Hp-s1 Ganglioside Suppresses Proinflammatory Responses by Inhibiting MyD88-Dependent NF-κB and JNK/p38 MAPK Pathways in Lipopolysaccharide-Stimulated Microglial Cells.

Hp-s1 ganglioside is isolated from the sperm of sea urchin (Hemicentrotus pulcherrimus). In addition to neuritogenic activity, the biological function of Hp-s1 in neuroinflammation is unknown. In this study, we investigated the anti-neuroinflammatory effect of Hp-s1 on lipopolysaccharide (LPS)-stimulated microglial cells. MG6 microglial cells were stimulated with LPS in the presence or absence of different Hp-s1 concentrations. The anti-inflammatory effect and underlying mechanism of Hp-s1 in LPS-activated microglia cells were assessed through a Cell Counting kit-8 assay, Western blot analysis, and immunofluorescence. We found that Hp-s1 suppressed not only the expression of inducible nitric oxide synthase and cyclooxygenase-2 but also the expression of proinflammatory cytokines, such as TNF-α, IL-1ß, and IL-6. Hp-s1 inhibited the LPS-induced NF-κB signaling pathway by attenuating the phosphorylation and translocation of NF-κB p65 and by disrupting the degradation and phosphorylation of inhibitor κB-α (IκBα). Moreover, Hp-s1 inhibited the LPS-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Hp-s1 also reduced the expression of myeloid differentiation factor 88 (MyD88) and TNF receptor-associated factors 6 (TRAF6), which are prerequisites for NF-κB and MAPKs activation. These findings indicated that Hp-s1 alleviated LPS-induced proinflammatory responses in microglial cells by downregulating MyD88-mediated NF-κB and JNK/p38 MAPK signaling pathways, suggesting further evaluation as a new anti-neuroinflammatory drug.

A novel interaction of nucleophosmin with BCL2-associated X protein regulating death evasion and drug sensitivity in human hepatoma cells.

UNLABELLED: Death evasion is crucial for both carcinogenesis and resistance to anticancer therapies. Recently, we identified nucleophosmin (NPM) as a key factor counteracting death stimuli in human hepatocellular carcinoma (HCC) cells. Here we report the identification of a novel NPM-BCL2-associated X protein (BAX) pathway orchestrating death evasion in human HCC cells. Silencing of NPM expression significantly sensitized HCC cells-particularly those bearing inactivated p53 gene (Huh7, Hep3B, and Mahlavu)-to ultraviolet irradiation, mitomycin C, doxorubicin, cisplatin, sorafenib, and lapatinib. This sensitizing effect was not changed further, as p53 expression had been simultaneously silenced. Following cell stress, NPM and BAX were induced and exported out of the nucleoli and nucleus, respectively. BAX was translocated to cytoplasm in cells with relatively high NPM level, or accumulated in the mitochondria in cells with relatively low NPM level and undergoing apoptosis. Subcellular fractionation revealed that silencing of NPM expression greatly enhanced mitochondrial translocation and oligomerization of BAX in Huh7 and Mahlavu cells. In situ proximity ligation assays and reciprocal co-immunoprecipitation revealed a direct interaction between NPM and BAX in the cytoplasm. Silencing of BAX expression abolished the sensitization effect exerted by silencing of NPM in HCC cells. Clinically, up-regulation of NPM was significantly associated with advanced tumor stage and poor prognosis. CONCLUSION: By directly blockading BAX mitochondrial translocation and activation, NPM helps human HCC cells evade death induction independently of p53-mediated cell death. Silencing of NPM significantly sensitized HCC cells to anticancer therapies. NPM is a potential cotarget in combination with other therapies for HCC, particularly those that harbor inactivated p53 gene. Our findings are of clinical significance because NPM up-regulation and p53 mutations are usually found in advanced human cancers, including HCC.

First total synthesis of ganglioside DSG-A possessing neuritogenic activity.

The first total synthesis of ganglioside DSG-A (1) is achieved via chemoselective glycosylation and a [1 + 1 + 2] synthetic strategy. We have also developed an efficient method that can be handled on large scale (50 g) for the synthesis of the phytosphingosine.

The total synthesis of a ganglioside Hp-s1 analogue possessing neuritogenic activity by chemoselective activation glycosylation.

