C-Type Lectin Receptor DCAR Recognizes Mycobacterial Phosphatidyl-Inositol Mannosides to Promote a Th1 Response during Infection.

Phosphatidyl-inositol mannosides (PIM) are glycolipids unique to mycobacteria and other related bacteria that stimulate host immune responses and are implicated in mycobacteria pathogenicity. Here, we found that the FcRγ-coupled C-type lectin receptor DCAR (dendritic cell immunoactivating receptor; gene symbol Clec4b1) is a direct receptor for PIM. Mycobacteria activated reporter cells expressing DCAR, and delipidation of mycobacteria abolished this activity. Acylated PIMs purified from mycobacteria were identified as ligands for DCAR. DCAR was predominantly expressed in small peritoneal macrophages and monocyte-derived inflammatory cells in lungs and spleen. These cells produced monocyte chemoattractant protein-1 (MCP-1) upon PIM treatment, and absence of DCAR or FcRγ abrogated MCP-1 production. Upon mycobacterial infection, Clec4b1-deficient mice showed reduced numbers of monocyte-derived inflammatory cells at the infection site, impaired IFNγ production by T cells, and an increased bacterial load. Thus, DCAR is a critical receptor for PIM that functions to promote T cell responses against mycobacteria.

Oleanolic Acid Glycosides from Scabiosa caucasica and Scabiosa ochroleuca: Structural Analysis and Cytotoxicity.

In the field of research on medicinal plants from the Armenian flora, the phytochemical study of two Scabiosa L. species, S. caucasica M. Bieb. and S. ochroleuca L. (Caprifoliaceae), has led to the isolation of five previously undescribed oleanolic acid glycosides from an aqueous-ethanolic extract of the roots: 3-O-α-L-rhamnopyranosyl-(1→3)-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl ester, 3-O-ß-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl ester, 3-O-ß-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid, 3-O-ß-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-ß-D-xylopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl ester, 3-O-α-L-rhamnopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl ester. Their full structural elucidation required extensive 1D and 2D NMR experiments, as well as mass spectrometry analysis. For the biological activity of the bidesmosidic saponins and the monodesmosidic saponin, their cytotoxicity on a mouse colon cancer cell line (MC-38) was evaluated.

Harringtonine Ester Derivatives with Enhanced Antiproliferative Activities against HL-60 and HeLa Cells.

Harringtonine (HT), produced from Cephalotaxus species, is known to exhibit potent antiproliferative activity against myeloid leukemia cells by inhibiting protein synthesis. A previous study using acute promyelocytic leukemia (HL-60) cells raised the possibility that the C-5' methyl group of HT plays an important role in regulating leukemia cell line antiproliferative activity. In order to investigate the effect of hydrocarbon chains at C-5' on the resultant activity, the C-5' methyl group was replaced with various straight- and branched-chain hydrocarbons using the corresponding alcohols, and their antiproliferative activity against HL-60 and HeLa cells was investigated. As a result, 4'-n-heptyl-4'-demethylharringtonine (1f, n-heptyl derivative) showed the most potent cytotoxicity among the HT ester derivatives produced, with IC50 values of 9.4 nM and 0.4 µM for HL-60 and HeLa cells, respectively. Interestingly, the cytotoxicity of derivative 1f against HL-60 and HeLa cells respectively was ∼5 (IC50 = 50.5 nM) and ∼10 times (IC50 = 4.0 µM) those of HT and ∼2 (IC50 = 21.8 nM) and ∼4 times (IC50 = 1.7 µM) more than homoharringtonine (HHT). These results demonstrate the potential of the derivative 1f as a lead compound against leukemia.

Activation of a Sweet Taste Receptor by Oleanane-Type Glycosides from Wisteria sinensis.

The phytochemical study of Wisteria sinensis (Sims) DC. (Fabaceae), commonly known as the Chinese Wisteria, led to the isolation of seven oleanane-type glycosides from an aqueous-ethanolic extract of the roots. Among the seven isolated saponins, two have never been reported before: 3-O-α-L-rhamnopyranosyl-(1→2)-ß-D-glucopyranosyl-(1→2)-ß-D-glucuronopyranosyl-22-O-acetylolean-12-ene-3ß,16ß,22ß,30-tetrol, and 3-O-ß-D-xylopyranosyl-(1→2)-ß-D-glucuronopyranosylwistariasapogenol A. Based on the close structures between the saponins from W. sinensis, and the glycyrrhizin from licorice, the stimulation of the sweet taste receptor TAS1R2/TAS1R3 by these glycosides was evaluated.

