Structural basis for the substrate specificity of an S-formylglutathione hydrolase derived from Variovorax sp. PAMC 28711.

S-Formylglutathione hydrolase was originally known to catalyze the hydrolysis of S-formylglutathione to formate and glutathione. However, this enzyme has a broader esterase activity toward substrates containing thioester and ester bonds. In a previous study, we identified a new S-formylglutathione hydrolase (VaSFGH) gene in the Antarctic bacterium Variovorax sp. PAMC 28711, and recombinant VaSFGH protein was purified and characterized. Previous enzyme activity assays showed that VaSFGH has high activity, especially toward short-chain p-nitrophenyl esters (C2-C4). In this study, we determined the crystal structure of substrate-free VaSFGH at a resolution of 2.38 Å. In addition, p-nitrophenyl ester-bound VaSFGH structure models were generated by molecular docking simulations to obtain structural evidence of its substrate specificity. Comparative structural analysis of the apo-form and p-nitrophenyl ester-bound VaSFGH model structures revealed that large substrates could not bind inside the hydrophobic substrate-binding pocket because of the intrinsically static and relatively small substrate-binding pocket size of VaSFGH. This study provides useful information for further protein engineering of SFGHs for industrial use.

Comparison of Fatty Acid Contents and MMP-1 Inhibitory Effects of the Two Antarctic Fish, Notothenia rossii and Champsocephalus gunnari.

Total fatty-acid (FA) contents of different organs (stomach, liver, brain, and skin) of two Antarctic fish, marbled rockcod (Notothenia rossii) and mackerel icefish (Champsocephalus gunnari), were examined using gas chromatography-mass spectrometry (GC-MS). N. rossii possessed higher contents of total omega-3, where eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the most represented omega-3 FAs, were distributed throughout all parts of the fish. The highest level of EPA was observed in the skin and that of DHA was observed in the brain of N. rossii. C. gunnari showed organ peculiarity in that most of the omega-3 FAs were found in stomach and skin. Specifically, the highest levels of EPA and DHA were both observed in the stomach. Although N. rossii and C. gunnari both inhabit the Antarctic Southern Oceans, their characteristics in terms of the composition of fatty acids were shown to vary. The extracts were also evaluated for matrix metalloproteinase-1 (MMP-1)-inhibitory activities in UVB-induced human dermal fibroblasts, where extracts of the skin and liver of N. rossii showed the most significant inhibition upon MMP-1 production. These findings provide experimental evidence that the extracts of the Antarctic fish could be utilized as bioactive nutrients, particularly in the enhancement of skin health.

Bioactive Terphenyls Isolated from the Antarctic Lichen Stereocaulon alpinum.

Three p-terphenyls (2-4)-2-hydroxy-3,5-dimethoxy-p-terphenyl (2), 2-hydroxy-3,6-dimethoxy-p-terphenyl (3), and 2,3,5,6-tetramethoxy-p-terphenyl (4)-were isolated for the first time as natural products along with seven known compounds (1, 5-10) from the Antarctic lichen Stereocaulon alpinum. Structures of the new compounds were elucidated by comprehensive analyses of 1D and 2D NMR and HREIMS experiments. Compound 3 exhibited cytotoxicity against HCT116 cells with the IC50 value of 3.76 ± 0.03 µM and also inhibited NO production in LPS-induced RAW264.7 macrophages with the IC50 value of 22.82 ± 0.015 µM.

Compositional variation of atranorin-related components of lichen Myelochroa leucotyliza dependent on extraction solvent and their quantitative analysis by qHNMR.

