Application of capillary electrophoresis mass spectrometry to the characterization of bacterial lipopolysaccharides

Capillary electrophoresis (CE) is a high-resolution technique for the separation of complex biological mixtures and has been widely applied to biological analyses. The coupling of capillary electrophoresis with mass spectrometry (MS) provides a powerful approach for rapid identification of target analytes present at trace levels in biological matrices, and for structural characterization of complex biomolecules. Here we review the analytical potential of combined capillary electrophoresis electrospray mass spectrometry (CE-MS) for the analysis of bacterial lipopolysaccharides (LPS). This hyphened methodology facilitates the determination of closely related LPS glycoform and isoform families by exploiting differences in their unique molecular conformations and ionic charge distributions by electrophoretic separation. On-line CE-MS also provides an additional avenue to improve detection limits, which has been successfully applied to directly probe oligosaccharide LPS glycoform populations of bacteria isolated from infected animal models without the need for further passage.     

Production of Glycolipid Biosurfactant from Sponge-Associated Marine Actinobacterium <Emphasis Type="Italic">Brachybacterium paraconglomeratum</Emphasis> MSA21

Potential biosurfactant producers and economic production processes are major considerations for commercialization of biosurfactants. The present study was aimed at exploring marine Actinobacteria for the production of biosurfactants using industrial and agro-industrial wastes under solid state culture (SSC). A biosurfactant producer, Brachybacterium paraconglomeratum MSA21 was isolated from a marine sponge. The strain MSA21 effectively utilized tannery pre-treated effluent as the substrate for the production of a biosurfactant under SSC. The critical control factors influence the production of biosurfactant includes glucose, yeast extract, copper sulfate and inoculum size. The glucose and yeast extract interactively increase the production maxima over a stable area. The surface active compound was characterized as a glycolipid derivative with a hydrophilic part of methyl-2-oxopropyl furan and a hydrophobic dodecanoic acid, methyl ester. The MSA21 biosurfactant displayed antibiotic activity. The domain ketosynthase in MSA21 showed that the polyketide synthase gene might be involved in the synthesis of antimicrobial compounds. The strain B. paraconglomeratum MSA21 could be used for the production of a biosurfactant as a green alternative to replace chemical surfactants.     

Cooperation of phosphatidylcholine with endogenous lipids of wheat flour for an increase in dough volume

Phosphatidylcholine (PC) increases the gas-retaining ability of dough, the dough volume on fermentation and the loaf volume of bread. The cooperation of wheat flour endogenous lipids with PC was examined. More than 90% of the total wheat flour lipids were extracted with chloroform, the extracted lipids comprising glycolipids (33 wt%), non-polar lipids (56 wt%), and phospholipids (11 wt%). The increase in the specific volume of dough with delipidated wheat flour by the addition of PC was smaller than the increase in the specific volume of dough with native wheat flour. The addition of the extracted lipids to delipidated wheat flour restored the increase in dough volume by the addition of PC. The glycolipid fraction of the extracted lipids was most effective for enhancing the action of PC. The results suggest that interaction of PC with wheat flour glycolipids may synergistically increase foam stability to enhance the gas-retaining stability of dough.     

In Vitro Apoptosis Induction in a Human Prostate Cancer Cell Line by Thermotolerant Glycolipid from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> SV1

A highly potent glycolipid was isolated from a crude oil‐contaminated soil bacterium Bacillus licheniformis SV1 (NCBI GenBank Acc. No. KX130852) when grown on a modified mineral salt medium supplemented with 2% oleic acid. The maximum reduction in surface tension of cell‐free broth from 71 ± 0.812 to 25.919 ± 0.984 mN/m with 89 ± 0.346% emulsification activity was recorded after 120 h of growth. Glycolipid was purified using chromatographic techniques and the presence of aliphatic chain (C–H stretch) and OH‐band was revealed by NMR, GC–MS and FTIR analysis. Stability of glycolipid up to 250 °C and its complete decomposition at 507 °C was recorded by thermogravimetric analysis (TGA). MTT assay (IC₅₀ = 0.473 ± 0.048 mg/ml) along with 4′,6‐diamidino‐2‐phenylindole (DAPI) and acridine orange and ethidium bromide (AO/EtBr) staining validated that glycolipid induces cell apoptosis in human prostate cancer cell line (PC‐3). Furthermore, potent anti‐cancerous compounds such as 9‐octadecanoic acid (22.55%), linoleic acid methyl ester (2.2%) and palmitic acid (1.18%) were also detected in the GC–MS spectra of the purified glycolipid.     