The total synthesis of ganglioside 2, an analogue of the ganglioside Hp-s1 (1) which displays neuritogenic activity toward the rat pheochromocytoma cell line PC-12 cell in the presence of nerve growth factor (NGF) with an effect (34.0%) greater than that of the mammalian ganglioside GM 1 (25.4%), was accomplished by applying a chemoselective-activation glycosylation strategy. Moreover, we also demonstrate that the synthesized ganglioside 2 exhibited neuritogenic activity toward the human neuroblastoma cell line SH-SY5Y without the presence of NGF.

Total Synthesis of the Echinodermatous Ganglioside LLG-3 Possessing the Biological Function of Promoting the Neurite Outgrowth.

A total synthesis of echinodermatous ganglioside LLG-3 with neuritogenic activity was accomplished by a convergent strategy. The synthesis of 2-hydroxyethyl 8-O-Me-α-sialoside 2 was started from the phenyl 7,8-di-O-Pico-thiosialoside 5, which can be chemoselectively removed the picoloyl group, and then the methyl group in 8-O-MeNeu5Ac moiety was chemoselectively prepared using TMSCHN2/FeCl3. For preparation of the terminal disialic unit, oxidative amidation was initially utilized by our group to efficiently construct the α(2,11) linkage of 8-O-Me-Neu5Acα(2,11)Neu5Gc. Herein, we also demonstrate that the synthesized ganglioside LLG-3 exhibited the neuritogenic activity toward the primary cortical neurons and that biological activity is superior to that of ganglioside DSG-A.

Ganglioside Hp-s1 Analogue Inhibits Amyloidogenic Toxicity in Alzheimer's Disease Model Cells.

Alzheimer's disease (AD) is characterized by extracellular deposition of amyloid plaques, which are predominantly composed of amyloid-ß (Aß) peptide derived from amyloid precursor protein (APP) cleavage. APP interacts with tropomyosin receptor kinase A, a neurotrophic receptor associated with gangliosides and mediating neuronal survival and differentiation through the extracellular signal-regulated protein kinase (ERK) pathway. The ganglioside Hp-s1's analogue Hp-s1A exerts neuritogenic activity; however, its effect on AD pathology remains unknown. To test the hypothesis that Hp-s1A is a potential candidate to treat AD, we established the AD-modeled cell line by expressing human Swedish and Indiana APP gene (APP-Swe/Ind) in N2a mouse neuroblastoma cells. The cells were treated with Hp-s1A or monosialoganglioside GM1 for comparison. The AD model cells expressing APP-Swe/Ind exhibited a significant reduction in viability, as well as neurite outgrowth rate, in comparison to the control cells expressing APP-695. APP C-terminal fragment-ß (CTFß) and Aß42 were increased in the AD cell lysates and the culture media, respectively. With the treatment of either Hp-s1A or GM1 at 1 µM, the AD model cells showed a significant increase in viability; however, only Hp-s1A reduced CTFß levels in these cells. Further analysis of the culture media revealed that Hp-s1A also reduced Aß42 production from AD model cells. The phosphorylation of ERK was elevated and the neurite outgrowth rate was restored with Hp-s1A treatment. In conclusion, the ganglioside analogue Hp-s1A inhibited amyloidogenic processing of APP and promoted neurotrophic activity and survival of AD model cells. Hp-s1A has great potential in AD therapeutic development.

(2S)-2-(3-Oxo-1,4-dioxaspiro-[4.5]decan-2-yl)ethanoic acid.

The title compound, C(10)H(14)O(5), is an inter-mediate in our study of the asymmetric synthesis of α-hydroxy-alkanoic acids. The structure consists of 1,4-dioxaspiro[4,5]decane skeleton formed when the cyclohexylidene group binds to both of the hydroxyl groups of carboxylic groups of the starting malic acid. The six-membered ring adopts a chair conformation.

Methyl (2'S,3'S)-3,4-O-(2',3'-dimethoxy-butane-2',3'-di-yl)-α-l-rhamnopyran-oside: a glycosyl acceptor.

