Macrophage activation in vitro by chemically cross-linked (1----3)-beta-D-glucans

The chemical cross-linking of soluble (1----3)-beta-D-glucans having molecular weights of 21000 (CL 3 h) and 6400 (CL 6 h), and laminarin (CL LAMI), which showed negligible biological activity, by epichlorohydrin provided rigid particles. These particles showed no gel-to-sol transition upon the addition of sodium hydroxide. We compared the effects of chemical cross-linking on the biological activities of glucans. The alternative complement pathway was not activated by any of the cross-linked glucans. Glucose consumption, lysosomal enzyme release, and interleukin-1 production by mouse resident peritoneal macrophages incubated in vitro were strongly induced by CL 3 h, CL 6 h and CL LAMI. However, cross-linked dextran, Sephadex, did not exhibit any of these biological activities. These results suggested that chemical cross-linking of (1----3)-beta-D-glucans enhances macrophage activities without opsonization by complement components.     

Lipid A and related compounds. XXVI. Syntheses of biologically active penta-O-acetyl-N-glycoloylneuraminyl- and penta-O-acetyl-3-deoxy-D-galacto-2- nonulopyranosonic acid-(alpha 2----6)-D-glucosamine-4-phosphate analogs of lipid A

The syntheses of novel penta-O-acetyl-N-glycoloylneuraminyl- and penta-O-acetyl-3-deoxy-D-glycero-D-galacto-2-nonulopyranosonic acid-(alpha 2----6)-D-glucosamine-4-phosphate analogues of lipid A containing sialic acid in place of 3-deoxy-D-manno-2-octulosonic acid are described. Preliminary examination of the biological activity revealed that two synthetic disaccharides showed mitogenic activities and weak antitumor activities.     

Intravenously administered (1----3)-beta-D-glucan, SSG, obtained from Sclerotinia sclerotiorum IFO 9395 augments murine peritoneal macrophage functions in vivo.

Effect of intravenously (i.v.) or intraperitoneally (i.p.) administered (1----3)-beta-D-glucan, SSG, obtained from Sclerotinia sclerotiorum IFO 9395 on the murine peritoneal macrophage (PM) functions were examined. A single i.v. administration of SSG increased the number of PMs at a dose of 250 micrograms/mouse, and the peak appeared 4 d after administration. However, no special change was observed on peritoneal exude cell (PEC) populations. These PMs showed augmented lysosomal enzyme activity and the peaks appeared in 2 phases, on days 2 and 10. In contrast, SSG administered i.p. (250 micrograms/mouse) increased the number of PMs and enhanced the lysosomal enzyme activity of PMs from day 4, and a broad peak appeared until days 8--12. The populations of PECs were also changed by i.p. injection of SSG. Additionally, SSG administered i.v. enhanced phagocytic activity, H2O2 production and interleukin 1 (IL-1) production, and the kinetics of the activation differed depending on the activities. These data suggest that the effects of SSG on macrophage functions are different depending on administration routes, and there are some different mechanisms in the activation of macrophages in vivo by SSG.     

Separation of the hot water extracts of sclerotia of Sclerotinia sclerotiorum IFO 9395 into mitogenic and antitumor-active subfractions

The hot water extract of sclerotia of Sclerotinia sclerotiorum IFO 9395 (TSHW) was divided into representative fractions by ammonium sulfate and ethanol precipitations, and (1----3)-beta-D-glucanase treatment. The ammonium sulfate and ethanol precipitations gave a (1----3)-beta-D-glucan fraction (TSG) and a mannan fraction (TSM). After the degradation of (1----3)-beta-D-glucan in TSHW by (1----3)-beta-D-glucanase treatment, a water-insoluble protein fraction (EDP) and supernatant (EDS) were obtained. Among these fractions, the mitogenic and antitumor activities were mainly observed in EDP and TSG, respectively. On the other hand, the stimulatory effect on the reticuloendothelial system was mainly found in EDP and EDS, and a weak effect was observed in TSG. These findings suggest that the mitogenic and antitumor activities of TSHW were mainly due to the protein and (1----3)-beta-D-glucan, respectively, and that the mitogenic substance (EDP) is tightly bound to (1----3)-beta-D-glucan (TSG) in TSHW, accounting for its solubility in aqueous solution.     

