Discrimination between endotoxin and (1----3)-beta-D-glucan using turbidimetric kinetic assay with Limulus amebocyte lysate

A procedure for the Limulus amebocyte lysate (LAL) test discriminating between endotoxin and (1----3)-beta-D-glucan based on the turbidimetric kinetic method was proposed. Endotoxin and (1----3)-beta-D-glucan, which are elicitors of the activation of LAL, showed different reaction courses with this lysate. To analyze the difference in the reactions, two parameters, the maximum differential coefficient of the reaction (Dmax) and the reaction time required to obtain Dmax (Tp) were defined. The logarithmic plottings of Tp versus Dmax (Tp-Dmax plot) discriminated between endotoxin and (1----3)-beta-D-glucan. Endotoxin was measured with a standard curve plotting logarithmic endotoxin concentration versus Dmax (ET-Dmax plot). The endotoxin calculated from Dmax was less influenced by (1----3)-beta-D-glucan than that calculated from the usual gelation time. A small amount of endotoxin in a sample could be concealed by the addition of polymyxin B, which inhibited the activation of LAL by endotoxin. (1----3)-beta-D-glucan was measured without being affected by the presence of a small amount of endotoxin using LAL with polymyxin B. The following procedure is proposed as a LAL test to discriminate between endotoxin and (1----3)-beta-D-glucan. (1) Identify the main substance (endotoxin or (1----3)-beta-D-glucan) triggering the activation of LAL using the Tp-Dmax plot. (2) Use the appropriate method to measure the main substance: the ET-Dmax plot for endotoxin or the LAL with polymyxin B for (1----3)-beta-D-glucan.     

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.     

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.     

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.     

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.     

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.     

Antifungal activity of bivittoside-D from Bohadschia vitiensis (Semper)

This study was carried out to investigate the antifungal activity of Bohadschia vitiensis Semper whole body extracts, followed by isolation and characterisation of bioactive molecules. The methanol extract of the B. vitiensis showed promising activity in in?vitro models against Candida albicans, Cryptococcus neoformans, Sporothrix schenckii, Trichophyton mentagrophytes, Aspergillus fumigatus and Candida parapsilosis. The antifungal activity was found in aqueous fraction against C. albicans, C. neoformans, S. schenckii, T. mentagrophytes and A. fumigatus. The major compound was purified from the aqueous fraction and was identified as bivittoside-D isolated earlier from the animal. It showed promising results against C. neoformans, C. neoformans, S. schenckii, T. mentagrophytes, A. fumigatus and C. parapsilosis.     

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.     

In vitro fungicidal photodynamic effect of hypericin on Candida species

Hypericin is a natural photosensitizer considered for the new generation of photodynamic therapy (PDT) drugs. The aim of this study was to evaluate the in vitro fungicidal effect of hypericin PDT on various Candida spp., assessing its photocytotoxicity to keratinocytes (HaCaT) and dermal fibroblasts (hNDF) to determine possible side effects. A 3 log fungicidal effect was observed at 0.5 McFarland for two Candida albicans strains, Candida parapsilosis and Candida krusei with hypericin concentrations of 0.625, 1.25, 2.5 and 40 μm, respectively, at a fluence of 18 J cm(-2) (LED lamp emitting at 602 ± 10 nm). To obtain a 6 log reduction, significantly higher hypericin concentrations and light doses were needed (C. albicans 5 μM, C. parapsilosis 320 μM and C. krusei 320 μM; light dose 37 J cm(-2)). Keratinocytes and fibroblasts can be preserved by keeping the hypericin concentration below 1 μm and the light dose below 37 J cm(-2). C. albicans appears to be suitable for treatment with hypericin PDT without significant damage to cutaneous cells.     

Inhibition of experimental pulmonary metastasis of Lewis lung carcinoma by orally administered beta-glucan in mice

The inhibitory effect on experimental pulmonary metastasis of Lewis lung carcinoma (3LL) of SSG, a (1----3)-beta-D-glucan obtained from the fungus Sclerotinia sclerotiorum IFO 9395, administered orally was examined in mice. Oral administration of SSG for 10 consecutive days just after the intravenous implantation of tumor cells significantly inhibited the experimental pulmonary metastasis of 3LL at a dose of 2000 micrograms. However, SSG administered orally involving other timings was less effective. In comparison with oral administration, SSG was effective when administered intraperitoneally for 10 consecutive days at a dose of 200 micrograms. These results suggest that SSG given by both parenteral and nonparenteral routes is effective in the inhibition of experimental pulmonary metastasis of tumors.     

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.     

