of iNOS in a concentration dependent manner in RAW264.7 cells. Western blotting analysis showed that the regulation of NO and cytokines was mediated through mitogen-activated protein kinase (MAPK) and PI3K/Akt signaling pathways. CONCLUSION: This study demonstrated that OSP was with a capacity to activate macrophage cells RAW264.7 for an improvement of immunomodulation activities, which was through regulation of inflammatory mediators via MAPK and PI3K/Akt signaling pathways.

Statistical Optimization of Polysaccharides Production by the Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Agaricomycetes) in Solid-State Fermentation Using Highland Barley Grains.

Ganoderma lucidum is a widely used medicinal mushroom in traditional Chinese medicine that creates a diverse set of bioactive compounds. Highland barley, a typical nutrition-balanced crop, is not convenient for direct consumption but its nutritional characteristics meet the modern requirements of health food. In the present study, barley grains were used as substrates on solid-state fermentation (SSF) of G. lucidum. Statistical optimization of media composition was employed for enhancing the production of polysaccharides. Peptone, medlar, and KH2PO4 were identified as the most important components for producing polysaccharide. Based on the one-factor-at-a-time experimentation, a quadratic regression model with the polysaccharide contents as the response value was established by central composite design (CCD). The results showed that the predicted variables were 2.38% peptone, 1.14% medlar, and 0.25% KH2PO4 for the maximum yield of predicted polysaccharides of 11.64% in the fermented substrate. The experimental polysaccharides obtained using the predicted optimum media composition constituted 11.61% of the fermented substrate, which validates the high degree of accuracy of optimization for enhanced production of polysaccharides by SSF. This study suggested that in the process of barley grains fermentation by G. lucidum for polysaccharides production, the optimized SSF substrate consists of 71.4% barley grains, 2.38% peptone, 1.14% medlar, 0.25% KH2PO4, 2.5% glucose, 0.25% MgSO4 · 7H2O, and 1% CaCO3. According to this study, the strain G. lucidum 00679 showed a good fermentation property by optimizing the media. It might be a candidate industrial strain for further process optimization and scale-up study.

Optimization of the fermentation parameters for the production of Ganoderma lucidum immunomodulatory protein by Pichia pastoris.

In order to obtain a better fermentation parameter for the production of recombinant Ganoderma lucidum immunomodulatory protein (rFIP-glu), an engineered Pichia pastoris GS115 was investigated on the fermentation time, temperature, methanol concentration and initial pH of media, while immunomodulatory activities of the rFIP-glu was confirmed. L9(33) orthogonal experiment were firstly employed to optimize various fermentation parameters in the shake-flask level. The optimized fermentation parameters were subsequently verified in a 5 L fermenter. Biological activities including cell viability and tumor necrosis factor-alpha (TNF-α) mRNA of the rFIP-glu were evaluated on murine macrophage RAW264.7 cells. The results showed that the yield of rFIP-glu was up to 368.71 µg/ml in the shake-flask, and 613.47 µg/ml in the 5 L fermenter, when the Pichia pastoris was incubated in basic media with the methanol concentration 1.0% and initial pH 6.5, and with constant shaking at 280 rpm for 4 days at 26 °C. In vitro assays of biological activity indicated that rFIP-glu had significant toxicity against RAW264.7 cells, and possessed the ability to induce TNF-α mRNA expression in macrophage RAW264.7 cells. In conclusion, engineered P. pastoris showed a good fermentation property under the optimum fermentation parameters. It could be a candidate industrial strain for further study.

Ganoderma proteins and their potential applications in cosmetics.

Ganoderma have been regarded as a traditional source of natural bioactive compounds for centuries and have recently been exploited for potential components in the cosmetics industry. Besides Ganoderma polysaccharides and triterpenes, multiple proteins have been found in Ganoderma. With the in-depth study of these proteins, various pharmacological functions of Ganoderma have become important in the discovery and development of new products. In the review, we summarized and discussed the kinds and characteristics of Ganoderma proteins, especially on fungal immunomodulatory proteins (FIPs) which can be potentially developed into cosmeceuticals or nutricosmetics and are a suitable target for production using established biotechnological methods. Furthermore, we discuss their pharmacological activities of the proteins with a focus on their pharmacological functions related to cosmetics, such as antioxidant activity, inhibition of melanin, antibacterial activity, and regulation of inflammatory mediators. Numerous other questions also are addressed before the proteins can be widely accepted and used as cosmetic additives.

Immunomodulatory activity of Ganoderma lucidum immunomodulatory protein via PI3K/Akt and MAPK signaling pathways in RAW264.7 cells.