Carpachromene Ameliorates Insulin Resistance in HepG2 Cells via Modulating IR/IRS1/PI3k/Akt/GSK3/FoxO1 Pathway

Insulin resistance contributes to several disorders including type 2 diabetes and cardiovascular diseases. Carpachromene is a natural active compound that inhibits α-glucosidase enzyme. The aim of the present study is to investigate the potential activity of carpachromene on glucose consumption, metabolism and insulin signalling in a HepG2 cells insulin resistant model. A HepG2 insulin resistant cell model (HepG2/IRM) was established. Cell viability assay of HepG2/IRM cells was performed after carpachromene/metformin treatment. Glucose concentration and glycogen content were determined. Western blot analysis of insulin receptor, IRS1, IRS2, PI3k, Akt, GSK3, FoxO1 proteins after carpachromene treatment was performed. Phosphoenolpyruvate carboxykinase (PEPCK) and hexokinase (HK) enzymes activity was also estimated. Viability of HepG2/IRM cells was over 90% after carpachromene treatment at concentrations 6.3, 10, and 20 µg/mL. Treatment of HepG2/IRM cells with carpachromene decreased glucose concentration in a concentration- and time-dependant manner. In addition, carpachromene increased glycogen content of HepG2/IRM cells. Moreover, carpachromene treatment of HepG2/IRM cells significantly increased the expression of phosphorylated/total ratios of IR, IRS1, PI3K, Akt, GSK3, and FoxO1 proteins. Furthermore, PEPCK enzyme activity was significantly decreased, and HK enzyme activity was significantly increased after carpachromene treatment. The present study examined, for the first time, the potential antidiabetic activity of carpachromene on a biochemical and molecular basis. It increased the expression ratio of insulin receptor and IRS1 which further phosphorylated/activated PI3K/Akt pathway and phosphorylated/inhibited GSK3 and FoxO1 proteins. Our findings revealed that carpachromene showed central molecular regulation of glucose metabolism and insulin signalling via IR/IRS1/ PI3K/Akt/GSK3/FoxO1 pathway.     

Polysaccharides in fungi. XXIX. Structural features of two antitumor polysaccharides from the fruiting bodies of Armillariella tabescens.

An antitumor polysaccharide containing peptide moieties AT-HW ([alpha]D + 31 degrees in water) and an antitumor polysaccharide AT-AL ([alpha]D + 209 degrees in 1 M sodium hydroxide) were isolated from hot-water extract and the alkaline extract of the fruiting bodies of Armillariella tabescens, respectively. Chemical structures of AT-HW and AT-AL were investigated by a combination of chemical and spectroscopic methods. The results indicate that the major constituent of AT-HW (molecular weight, 105000), a heteroglycan, is composed primarily of beta-(1----6)-linked D-glucopyranosyl and D-galactopyranosyl residues, and contains their branched residues and terminal sugar (gluco-, manno-, and fucopyranose) residues, in addition to beta-(1----3)-linked D-glucopyranosyl residues, while AT-AL (molecular weight, 93000) is chiefly composed of alpha-(1----3)-linked D-glucopyranosyl residues.     

Physicochemical characteristics and antitumor activities of a highly branched fungal (1----3)-beta-D-glucan, OL-2, isolated from Omphalia lapidescens.