INTRODUCTION: Quantitative nuclear magnetic resonance (qNMR) is one of the effective and reliable quantification tools for natural product research. Myelochroa leucotyliza belongs to the genus Myelochroa, a common foliose lichen genus found in the Korean Peninsula, and has not been quantitatively analysed using NMR. Previous chemical studies on M. leucotyliza have been limited to the main components by traditional thin-layer chromatography (TLC) experiments. OBJECTIVE: We explored the stability of atranorin, a major component of M. leucotyliza, in methanol and acetone using NMR and characterised the changes in the chemical profiles of the lichen extracts in methanol and acetone using qNMR. METHODOLOGY: Atranorin transformation in the presence of methanol was analysed using time-dependent proton (1 H)-NMR analysis (600 MHz NMR spectrometer). A 1 H qNMR (qHNMR) method was established using dimethyl sulfone as the internal standard for quantifying the selected components isolated from M. leucotyliza. Homogenous mixtures of the samples were dissolved in deuterated chloroform. RESULTS: Time-dependent 1 H-NMR experiments revealed that atranorin (5) from lichen M. leucotyliza decomposed into atraric acid (1) and methyl haemmatommate (2) in methanol. Four components were identified from M. leucotyliza: 1, 2, usnic acid (4), and 5, and their respective contents were determined using qHNMR. The percentages (w/w) of 1, 2, and 4 in the methanol extract were calculated as 5.66%, 0.69%, and 0.90%, while those of 1, 4, and 5 in the acetone extract were 1.70%, 1.68%, and 19.11%, respectively. CONCLUSION: We used qHNMR to effectively analyse quantitative compositional variations in two different M. leucotyliza extracts and reliably determined the chemical conversion of the unstable compound atranorin.

Anticancer Activity of 2-O-caffeoyl Alphitolic Acid Extracted from the Lichen, Usnea barbata 2017-KL-10.

Colorectal cancer is one of the life-threatening ailments causing high mortality and morbidity worldwide. Despite the innovation in medical genetics, the prognosis for metastatic colorectal cancer in patients remains unsatisfactory. Recently, lichens have attracted the attention of researchers in the search for targets to fight against cancer. Lichens are considered mines of thousands of metabolites. Researchers have reported that lichen-derived metabolites demonstrated biological effects, such as anticancer, antiviral, anti-inflammatory, antibacterial, analgesic, antipyretic, antiproliferative, and cytotoxic, on various cell lines. However, the exploration of the biological activities of lichens' metabolites is limited. Thus, the main objective of our study was to evaluate the anticancer effect of secondary metabolites isolated from lichen (Usnea barbata 2017-KL-10) on the human colorectal cancer cell line HCT116. In this study, 2OCAA exhibited concentration-dependent anticancer activities by suppressing antiapoptotic genes, such as MCL-1, and inducing apoptotic genes, such as BAX, TP53, and CDKN1A(p21). Moreover, 2OCAA inhibited the migration and invasion of colorectal cancer cells in a concentration-dependent manner. Taken together, these data suggest that 2OCAA is a better therapeutic candidate for colorectal cancer.

Crystal structure of a transcription factor, GerE (PaGerE), from spore-forming bacterium Paenisporosarcina sp. TG-14.

In cold and harsh environments such as glaciers and sediments in ice cores, microbes can survive by forming spores. Spores are composed of a thick coat protein, which protects against external factors such as heat-shock, high salinity, and nutrient deficiency. GerE is a key transcription factor involved in spore coat protein expression in the mother cell during sporulation. GerE regulates transcription during the late sporulation stage by directly binding to the promoter of cotB gene. Here, we report the crystal structure of PaGerE at 2.09 Šresolution from Paenisporosarcina sp. TG-14, which was isolated from the Taylor glacier. The PaGerE structure is composed of four α-helices and adopts a helix-turn-helix architecture with 68 amino acid residues. Based on our DNA binding analysis, the PaGerE binds to the promoter region of CotB to affect protein expression. Additionally, our structural comparison studies suggest that DNA binding by PaGerE causes a conformational change in the α4-helix region, which may strongly induce dimerization of PaGerE.

The Comprehensive Roles of ATRANORIN, A Secondary Metabolite from the Antarctic Lichen Stereocaulon caespitosum, in HCC Tumorigenesis.

Hepatocellular carcinoma (HCC) is one of the most deadly genetic diseases, but surprisingly chemotherapeutic approaches against HCC are only limited to a few targets. In particular, considering the difficulty of a chemotherapeutic drug development in terms of cost and time enforces searching for surrogates to minimize effort and maximize efficiency in anti-cancer therapy. In spite of the report that approximately one thousand lichen-derived metabolites have been isolated, the knowledge about their functions and consequences in cancer development is relatively limited. Moreover, one of the major second metabolites from lichens, Atranorin has never been studied in HCC. Regarding this, we comprehensively analyze the effect of Atranorin by employing representative HCC cell lines and experimental approaches. Cell proliferation and cell cycle analysis using the compound consistently show the inhibitory effects of Atranorin. Moreover, cell death determination using Annexin-V and (Propidium Iodide) PI staining suggests that it induces cell death through necrosis. Lastly, the metastatic potential of HCC cell lines is significantly inhibited by the drug. Taken these together, we claim a novel functional finding that Atranorin comprehensively suppresses HCC tumorigenesis and metastatic potential, which could provide an important basis for anti-cancer therapeutics.

