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Single-stranded DNA-binding proteins (SSBs) are essential to cells because they participate in DNA metabolic processes, such as DNA replication, repair, and recombination. Some bacteria possess more than one paralogous SSB. Three similar SSBs, namely, SsbA, SsbB, and SsbC, are found in Staphylococcus aureus. Whether the FDA-approved clinical drug 5-fluorouracil (5-FU) that is used to target the enzyme thymidylate synthase for anticancer therapy can also bind to SSBs remains unknown. In this study, we found that 5-FU could form a stable complex with S. aureus SsbB (SaSsbB). We cocrystallized 5-FU with SaSsbB and solved complex structures to assess binding modes. Two complex forms of the structures were determined, namely, the individual asymmetric unit (two SaSsbB monomers) containing one (PDB entry 7D8J) or two 5-FU molecules (PDB entry 7DEP). The locations of 5-FU in these two SaSsbB complexes were similar regardless of the binding ratio. The structures revealed that residues T12, K13, T30, F48, and N50 of SaSsbB were involved in 5-FU binding. The mutations of T12, K13, and F48 caused the low 5-FU binding activity of SaSsbB, a result consistent with the structural analysis results. Taken together, the complexed structure and the binding mode analysis of SaSsbB extended the anticancer drug 5-FU interactome to include the oligonucleotide/oligosaccharide-binding fold protein.

Single-stranded DNA (ssDNA)-binding proteins (SSBs) play a central role in cells by participating in DNA metabolism, including replication, repair, recombination, and replication fork restart. SSBs are essential for cell survival and thus an attractive target for potential anti-pathogen chemotherapy. In this study, we determined the crystal structure and examined the size of the ssDNA-binding site of an SSB from Salmonella enterica serovar Typhimurium LT2 (SeSSB), a ubiquitous opportunistic pathogen which is highly resistant to antibiotics. The crystal structure was solved at a resolution of 2.8 Å (PDB ID 7F25), indicating that the SeSSB monomer possesses an oligonucleotide/oligosaccharide-binding (OB) fold domain at its N-terminus and a flexible tail at its C-terminus. The core of the OB-fold in the SeSSB is made of a six-stranded β-barrel capped by an α-helix. The crystal structure of the SeSSB contained two monomers per asymmetric unit, which may indicate the formation of a dimer. However, the gel-filtration chromatography analysis showed that the SeSSB forms a tetramer in solution. Through an electrophoretic mobility shift analysis, we characterized the stoichiometry of the SeSSB complexed with a series of ssDNA dA homopolymers, and the size of the ssDNA-binding site was determined to be around 22 nt. We also found the flavanonol taxifolin, also known as dihydroquercetin, capable of inhibiting the ssDNA-binding activity of the SeSSB. Thus, this result extended the SSB interactome to include taxifolin, a natural product with a wide range of promising pharmacological activities.

Certain herbs used in traditional Chinese medicine may produce a growth-enhancing effect by promoting the secretion of growth hormone (GH) by the pituitary gland or mimicking the function of GH. In this study, we aimed to identify herbs that could serve as GH alternatives. A reporter gene assay for GH was developed, and 100 different herbal extracts were assayed. We found that Rhizoma Anemarrhenae (RA) water extracts exhibited transactivation activities that stimulate the activation of signal transducer and activator of transcription 5 (STAT5). The growth-promoting effect of RA in NB2-11 cells was inhibited by co-treatment with GH receptor (GHR)-Fc fusion protein. Unlike GH, RA extracts did not enhance the growth of B16F10 melanoma cells. The activation of the Janus kinase 2-STAT5 signaling pathway was confirmed in both NB2-11 cells and WI-38 human normal lung fibroblasts; the activation was inhibited by co-treatment with GHR-Fc fusion protein. Docking analysis of the active ingredients of RA, including mangiferin, neomangiferin, isomangiferin, anemarsaponin E, 7-O-methylmangiferin, officinalisinin I, timosaponin BII, timosaponin AI, and timosaponin AIII, using SWISSDOCK indicated a direct interaction of these compounds with GHR. The growth-promoting effects and activation of STAT5 were also confirmed. Moreover, we found that RA extract significantly increased the height of the tibial growth plate and stimulated the production of insulin-like growth factor 1 in the serum, liver, and muscle tissues. Our findings provide evidence that herbal extracts, particularly, RA extracts, can promote growth by mimicking GH bioactivity.

Background: Dopamine has been suggested to be a stop signal for eye growth and affects the development of myopia. Acupuncture is known to increase dopamine secretion and is widely used to treat myopia clinically. Objective: The aim of this study was to determine if acupuncture inhibits myopia progression in form deprived Syrian hamsters by inducing rises in dopamine content that in turn suppress inflammasome activation. Methods: Acupuncture was applied at LI4 and Taiyang every other day for 21 days. The levels of molecules associated with the dopamine signaling pathway, inflammatory signaling pathway and inflammasome activation were determined. A dopamine agonist (apomorphine) was used to evaluate if activation of the dopaminergic signaling pathway suppresses myopia progression by inhibiting inflammasome activation in primary retinal pigment epithelial (RPE) cells. A dopamine receptor 1 (D1R) inhibitor (SCH39166) was also administered to the hamsters. Results: Acupuncture inhibited myopia development by increasing dopamine levels and activating the D1R signaling pathway. Furthermore, we also demonstrated that nucleotide-binding oligomerization domain (NOD)-, leucine-rich repeat (LRR)- and pyrin domain-containing protein 3 (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome activation was inhibited by activation of the D1R signaling pathway. Conclusion: Our findings suggest that acupuncture inhibits myopia development by suppressing inflammation, which is initiated by activation of the dopamine—D1R signaling pathway.