Physicochemical properties and antitumor activities of a fungal (1----3)-beta-D-glucan, OL-2, isolated from Leiwan (Omphalia lapidescens) were examined. OL-2 showed sharp signals on carbon-13 nuclear magnetic resonance spectrum in dimethylsulfoxide-d6 as a solvent, and these signals were significantly reduced by the addition of distilled water to the concentration of 20%. This phenomenon is consistent with the general property of the gel forming (1----3)-beta-D-glucan. Binding of OL-2 to Congo red induced a significant change of lambda max to a longer wavelength, and the concentration to induce gel to sol transition was about 0.7 N; in contrast, the concentration was about 0.2 N in the cases of SPG and curdlan. These observations suggested that the gel structure would be significantly stabilized in the case of OL-2. OL-2 showed no or low antitumor activity against the solid form of Sarcoma 180 by intraperitoneal and intralesional administrations; however, it was effective on the ascites form of Sarcoma 180. Of interest, OL-2 also showed significant antitumor activity against the ascites form of MH-134 when administered with 5-fluorouracil. These results indicated that OL-2 showed characteristic features regarding its physicochemical properties and antitumor activity.     

Polysaccharides in fungi. XXX. Antitumor and immunomodulating activities of two polysaccharides from the fruiting bodies of Armillariella tabescens.

The effects of two polysaccharides, AT-HW and AT-AL obtained from the fruiting bodies of Armillariella tabescens on murine sarcoma 180 tumor and peritoneal macrophages were examined at intraperitoneal administration. AT-HW from the hot-water extract and AT-AL from the alkaline extract significantly inhibited the tumor, and the results of different administration schedule and phagocytic system blockade suggested that the mechanism of AT-AL differed from that of AT-HW and branched (1----3)-beta-D-glucans. AT-HW and AT-AL showed reticuloendothelial system-potentiating activity, increased the number of peritoneal exudate cells, activated on macrophages (acid phosphatase activity, glucose consumption, superoxide anion production), and enhanced mitogenic reaction, although AT-HW did not produce superoxide anion in vitro.     

Study of complement activation on well-defined surfaces using surface plasmon resonance

It has been accepted that covalent immobilization of C3b on artificial materials is the most important step to initiate the complement activation. However, there are few studies that have directly demonstrated covalent immobilization of C3b on artificial surfaces. In this study, model thin layers were prepared by the self-assembled monolayer method to produce a surface covered with hydroxyl or methyl groups using mercaptododecane (CH₃-SAM) and mercaptoundecanol (OH-SAM). Interactions of the complement system with the model surfaces were studied using a surface plasmon resonance instrument. The OH-SAM immobilized C3b, resulting in activating of the complement system through the alternative pathway in Veronal-buffered saline, but this surface did not activate the classical pathway. However, the OH-SAM could not activate the alternative pathway in Veronal-buffered saline containing 10 mM EGTA and 2 mM MgCl₂ that is believed not to interfere with the activation of the alternative pathway. The hydrophobic CH₃-SAM surface could not activate the classical pathway, but activated the alternative pathway, although the extent was small.     

Conformation-dependent change in antitumor activity of linear and branched (1----3)-beta-D-glucans on the basis of conformational elucidation by carbon-13 nuclear magnetic resonance spectroscopy.

The antitumor activity of (1----3)-beta-D-glucans was tested in order to clarify its conformation-dependent response together with conformational elucidation by carbon-13 nuclear magnetic resonance (13C-NMR) spectroscopy. It was shown that the following three conformations, single chain, single helix and triple helix, are readily distinguished by the high-resolution solid-state 13C-NMR method. It turned out that preparations of linear (1----3)-beta-D-glucans of a triple helical conformation were ineffective in the inhibition of tumor growth. These linear (1----3)-beta-D-glucans were converted to an effective form in the inhibition of tumor growth when they were lyophilized from dimethyl sulfoxide (DMSO) solutions as a result of a conformational change from the triple helical to the single chain forms. They were not effective, however, when assayed in DMSO solution. In contrast, it was found that a branched (1----3)-beta-D-glucan is effective not only in either saline solutions of the triple helical sample or the lyophilized sample from DMSO, but also in DMSO solution. The aforementioned drastic change in antitumor activity was interpreted in terms of resulting conformational changes as analyzed by the 13C-NMR method.     