The title compound, C(13)H(24)O(7), is the product of the ketalization of methyl l-(+)-rhamnopyran-oside with 2,3-butane-dione. It crystallizes with two mol-ecules in the asymmetric unit, which are connected by O-H⋯O hydrogen bonds. The C-3,4 diequatorial hydroxy groups of the methyl l-(+)-rhamnopyran-oside were protected, leaving the C-2 hydroxy group free. The l-(+)-rhamnopyran-oside and 2',3'-dimethoxy-butane-2',3'-diyl rings adopt chair conformations and all meth-oxy groups are in axial positions. The absolute configuration was assumed from the synthesis.

Assistance of the C-7,8-Picoloyl Moiety for Directing the Glycosyl Acceptors into the α-Orientation for the Glycosylation of Sialyl Donors.

An efficient α-sialylation method for many primary hydroxyl acceptors that include 6-OH glycosides has been developed. 7,8-Di-O-picoloyl sialyl glycoside was used as the glycosyl donor, and α-glycoconjugation was controlled by using the 7,8-di-O-picoloyl moiety in CH2Cl2. The methodology was successfully applied to the total synthesis of ganglioside Hp-s1 possessing neuritogenic activity.

A novel sensitive pathogen detection system based on Microbead Quantum Dot System.

A fast and accurate detection system for pathogens can provide immediate measurements for the identification of infectious agents. Therefore, the Microbead Quantum-dots Detection System (MQDS) was developed to identify and measure target DNAs of pathogenic microorganisms and eliminated the need of PCR amplifications. This nanomaterial-based technique can detect different microorganisms by flow cytometry measurements. In MQDS, pathogen specific DNA probes were designed to form a hairpin structure and conjugated on microbeads. In the presence of the complementary target DNA sequence, the probes will compete for binding with the reporter probes but will not interfere with the binding between the probe and internal control DNA. To monitor the binding process by flow cytometry, both the reporter probes and internal control probes were conjugated with Quantum dots that fluoresce at different emission wavelengths using the click reaction. When MQDS was used to detect the pathogens in environmental samples, a high correlation coefficient (R=0.994) for Legionella spp., with a detection limit of 0.1 ng of the extracted DNAs and 10 CFU/test, can be achieved. Thus, this newly developed technique can also be applied to detect other pathogens, particularly viruses and other genetic diseases.

Intra- and intermolecular hydrogen bonds enhance the fluoride-responsiveness of functionalized glycolipid-based gelators.

We propose a facile approach toward enhancing the efficiency of fluoride-responsive gels through the positioning of functionalized receptor units, allowing tunable intra- and intermolecular hydrogen bonding, in the gelator molecules. We prepared the new glycolipid-based gelator 2 and its hydroxy and methoxy derivatives 2a and 2b, respectively, to study the effects of three types modes of supramolecular assembly: solely intermolecular hydrogen bonding in 2, solely intramolecular hydrogen bonding in 2b, and both inter- and intramolecular hydrogen bonding in 2a. 1H NMR spectra confirmed the self-assembly interactions of these glycolipid-based gelators. We measured the minimum gel concentrations and sol-gel transitions and recorded X-ray diffraction patterns and electron micrographs to characterize the gelation behavior and structural organization of each of these supramolecular gels. Among these three gelators, only 2 and 2a could form organogels in the test solvents, indicating that intermolecular hydrogen bonding plays a determinant role in the supramolecular assemblies leading to gelation. The self-assembly of 2 resulted in a bilayer-packed lamellar structure within ribbon-like fibers, whereas that of 2a resulted in hexagonally packed cylindrical micelles within tree-like fibers. A minimum amount of 0.3 equivalent of F- was required for complete disruption of the gel formed from 2a, which was approximately four times lower than that required for the gel formed from 2. Thus, the incorporation of a ß-hydroxy motif-the only difference in the chemical structures of 2 and 2a-led to interesting variations in the resulting gel morphologies and enhanced the gel's fluoride-responsiveness.

Antioxidant and semicarbazide-sensitive amine oxidase inhibitory activities of glucuronic acid hydroxamate.