C4-monomethylsterol ß-glucoside and its synthase in Aurantiochytrium limacinum mh0186.

Thraustochytrids, unicellular marine protists, synthesize polyunsaturated fatty acids (PUFAs) and PUFA-containing phospholipids; however, little is known about their glycolipids and their associated metabolism. Here, we report two glycolipids (GL-A, B) and their synthases in Aurantiochytrium limacinum mh0186. Two glycolipids were purified from A. limacinum mh0186, and they were determined by gas chromatography, mass spectrometry and 2D nuclear magnetic resonance to be 3-O-ß-D-glucopyranosyl-stigmasta-5,7,22-triene (GL-A) and 3-O-ß-D-glucopyranosyl-4α-methyl-stigmasta-7,22-diene (GL-B), both of which are sterol ß-glucosides (ß-SGs); the structure of GL-B has not been reported thus far. Seven candidate genes responsible for the synthesis of these ß-SGs were extracted from the draft genome database of A. limacinum using the yeast sterol ß-glucosyltransferase (SGT; EC 2.4.1.173) sequence as a query. Expression analysis using Saccharomyces cerevisiae revealed that two gene products (AlSGT-1 and 2) catalyze the transfer of glucose from uridine diphosphate (UDP)-glucose to sterols, generating sterylglucosides (SGs). Compared to AlSGT-1, AlSGT-2 exhibited wide specificity for sterols and used C4-monomethylsterol to synthesize GL-B. The disruption of alsgt-2 but not alsgt-1 in strain mh0186 resulted in a decrease in the total SG and an almost complete loss of GL-B, indicating that AlSGT-2 is responsible for the synthesis of ß-SGs in A. limacinum mh0186, especially GL-B, which possesses a unique sterol structure.

Effects of Ingesting Fucoidan Derived from Cladosiphon okamuranus Tokida on Human NK Cells: A Randomized, Double-Blind, Parallel-Group, Placebo-Controlled Pilot Study.

The aim of this study was to evaluate the effects of ingesting fucoidan derived from Okinawa mozuku (Cladosiphon okamuranus) on natural killer (NK) cell activity and to assess its safety in healthy adults via a randomized, double-blind, parallel-group, placebo-controlled pilot study. Subjects were randomly divided into two groups-a placebo group (ingesting citric acid, sucralose, and caramel beverages; n = 20; 45.5 ± 7.8 years (mean ± standard deviation)) and a fucoidan group (3.0 g/day from beverages; n = 20; 47.0 ± 7.6 years); after 12 weeks, blood, biochemical, and immunological tests were performed. Clinically adverse events were not observed in any of the tests during the study period. In addition, adverse events due to the test food were not observed. In the immunological tests, NK cell activity was significantly enhanced at 8 weeks in the fucoidan group, compared to before ingestion (0 weeks). In addition, a significantly enhanced NK cell activity was observed in male subjects at 8 weeks, compared with the placebo group. These results confirm that Okinawa mozuku-derived fucoidan enhances NK cell activity and suggest that it is a safe food material.

Lipoteichoic acid anchor triggers Mincle to drive protective immunity against invasive group A Streptococcus infection.

Group A Streptococcus (GAS) is a Gram-positive bacterial pathogen that causes a range of diseases, including fatal invasive infections. However, the mechanisms by which the innate immune system recognizes GAS are not well understood. We herein report that the C-type lectin receptor macrophage inducible C-type lectin (Mincle) recognizes GAS and initiates antibacterial immunity. Gene expression analysis of myeloid cells upon GAS stimulation revealed the contribution of the caspase recruitment domain-containing protein 9 (CARD9) pathway to the antibacterial responses. Among receptors signaling through CARD9, Mincle induced the production of inflammatory cytokines, inducible nitric oxide synthase, and reactive oxygen species upon recognition of the anchor of lipoteichoic acid, monoglucosyldiacylglycerol (MGDG), produced by GAS. Upon GAS infection, Mincle-deficient mice exhibited impaired production of proinflammatory cytokines, severe bacteremia, and rapid lethality. GAS also possesses another Mincle ligand, diglucosyldiacylglycerol; however, this glycolipid interfered with MGDG-induced activation. These results indicate that Mincle plays a central role in protective immunity against acute GAS infection.