Total Syntheses of Lobaric Acid and Its Derivatives from the Antarctic Lichen Stereocaulon alpinum.

The first total syntheses of the natural products lobaric acid (1) and its derivatives isolated from the Antarctic lichen Stereocaulon alpinum are reported in this study. Lobarin (3), with a pseudodepsidone structure, was synthesized first in 11 steps by utilizing an Ullmann aryl ether coupling reaction, and lobaric acid was synthesized in an additional three steps by a seven-membered lactonization reaction. Various derivatives were also obtained from the prepared lobaric acid, and the synthetic compounds exhibited significant PTP1B inhibitory activities.

Cholinesterase inhibitory alkaloids from the rhizomes of Coptis chinensis.

Coptis chinensis has been used as a medicinal herb in traditional oriental medicine. In this study, chemical investigation of a water extract of C. chinensis identified two new quaternary protoberberines (1, 2), a new tricyclic amide (3), together with five known compounds. Their chemical structures were elucidated by analysis with 1D and 2D NMR and high-resolution mass spectroscopy, as well as by comparison with those reported in the literature. Compounds 4, 5, and 7 showed potent inhibition against acetylcholinesterase (AChE) with IC50 values of 1.1, 5.6, and 12.9 µM, respectively. Compounds 2 and 4 showed inhibition of butyrylcholinesterase (BChE) with IC50 values of 11.5 and 27.8 µM, respectively. The kinetic activities were investigated to find out the type of enzyme inhibition involved. The types of AChE inhibition shown by compounds 5 and 7 were noncompetitive; BChE inhibition by compound 2 was also noncompetitive.

Passive warming effect on soil microbial community and humic substance degradation in maritime Antarctic region.

Although the maritime Antarctic has undergone rapid warming, the effects on indigenous soil-inhabiting microorganisms are not well known. Passive warming experiments using open-top chamber (OTC) have been performed on the Fildes Peninsula in the maritime Antarctic since 2008. When the soil temperature was measured at a depth of 2-5 cm during the 2013-2015 summer seasons, the mean temperature inside OTC (OTC-In) increased by approximately 0.8 °C compared with outside OTC (OTC-Out), while soil chemical and physical characteristics did not change. Soils (2015 summer) from OTC-In and OTC-Out were subjected to analysis for change in microbial community and degradation rate of humic substances (HS, the largest pool of recalcitrant organic carbon in soil). Archaeal and bacterial communities in OTC-In were minimally affected by warming compared with those in OTC-Out, with archaeal methanogenic Thermoplasmata slightly increased in abundance. The abundance of heterotrophic fungi Ascomycota was significantly altered in OTC-In. Total bacterial and fungal biomass in OTC-In increased by 20% compared to OTC-Out, indicating that this may be due to increased microbial degradation activity for soil organic matter (SOM) including HS, which would result in the release of more low-molecular-weight growth substrates from SOM. Despite the effects of warming on the microbial community over the 8-years-experiments warming did not induce any detectable change in content or structure of polymeric HS. These results suggest that increased temperature may have significant and direct effects on soil microbial communities inhabiting maritime Antarctic and that soil microbes would subsequently provide more available carbon sources for other indigenous microbes.

Anti-Cancer Activity of Lobaric Acid and Lobarstin Extracted from the Antarctic Lichen Stereocaulon alpnum.

