Yu-Liang Yang

Asperjinone, a nor-neolignan, and terrein, a suppressor of ABCG2-expressing breast cancer cells, from thermophilic Aspergillus terreus.

Breast cancer cells express ABCG2 transporters, which mediate multidrug resistance. Discovering a novel compound that can suppress ABCG2 expression and restore drug sensitivity could be the key to improving breast cancer therapeutics. In the current work, one new nor-neolignan, asperjinone (1), as well as 12 other known compounds, was isolated from Aspergillus terreus. The structure of the new isolate was determined by spectroscopic methods. Among these isolates, terrein (2) displayed strong cytotoxicity against breast cancer MCF-7 cells. Treatment with terrein (2) significantly suppressed growth of ABCG2-expressing breast cancer cells. This suppressive effect was achieved by inducing apoptosis via activating the caspase-7 pathway and inhibiting the Akt signaling pathway, which led to a decrease in ABCG2-expressing cells and a reduction in the side-population phenotype.

A novel exopolysaccharide from the biofilm of Thermus aquaticus YT-1 induces the immune response through Toll-like receptor 2.

Bacterial polysaccharides are known to induce the immune response in macrophages. Here we isolated a novel extracellular polysaccharide from the biofilm of Thermus aquaticus YT-1 and evaluated its structure and immunomodulatory effects. The size of this polysaccharide, TA-1, was deduced by size-exclusion chromatography as 500 kDa. GC-MS, high performance anion-exchange chromatography with pulsed amperometric detection, electrospray ionization-MS/MS, and NMR revealed the novel structure of TA-1. The polysaccharide is composed of tetrasaccharide-repeating units of galactofuranose, galactopyranose, and N-acetylgalactosamine (1:1:2) and lacked acidic sugars. TA-1 stimulated macrophage cells to produce the cytokines TNF-α and IL-6. Screening of Toll-like receptors and antibody-blocking experiments indicated that the natural receptor of TA-1 in its immunoactivity is TLR2. Recognition of TA-1 by TLR2 was confirmed by TA-1 induction of IL-6 production in peritoneal macrophages from wild-type mice but not from TLR2−/− mice. TA-1, as a TLR2 agonist, could possibly be used as an adjuvant and could enhance cytokine release, which increases the immune response. Furthermore, TA-1 induced cytokine release is dependent on MyD88/TIRAP.

Structural elucidation of phosphoglycolipids from strains of the bacterial thermophiles Thermus and Meiothermus.

The structures of two major phosphoglycolipids from the thermophilic bacteria Thermus oshimai NTU-063, Thermus thermophilus NTU-077, Meiothermus ruber NTU-124, and Meiothermus taiwanensis NTU-220 were determined using spectroscopic and chemical analyses to be 2′-O-(1,2-diacyl-sn-glycero-3-phospho) -3′-O-(α-N-acetyl-glucosaminyl)-N-glyceroyl alkylamine [PGL1 (1)] and the novel structure 2′-O-(2-acylalkyldio-1-O-phospho)-3′-O-(α-N-acetylglucosaminyl)-N-glyceroyl alkylamine [PGL2 (2)]. PGL2 (2) is the first phosphoglycolipid identified with a 2-acylalkyldio-1-O-phosphate moiety. The fatty acids of the phosphoglycolipids are mainly iso-C15:0, -C16:0, and -C17:0 and anteiso-C15:0 and -C17:0. The ratios of PGL2 (2) to PGL1 (1) are significantly altered when grown at different temperatures for three strains, T. thermophilus NTU-077, M. ruber NTU-124, and M. taiwanensis NTU-220, but not for T. oshimai NTU-063. Accordingly, the ratios of iso- to anteiso-branched fatty acids increase when grown at the higher temperature.