Triterpenoid Saponins from the Cultivar "Green Elf" of Pittosporum tenuifolium.

Four oleanane-type glycosides were isolated from a horticultural cultivar "Green Elf" of the endemic Pittosporum tenuifolium (Pittosporaceae) from New Zealand: three acylated barringtogenol C glycosides from the leaves, with two previously undescribed 3-O-ß-d-glucopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-ß-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C, 3-O-ß-d-galactopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-ß-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C, and the known 3-O-ß-d-glucopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-ß-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C (Eryngioside L). From the roots, the known 3-O-ß-d-glucopyranosyl-(1→2)-ß-d-galactopyranosyl-(1→2)-ß-d-glucuronopyranosyloleanolic acid (Sandrosaponin X) was identified. Their structures were elucidated by spectroscopic methods including 1D- and 2D-NMR experiments and mass spectrometry (ESI-MS). According to their structural similarities with gymnemic acids, the inhibitory activities on the sweet taste TAS1R2/TAS1R3 receptor of an aqueous ethanolic extract of the leaves and roots, a crude saponin mixture, 3-O-ß-d-glucopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-ß-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C, and Eryngioside L were evaluated.

Identification of lipophilic ligands of Siglec5 and -14 that modulate innate immune responses.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of cell-surface immune receptors that bind to sialic acid at terminal glycan residues. Siglecs also recognize nonsialic acid ligands, many of which remain to be characterized. Here, we found that Siglec5 and Siglec14 recognize lipid compounds produced by Trichophyton, a fungal genus containing several pathogenic species. Biochemical approaches revealed that the Siglec ligands are fungal alkanes and triacylglycerols, an unexpected finding that prompted us to search for endogenous lipid ligands of Siglecs. Siglec5 weakly recognized several endogenous lipids, but the mitochondrial lipid cardiolipin and the anti-inflammatory lipid 5-palmitic acid-hydroxystearic acid exhibited potent ligand activity on Siglec5. Further, the hydrophobic stretch in the Siglec5 N terminus region was found to be required for efficient recognition of these lipids. Notably, this hydrophobic stretch was dispensable for recognition of sialic acid. Siglec5 inhibited cell activation upon ligand binding, and accordingly, the lipophilic ligands suppressed interleukin-8 (IL-8) production in Siglec5-expressing human monocytic cells. Siglec14 and Siglec5 have high sequence identity in the extracellular region, and Siglec14 also recognized the endogenous lipids. However, unlike Siglec5, Siglec14 transduces activating signals upon ligand recognition. Indeed, the endogenous lipids induced IL-8 production in Siglec14-expressing human monocytic cells. These results indicated that Siglec5 and Siglec14 can recognize lipophilic ligands that thereby modulate innate immune responses. To our knowledge, this is the first study reporting the binding of Siglecs to lipid ligands, expanding our understanding of the biological function and importance of Siglecs in the innate immunity.

Intracellular metabolite ß-glucosylceramide is an endogenous Mincle ligand possessing immunostimulatory activity.

Sensing and reacting to tissue damage is a fundamental function of immune systems. Macrophage inducible C-type lectin (Mincle) is an activating C-type lectin receptor that senses damaged cells. Notably, Mincle also recognizes glycolipid ligands on pathogens. To elucidate endogenous glycolipids ligands derived from damaged cells, we fractionated supernatants from damaged cells and identified a lipophilic component that activates reporter cells expressing Mincle. Mass spectrometry and NMR spectroscopy identified the component structure as ß-glucosylceramide (GlcCer), which is a ubiquitous intracellular metabolite. Synthetic ß-GlcCer activated myeloid cells and induced production of inflammatory cytokines; this production was abrogated in Mincle-deficient cells. Sterile inflammation induced by excessive cell death in the thymus was exacerbated by hematopoietic-specific deletion of degrading enzyme of ß-GlcCer (ß-glucosylceramidase, GBA1). However, this enhanced inflammation was ameliorated in a Mincle-deficient background. GBA1-deficient dendritic cells (DCs) in which ß-GlcCer accumulates triggered antigen-specific T-cell responses more efficiently than WT DCs, whereas these responses were compromised in DCs from GBA1 × Mincle double-deficient mice. These results suggest that ß-GlcCer is an endogenous ligand for Mincle and possesses immunostimulatory activity.