Structure and antitumor activity of the less-branched derivatives of an alkali-soluble glucan isolated from Omphalia lapidescens. (Studies on fungal polysaccharide. XXXVIII).

The structure and antitumor activity of Smith-type degradation products (OL-2-I, OL-2-II and OL-2-III) of an alkali-soluble glucan, OL-2, isolated from a crude fungal drug "Leiwan" (Omphalia lapidescens) were investigated. Methylation analysis suggested that OL-2-I was a (1----3)-beta-D-glucan with approximately one branch at every three main chain glucosyl units at each C-6 position; OL-2-II was a (1----3)-beta-D-glucan with approximately one branch at twenty four main chain glucosyl units at each C-6 position (number of all main chain glucosyl units is on average). OL-2-I, OL-2-II and OL-2-III which were Smith-type degradation products of OL-2, showed potent antitumor activity against the solid form of sarcoma 180 in ICR mice. These results indicated that the degree of beta-linked branching at position 6 was remarkably related to the antitumor activity.     

Mode of complement activation by acidic heteroglycans from the leaves of Artemisia princeps PAMP.

The mode of action of the anti-complementary acidic heteroglycans, AAF-IIb-2 and IIb-3 which consisted of rhamnogalacturonan core and arabinogalactan moieties, purified from the leaves of Artemisia princeps PAMP (Japanese name = Gaiyo) were investigated. The anti-complementary activities of AAF-IIb-2 and IIb-3 were reduced partially in the absence of Ca2+ ions. A marked consumption of C4 was observed to have occurred when serum was incubated with both polysaccharides in the presence of Ca2+ ions. AAF-IIb-2 showed more potent C4 consumption than IIb-3. After the incubation of the serum with AAF-IIb-2 in the absence of Ca2+ ions, a cleavage of C3 in the serum was detected by immunoelectrophoresis. AAF-IIb-2 showed more significant consumption of the complement than IIb-3 when rabbit erythrocytes were used in the assay system in the absence of Ca2+ ions. These results indicate that AAF-IIb-2 activates the complement via both the alternative and classical pathways, whereas IIb-3 mainly activates the complement via the classical pathway. The absorption of serum with Protein A-Sepharose results in a decrease of the activity of AAF-IIb-2 and IIb-3. However, the decrease of the activity was restored by the replacement of the immunoglobulin G (IgG) fraction after its recovery from the Protein A-Sepharose. These results suggest that IgG dependent mechanisms are both involved in the anti-complementary activity of AAF-IIb-2 and IIb-3.     

Synthesis and antitumor activities of mitomycin C (1----3)-beta-D-glucan conjugate.

The conjugate of mitomycin C (MMC) with linear (1----3)-beta-D-glucan from Alcaligenes faecalis var. myxogenes IFO 13140 was synthesized and its antitumor activities investigated. The conjugate (MMC-carboxymethylated linear (1----3)-beta-D-glucan (CMPS)) was obtained by treatment of CMPS with MMC in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. In vitro cytotoxicity of MMC-CMPS against L1210 leukemia cells was similar to that of MMC. In i.p.-i.p. system in vivo against P388 leukemia in mice, the maximum increase of MMC-CMPS conjugate in life span (ILSmax) was higher than that of MMC but the therapeutic index was reduced. However, the antitumor activity of MMC-CMPS conjugate against subcutaneously implanted sarcoma 180 solid tumor in mice by i.p. administration was similar to that of MMC at a dose of 1.5 mg eq MMC/kg/d x 7 and the reduction of the number of leukocytes caused by MMC was suppressed by attaching MMC to CMPS. In addition, on assay using serum of sarcoma 180 solid tumor-bearing mice with injection of MMC-CMPS conjugate, a drastic loss of tumor cells and an increase in polymorphonuclear leukocytes (PMN) were observed. This result suggested that MMC-CMPS conjugate induced tumor-regressing factor similar to CMPS.     