The antioxidant and inhibitory activities of glucuronic acid (GluA) and its synthesized 1-methoxyl-6-glucuronic acid hydroxamate (GluA-NHOH) against semicarbazide-sensitive amine oxidase (SSAO) were investigated. The scavenging activities of GluA-NHOH against 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radicals (half-inhibition concentration [IC50], 152µM) and hydroxyl radicals (IC50, 70nM) were determined. The protective activities of GluA-NHOH against hydroxyl radical-mediated calf thymus DNA damage and peroxynitrite-mediated dihydrorhodamine oxidation were also investigated. GluA-NHOH, but not GluA, exhibited dose-dependent protective activities. GluA-NHOH, but not GluA, effectively inhibited bovine SSAO activities in a dose-dependent manner, and was further confirmed by AO activity stains on an acrylamide gel. GluA-NHOH showed mixed noncompetitive inhibition of bovine SSAO with respect to benzylamine (substrate) and benzylamine-SSAO (substrate-enzyme complex). The Vmax' and Km' values were reduced in the presence of GluA-NHOH (0.04mM).

Diels-Alder reactions of 4-triflyloxy-2,6,6-trimethyl-2,4-cyclohexadienone. An expedient methodology for the synthesis of bicyclo[2.2.2]oct-5-en-2-ones and bicyclo[2.2.2]octa-5,7-dien-2-ones.

The synthesis of 4-triflyloxy-2,6,6-trimethyl-2,4-cyclohexadienone (13), bicyclo[2.2.2]octenones 1a-j and 15a-j, and bicyclo[2.2.2]octadienones 2a-f, 6a-d, and 11a-f is described. The 2,4-cyclohexadienones 4 and 13 were used for the first time as nondimerizing and easily accessible alternatives to 2,6,6-trimethyl-2,4-cyclohexadienone 12 in Diels-Alder reactions with acetylene derivatives 5a-d to prepare the adducts 6a-d and 11a-e in excellent yields. Compounds 11a-d were initially prepared by the alcoholysis of 6a-d to afford bicyclo[2.2.2]octene-2,5-diones 7a-dfollowed by treatment of 7a-d with N-phenyltriflimide in the presence of LHMDS at -78 degrees C. Diels-Alder reaction of 13 with an acetylene equivalent, phenyl vinyl sulfoxide, was also studied. A detailed study of the Diels-Alder reactions of various olefinic dienophiles 14a-j with 13 has been carried out to furnish cycloadducts 15a-j in high yields. Reductive removal of triflyloxy group of vinyl triflates 11a-f and 15a-j was performed in the presence of [Pd(PPh(3))(2)Cl(2)-Bu(3)N-HCO(2)H] to obtain the desired bicyclo[2.2.2]octadienones 2a-f and bicyclo[2.2.2]octenones 1a-j, respectively, in good overall yields.

Studies on the immuno-modulating and anti-tumor activities of Ganoderma lucidum (Reishi) polysaccharides.

We describe here the isolation of Reishi polysaccharides for the study of their effect on cytokine expression in mouse splenocytes. A fraction (F3) has been shown to activate the expression of IL-1, IL-6, IL-12, IFN-gamma, TNF-alpha, GM-CSF, G-CSF, and M-CSF, and from this three subfractions have been prepared where F3G1 activates IL-1, IL-12, TNF-alpha, and G-CSF, F3G2 activates all the cytokines as F3 does, and F3G3 activates only IL-1 and TNF-alpha. Together with previous studies, the mode of action on macrophages has been proposed where F3 binds to TLR4 receptor and activates extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 to induce IL-1 expression.

Synthesis of alpha-(2-->5)Neu5Gc oligomers.

A facile synthesis of the sialic acid oligomers alpha-(2-->5)Neu5Gc (1) is presented. Monosaccharides 2-4 with suitable functionality were used as the building blocks. After selective removal of the paired carboxyl and amine protecting groups, the fully protected oligomers were assembled through consecutive coupling of the building blocks by well established peptide coupling techniques. By this approach, fully protected oligomers as large as an octasaccharide were synthesized. Deprotection of these fully protected oligomers was conducted in two steps (LiCl in refluxing pyridine and 0.1 n NaOH) to afford the desired products in high yield. Enzymatic degradation of the octamer with neuraminidase, monitored by capillary electrophoresis (CE), was also accomplished. The stepwise exo-cleavage adducts were all well separated and identified in the CE spectrum. The strategy described here for solution-phase synthesis also provides the basis for future solid-phase synthesis of poly-alpha-(2-->5)Neu5Gc.