Evaluation of the Immunomodulatory Effects of Fucoidan Derived from Cladosiphon Okamuranus Tokida in Mice.

Okinawa mozuku (Cladosiphon okamuranus Tokida) is an edible seaweed classified as brown algae and is a native species of the Ryukyu Islands in Japan. In recent years, the genomic decoding of Okinawa mozuku has been completed. Previous studies on the anti-inflammatory, antiviral, and antitumor properties of Okinawa mozuku have suggested that it affects the regulation of cellular and humoral immunity. The aim of the present study was to examine the immunoregulatory effect of fucoidan derived from Okinawa mozuku in mice. A product containing fucoidan (purity, 88.3%; molecular weight, 49.8 kDa) was developed from Okinawa mozuku and tested for its immunoregulatory effects in mice. The experimental animals were 8-week-old female BALB/c mice to which fucoidan (0, 102.5, 205.0, 410.0, and 1025.0 mg/kg) was administered orally continuously for six weeks. Immune cell proliferation, cytokine production, macrophage phagocytosis, and serum antibody concentration were measured. We found that immune cell proliferation, interleukin (IL)-2, macrophage phagocytes, and serum antibodies (IgM, -G, -A) increased significantly, but IL-4, -5, and IgE decreased significantly. These results indicated that fucoidan modulated cellular and humoral immunity.

Melanogenesis Inhibitors from the Endophytic Fungus Aspergillus amstelodami.

Two new compounds, named 3,4-dimethoxyphenyl α-d-ribofuranoside (1) and 3ß-(ß-d-glucopyranosyloxy)olean-12-ene-23,28,30-trioic acid (2), together with thirteen known compounds, were isolated from the white beans culture of the marine derived endophytic fungus Aspergillus amstelodami. Structure elucidation of the new compounds was carried out by one-, two-dimensional spectroscopy, and high resolution electrospray ionization mass. The antimelanogenic and anti-allergic activity of the isolated compounds were investigated. Compounds 4, 7, 1, 3, 11, 6 and 9 selectively suppressed melanin production in B16 melanoma cells, using arbutin as a positive control. Their IC50 values were 30.8±5.57, 38.5±6.08, 52.6±6.64, 98.0±1.16, 100.4±3.05, 112.0±0.22 and 144.7±2.35 µm, respectively, while that of arbutin was 151.7±1.27 µm. The tested compounds did not show any significant anti-allergic activity in RBL-2H3 cells, as compared to quercetin.

Uniqueness of Entamoeba sulfur metabolism: sulfolipid metabolism that plays pleiotropic roles in the parasitic life cycle.

Sulfur metabolism is ubiquitous and terminally synthesizes various biomolecules that are crucial for organisms, such as sulfur-containing amino acids and co-factors, sulfolipids and sulfated saccharides. Entamoeba histolytica, a protozoan parasite responsible for amoebiasis, possesses the unique sulfur metabolism features of atypical localization and its terminal product being limited to sulfolipids. Here, we present an overall scheme of E. histolytica sulfur metabolism by relating all sulfotransferases and sulfatases to their substrates and products. Furthermore, a novel sulfur metabolite, fatty alcohol disulfates, was identified and shown to play an important role in trophozoite proliferation. Cholesteryl sulfate, another synthesized sulfolipid, was previously demonstrated to play an important role in encystation, a differentiation process from proliferative trophozoite to dormant cyst. Entamoeba survives by alternating between these two distinct forms; therefore, Entamoeba sulfur metabolism contributes to the parasitic life cycle via its terminal products. Interestingly, this unique feature of sulfur metabolism is not conserved in the nonparasitic close relative of Entamoeba, Mastigamoeba, because lateral gene transfer-mediated acquisition of sulfatases and sulfotransferases, critical enzymes conferring this feature, has only occurred in the Entamoeba lineage. Hence, our findings suggest that sulfolipid metabolism has a causal relationship with parasitism.

