Phosphatidylglucoside exists as a single molecular species with saturated fatty acyl chains in developing astroglial membranes

We previously found that phosphatidylglucoside (PtdGlc), a novel glycolipid expressed in HL60 cells, plays a role in forming signaling microdomains involved in cellular differentiation. Because cells contain minute levels of PtdGlc, pure PtdGlc is very difficult to isolate. Thus, its complete structure has never been assessed. To aid in analyzing PtdGlc, we generated a PtdGlc-specific monoclonal antibody, DIM21, by immunizing mice with detergent-insoluble membranes isolated from HL60 cells [Yamazaki, Y., et al. (2006) J. Immunol. Methods 311, 106-116]. DIM21 immunostaining of murine CNS tissues revealed stage- and cell type-specific localization of the DIM21 antigen during development, with especially high levels of expression in radial glia/astroglia. DIM21 immunostained cultured hippocampal astroglia in a punctate fashion. To characterize the structure of PtdGlc, we isolated DIM21 antigen from fetal brains. Using successive column chromatography, we purified two previously unrecognized glycolipids, PGX-1 and PGX-2, from embryonic day 21 rat brains. DIM21 reacted more strongly to PGX-2 than to PGX-1. Structural analyses with 600 MHz (1)H NMR, FT-ICR mass spectrometry, and GC revealed that PGX-1 is phosphatidyl beta-d-(6-O-acetyl)glucopyranoside and PGX-2 is phosphatidyl beta-d-glucopyranoside. The yields of PGX-1 and PGX-2 were approximately 250 +/- 150 and 440 +/- 270 nmol/g of dried brains, respectively. Surprisingly, both glycolipids were composed exclusively of C18:0 at the C1 position and C20:0 at the C2 position of the glycerol backbone. This saturated fatty acyl chain composition comprising a single molecular species rarely occurs in known mammalian lipids and provides a molecular basis for why PtdGlc resides in raftlike lipid microdomains.     

Phosphatidylglucoside: its structure, thermal behavior, and domain formation in plasma membranes

Phosphatidylglucoside (PtdGlc) is a unique glyco-glycerophospholipid that is found in both bacterial and mammalian cells. The discovery of PtdGlc in mammalian cells is relatively recent (Nagatsuka et al., 2001. FEBS Lett. 497, 141-147). Chemical structural analysis of the PtdGlc found in mammalian organs and cultured cells showed that PtdGlc is composed exclusively of a single pair of saturated fatty acid chains; the sn-1 chain is stearic acid (C18:0) and the sn-2 chain is arachidic acid (C20:0). PtdGlc forms distinct domains, which are different from cholesterol-based sphingolipid domains, on the outer leaflet of the plasma membrane. In this review, we summarize recent studies of PtdGlc. Special attention is paid to the thermal behavior of PtdGlc in a pure system and in mixtures with other lipid components that may relate to the formation of PtdGlc domains in biomembranes. Finally, we discuss proposed biological functions of PtdGlc based on recent experimental results.     

A fast and simple assay for busulfan in serum or plasma by liquid chromatography-tandem mass spectrometry using turbulent flow online extraction technology

Busulfan is used in myeloablative preparation regimens for hematopoietic bone marrow transplantation. Due to its narrow therapeutic range therapeutic drug monitoring of busulfan is recommended. In this study a fast and simple method for measuring busulfan in serum or plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed utilizing turbulent flow online extraction technology. Serum or plasma was mixed with acetonitrile containing d(8)-busulfan. After centrifugation the supernatant was injected onto a turbulent flow preparatory column then transferred to a C18 analytical column monitored by a tandem mass spectrometer set at positive electrospray ionization. The analytical cycle time was 4.0min. The method was linear from 0.15 to 41.90μmol/L with an accuracy of 87.9-103.0%. Inter- and intra-assay CVs across four concentration levels were 2.1-7.8%. No significant carryover or ion suppression was observed. No interference was observed from commercial control materials containing more than 100 compounds. Comparison with a well established LC-MS/MS method using patient specimens (n=45) showed a mean bias 1.3% with Deming regression of slope 1.02, intercept -0.02μmol/L, and a linear correlation coefficient 0.9883. The LC-MS/MS method coupled with turbulent flow online sample cleaning technology described here offers reliable busulfan quantitation in serum or plasma with minimum manual sample preparation and was fully validated for clinical use.     

C6 glioma cells produce basic fibroblast growth factor that can stimulate their own proliferation

With purified preparations of basic fibroblast growth factor (bFGF), we studied the effect of its growth-promoting activity on C6 glioma cells. We also examined with its antibody whether the cultured glioma cells could produce it. It was shown that bFGF stimulated the DNA synthesis and proliferation of C6 glioma cells in serum-free medium, and that the activity was potentiated by heparin, the bFGF concentrations for half-maximal stimulation being 0.2 and 5 ng/ml in the presence and absence of heparin, respectively. This effect of heparin was dose-dependent and was half-maximal at 0.5 microgram/ml. Next, we raised the antiserum against bFGF and detected a single immunoreactive band from extracts of C6 glioma cells by immunoblot analysis. The immunoreactive substance was partially purified on a heparin-Sepharose column and was shown to stimulate the DNA synthesis of C6 glioma cells. On the basis of its immunoreactivity, molecular weight, affinity for heparin, and growth-promoting activity, this substance was identified as bFGF. The content of bFGF in the cells was elevated as the cell density increased, but no immunoreactivity was detected in the conditioned medium of the cells. These results suggest that C6 glioma cells produce and store bFGF which is potent in stimulating their own growth.     