Lobaric acid and lobarstin, secondary metabolites derived from the antarctic lichen Stereocaulon alpnum, exert various biological activities, including antitumor, anti-proliferation, anti-inflammation, and antioxidant activities. However, the underlying mechanisms of these effects have not yet been elucidated in human cervix adenocarcinoma and human colon carcinoma. In the present study, we evaluated the anticancer effects of lobaric acid and lobarstin on human cervix adenocarcinoma HeLa cells and colon carcinoma HCT116 cells. We show that the proliferation of Hela and HCT116 cells treated with lobaric acid and lobarstin significantly decreased in a dose- and time-dependent manner. Using flow cytometry analysis, we observed that the treatment with these compounds resulted in significant apoptosis in both cell lines, following cell cycle perturbation and arrest in G2/M phase. Furthermore, using immunoblot analysis, we investigated the expression of cell cycle and apoptosis-related marker genes and found a significant downregulation of the apoptosis regulator B-cell lymphoma 2 (Bcl-2) and upregulation of the cleaved form of the poly (ADP-ribose) polymerase (PARP), a DNA repair and apoptosis regulator. These results suggest that lobaric acid and lobarstin could significantly inhibit cell proliferation through cell cycle arrest and induction of apoptosis via the mitochondrial apoptotic pathway in cervix adenocarcinoma and colon carcinoma cells. Taken together, our data suggests that lobaric acid and lobarstin might be novel agents for clinical treatment of cervix adenocarcinoma and colon carcinoma.

Bioactive polyprenylated benzophenone derivatives from the fruits extracts of Garcinia xanthochymus.

Two new polycyclic prenylated xanthones (1 and 2) and a new phenylpropanoid glycoside (3), along with seven known compounds (4-10) were isolated from the fruits of Garcinia xanthochymus. The structures were elucidated by 1D- and 2D-NMR, and HRMS experiments. The isolates were evaluated for their inhibitory effects against the viability of U251MG glioblastoma and MDA-MB-231 breast cancer cells that harbor an aberrantly active signal transducer and exhibit activation of transcription 3 (STAT3), and compared to normal NIH3T3 mouse fibroblasts. Among the isolates, compounds 1, 2, 5, and 6-9 inhibited the viability of glioma cancer cells with IC50 values in the range of 1.6-6.5µM. Furthermore, treatment of U251MG with 6 and 7 inhibited intracellular STAT3 tyrosine phosphorylation and glioma cell migration in vitro, respectively.

Bioactivities of ethanol extract from the Antarctic freshwater microalga, Chloromonas sp.

Cancer is the principal cause of human death and occurs through highly complex processes that involve the multiple coordinated mechanisms of tumorigenesis. A number of studies have indicated that the microalgae extracts showed anticancer activity in a variety of human cancer cells and can provide a new insight in the development of novel anti-cancer therapy. Here, in order to investigate molecular mechanisms of anticancer activity in the Antarctic freshwater microalga, Chloromonas sp., we prepared ethanol extract of Chloromonas sp. (ETCH) and performed several in vitro assays using human normal keratinocyte (HaCaT) and different types of cancer cells including cervical, melanoma, and breast cancer cells (HeLa, A375 and Hs578T, respectively). We revealed that ETCH had the antioxidant capacity, and caused significant cell growth inhibition and apoptosis of cancer cells in a dose-dependent manner, whereas it showed no anti-proliferation to normal cells. In addition, ETCH had a significant inhibitory effect on cell invasion without the cytotoxic effect. Furthermore, ETCH-induced apoptosis was mediated by increase in pro-apoptotic proteins including cleaved caspase-3 and p53, and by decrease in anti-apoptotic protein, Bcl-2 in ETCH-treated cancer cells. Taken together, this work firstly explored the antioxidant and anticancer activities of an Antarctic freshwater microalga, and ETCH could be a potential therapeutic candidate in the treatment of human cancer.

Comprehensive Expression Profiling and Functional Network Analysis of Porphyra-334, One Mycosporine-Like Amino Acid (MAA), in Human Keratinocyte Exposed with UV-radiation.