Bringing microbial interactions to light using imaging mass spectrometry

Covering: up to the end of September 2013 Microorganisms are a plentiful resource for natural products research. Traditionally, natural products discovery from microbial sources depends on the screening of target-mediated inhibition. The natural products identified through this strategy usually correlate to significant microbial phenotypes. However, the target-mediated transcriptions deduced from low concentrations of natural products sometimes do not generate an obvious phenotype. The better understanding of the true biological roles of those microbial natural products will permit the application of rational approaches to the more effective exploitation of their use. Imaging mass spectrometry (IMS) has been developed and applied in many fields for decades. However, the applications of IMS on microbial natural products research have just been recently reported. IMS is one of few tools capable of revealing both phenotype and relevant and irrelevant chemotypes of microorganisms. In this review, we summarize the latest applications of IMS technologies. The challenges and prospect of improvement and application of IMS to microbial natural products research are discussed as well.

Synthesis and Biological Evaluation of Polyenylpyrrole Derivatives as Anticancer Agents Acting through Caspases-Dependent Apoptosis

A class of polyenylpyrroles and their analogues were designed from a hit compound identified in a fungus. The compounds synthesized were evaluated for their cell cytotoxicity against human non-small-cell lung carcinoma cell lines A549. Two compounds were found to exhibit high cytotoxicity against A549 cells with IC(50) of 0.6 and 0.01 μM, respectively. The underlying mechanisms for the anticancer activity were demonstrated as caspases activation dependent apoptosis induction through loss of mitochondrial membrane potential, release of cytochrome c, increase in B-cell lymphoma-2-associated X protein (Bax) level, and decrease in B-cell lymphoma-2 (Bcl-2) level. The two compounds were nontoxic to normal human lung Beas-2b cells (IC(50) > 80 μM), indicating that they are highly selective in their cytotoxicity activities. Furthermore, one compound showed in vivo anticancer activity in human-lung-cancer-cell-bearing mice. These results open promising insights on how these conjugated polyenes mediate cytotoxicity and may provide a molecular rationale for future therapeutic interventions in carcinogenesis.

Structure and Immunological Characterization of the Capsular Polysaccharide of a Pyrogenic Liver Abscess Caused by Klebsiella pneumoniae ACTIVATION OF MACROPHAGES THROUGH TOLL-LIKE RECEPTOR 4

The active components of a primary pyrogenic liver abscess (PLA) Klebsiella pneumoniae in stimulating cytokine expression in macrophages are still unclear. The capsular polysaccharide (CPS) of PLA K. pneumoniae is important in determining clinical manifestations, and we have shown that it consists of repeating units of the trisaccharide (→3)-β-d-Glc-(1→4)-[2,3-(S)-pyruvate]-β-d-GlcA-(1→4)-α-l-Fuc-(1→) and has the unusual feature of extensive pyruvation of glucuronic acid and acetylation of C2-OH or C3-OH of fucose. We demonstrated that PLA K. pneumoniae CPS induces secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by macrophages through Toll-like receptor 4 (TLR4) and that this effect was lost when pyruvation and O-acetylation were chemically destroyed. Furthermore, expression of TNF-α and IL-6 in PLA K. pneumoniae CPS-stimulated macrophages was shown to be regulated by the TLR4/ROS/PKC-δ/NF-κB, TLR4/PI3-kinase/AKT/NF-κB, and TLR4/MAPK signaling pathways.

Discovery of New Natural Products by Intact‐Cell Mass Spectrometry and LC‐SPE‐NMR: Malbranpyrroles, Novel Polyketides from Thermophilic Fungus Malbranchea sulfurea

Six photosensitive polyketides, malbranpyrroles A-F, were discovered from the thermophilic fungus Malbranchea sulfurea by using intact-cell desorption/ionization on silicon mass (ICD-MS) and LC-SPE-NMR. These two strategies facilitate the searching and structural determination of unstable natural products. The ICD-MS indicated that only brown hyphae of M. sulfurea can produce malbranpyrroles. The biosynthetic pathway of malbranpyrroles was evidenced by 13C isotope precursors and amino acid feeding experiments. The cytotoxicity data revealed that the conformation of the conjugated system in malbranpyrroles does not affect cytotoxic potency against cancer cell lines. In addition, the chlorine atom was shown to be the pharmacophore for cytotoxicity.

Polysaccharides from Dioscorea batatas induce tumor necrosis factor-alpha secretion via Toll-like receptor 4-mediated protein kinase signaling pathways.