Ganoderma lucidum immunomodulatory protein (FIP-glu) is an active ingredient with potential immunoregulatory functions. The study was conducted to explore the immunomodulatory activities of recombinant FIP-glu (rFIP-glu) and its possible mechanism in macrophage RAW264.7 cells. In vitro assays of biological activity indicated that rFIP-glu significantly activated RAW264.7 cells and possessed proinflammatory and anti-inflammatory abilities. RNA sequencing analysis and Western blot analysis showed that macrophage activation involved PI3K/Akt and MAPK pathways. Furthermore, real-time quantitative polymerase chain reaction indicated that the PI3K inhibitor LY294002 blocked the messenger RNA (mRNA) levels of MCP-1 (CCL-2), the MEK1/2 inhibitor U0126 reduced the mRNA levels of TNF-α and MCP-1 (CCL-2), and the JNK1/2/3 inhibitor SP600125 prevented the upregulation of inducible nitric oxide synthase mRNA in rFIP-glu-induced cells. rFIP-glu did not mediate these inflammatory effects through a general pathway but rather through a different pathway for a different inflammatory mediator. These data imply that rFIP-glu possessed immunomodulatory activity in macrophages, which was mediated through PI3K/Akt and MAPK pathways.

Composition and predictive functional analysis of bacterial communities inhabiting Chinese Cordyceps insight into conserved core microbiome.

BACKGROUND: Over the past few decades, most attention to Chinese Cordyceps-associated endogenous microorganism was focused on the fungal community that creates critical bioactive components. Bacterial community associated with Chinese Cordyceps has been previously described; however, most studies were only presenting direct comparisons in the Chinese Cordyceps and its microenvironments. In the current study, our objectives were to reveal the bacterial community structure composition and predict their function. RESULTS: We collected samples of Chinese Cordyceps from five sites located in the Qinghai-Tibet Plateau and used a high throughput sequencing method to compare Chinese Cordyceps-associated bacterial community composition and diversity quantitatively across sites. The results indicated that for the Chinese Cordyceps-associated bacterial community there is no single core microbiome, which was dominated by the both Proteobacteria and Actinobacteria. Predictive functional profiling suggested a location specific function pattern for Chinese Cordyceps and bacteria in the external mycelial cortices involved in the biosynthesis of active constituents. CONCLUSIONS: This study is firstly used high throughput sequencing method to compare the bacterial communities inhabiting Chinese Cordyceps and its microhabitat and to reveal composition functional capabilities of the bacteria, which will accelerate the study of the functions of bacterial communities in the micro-ecological system of Chinese Cordyceps.

Fungal immunomodulatory proteins: characteristic, potential antitumor activities and their molecular mechanisms.

During recent decades, >30 fungal immunomodulatory proteins (FIPs) have been found in a range of mushrooms and other fungi. Various pharmacological functions of FIPs have become important in the discovery and development of new drugs. In this review, we discuss some important factors, focusing on the use of amino acid sequence data to predict structural and physicochemical properties. We also discuss pharmacologic activities and possible mechanisms of the proteins with a focus on antitumor activities. Numerous other questions must also be addressed before FIPs can be widely accepted and used as antitumor agents.

Cloning and Characterization of Promoters of the Fungal Immunomodulatory Protein Genes from Ganoderma spp. (Agaricomycetes) and Their Response to Methyl Jasmonate and Salicylic Acid.

Ganoderma mushrooms for medicinal use contain various bioactive compounds, but the genetic elements available for these medicinal mushrooms are still limited. In this study we cloned and analyzed the promoters of fungal immunomodulatory protein (FIP) genes from G. lucidum and G. atrum. FIP gene expression was induced by different concentrations of methyl jasmonate (MeJA) and salicylic acid (SA), and messenger RNA expression was detected by quantitative reverse-transcription polymerase chain reaction. The results provided 5' upstream sequences of FIP genes from G. lucidum and G. atrum. Sequence analysis showed that the FIP-glu promoter sequence contained 11 CAAT boxes, 3 TATA boxes, 3 MeJA-responsive elements, 3 MYB binding site (MBS) motifs, 1 abscisic acid responsive element, 1 TGA, 1 anaerobic inducible element, 2 circadian elements, 1 fungal elicitor, 1 meristem-specific activation element, 3 Skn-1 motifs, and several light-responsive elements. The 5' flanking region of FIP-gat included 9 CAAT boxes, 4 TATA boxes, 3 MeJA-responsive elements, 1 AuxRR core, 1 GC motif, 1 MBS, 1 fungal elicitor, 1 meristem-specific activation element, 3 Skn-1 motifs, and several light-responsive elements. On the transcriptional level, both FIP-glu and FIP-gat reached their highest expression after treatment with MeJA at 500 µmol/L. FIP-glu expression depended on the concentration of SA (0-1000 mg/L); the expression of the FIP-gat gene was highest at a concentration of 100 mg MeJA/L. This research lays the foundation to use Ganoderma mycelia as bioreactors for producing FIPs.