Genotypic dependent effect of exogenous glutathione on Cd-induced changes in proteins, ultrastructure and antioxidant defense enzymes in rice seedlings

Greenhouse hydroponic experiments were conducted using Cd-sensitive (cv. Xiushui63) and tolerant (Bing97252) rice genotypes to evaluate how different genotypes responded to Cd toxicity in presence of glutathione (GSH). Results showed that GSH alleviates Cd-toxicity, ameliorates Cd-induced damages on leaf/root ultrastructures. Nine proteins in roots were identified, using 2-DE coupled with mass spectrometry, whose expression were down-regulated in Xiushui63, up-regulated/unchanged in Bing97252 by Cd; coinstantaneously enhanced/unchanged in Cd + GSH over Cd alone treatment in both genotypes. They are l-ascorbate peroxidase, putative short-chain dehydrogenase/reductase, Glycolipid transfer protein, elongation factor, Os04g0652700, carbonic anhydrase, Os08g0374000, chitinase, and putative disease resistance response protein. Eight proteins in leaves with expression of increase in Bing97252 but down-regulate/unchange in Xiushui63, categorized as four groups of their functions: carbon metabolism, TCA cycle, photorespiration and RNA processing. Furthermore, we identified eight proteins with repressed expression in Cd-treated and up-regulated in Cd + GSH-treated rice leaves of Xiushui63.     

Lipidomic Analysis of Clostridium cadaveris and Clostridium fallax

The lipidomes of Clostridium fallax and Clostridium cadaveris were studied using thin-layer chromatography (TLC) and normal phase liquid chromatography/mass spectrometry (NPLC/MS). Both species contain diradylglycerol (DRG), monohexosyldiradylglycerol (MHDRG), monohexosyl monoacylglycerol (MHMAG), phosphatidylglycerol (PtdGro), and phosphatidylethanolamine (PtdEtn). DRG, MHDRG, PtdEtn, and PtdGro are present in both diacyl and alk-1-enyl acyl (plasmalogen) forms. Both species contain cardiolipin (Ptd₂Gro), which is present in tetraacyl, monoalkenyl-triacyl, and dialkenyl-diacyl forms. Both species contain small amounts of phosphatidylcholine (PtdCho). The presence of octadecadienoic (18:2) acyl chains in some PtdCho species indicates that they arise from the medium because no 18:2 is seen in the other lipids and clostridia generally lack the capacity to synthesize polyunsaturated fatty acids. The major lipidomic differences between these two species are that C. fallax contains a glycerolacetal of plasmenylethanolamine while C. cadaveris contains an ethanolamine-phosphate-modified diacylglycerol. The significance of these lipid compositions is discussed.     

Distribution of Glycolipid and Unsaturated Fatty Acids in Human Hair

It has been recognized that human hair lipids play crucial roles in the integrity of cells and matrices, while the details of distribution and structure of the minor lipids are hardly known. Here we investigated the lipids at the hair surface, at the interface between cuticle and cortex and in the interior of hair (cortex, medulla and melanin granules). Hair lipids and fatty acids and their metabolites were detected and characterized by using infrared spectroscopy and several mass spectrometry techniques (FTIR, ToF–SIMS, GCMS, and ESI–MS). As a result, it was found that unsaturated fatty acids were present more in the cortex of hair than at the hair surface. At the interface between cuticle and cortex, it is suggested that steryl glycoside‐like lipids containing N‐acetylglucosamine were present, and contributing to the adhesion between the cuticle and cortex of hair. Oxidative metabolites derived from integral fatty acids such as linoleic and alpha‐linolenic acids were found in the hair bulb and melanin granules. Especially the oxidative metabolites of alpha‐linolenic acid were integrated into the lipids non‐covalently and tightly bound to melanin granules (namely, melanin lipids) and suggested as being involved in the biosynthetic processes of melanosome.     