Single-stranded DNA (ssDNA)-binding protein (SSB) plays a crucial role in DNA replication, repair, and recombination as well as replication fork restarts. SSB is essential for cell survival and, thus, is an attractive target for potential antipathogen chemotherapy. Whether naturally occurring products can inhibit SSB remains unknown. In this study, the effect of the flavonols myricetin, quercetin, kaempferol, and galangin on the inhibition of Pseudomonas aeruginosa SSB (PaSSB) was investigated. Furthermore, SSB was identified as a novel quercetin-binding protein. Through an electrophoretic mobility shift analysis, myricetin could inhibit the ssDNA binding activity of PaSSB with an IC50 of 2.8 ± 0.4 μM. The effect of quercetin, kaempferol, and galangin was insignificant. To elucidate the flavonol inhibition specificity, the crystal structure of PaSSB complexed with the non-inhibitor quercetin was solved using the molecular replacement method at a resolution of 2.3 Å (PDB entry 7VUM) and compared with a structure with the inhibitor myricetin (PDB entry 5YUN). Although myricetin and quercetin bound PaSSB at a similar site, their binding poses were different. Compared with myricetin, the aromatic ring of quercetin shifted by a distance of 4.9 Å and an angle of 31o for hydrogen bonding to the side chain of Asn108 in PaSSB. In addition, myricetin occupied and interacted with the ssDNA binding sites Lys7 and Glu80 in PaSSB whereas quercetin did not. This result might explain why myricetin could, but quercetin could not, strongly inhibit PaSSB. This molecular evidence reveals the flavonol inhibition specificity and also extends the interactomes of the natural anticancer products myricetin and quercetin to include the OB-fold protein SSB.

PriB is a primosomal protein required for the replication fork restart in bacteria. Although PriB shares structural similarity with SSB, they bind ssDNA differently. SSB consists of an N-terminal ssDNA-binding/oligomerization domain (SSBn) and a flexible C-terminal protein–protein interaction domain (SSBc). Apparently, the largest difference in structure between PriB and SSB is the lack of SSBc in PriB. In this study, we produced the chimeric PriB-SSBc protein in which Klebsiella pneumoniae PriB (KpPriB) was fused with SSBc of K. pneumoniae SSB (KpSSB) to characterize the possible SSBc effects on PriB function. The crystal structure of KpSSB was solved at a resolution of 2.3 Å (PDB entry 7F2N) and revealed a novel 114-GGRQ-117 motif in SSBc that pre-occupies and interacts with the ssDNA-binding sites (Asn14, Lys74, and Gln77) in SSBn. As compared with the ssDNA-binding properties of KpPriB, KpSSB, and PriB-SSBc, we observed that SSBc could significantly enhance the ssDNA-binding affinity of PriB, change the binding behavior, and further stimulate the PriA activity (an initiator protein in the pre-primosomal step of DNA replication), but not the oligomerization state, of PriB. Based on these experimental results, we discuss reasons why the properties of PriB can be retrofitted when fusing with SSBc.

Background The increased global incidence of myopia requires the establishment of therapeutic approaches. This study aimed to investigate the effect of Fallopia Japonica (FJ) and Prunella vulgaris (PV) extract on myopia caused by monocular form deprivation (MFD). Methods We used human retinal pigment epithelial cell to study the molecular mechanisms on how FJ extract (FJE) and PV extract (PVE) lowering the inflammation of the eye. The effect of FJE and PVE in MFD induced hamster model and explore the role of inflammation cytokines in myopia. Results FJE + PVE reduced IL-6, IL-8, and TNF-α expression in RPE cells. Furthermore, FJE and PVE inhibited inflammation by attenuating the phosphorylation of protein kinase B (AKT), and nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) pathway. In addition, we report two resveratrol + ursolic acid compounds from FJ and PV and their inhibitory activities against IL-6, IL-8, and TNF-α expression levels in RPE cells treated with IL-6 and TNF-α. FJE, PVE, and FJE + PVE were applied to MFD hamsters and their axial length was measured after 21 days. The axial length showed statistically significant differences between phosphate-buffered saline- and FJE-, PVE-, and FJE + PVE-treated MFD eyes. FJE + PVE suppressed expressions of IL-6, IL-8, and TNF-α. They also inhibited myopia-related transforming growth factor-beta (TGF)-β1, matrix metalloproteinase (MMP)-2, and NF-κB expression while increasing type I collagen expression. Conclusions Overall, these results suggest that FJE + PVE may have a therapeutic effect on myopia and be used as a potential treatment option.