C-type Lectin Mincle Recognizes Glucosyl-diacylglycerol of Streptococcus pneumoniae and Plays a Protective Role in Pneumococcal Pneumonia.

Among various innate immune receptor families, the role of C-type lectin receptors (CLRs) in lung protective immunity against Streptococcus pneumoniae (S. pneumoniae) is not fully defined. We here show that Mincle gene expression was induced in alveolar macrophages and neutrophils in bronchoalveolar lavage fluids of mice and patients with pneumococcal pneumonia. Moreover, S. pneumoniae directly triggered Mincle reporter cell activation in vitro via its glycolipid glucosyl-diacylglycerol (Glc-DAG), which was identified as the ligand recognized by Mincle. Purified Glc-DAG triggered Mincle reporter cell activation and stimulated inflammatory cytokine release by human alveolar macrophages and alveolar macrophages from WT but not Mincle KO mice. Mincle deficiency led to increased bacterial loads and decreased survival together with strongly dysregulated cytokine responses in mice challenged with focal pneumonia inducing S. pneumoniae, all of which was normalized in Mincle KO mice reconstituted with a WT hematopoietic system. In conclusion, the Mincle-Glc-DAG axis is a hitherto unrecognized element of lung protective immunity against focal pneumonia induced by S. pneumoniae.

Sodium-Periodate-Mediated Harringtonine Derivatives and Their Antiproliferative Activity against HL-60 Acute Leukemia Cells.

Harringtonine (HT) is a naturally occurring alkaloid isolated from the plant genus Cephalotaxus. It possesses antileukemic activity and has been clinically utilized for the treatment of acute leukemia and lymphoma. Sodium periodate (NaIO4) was reacted with HT to produce five HT derivatives including four novel compounds. Their antiproliferative activity against HL-60 acute promyelocytic leukemia cells revealed that the presence of the C-5' methyl group enhances the antiproliferative activity because the IC50 values of the HT derivatives, including HT1 (5'-de-O-methylharringtonine), were at least 2000 times higher (>100 µM) than that of HT (∼47 nM). In addition, an indirect competitive enzyme-linked immunosorbent assay (icELISA) using a monoclonal antibody against HT (mAb 1D2) revealed that these antiproliferative activities were related to their cellular uptake. These results indicated that esterification of HT1 at the C-4' carboxylic acid group may enhance the antiproliferative activity of HT.

Entamoeba mitosomes play an important role in encystation by association with cholesteryl sulfate synthesis.

Hydrogenosomes and mitosomes are mitochondrion-related organelles (MROs) that have highly reduced and divergent functions in anaerobic/microaerophilic eukaryotes. Entamoeba histolytica, a microaerophilic, parasitic amoebozoan species, which causes intestinal and extraintestinal amoebiasis in humans, possesses mitosomes, the existence and biological functions of which have been a longstanding enigma in the evolution of mitochondria. We previously demonstrated that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function of E. histolytica mitosomes. However, because the final metabolites of sulfate activation remain unknown, the overall scheme of this metabolism and the role of mitosomes in Entamoeba have not been elucidated. In this study we purified and identified cholesteryl sulfate (CS) as a final metabolite of sulfate activation. We then identified the gene encoding the cholesteryl sulfotransferase responsible for synthesizing CS. Addition of CS to culture media increased the number of cysts, the dormant form that differentiates from proliferative trophozoites. Conversely, chlorate, a selective inhibitor of the first enzyme in the sulfate-activation pathway, inhibited cyst formation in a dose-dependent manner. These results indicate that CS plays an important role in differentiation, an essential process for the transmission of Entamoeba between hosts. Furthermore, we show that Mastigamoeba balamuthi, an anaerobic, free-living amoebozoan species, which is a close relative of E. histolytica, also has the sulfate-activation pathway in MROs but does not possess the capacity for CS production. Hence, we propose that a unique function of MROs in Entamoeba contributes to its adaptation to its parasitic life cycle.

Sodium periodate-mediated conjugation of harringtonine enabling the production of a highly specific monoclonal antibody, and the development of a sensitive quantitative analysis method.