Mycosporine-like amino acids (MAAs) have been highlighted as pharmacologically active secondary compounds to protect cells from harmful UV-radiation by absorbing its energy. Previous studies have mostly focused on characterizing their physiological properties such as antioxidant activity and osmotic regulation. However, molecular mechanisms underlying their UV-protective capability have not yet been revealed. In the present study, we investigated the expression profiling of porphyra-334-modulated genes or microRNA (miRNAs) in response to UV-exposure and their functional networks, using cDNA and miRNAs microarray. Based on our data, we showed that porphyra-334-regulated genes play essential roles in UV-affected biological processes such as Wnt (Wingless/integrase-1) and Notch pathways which exhibit antagonistic relationship in various biological processes; the UV-repressed genes were in the Wnt signaling pathway, while the activated genes were in the Notch signaling. In addition, porphyra-334-regulated miRNAs can target many genes related with UV-mediated biological processes such as apoptosis, cell proliferation and translational elongation. Notably, we observed that functional roles of the target genes for up-regulated miRNAs are inversely correlated with those for down-regulated miRNAs; the former genes promote apoptosis and translational elongation, whereas the latter function as inhibitors in these processes. Taken together, these data suggest that porphyra-334 protects cells from harmful UV radiation through the comprehensive modulation of expression patterns of genes involved in UV-mediated biological processes, and that provide a new insight to understand its functional molecular networks.

Anticancer activities of ethanol extract from the Antarctic freshwater microalga, Botryidiopsidaceae sp.

BACKGROUND: Cancer is a leading cause of human death around the world and occurs through the highly complex coordination of multiple cellular pathways. Recent studies have revealed that microalgal extracts exhibit considerable pharmaceutical activities, including those against various cancer cells. Thus, microalgae are promising candidates as novel cancer therapeutic drugs. In this study, we evaluated the biological functions of the ethanolic extract of the Antarctic freshwater microalga, Bo tryidiopsidaceae sp., such as its antioxidant, anti-proliferative, apoptotic and anti-invasive properties. METHODS: To estimate antioxidant capacity of ethanol extract of Bo tryidiopsidaceae sp. (ETBO), free radical 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays were used. The anti-proliferative activity of ETBO was assessed in several cancer cell lines (A375, Hs578T and HeLa) and non-tumorigenic keratinocyte cells (HaCaT), using MTT assay. In addition, Annexin V binding was performed to detect ETBO-induced apoptotic cells, and the expression levels of apoptosis-regulating proteins, caspase-3, p53, and Bcl-2, were determined by western blot. Boyden chamber assays were used to determine anti-migratory and anti-invasive properties of ETBO. RESULTS: ETBO exhibited antioxidant activity and concentration-dependent anticancer activities, such as anti-proliferation and pro-apoptotic activities against cancer cells. Furthermore, the expression of the apoptosis-inducing proteins, p53 and caspase-3, significantly increased in response to ETBO, whereas the expression of the anti-apoptotic protein, Bcl-2, decreased. These data imply that ETBO induces apoptosis by caspase activation through the modulation of pro-apoptotic and anti-apoptotic gene, p53 and Bcl-2, respectively. In addition, ETBO significantly inhibited migration and invasion of cervical cancer cells in a concentration-dependent manner. CONCLUSION: In this study, ETBO exhibited considerable anticancer activities, such as inhibition of proliferation, invasion, and migration, as well as induction of apoptosis. These data suggest that ETBO is a promising therapeutic agent in cancer therapy and drug discovery.

Anticancer Activity of Ramalin, a Secondary Metabolite from the Antarctic Lichen Ramalina terebrata, against Colorectal Cancer Cells.

Colorectal cancer is a leading cause of death worldwide and occurs through the highly complex coordination of multiple cellular pathways, resulting in carcinogenesis. Recent studies have increasingly revealed that constituents of lichen extracts exhibit potent pharmaceutical activities, including anticancer activity against various cancer cells, making them promising candidates for new anticancer therapeutic drugs. The main objective of this study was to evaluate the anticancer capacities of ramalin, a secondary metabolite from the Antarctic lichen Ramalina terebrata, in the human colorectal cancer cell line HCT116. In this study, ramalin displayed concentration-dependent anticancer activity against HCT116 cells, significantly suppressing proliferation and inducing apoptosis. Furthermore, ramalin induced cell cycle arrest in the gap 2/mitosis (G2/M) phase through the modulation of hallmark genes involved in the G2/M phase transition, such as tumour protein p53 (TP53), cyclin-dependent kinase inhibitor 1A (CDKN1A), cyclin-dependent kinase 1 (CDK1) and cyclin B1 (CCNB1). At both the transcriptional and translational level, ramalin caused a gradual increase in the expression of TP53 and its downstream gene CDKN1A, while decreasing the expression of CDK1 and CCNB1 in a concentration-dependent manner. In addition, ramalin significantly inhibited the migration and invasion of colorectal cancer cells in a concentration-dependent manner. Taken together, these data suggest that ramalin may be a therapeutic candidate for the targeted therapy of colorectal cancer.