The bioactive polysaccharides (named ZPF1) from yam ( Dioscorea batatas) were chemically determined, suggesting repeating beta-1,4-mannan as mainly having a feature of acetylation on C2-OH and C3-OH, around 28%. The ZPF1 participated in the stimulation of murine wild-type macrophages predominantly in tumor necrosis factor-alpha (TNFalpha). Toll-like receptor 4 is proved to be one of the cellular receptors in ZPF1-mediated TNFalpha secretion. Reactive oxygen species transmission and PI3-kinase are found necessary for regulating TNFalpha secretion by ZPF1 stimulation. Moreover, we found that extracellular signal-regulated kinase 1/2, Jun N-terminal kinase 1/2, and p38 mitogen-activated protein kinase play important roles in the regulation of TNFalpha secretion in ZPF1-stimulated macrophages.

Polyenylpyrrole Derivatives Inhibit NLRP3 Inflammasome Activation and Inflammatory Mediator Expression by Reducing Reactive Oxygen Species Production and Mitogen-Activated Protein Kinase Activation

Two polyenylpyrroles from a soil ascomycete Gymnoascus reessii were previously identified as hit compounds in screening for cytotoxicity against lung cancer cells. These compounds and various analogs, which have been previously synthesized and tested for anti-lung cancer cell activity, were tested for anti-inflammatory activity. After preliminary screening for cytotoxicity for RAW 264.7 murine macrophage cells, the non-toxic compounds were tested for anti-inflammatory activity using lipopolysaccharide (LPS)-activated RAW 264.7 cells. Compounds 1h, 1i, and 1n reduced LPS-induced nitric oxide (NO) production, with respective ED50 values of 15 ± 2, 16 ± 2, and 17 ± 2 µM. They also reduced expression of inducible NO synthase and interleukin-6 (IL-6) without affecting cyclooxygenase-2 expression. Compound 1h also reduced secretion of IL-6 and tumor necrosis factor-α by LPS-activated J774A.1 murine macrophage cells, primary mice peritoneal macrophages, and JAWSII murine bone marrow-derived dendritic cells and reduced NLRP3 inflammasome-mediated interleukin-1β (IL-1β) secretion by LPS + adenosine triphosphate-activated J774A.1 and JAWSII cells. The underlying mechanisms for the anti-inflammatory activity of compound 1h were found to be a decrease in LPS-induced reactive oxygen species (ROS) production, mitogen-activated protein kinase phosphorylation, and NF-κB activation and a decrease in ATP-induced ROS production and PKC-α phosphorylation. These results provide promising insights into the anti-inflammatory activity of these conjugated polyenes and a molecular rationale for future therapeutic intervention in inflammation-related diseases. They also show how compound 1h regulates inflammation and suggest it may be a new source for the development of anti-inflammatory agents to ameliorate inflammation- and NLRP3 inflammasome-related diseases.

Oligosaccharide and Peptidoglycan of Ganoderma lucidum Activate the Immune Response in Human Mononuclear Cells

The acid-hydrolyzed fragments of Ganoderma lucidum polysaccharides (GLPS) obtained by Smith degradation were separated by size-exclusion chromatography into two major water-soluble fractions: peptidoglycans (GLPS-SF1) and oligosaccharides (GLPS-SF2). Both fractions induced CD69 in human peripheral blood mononuclear cells (hPB-MNCs), and they displayed distinct immunomodulating properties. GLPS-SF1, with a molecular weight of around 20 kDa, were heterogeneous peptidoglycans composed of glucose/mannose (4:1) that exhibited biological activities with Th1 cytokines IL-12, IL-2, TNF-α, and IFN-γ in hPB-MNCs and stimulated macrophage cytokine expression via Toll-like receptor 4 (TLR4) signaling. For GLPS-SF2, with a molecular weight of around several kilodaltons, its sugar sequence was elucidated by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy as [-α-1,4-Glc-(β-1,4-GlcA)(3)-](n). This oligosaccharide displayed specific immune property with low monocyte induction, greatly stimulated cell activation and proliferation of NK and T cells. This oligosaccharide isolated from G. lucidum polysaccharides with internal glucuronic acids/glucose repeat unit in a 3:1 ratio may be responsible for the active stimulation of NK and T cells.