Single-stranded DNA-binding protein (SSB) is essential to cells as it participates in DNA metabolic processes, such as DNA replication, repair, and recombination. Escherichia coli SSB (EcSSB) tetramer cooperatively binds and wraps ssDNA in two major binding modes. In this study, we report the complex crystal structure of Pseudomonas aeruginosa SSB (PaSSB) with ssDNA dT20 at 2.39 A resolution (PDB entry 6JDG) that revealed a new binding mode, namely, (SSB)3:1. In the (SSB)65 mode revealed by the EcSSB–dC35 complex structure, all four subunits fully participate in the binding to ssDNA. However, only three subunits in the PaSSB tetramer can participate in wrapping ssDNA in the (SSB)3:1 mode. The bound ssDNA in the PaSSB–ssDNA complex adopts an Ω-shaped conformation rather than a χ-shaped conformation in the (SSB)65 mode possibly due to the disability of Phe60. Phe60 is known to play a critical role in defining DNA-binding paths and promoting the wrapping of ssDNA around SSB tetramers. However, it is not important in the (SSB)3:1 mode. The ssDNA binding path revealed by our structural evidence suggests that ssDNA occupies half of the binding sites of the two subunits and slightly comes into contact with the ssDNA binding sites of the third subunit. Accordingly, we propose and sketch a possible wrapping mechanism of SSB via this novel ssDNA-binding mode, (SSB)3:1.

Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway for pyrimidine nucleotides, and an attractive target for potential anticancer chemotherapy. By screening plant extracts and performing GC–MS analysis, we identified and characterized that the potent anticancer drug plumbagin (PLU), isolated from the carnivorous plant Nepenthes miranda, was a competitive inhibitor of DHOase. We also solved the complexed crystal structure of yeast DHOase with PLU (PDB entry 7CA1), to determine the binding interactions and investigate the binding modes. Mutational and structural analyses indicated the binding of PLU to DHOase through loop-in mode, and this dynamic loop may serve as a drug target. PLU exhibited cytotoxicity on the survival, migration, and proliferation of 4T1 cells and induced apoptosis. These results provide structural insights that may facilitate the development of new inhibitors targeting DHOase, for further clinical anticancer chemotherapies.

Resveratrol is a key component of red wine and other grape products. Recent studies have characterized resveratrol as a polyphenol, and shown its beneficial effects on cancer, metabolism, and infection. This study aimed to obtain insights into the biological effects of resveratrol on myopia. To this end, we examined its anti-inflammatory influence on human retinal pigment epithelium cells and in a monocular form deprivation (MFD)-induced animal model of myopia. In MFD-induced myopia, resveratrol increased collagen I level and reduced the expression levels of matrix metalloproteinase (MMP)2, transforming growth factor (TGF)-β, and nuclear factor (NF)-κB expression levels. It also suppressed the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β. Resveratrol exhibited no significant cytotoxicity in ARPE-19 cells. Downregulation of inflammatory cytokine production, and inhibition of AKT, c-Raf, Stat3, and NFκB phosphorylation were observed in ARPE-19 cells that were treated with resveratrol. In conclusion, the findings suggest that resveratrol inhibits inflammatory effects by blocking the relevant signaling pathways, to ameliorate myopia development. This may make it a natural candidate for drug development for myopia.

BackgroundThe increased global incidence of myopia requires the establishment of therapeutic approaches. Previous studies have suggested that inflammation plays an important role in the development and progression of myopia. This study aimed to investigate the effect of Fallopia Japonica (FJ) and Prunella Vulgaris (PV) extract on myopia caused by monocular form deprivation (MFD) and elucidate the underlying mechanisms. MethodsWe used human retinal pigment epithelial cell to study the molecular mechanisms on how FJE and PVE lowering the inflammation of the eye. The effect of FJE and PVE in MFD induced hamster model and explore the role of inflammation cytokines in myopia.ResultsMyopia progression was enhanced upon tumor necrosis factor (TNF)-α or interleukin (IL)-6 administration. Expression levels of IL-6, IL-8, and TNF-α were upregulated in retinal pigment epithelium (RPE) cells treated with IL-6 and TNF-α. FJ extract (FJE) + PV extract (PVE) reduced IL-6, IL-8, and TNF-α expression in RPE cells. Furthermore, FJE and PVE inhibited inflammation by attenuating the phosphorylation of protein kinase B (AKT), and nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) pathway. In addition, we report two resveratrol + ursolic acid compounds from FJ and PV and their inhibitory activities against IL-6, IL-8, and TNF-α expression levels in RPE cells treated with IL-6 and TNF-α. FJE, PVE, and FJE + PVE were applied to MFD hamsters and their axial length was measured after 21 days. The axial length showed statistically significant differences between phosphate-buffered saline- and FJE-, PVE-, and FJE + PVE-treated MFD eyes. FJE + PVE suppressed expressions of IL-6, IL-8, and TNF-α. They also inhibited myopia-related transforming growth factor-beta (TGF)-β1, matrix metalloproteinase (MMP)-2, and NF-κB expression while increasing type Ⅰ collagen expression. ConclusionOverall, these results suggest that FJE + PVE may have a therapeutic effect on myopia and be used as a potential treatment option.

Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway of pyrimidine nucleotides and considered an attractive target for potential antimalarial, anticancer, and antipathogen chemotherapy. Whether the FDA-approved clinical drug 5-fluorouracil (5-FU) that is used to target the enzyme thymidylate synthase for anticancer therapy can also bind to DHOase remains unknown. Here, we report the crystal structures of DHOase from Saccharomyces cerevisiae (ScDHOase) complexed with malate, 5-FU, and 5-aminouracil (5-AU). ScDHOase shares structural similarity with Escherichia coli DHOase. We also characterized the binding of 5-FU and 5-AU to ScDHOase by using the fluorescence quenching method. These complexed structures revealed that residues Arg18, Asn43, Thr106, and Ala275 of ScDHOase were involved in the 5-FU (PDB entry 6L0B) and 5-AU binding (PDB entry 6L0F). Overall, these results provide structural insights that may facilitate the development of new inhibitors targeting DHOase and constitute the 5-FU and 5-AU interactomes for further clinical chemotherapies.