Chemical constituents of the methanolic extract of leaves of Leiothrix spiralis Ruhland and their antimicrobial activity

Chemical fractionation of the methanolic extract of leaves of Leiothrix spiralis Ruhland afforded the flavonoids luteolin-6-C-β-D-glucopyranoside (1), 7-methoxyluteolin-6-C-β-D-glucopyranoside (2), 7-methoxyluteolin-8-C-β-D-glucopyranoside (3), 4'-methoxyluteolin-6-C-β-D-glucopyranoside (4), and 6-hydroxy-7-methoxyluteolin (5), and the xanthones 8-carboxymethyl-1,5,6-trihydroxy-3-methoxyxanthone (6), 8-carboxy-methyl-1,3,5,6-tetrahydroxyxanthone (7). Methanolic extract, fractions, and isolated compounds of the leaves of L. spiralis were assayed against Gram-positive (Staphylococcus aureus, Bacillus subtilis and Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella setubal and Helicobacter pylori) and fungi (the yeasts Candida albicans, C. tropicalis, C. krusei and C. parapsilosis). We observed the best minimum inhibitory concentration values for the methanolic extract against Candida parapsilosis, for the fraction 5 + 6 against Gram-negative bacteria E. coli and P. aeruginosa, and compound 7 against all tested Candida strains. The methanolic extract contents suggest that this species may be a promising source of compounds to produce natural phytomedicines.     

Study on the constituents of Desmodium styracifolium

Two triterpenoid saponins (1 and 2) were isolated from Desmodii Herba [the dried whole plants of Desmodium styracifolium (Osbeck) Merr. (Leguminosae)] and their chemical structures were characterized as soyasaponin I and a new saponin, 3-O-[alpha-L-rhamnopyranosyl-(1----2)-beta-D-galactopyranosyl- (1----2)-beta-D-glucuronopyranosyl]soyasapogenol E, respectively, by chemical and spectroscopic means.     

Separation of phenotypically indistinguishable Candida species, C. orthopsilosis, C. metapsilosis and C. parapsilosis, by capillary electromigration techniques

At the current state of laboratory diagnostics, methods for fast identification of phenotypically indistinguishable species are difficult or inaccurate. An example is represented by Candida parapsilosis, which is the second most common yeast species isolated from bloodstream infections. C. parapsilosis comprises a complex of three genetically distinct groups. Genotypes II and III have been designated as the separate species Candida orthopsilosis and Candida metapsilosis, phenotypically indistinguishable. The considerable genetic variability of these newly described yeasts species has caused difficulties in the development of molecular techniques for their precise identification. Similarly, the detection of biofilm formation, which is considered as an important yeast virulence factor, is accompanied by difficulties. In this study we optimize the first precise and reproducible method for the separation and possible identification of C. orthopsilosis, C. metapsilosis and C. parapsilosis as well as the detection of their ability to form biofilm. The method is based on capillary isoelectric focusing and capillary electrophoresis with UV detection. In capillary isoelectric focusing, very narrow pH gradients were established. With such gradients, differences in isoelectric points of biofilm-negative and biofilm-positive species calculated from the migration times of the selected pI markers were below 0.03 pI units. In the capillary zone electrophoresis narrow zones of the cells of Candida species were detected with sufficient resolution. The values of the isoelectric point and the migration velocities of the examined species were independent on the origin of the tested strains. Capillary isoelectric focusing was examined also for the separation and detection of the cultivated biofilm-negative C. parapsilosis in the blood serum.     

布洛芬-扑热息痛孪药的合成

以布洛芬（IPF）和扑热息痛（PCM）为原料,无水丙酮和乙酸乙酯为溶剂,二环己基碳二亚胺（DCC）为脱水剂,4-二甲氨基吡啶（DMAP）为催化剂,合成了布洛芬-扑热息痛孪药（PHI）,以重结晶法对目标产物进行纯化,产物结构经IR和1H NMR确定。 探讨了原料比例、脱水剂用量、催化剂用量、反应时间和溶媒用量对目标物产率的影响,并采用正交试验筛选最优合成工艺,最终确定的最优反应条件为:n(PCM)∶n(IPF)=1∶1.5,催化剂的最佳用量为布洛芬质量的10%,反应时间为8 h,溶媒体积为70 mL（V(乙酸乙酯)∶V(丙酮)=1∶1）,在该条件下,目标物PHI的产率为66.53%。     

Chemical composition and anticandidal properties of the essential oil isolated from aerial parts of Cotula cinerea: a rare and threatened medicinal plant in Morocco

The chemical composition and anticandidal properties of the essential oil of Moroccan Cotula cinerea aerial parts have been examined. GC-MS data were used to identify 24 constituents. Oxygenated monoterpenes constituted the main fraction with trans-thujone (41.4%), cis-verbenyl acetate (24.7%), 1,8-cineole (8.2%) and camphor (5.5%) as the major components. The anticandidal activity of the essential oil was evaluated using a panel of human pathogenic fungi (Candida albicans CCMM L4 and CCMM L5, C. krusei CCMM L10, C. glabrata CCMM L7 and C. parapsilosis CCMM L18). The oil showed high anticandidal activity against all investigated strains with minimal inhibitory concentrations of 3.2 to 4.7 mg/mL depending on the tested yeast and 5.9 mg/mL as a minimal candidicidal concentration value. These findings add significant information to the pharmacological activity of Cotula cinerea essential oil, which may present a good alternative to antibiotics for the treatment of resistant strains of Candida.     