Withanolides derived from Physalis peruviana (Poha) with potential anti-inflammatory activity.

Three new withanolides, physaperuvin G (1), with physaperuvins I (2), and J (3), along with seven known derivatives (4-10), were isolated from the aerial parts of Physalis peruviana. The structures of 1-3 were determined by NMR, X-ray diffraction, and mass spectrometry. Compounds 1-10 were evaluated in lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cells. Compounds 4, 5, and 10 with potent nitric oxide inhibitory activity in LPS-activated RAW 264.7 cells, with IC50 values in the range of 0.32-7.8µM. In addition, all compounds were evaluated for potential to inhibit tumor necrosis factor-alpha (TNF-α)-activated nuclear factor-kappa B (NF-κB) activity with transfected human embryonic kidney cells 293. Compounds 4-7 inhibited TNF-α-induced NF-κB activity with IC50 values in the range of 0.04-5.6µM.

Anti-inflammatory and Quinone Reductase Inducing Compounds from Fermented Noni (Morinda citrifolia) Juice Exudates.

A new fatty acid ester disaccharide, 2-O-(ß-d-glucopyranosyl)-1-O-(2E,4Z,7Z)-deca-2,4,7-trienoyl-ß-d-glucopyranose (1), a new ascorbic acid derivative, 2-caffeoyl-3-ketohexulofuranosonic acid γ-lactone (2), and a new iridoid glycoside, 10-dimethoxyfermiloside (3), were isolated along with 13 known compounds (4-16) from fermented noni fruit juice (Morinda citrifolia). The structures of the new compounds, together with 4 and 5, were determined by 1D and 2D NMR experiments, as well as comparison with published values. Compounds 2 and 7 showed moderate inhibitory activities in a TNF-α-induced NF-κB assay, and compounds 4 and 6 exhibited considerable quinone reductase-1 (QR1) inducing effects.

Determination of the absolute configuration of chaetoviridins and other bioactive azaphilones from the endophytic fungus Chaetomium globosum.

Chemical investigation of an endophytic fungus Chaetomium globosum isolated from leaves of Wikstroemia uva-ursi led to the isolation of two new azaphilones, chaetoviridins J and K (1 and 3), along with five known derivatives (2 and 4-7). The structures of azaphilones were determined by NMR, X-ray diffraction, Mosher's method, and CD analysis. The isolated compounds were evaluated for their cancer chemopreventive-potential based on their abilities to inhibit tumor necrosis factor alpha (TNF-α)-induced nuclear factor-kappa B (NF-κB). Compounds 4, 5, 7, and synthetic 8 and 9 inhibit nitric oxide (NO) production with IC50 values in the range of 0.3-5.8 µM. Compounds 4, 5, and 9 also displayed (TNF-α)-induced NF-κB activity with IC50 values in the range of 0.9-5.1 µM.

Herbicidin congeners, undecose nucleosides from an organic extract of Streptomyces sp. L-9-10.

Four new undecose nucleosides (herbicidin congeners), three known herbicidins, and 9-(ß-d-arabinofuranosyl)hypoxanthine (Ara-H) were isolated from the organic extract of a fermentation culture of Streptomyces sp. L-9-10 using proton NMR-guided fractionation. Their structures were elucidated on the basis of comprehensive 1D and 2D NMR and mass spectrometry analyses. These structures included 2'-O-demethylherbicidin F (1), 9'-deoxy-8',8'-dihydroxyherbicidin B (2), 9'-deoxy-8'-oxoherbicidin B (2a), and the 8'-epimer of herbicidin B (3). This is the first detailed assignment of proton and carbon chemical shifts for herbicidins A, B, and F. The isolated compounds were evaluated for cancer chemopreventive potential based on inhibition of tumor necrosis factor alpha (TNF-α)-induced nuclear factor-kappa B (NF-κB) activity.