The formation of foam cells, which are macrophages that have engulfed oxidized low-density lipoprotein (OxLDL), constitutes the first stage in the development of atherosclerosis. Previously, we found that knocking down galectin-12, a negative regulator of lipolysis, leads to reduced secretion of monocyte chemoattractant protein-1 (MCP-1), a chemokine that plays an important role in atherosclerosis. This prompted us to study the role of galectin-12 in atherosclerosis. With that aim, we examined foam cell formation in Gal12‒/‒ murine macrophages exposed to OxLDL and acetylated LDL (AcLDL). Then, we generated an LDL receptor and galectin-12 double knockout (DKO) mice and studied the effect of galectin-12 on macrophage function and atherosclerosis. Lastly, we evaluated the role of galectin-12 in human THP-1 macrophages using a doxycycline-inducible conditional knockdown system. Galectin-12 knockout significantly inhibited foam cell formation in murine macrophages through the downregulation of cluster of differentiation 36 (CD36), and the upregulation of ATP Binding Cassette Subfamily A Member 1 (ABCA1), ATP Binding Cassette Subfamily G Member 1 (ABCG1), and scavenger receptor class B type 1 (SRB1). Consistent with this, galectin-12 knockdown inhibited foam cell formation in human macrophages. In addition, the ablation of galectin-12 promoted M2 macrophage polarization in human and murine macrophages as evidenced by the upregulation of the M2 marker genes, CD206 and CD163, and downregulation of the M1 cytokines, tumor necrosis factor &alpha, (TNF- &alpha, ), interleukin-6 (IL-6), and MCP-1. Moreover, the ablation of galectin-12 decreased atherosclerosis formation in DKO mice. Based on these results, we propose galectin-12 as a potential therapeutic target for atherosclerosis.

Water extracts from beefsteak plant stems (BS), leaves (BL), and ripe seeds (BR) were examined for their antioxidant activities and phenolic compounds. Based on superoxide anion radicals scavenging activity, the BS extract (61.8%) is the most effective one followed by BL and BR extract (60.8% and 33.3%, respectively). The antioxidant activity determined by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method revealed that the BS extract had the highest activity on DPPH free radicals (54.8%). In addition, the extracts of BS, BL and BR were effective in reducing capacity and chelating effect on ferrous ions in comparison with the control. Total phenolic compound and flavonoid content of the BS extract was found to be superior to other beefsteak plant extracts. The results indicated that the BS extract of beefsteak plant has effective antioxidant functions, especially in superoxide anion radical and DPPH radical scavenging activities and reducing power. It was of our paramount interest to further identify the specific antioxidant components in BS which may be a new health-care food supplement or functional food for special use in the future.

Dihydropyrimidinase is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. This enzyme is important in pyrimidine metabolism, and blocking its activity would be detrimental to cell survival. This study investigated the dihydropyrimidinase inhibition by plumbagin isolated from the extract of carnivorous plant Nepenthes miranda (Nm). Plumbagin inhibited dihydropyrimidinase with IC50 value of 58 ± 3 μM. Double reciprocal results of Lineweaver–Burk plot indicated that this compound is a competitive inhibitor of dihydropyrimidinase. Fluorescence quenching analysis revealed that plumbagin could form a stable complex with dihydropyrimidinase with the Kd value of 37.7 ± 1.4 μM. Docking experiments revealed that the dynamic loop crucial for stabilization of the intermediate state in dihydropyrimidinase might be involved in the inhibition effect of plumbagin. Mutation at either Y155 or K156 within the dynamic loop of dihydropyrimidinase caused low plumbagin binding affinity. In addition to their dihydropyrimidinase inhibition, plumbagin and Nm extracts also exhibited cytotoxicity on melanoma cell survival, migration, and proliferation. Further research can directly focus on designing compounds that target the dynamic loop in dihydropyrimidinase during catalysis.

Keratinocytes constitute the major cell type of epidermis, which participates in re-epithelialization during wound repair and the immune defense response to pathogens. The aim of the current study was to explore the differentially expressed genes and novel microRNA (miRNA) regulations that are potentially involved in diabetic keratinocytes through next-generation sequencing (NGS) and bioinformatics approaches. A total of 420 differentially expressed genes between normal and diabetic keratinocytes were identified, and systematic bioinformatics analyses indicated that these differentially expressed genes were functionally enriched in interferon-alpha signaling, viral defense response, and immune response. Additionally, the potential miR-340-3p-DTX3L interaction that has been systematically validated in miRNA prediction databases was proposed to participate in the disrupted skin homeostasis, altering the defense and immune response of diabetic skin. The findings may provide new insights into understanding the pathogenesis of epidermal pathologies in diabetic patients and targeting novel molecules to advance diabetic skin care in clinical practice.