Anti-Tumor Effect of Orally Administered Spinach Glycolipid Fraction on Implanted Cancer Cells, Colon-26, in Mice

We succeeded in purifying a major glycolipid fraction from a green vegetable, spinach. This fraction consists mainly of three glycolipids: monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), and sulfoquinovosyl diacylglycerol (SQDG). In a previous study, we found that the glycolipid fraction inhibited DNA polymerase activity, cancer cell growth and tumor growth with subcutaneous injection. We aimed to clarify oral administration of the glycolipid fraction, suppressing colon adenocarcinoma (colon-26) tumor growth in mice. A tumor graft study showed that oral administration of 20 mg/kg glycolipid fraction for 2 weeks induced a 56.1% decrease in the solid tumor volume (P < 0.05) without any side-effects, such as loss of body weight or major organ failure, in mice. The glycolipid fraction induced the suppression of colon-26 tumor growth with inhibition of angiogenesis and the expression of cell proliferation marker proteins such as Ki-67, proliferating cell nuclear antigen (PCNA), and Cyclin E in the tumor tissue. These results suggest that the orally administered glycolipid fraction from spinach could suppress colon tumor growth in mice by inhibiting the activities of neovascularization and cancer cellular proliferation in tumor tissue.     

黄精多糖预防小鼠糖脂代谢紊乱的作用研究

目的 观察黄精多糖（polygonatumsibiricum polysaccharides, PSP）对C57小鼠糖脂代谢功能的保护作用,根据中医药治未病的理念初步探讨黄精多糖对小鼠糖尿病的预防作用。方法 对C57小鼠进行黄精多糖灌胃干预,然后采用高脂饮食联合低剂量链脲佐菌素使小鼠糖脂代谢紊乱,32只小鼠随机分为正常组、PSP高剂量组（800 mg/kg）、PSP低剂量组(400 mg/kg)与模型组。实验后期测定各组小鼠体重、空腹血糖（fasting blood glucose ,FBG）、餐后血糖耐量曲线(oral glucose tolerance test ,OGTT)、空腹胰岛素(fasting blood insulin ,FINS),计算胰岛素抵抗指数(HOMA-IR);检测小鼠血清总胆固醇(total cholesterol ,TC)、总甘油三酯(total triglyceride , TG)、高密度脂蛋白(high density lipoprotein ,HDL-C)、低密度脂蛋白(low density lipoprotein ,LDL-C)以及肝脏TG的含量,观察肝脏组织油红O染色切片,利用实时定量逆转录聚合酶链反应测定肝脏组织中磷脂酰肌醇3-激酶（phosphoinositide-3-kinase ,PI3K）、蛋白激酶B(protein kinase B, AKT）信号分子的mRNA表达水平。结果 经黄精多糖的灌胃干预后,与模型组小鼠相比,PSP高剂量组小鼠的体重、FBG、FINS、HOMA-IR、TC、TG、HDL、LDL等代谢指标水平显著性降低(P<0.05),PSP低剂量组小鼠体重、糖耐量曲线面积以及肝脏TG水平显著性降低(P<0.05),其他代谢指标与模型组无显著性差异,PSP高、低剂量组小鼠肝脏脂质沉积现象明显改善且成剂量依赖性,PSP高剂量能够增强小鼠肝脏组织中PI3K/AKT信号通路中PI3K、AKT分子mRNA的表达水平。结论 黄精多糖的灌胃干预可以有效保护小鼠的糖脂代谢功能,从而预防小鼠糖尿病的发生发展,并且该种作用可能与增强PI3K、AKT信号分子的表达水平有关。