Preparation and antitumor activity of hydroxyethylated derivatives of 6-branched (1----3)-beta-D-glucan, SSG, obtained from the culture filtrate of Sclerotinia sclerotiorum IFO 9395

SSG is an antitumor branched (1----3)-beta-D-glucan obtained from the culture filtrate of Sclerotinia sclerotiorum IFO 9395. Hydroxyethylation of SSG higher than MS 0.45 (MS value represents molar ratio of hydroxyethyl group vs. glucosyl group) by ethyleneoxide in aqueous sodium hydroxide lose the antitumor activity. Degradation of branching point of hydroxyethylated SSG (HE-SSG) by the sequential treatments of periodate oxidation, borohydride reduction, and mild acid hydrolysis of these derivatives regenerated the antitumor activity. These results directly demonstrated that the branching point covered, at least a part of, the dormant active site of SSG.     

Polysaccharides in fungi. XXVI. Two branched (1----3)-beta-D-glucans from hot water extract of Yu ?r

Two water-insoluble glucans, U-3-N ([alpha]D +1.0 degree, 0.5 M sodium hydroxide) and U-3-AP1 ([alpha]D +2.5 degrees, 1 M sodium hydroxide) were isolated from hot-water extract of the fruiting bodies of Y? ?r (Chinese name) (Auricularia sp.). U-3-N and U-3-AP1 were investigated by a combination of chemical and spectroscopic methods. The results indicated that U-3-N (molecular weight, 6.1 x 10(5)) was similar to beta-(1----6)-branched (1----3)-beta-D-glucan (N-5P: molecular weight, 5.6 x 10(5)) isolated from the alkaline extract of the fruiting bodies, and U-3-AP1 (molecular weight, 6.3 x 10(4)) was beta-(1----6)-branched (1----3)-beta-D-glucan containing beta-(1----6)-linked D-glucopyranosyl residues. U-3-N showed potent anti-tumor activity against the solid form of sarcoma 180, although U-3-AP1 had little effect on the tumor.     

Natural Compound 3β,7β,25-trihydroxycucurbita-5,23(E)-dien-19-al from Momordica charantia Acts as PPARγ Ligand

Momordica charantia is a popular vegetable associated with effective complementary and alternative diabetes management in some parts of the world. However, the molecular mechanism is less commonly investigated. In this study, we investigated the association between a major cucurbitane triterpenoid isolated from M. charantia, 3β,7β,25-trihydroxycucurbita-5,23(E)-dien-19-al (THCB) and peroxisome proliferator activated receptor gamma (PPARγ) activation and its related activities using cell culture and molecular biology techniques. In this study, we report on both M. charantia fruit crude extract and THCB in driving the luciferase activity of Peroxisome Proliferator Response Element, associated with PPARγ activation. Other than that, THCB also induced adipocyte differentiation at far less intensity as compared to the full agonist rosiglitazone. In conjunction, THCB treatment on adipocytes also resulted in upregulation of PPAR gamma target genes expression; AP2, adiponectin, LPL and CD34 at a lower magnitude compared to rosiglitazone’s induction. THCB also induced glucose uptake into muscle cells and the mechanism is via Glut4 translocation to the cell membrane. In conclusion, THCB acts as one of the many components in M. charantia to induce hypoglycaemic effect by acting as PPARγ ligand and inducing glucose uptake activity in the muscles by means of Glut4 translocation.     

Isolation of mitogenic substance from sclerotia of Sclerotinia sclerotiorum IFO 9395 extracted with phosphate buffer.