Metastatic lung cancer is a leading cause of mortality and has a mortality rate of ≥90%. Isolinderalactone (ILL) is a sesquiterpene lactone compound that has been used in traditional Chinese medicine. Research has demonstrated that ILL has anti-inflammatory and anti-proliferative properties; however, to the best of our knowledge, studies investigating whether ILL can inhibit lung cancer cell metastasis have not been conducted. In the present study, 1–10 µM ILL was applied in the culturing of the A549 lung cancer cell line to investigate the effects of ILL on the invasion and migration of lung cancer cells, including whether the possible mechanisms of ILL are associated with the expression of matrix metalloproteinase (MMP)-2 and NME/NM23 nucleoside diphosphate kinase 1 (NM23-H1) genes. The results of the present study indicated that ILL inhibited the invasion and migration of the A549 cancer cells and exhibited a dose-response association. ILL also significantly inhibited the protein expression and activity of MMP-2 (P

The antioxidant properties of perilla seed, leaf and stalk extracts were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radicals scavenging activities, reducing power and metal chelating ability. The DPPH radical scavenging activity of perilla leaf extract was 57.5%, a value which was higher than scavenging activity of other perilla extracts at 15.0 μg/ml. Based on superoxide radical scavenging effect, the perilla leaf extract was the most effective (64.2%) one followed by perilla seed extract (55.1%) and perilla stalk extract (20.1%) at 10.0 μg/ml. Reducing powers of perilla seed, leaf and stalk extracts were excellent. Chelating effects of perilla seed, leaf and stalk extracts were increased with the increasing concentration. The data shows that perilla leaf extract has effective antioxidant functions, especially in DPPH and superoxide radicals scavenging activities and reducing power. The results indicate that the 50% aqueous-methanol extracts of perilla leaf can be used as a new functional food and pharmaceutical agent. Key words: Perilla, scavenging activity, reducing power, chelating effect, phenolics compound.

The aim of the present study was to determine whether Lactobacillus salivarius (LS) and Lactobacillus johnsonii (LJ) prevent alcoholic liver damage in HepG2 cells and rat models of acute alcohol exposure. In this study, heat-killed LS and LJ were screened from 50 Lactobacillus strains induced by 100 mM alcohol in HepG2 cells. The severity of alcoholic liver injury was determined by measuring the levels of aspartate transaminase (AST), alanine transaminase (ALT), gamma-glutamyl transferase (γ-GT), lipid peroxidation, triglyceride (TG) and total cholesterol. Our results indicated that heat-killed LS and LJ reduced AST, ALT, γ-GT and malondialdehyde (MDA) levels and outperformed other bacterial strains in cell line studies. We further evaluated these findings by administering these strains to rats. Only LS was able to reduce serum AST levels, which it did by 26.2%. In addition LS significantly inhibited serum TG levels by 39.2%. However, both strains were unable to inhibit ALT levels. In summary, we demonstrated that heat-killed LS and LJ possess hepatoprotective properties induced by alcohol both in vitro and in vivo.

Scavenging activities on superoxide anion and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals of Antrodia cinnamomea fermentation filtrate from the submerged culture were determined. We prove that controlling the medium composition can dramatically enhance the antiradical activity. The C/N ratio was available utilized preferentially for the scavenging activity on superoxide anion radical, rather than for DPPH radical. A relatively high glucose concentration (8%) was favorable for superoxide anion radical-scavenging activity. Flushing the culture medium with yeast extract (0.8%) had a stimulating effect on superoxide anion radical-scavenging. In addition, peptone appeared to be significantly inhibitory for superoxide anion radical. Phosphate ion, magnesium ion and thiamine were probably not essential for DPPH radical-scavenging activity. By optimizing the effects of additional nutrition, the scavenging effects on superoxide anion and DPPH radicals increased to 61.43 ± 1.64% and 27.24 ± 0.95%, respectively. PRACTICAL APPLICATION Antrodia cinnamomea is a mushroom used as a traditional health care in Taiwan. Fruit bodies of A. cinnamomea are expensive and scarce, partially due to their rareness and difficulty of cultivation. Thus, both the mycelia and fermentation filtrate of A. cinnamomea from the submerged culture are mainly prepared for use in the formulation of nutraceuticals and functional foods. As previously studied, A. cinnamomea may constitute a good source of healthy compounds, namely polysaccharides, suggesting that it could be useful in the prevention of diseases in which free radicals are implicated. Presented is a study about how controlling the mushroom culturing composition of the medium can dramatically enhance the free radical-scavenging activity. A. cinnamomea fermentation filtrate from the submerged culture prepared may be a new health care food supplement or functional food for special use in the future.

The antioxidant activities of fermented filtrates and ethanolic extracts from Antrodia cinnamomea strains as TFRI B159, B179, B235 and B489 in submerged culture were determined. The antioxidant activities of A. cinnamomea strains, including the scavenging activity on superoxide anion and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, the reducing power and the metal chelating activity, were evaluated. Total polysaccharides were the major naturally occurring antioxidant components found in the fermented filtrates from A. cinnamomea strains. These results indicated that all kinds of A. cinnamomea fermented filtrates and ethanolic extracts have effective antioxidant function, especially in scavenging activity on superoxide anion radical and the reducing power. The antioxidant capacity of ethanolic extract from A. cinnamomea TFRI B235 was effective than that of other A. cinnamomea strains. The maximum scavenging effect on superoxide anion radical, DPPH radical scavenging activity, reducing power and the chelating effect on ferrous ions, found to be 67.66 ± 1.02%, 22.31 ± 1.66%, 0.80 ± 0.01 and 52.65 ± 0.77%, respectively. PRACTICAL APPLICATION Antrodia cinnamomea, one of the best known traditional Chinese medicines and health food, has been highly valued for the treatment of a wide range of diseases and reported to have antioxidant properties. The result suggested that all kinds of A. cinnamomea fermented filtrates and ethanolic extracts possessed good antioxidant properties, expect for scavenging ability on DPPH radicals. We aimed to investigate the antioxidant activities and the free radical scavenging effects of fermented filtrate and ethanolic extract from A. cinnamomea strains to determine whether filtrates and extracts could have the potential ability to prevent free-radical-related diseases. Therefore, in addition to their therapeutic effects, A. cinnamomea in human diets might serve as possible protective agents to help humans reduce oxidative damage. The fermented filtrates and ethanolic extracts of A. cinnamomea in submerged culture prepared could be developed as a new dietary supplement and functional food.