近平滑假丝酵母细胞催化乙酰基三甲基硅烷不对称还原反应

 采用近平滑假丝酵母细胞用于催化乙酰基三甲基硅烷不对称还原反应, 可高选择性地生成 (R)-1-三甲基硅乙醇. 结果表明, 固定化于海藻酸钙的细胞催化该反应的产物收率比游离细胞的高. 不同辅底物对该反应的影响显著, 以葡萄糖为辅底物时, 反应的初速率较快, 产物收率较高. 该反应的最适条件为: 辅底物 (葡萄糖) 浓度 110 mmol/L, 振荡速度 180 r/min, 缓冲液 pH 值 6.0, 反应温度 30 oC, 底物浓度 20 mmol/L. 在此反应条件下反应的初速率、产物收率和产物的 ee 值分别为 11.4 μmol/h, 96.5% 和 99.9%.     

Inactivation of <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilms on Polymethyl Methacrylate and Enhancement of the Drug Susceptibility by Cold Ar/O<Subscript>2</Subscript> Plasma Jet

The antimicrobial effects of the cold plasma for the dental pathogenic microorganism propose a promising approach to the Denture Stomatitis (DS) treatment. However, it is crucial to understand that the complexity of the biofilm microenvironment may compromise the efficiency of the therapy. As one of the major issue for DS, Candida albicans biofilms (ATCC10231) formed on denture base resins were treated by cold Ar/O₂ (2 %) plasma jet. Spatial viability of the biofilms was investigated with confocal scanning laser microscopy through evaluating their inside cross-section properties. Results showed Candida albicans biofilms with thickness of ~100 µm was completely inactivated by 8 min plasma treatment. Morphology change of the fungi was also observed by the scanning electron microscopy. Drug susceptibilities, the sessile minimum inhibitory concentration (SMIC50) of the biofilm for amphotericin B and fluconazole were decreased from >32 and >256 µg/mL to 8 and 64 µg/mL after 1 min’s plasma treatment, respectively. The reactive species produced from plasma were monitored by optical emission spectroscopy. The successfully inactivation of Candida albicans biofilms and the significant enhancement of its drug susceptibilities induced by the plasma released reactive species propose a promising strategy for the treatment of DS caused by drug-resistant Candida albicans biofilms.     

Non-Thermal Plasma Assisted Regeneration of Acetone Adsorbed TiO2 Surface

Improvement of indoor air quality regarding volatile organic compounds (VOCs) requires the development of innovative oxidation processes. This paper investigates the coupling of a metal oxide sorbent with non-thermal plasma (NTP) in an especially designed reactor. TiO₂ was selected as model sorbent and acetone was used as model VOC. The analyses of gas phase species at the reactor downstream have been performed using FTIR spectroscopy. In a first step, acetone adsorption on TiO₂ surface under dry air was characterized in terms of total amount adsorbed, as well as reversibly and irreversibly adsorbed fractions. Obtained results were compared and discussed with literature in terms of acetone reactive adsorption on TiO₂ surface. Mesityloxide was proposed as the major compound in the irreversibly adsorbed fraction. In a second time, acetone saturated TiO₂ surface was exposed to NTP surface discharge. Irrespectively of the injected power, <30 % of the initially adsorbed acetone has been recovered as CO, CO₂ and desorbed acetone. Finally, thermal desorptions have been performed. They evidenced that (1) NTP treatment modifies the nature of the adsorbed organic species, (2) mineralization rate is considerably improved. Based on desorbed species temporal profile analysis, carboxylates and more especially formates are suggested as major adsorbed species after NTP treatment (P_inj > 0.2 W). This hypothesis has been evaluated and confirmed. This paper finally evidenced that NTP can be used as an efficient pretreatment technique to promote the mineralization of adsorbed acetone for further thermal treatment.     

Mitogenic and colony-stimulating factor-inducing activities of polysaccharide fractions from the fruit bodies of Dictyophora indusiata Fisch

Biological effects (mitogenic and colony-stimulating factor (CSF)-inducing activities) of five homogeneous polysaccharides and a conjugated polysaccharide fraction isolated from the fruit bodies of Dictyophora indusiata Fisch. were investigated. Fucomammogalactan (T-3-Ad) and conjugated polysaccharide fraction (T-2-A) exhibited significant mitogenic and CSF-inducing activities. Among two beta-(1----6)-branched (1----3)-beta-D-glucans (T-4-N and T-5-N), only T-4-N showed both mitogenic and CSF-inducing effects. Partially O-acetylated (1----3)-alpha-D-mannans (T-2-HN and T-3-M') did not show these effects.