The buffer extracts (3S) of sclerotia of Sclerotinia sclerotiorum IFO 9395 contained mitogenic substance(s) to murine splenocytes (Shinohara et al. Chem. Pharm. Bull., 38, 2219 (1990)). Although the native 3S was slightly mitogenic, heating of 3S induced significant mitogenic activity. To isolate the mitogen, we separated 3S by ion-exchange and gel filtration chromatographies. The isolated mitogen, named sclerogen, has a molecular mass of 32 kilodaltons (kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the isoelectric point (pI) of 5.9 by chromatofocusing. Sclerogen was significantly mitogenic in vitro against murine splenocytes after heat denaturation, and also showed the augmentation of the primary mixed lymphocyte reaction (MLR) in vitro. However, sclerogen did not show the activation of an alternative pathway of complement and hemagglutination activity. These results suggest that sclerogen is a unique mitogen which differs from lectins and shows mitogenicity after heat denaturation.     

Covalent attachment of lysozyme to cotton/cellulose materials: protein verses solid support activation

Covalent attachment of enzymes to cellulosic materials like cotton is of interest where either release or loss of enzyme activity over time needs to be avoided. The covalent attachment of an enzyme to a cellulosic substrate requires either activation of a protein side chain or an organic functional group on the cellulosic substrate. Use of a water soluble carbodiimide to create an amide linkage as the covalent attachment between the enzyme and substrate represents an aqueous-based alternative which may be preferred for textile processes. Here we describe an amide bond-mediated lysozyme immobilization applied to cotton where either the carboxylate side chains of the protein or pendant carboxylates in a citrate, cross-linked cotton support are activated as the O-acyl-isourea intermediate, and the reactive amino nucleophiles are derived from amino-silanized cotton and the protein’s amino side chains, respectively. A comparison is made of the two activation approaches to covalently link lysozyme to two different cotton fabrics using the water soluble carbodiimide 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluene sulfonate. A comparison of the resulting enzyme activities of lysozyme on two different cotton supports showed that linking lysozyme to citrate crosslinked cotton gave higher activity than on aminosilanized cotton. The lysozyme-cellulose conjugate formed on the citrate crosslinked nonwoven cotton fabric gave the highest yield and antimicrobial activity.     

Biosynthesis, total syntheses, and antitumor activity of tanshinones and their analogs as potential therapeutic agents

Tanshinones are a series of abietane diterpenes, isolated exclusively from Salvia miltiorrhiza and related species. More than 40 tanshinones and their analogs have been isolated since the 1930s. Their biosynthetic pathway correlates with the MEP/DOXP pathway, and many key enzymes, such as mCPS, are responsible for establishing their molecular scaffolds and stereospeci?city. Because of their unique structural characteristics and promising biological activities, total syntheses of various tanshinones have attracted the interest of many synthetic chemists, including R. H. Thomson, H. Kakisawa, R. L. Danheiser, Y. Inouye and J. K. Snyder. Tanshinones and their analogs exhibit interesting and broad antitumor activity in various cell and animal models. Most recently, the tanshinone analog neo-tanshinlactone has shown potent and selective activity against breast cancer. This review will discuss the biosynthesis, total syntheses, and antitumor activities of tanshinones,especially neo-tanshinlactone and its analogs.     

Synthesis,Biological Evaluation and Molecular Docking of Novel Phenylpyrimidine Derivatives as Potential Anticancer Agents

Based on our previous researches, a novel phenylpyrimidine pharmacophore model was proposed and fifteen derivatives were synthesized and characterized by means of spectroscopy methods. The inhibitory effects of them were screened against HeLa cell line by virtue of MTT assay in vitro. The results indicate some of the phenylpyrimidine derivatives exhibit potent biological activities. Among them, compounds 6g and 6h exhibit the best activity at half maximal inhibitory concentrations of 1.5 and 2.8 μmol/L, respectively. These compounds also exhibit good activities against HepG2 cell line and MCF-7 cell line. FLT-3 kinase was screened as the most potent molecular target. Computational docking between compound 6g and FLT-3 was carried out to interpret the binding mode. The results show phenylpyrimidine derivatives have effective antitumor activities, which provides a base for further research of them as antitumor agents.