Effects of nutritional requirements for superoxide anion scavenging activity and reducing power by a newly screened Grifola frondosa TFRI1073 were studied in submerged cultures. The results clearly showed that the culture medium significantly affected superoxide anion scavenging activity and reducing power. In addition, both superoxide anion scavenging capability and reducing power were found to be greatly affected by varying the carbon source, the nitrogen source, the growth factor, and the mineral elements. The best superoxide anion scavenging activity could be obtained when the medium compositions (carbon source, nitrogen source, growth factor, and the mineral elements) were sucrose, ammonium chloride, nicotinic acid, K3PO4, and CuSO4; while a maximal reducing power could be achieved when the medium composition setting as: xylose, arginine, ascorbic acid, K2HPO4, and MgSO4. From a series experiments, the maximal superoxide anion scavenging activity and reducing power were 98.36±0.59% and 0.947±0.06 at 100 μg/mL, respectively. This study proved that modifying the composition of the medium can dramatically enhance the superoxide anion scavenging ability and reducing power by a newly screened G. frondosa TFRI1073.

The exopolysaccharide (EPS) extract of a newly screened Grifola frondosa TFRI1073 was evaluated antioxidant activity, including superoxide anion scavenging activity and reducing power, and the antiproliferative activities by using lung (A549 cells) and breast (MDA-MD-231 cells) cancer cell line. The exopolysaccharide of G. frondosa plays the important role on antioxidant and antiproliferative activities. Nutritional requirements for mycelial biomass and EPS production by G. frondosa were studied. Flushing the culture medium with carbon sources has a stimulating effect on both mycelial biomass and EPS production. In addition, nitrogen sources appeared to be an important and significant component for mycelial biomass. Vitamins were probably not essential for mycelial biomass and EPS production. Inorganic salts appeared to have a significant effect on both mycelial biomass and EPS productions. The effect of phosphate ion on mycelial biomass was better than on EPS production. The information generated in this study will facilitate physiological research of a newly screened G. frondosa TFRI1073, helping to develop the exopolysaccharide of a new dietary supplement and functional food from G. frondosa.

The effects of nutritional conditions on the scavenging capacity on superoxide anion radical and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical of ethanolic extracts from filtrates of Antrodia cinnamomea were investigated in shake-flask cultures. Culture medium significantly affected inhibition of superoxide anion generation and DPPH free radical scavenging activity. In contrast, both scavenging capabilities on superoxide anion radical and DPPH free radical were found to be greatly affected by varying the carbon source, the nitrogen source, the growth factors or the trace elements. The maximal inhibition of superoxide anion generation could be obtained when the culture medium compositions were: xylose, ammonium oxalate, nicotinic acid, NaH 2 PO 4 and CuSO 4 ; while a maximal DPPH free radical scavenging activity could be achieved when the medium composition setting as: fructose, peptone, nicotinic acid, KH 2 PO 4 and CaCl 2 (the scavenging effects on superoxide anion radical and DPPH free radical were increased to 96.3 ± 0.3% and 29.0 ± 1.0%, respectively). We proved that controlling the culturing conditions and modifying the composition of the medium can dramatically enhance the scavenging ability on superoxide anion radical and DPPH free radical of A. cinnamomea.

Submerged cultures were used to identify growth-limiting nutrients by Antrodia cinnamomea strains. The mycelial biomass and EPS production by A. cinnamomea BCRC 35396 were markedly higher than other A. cinnamomea strains. A relatively high C/N ratio was favorable for both the mycelial growth (5.41 g/l) and EPS production (0.55 g/l); the optimum ratio was 40. The glucose was available utilized preferentially for mycelial growth, rather than for EPS production. Flushing the culture medium with nitrogen had a stimulating effect on both mycelial growth and EPS production. In addition, peptone, yeast extract and malt extract appeared to be important and significant component for EPS production. Phosphate ion, magnesium ion and thiamine were probably not essential for mycelial growth. By optimizing the effects of additional nutrition, the results showed that 5% (w/v) glucose, 0.8% (w/v) peptone, 0.8% (w/v) yeast extract, 0.8% (w/v) malt extract, 0.03% (w/v) KH2PO4, 0.1% (w/v) MgSO4 .7H2O and 0.1% (w/v) thiamine could lead to the maximum production of EPS (1.36 g/l).