Harringtonine (HT) is a promising natural product that is mainly isolated from plants of the genus Cephalotaxus. Due to its remarkable antileukemic activities, HT has been utilized clinically in China for the treatment of acute promyelocytic leukemia (APL). No antibody that recognizes free HT has been reported to date due to the difficulty of preparing antigen conjugates in which haptens bind to a carrier protein. To overcome this difficulty, we focused on sodium periodate (NaIO4), which catalyzes unique oxidative reactions; the resulting conjugates enabled the production of a highly specific monoclonal antibody (MAb) against HT (MAb 1D2) and the establishment of an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the determination of HT. Further analysis revealed that MAb 1D2 was produced by the HT3 (8-carbonyl HT)-based conjugate antigen; HT3 was synthesized by a NaIO4-mediated oxidative reaction. The minimum detectable concentration for HT in the icELISA system was found to be 0.76 ng mL-1, which is approximately 13 to 160 times more sensitive than a conventional HPLC system. Several validation analyses revealed that the icELISA using MAb 1D2 is sufficiently accurate, reliable, and sensitive to assess small amounts of HT in plant samples.

Development of an immunoassay using an anti-wogonin glucuronide monoclonal antibody.

Wogonin 7-O-ß-D-glucuronide (Wgn) is a bioactive flavone present in the dried root of Scutellaria baicalensis Georgi. To generate a monoclonal antibody (MAb) against Wgn, BALB/c mice injected with Wgn-bovine serum albumin yielded splenocytes that we fused with SP2/0 myeloma cells using the polyethylene glycol method. We obtained a hybridoma designated 315A that produced a MAb reactive to Wgn. The anti-Wgn MAb 315A was applied to an indirect competitive enzyme-linked immunosorbent assay (icELISA) to quantify Wgn. Subsequent validation revealed that icELISA using the 315A anti-Wgn MAb is an accurate and reliable method for the quantification of Wgn in S. baicalensis.

A dihydrochalcone derivative and further steroidal saponins from Sansevieria trifasciata Prain.

Phytochemical investigation of the aerial parts of Sansevieria trifasciata, one of the most common Dracaenaceae plants, has resulted in the isolation of a new dihydrochalcone derivative named trifasciatine C (1), four previously unreported steroidal saponins as two pairs of inseparable regioisomers: trifasciatosides K/L (2/3), M/N (4/5), together with the known 1,2-(dipalmitoyl)-3-O-ß-D-galactopyranosylglycerol (6), aconitic acid (7), and 1-methyl aconitic acid (8). Their structures were elucidated mainly by extensive spectroscopic analysis (1D and 2D nuclear magnetic resonance) and high-resolution electronspray ionization-mass spectrometry, as well as chemical methods and comparison of their spectral data with those of related compounds. Compounds 2/3 and 4/5 were evaluated for their antiproliferative activity on Hela cells, and no significant effect was observed.

Human Mincle Binds to Cholesterol Crystals and Triggers Innate Immune Responses.

C-type lectin receptors (CLRs) are an emerging family of pattern recognition receptors that recognizes pathogens or damaged tissue to trigger innate immune responses. However, endogenous ligands for CLRs are not fully understood. In this study, we sought to identify an endogenous ligand(s) for human macrophage-inducible C-type lectin (hMincle). A particular fraction of lipid extracts from liver selectively activated reporter cells expressing hMincle. MS analysis determined the chemical structure of the active component as cholesterol. Purified cholesterol in plate-coated and crystalized forms activates reporter cells expressing hMincle but not murine Mincle (mMincle). Cholesterol crystals are known to activate immune cells and induce inflammatory responses through lysosomal damage. However, direct innate immune receptors for cholesterol crystals have not been identified. Murine macrophages transfected with hMincle responded to cholesterol crystals by producing pro-inflammatory cytokines. Human dendritic cells expressed a set of inflammatory genes in response to cholesterol crystals, and this was inhibited by anti-human Mincle. Importantly, other related CLRs did not bind cholesterol crystals, whereas other steroids were not recognized by hMincle. These results suggest that cholesterol crystals are an endogenous ligand for hMincle and that they activate innate immune responses.