Areca catechu is a common traditional Chinese medicinal plant used to treat dyspepsia, constipation, beriberi and oedema. The antiradical capacities of different extraction method of A. catechu extracts were evaluated by scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide radicals. Reducing power, total phenolic and flavonoid contents were also detected. Based on DPPH radical scavenging activity, the 100% methanolic extract (AME of 38.77%) is the most effective followed by 50% water/methanol (AWM) and 100% water (AWE) extracts (22.92 and 21.47%, respectively). In addition, the extracts of AME (78.04%), AWM (75.77%) and AWE (68.42%) were effective in superoxide radical scavenging activity in comparison with the control (ascorbic acid and gallic acid). The reducing power, total phenolic and flavonoid contents of the AME extract was found to be superior to other extracts. The results indicated that different extraction method of A. catechu extracts were related to their antiradical activities and reducing power. The present study suggests that A. catechu extracts are useful nutritional antioxidants for the nutraceutical industry. Keywords : Areca catechu , antioxidant activity, radical scavenging, reducing power

Our previous study demonstrated that quercetin-metabolite-enriched plasma (QP) but not quercetin itself upregulates peroxisome proliferator-activated receptor gamma (PPAR-γ) expression to induce G2/M arrest in A549 cells. In the present study, we incubated A549 cells with QP as well as quercetin-3-glucuronide (Q3G) and quercetin-3'-sulfate (Q3'S), two major metabolites of quercetin, to investigate the effects of quercetin metabolites on cell invasion and migration, the possible mechanisms and the role of PPAR-γ. We also compared the effects of QP with those of quercetin and troglitazone (TGZ), a PPAR-γ ligand. The results showed that QP significantly suppressed cell invasion and migration, as well as matrix metalloproteinases (MMPs)-2 activity and expression in a dose-dependent manner. The effects of 10% QP on those parameters were similar to those of 10μM quercetin and 20μM TGZ. However, QP and TGZ rather than quercetin itself increased the expressions of nm23-H1 and tissue inhibitor of metalloproteinase (TIMP-2). Furthermore, we demonstrated that Q3G and Q3'S also inhibited the protein expression of MMP-2. GW9662, a PPAR-γ antagonist, significantly diminished such an effect of Q3G and Q3'S. Silencing PPAR-γ expression in A549 cells also significantly diminished the suppression effect of Q3G and Q3'S on MMP-2 expression. Taken together, our study demonstrated that QP inhibited cell invasion and migration through nm23-H1/TIMP-2/MMP-2 associated mechanisms. The upregulation of PPAR-γ by quercetin metabolites such as Q3G and Q3'S could play an important role in the effects of QP.

Development of an efficient treatment for triple-negative breast cancer is an urgent issues. Compounds from plant extracts are a potential source of novel cancer treatment. Isolinderalactone, a kind of sesquiterpenoids compound, was purified from the root of Lindera strychnifolia and Neolitsea daibuensis and shows anti-inflammatory and anticancer capacity. In the present study, isolinderalactone induced apoptosis in MDA-MB-231 cells which is a kind of triple-negative breast cancer cell line through induction of an intrinsic mitochondria-mediated and caspase-independent cell death. Treatment of isolinderalactone increased the protein level of the suppressor of cytokine signaling 3 (SCOS3), decreased phosphorylation of the signal transducer and activator of transcription 3 (STAT3), and suppressed expression of the down-stream genes of the X-linked inhibitor of apoptosis protein in MDA-MB-231 cells. Our results further showed that the level of SOCS3 expression was induced by isolinderalactone due to inhibiting the microRNA hsa-miR-30c-5p (miR-30c) expression. In addition, intraperitoneal injection of isolinderalactone induced apoptosis in a xenograft breast tumor while it did not significantly affect the histology of liver, kidney and lung of the treated mice. In conclusion, isolinderalactone induces apoptosis in MDA-MB‑231 cells and suppresses STAT3 signaling pathway through regulation of SOCS3 and miR-30c. It may become a novel treatment for triple-negative breast cancer in the future.

This paper describes a study of the extracellular lipase synthesised by a submerged culture of Antrodia cinnamomea in two different media (modified ME and modified YM). Lipase production increased substantially upon the addition of glucose; when it comprised 8% of the modified ME and YM media, the lipase production increased remarkably from 0 to 3.35 mU/ml and from 0.59 to 6.17 mU/ml, respectively. The lipase activity decreased, however, beyond certain concentrations of glucose. The lipase synthesis in the modified YM medium was superior to that which occurred in the modified ME medium. In addition, the optimum values of pH and temperature for the crude lipase were 11.0 and 60 °C, respectively. The enzyme was stable at values of pH between 7.0 and 12.0 and at temperatures up to 80 °C. Thus, this novel lipase from A. cinnamomea is particularly alkaline-resistant and thermostable.

Phenolsulfotransferase(PST)catalyzessulfurylgrouptransferfromadenosine3-phosphate5-phosphosulfate (PAPS) to a variety of nucleophilic acceptors in biological systems. Physiological sulfation by PAPS resultsintheproductionofadenosine3,5-diphosphate(PAP).PAPmaybecomeastronginhibitororcofactor for the physiological or the transfer reactions, respectively. Several nucleotides other than PAP also serve as substrates, inhibitors or cofactors of PST catalyzed reactions. We are interested in the effects of these nucleotides on the PST catalyzed reactions and their possible physiological significance in biology. Several nucleotide inhibitors (such as adenosine 5-diphosphate and adenosine 5-triphosphate) of sulfotransferase in physiological reactions have been reported in the literature. However, our data suggests that they are not inhibitors for the transfer reaction or the reverse physiological reaction catalyzed by PST. The aim of this work is to show that the molecular docking analysis can be successfully used to underline the inhibition mechanism of these nucleotides. First, the selected compounds were subjected to a detailed docking analysis, by means of GEMDOCK, a program able to reveal the most likely binding mode for each ligand. By comparing these results with binding sites and binding compounds of the nucleotides with known X-ray structures, it is possible to highlight the site specificity and the inhibition mechanism of the select compounds. The results obtained by the above algorithm were further confirmed experimentally. In this paper, we show the effect of a variety of nucleotides on the activity of sulfotransferase in different docking conditions.

Known spectroscopic and kinetic data are used to formulate pathways of the physiological and transfer reactions and the substrate inhibition of phenol sulfotransferase. Kinetic mechanisms indicate that release of PAP from enzyme complex is required for the physiological reaction but not for the transfer reaction. The pathways explain rate difference between the physiological and transfer reactions since the release of PAP is the rate-limiting step of the former reaction. Two enzyme species of phenol sulfotransferase which distinguish the physiological and transfer reaction were found to involve the binding of PAP. Differences between two forms of phenol sulfotransferase, alpha and beta, indicate that they assemble through different folding process. It is demonstrated that only alpha enzyme renatures in the presence of PAP and beta enzyme renatures only in the absence of PAP in vitro. In the over-expressed system, formation of alpha and beta phenol sulfotransferase is also dependent on the availability of PAP in Escherichia coli. It is concluded that folding of phenol sulfotransferase is assisted by PAP to form alpha enzyme. In the absence of PAP, beta form of phenol sulfotransferase is produced.

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Lung cancer is currently the leading cause of cancer-related mortality worldwide. In Taiwan, lung cancer is also the type of malignancy that is the major cause of cancer-mortality. Investigating the mechanism of apoptosis of lung cancer cells is important in the treatment of lung cancer. In the present study, isolinderalactone was demonstrated to exhibit anticancer effects in A549 human non-small cell lung cancer cells. The effect of isolinderalactone on apoptosis, cell cycle distribution p21 levels and the Fas receptor and soluble Fas ligand (sFasL) were assayed in order to determine the mechanism underlying the anticancer effect of isolinderalactone. It was demonstrated that isolinderalactone may induce p21 expression and then cause the cell cycle arrest of A549 cells. The data of the present study also revealed that the Fas/sFasL apoptotic system is significant in the mechanism of isolinderalactone‑induced apoptosis of A549 cells. These novel findings demonstrated that isolinderalactone may cause the cell cycle arrest of A549 cells by induction of p21, and induce apoptosis of A549 human non-small-cell lung carcinoma cells through the Fas/sFasL apoptotic system.

This study investigated the anticancer effects of subamolide A (Sub-A), isolated fromCinnamomum subavenium, on human nonsmall cell lung cancer cell lines A549 and NCI-H460. Treatment of cancer cells with Sub-A resulted in decreased cell viability of both lung cancer cell lines. Sub-A induced lung cancer cell death by triggering mitotic catastrophe with apoptosis. It triggered oxidant stress, indicated by increased cellular reactive oxygen species (ROS) production and decreased glutathione level. The elevated ROS triggered the activation of ataxia-telangiectasia mutation (ATM), which further enhanced the ATF3 upregulation and subsequently enhanced p53 function by phosphorylation at Serine 15 and Serine 392. The antioxidant, EUK8, significantly decreased mitotic catastrophe by inhibiting ATM activation, ATF3 expression, and p53 phosphorylation. The reduction of ATM and ATF3 expression by shRNA decreased Sub-A-mediated p53 phosphorylation and mitotic catastrophe. Sub-A also caused a dramatic 70% reduction in tumor size in an animal model. Taken together, cell death of lung cancer cells in response to Sub-A is dependent on ROS generation, which triggers mitotic catastrophe followed by apoptosis. Therefore, Sub-A may be a novel anticancer agent for the treatment of nonsmall cell lung cancer.

Cellular redox signaling is important in diverse physiological and pathological processes. The activity of rat phenol sulfotransferase (rSULT1A1), which is important for the metabolism of hormone and drug, is subjected to redox regulation. Two cysteines, Cys232 and Cys66, nanometer away from each other and from the enzyme active site were proposed to form disulfide bond to regulate the activity of rSULT1A1. A nano switch, composed of a flexible loop from amino acid residues 59–70, explained how this long distance interaction between two cysteines can be achieved. The enzyme properties were investigated through site-directed muatagnesis, circular dichroism, enzyme kinetics and homologous modeling of the rSULT1A1 structures. We proposed that the formation of disulfide bond between Cys232 and Cys66 induced conformational changes of sulfotransferase, then in turn affected its nucleotide binding and enzyme activity. This discovery was extended to understand the possible redox regulation of other sulfotransferases from different organisms. The redox switch can be created in other redox-insensitive sulfotransferases, such as human phenol sulfotransferase (hSULT1A1) and human alcohol sulfotransferase (hSULT2A1), to produce mutant enzymes with redox regulation capacity. This study strongly suggested that redox regulation of drug and hormone metabolism can be significantly varied even though the sequence and structure of SULT1A1 of human and rat have a high degree of homology.