Quantification of sugar phosphate intermediates of the pentose phosphate pathway by LC-MS/MS: application to two new inherited defects of metabolism

We describe a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to quantify pentose phosphate pathway intermediates (triose-3-phosphates, tetrose-4-phosphate, pentose-5-phosphate, pentulose-5-phosphates, hexose-6-phosphates and sedoheptulose-7-phosphate (sed-7P)) in bloodspots, fibroblasts and lymphoblasts. Liquid chromatography was performed using an ion pair loaded C(18) HPLC column and detection of the sugar phosphates was carried out by tandem mass spectrometry using an electron ion spray source operating in the negative mode and multiple reaction monitoring. Reference values for the pentose phosphate pathway intermediates in blood spots, fibroblasts and lymphoblasts were established. The method was applied to cells from patients affected with a deficiency of transaldolase. The transaldolase-deficient cells showed an increased concentration of sedoheptulose-7-phosphate. (Bloodspots: 5.19 and 5.43 micromol/L [0.49-3.33 micromol/L]; fibroblasts 7.43 and 26.46 micromol/mg protein [0.31-1.14 micromol/mg protein]; lymphoblasts 16.03 micromol/mg protein [0.61-2.09 micromol/mg protein].) The method was also applied to study enzymes of the pentose phosphate pathway by incubating fibroblasts or lymphoblasts homogenates with ribose-5-phosphate or 6-phosphogluconate and the subsequent analysis of the formed sugar phosphates.     

Phosphatidylglucoside: a new marker for lipid rafts

Lipid rafts are functional microdomains enriched with sphingolipids and cholesterol. The fatty acyl chain composition of sphingolipids is a critical factor in the localization of lipids in lipid rafts. The recent studies suggest that lipid rafts are more heterogeneous than previously thought. In addition, our discovery of a new glycolipid, phosphatidylglucoside (PtdGlc), also supports the notion of raft heterogeneity. The complete structural characterization of PtdGlc shows that it consists solely of saturated fatty acyl chains: C18:0 at the sn-1 and C20:0 at the sn-2 positions of the glycerol backbone. This unique fatty acyl composition comprising a single molecular species rarely occurs in known mammalian lipids. Although the structure of PtdGlc is similar to that of phosphatidylinositol, PtdGlc localizes to the outer leaflet of the plasma membrane and is possibly involved in cell-cell interaction signaling in the central nervous system.     

Determination of talinolol in human plasma using automated on-line solid phase extraction combined with atmospheric pressure chemical ionization tandem mass spectrometry

A specific LC-MS/MS assay was developed for the automated determination of talinolol in human plasma, using on-line solid phase extraction system (prospekt 2) combined with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry. The method involved simple precipitation of plasma proteins with perchloric acid (contained propranolol) as the internal standard (IS) and injection of the supernatant onto a C8 End Capped (10 mmx2 mm) cartridge without any evaporation step. Using the back-flush mode, the analytes were transferred onto an analytical column (XTerra C18, 50 mmx4.6 mm) for chromatographic separation and mass spectrometry detection. One of the particularities of the assay is that the SPE cartridge is used as a column switching device and not as an SPE cartridge. Therefore, the same SPE cartridge could be used more than 28 times, significantly reducing the analysis cost. APCI ionization was selected to overcome any potential matrix suppression effects because the analyte and IS co-eluted. The mean precision and accuracy in the concentration range 2.5-200 ng/mL was found to be 103% and 7.4%, respectively. The data was assessed from QC samples during the validation phase of the assay. The lower limit of quantification was 2.5 ng/mL, using a 250 microL plasma aliquot. The LC-MS/MS method provided the requisite selectivity, sensitivity, robustness accuracy and precision to assess pharmacokinetics of the compound in several hundred human plasma samples.     

Characterization of pertussis toxoid by two-dimensional liquid chromatography-tandem mass spectrometry

Pertussis toxoid, an acellular pertussis vaccine prepared by hydrogen peroxide treatment in the presence of Fe³⁺, has not been well characterized. Because the toxoid has been a part of the DTaP vaccine for infants, it is of interest and significance to have a clear understanding of its structure. The five subunits of pertussis toxin (PT) have a combined molecular weight of approximately 95,000 Da. The peroxide treatment in toxoid formation introduces additional complexity into the protein sequence. To maximize sequence coverage, a two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) approach was used to analyze the tryptic digest of toxoid as a whole. An analytical-scale high-performance liquid chromatography (HPLC) instrument using a pentafluorophenyl (PFP) column was used as the first-dimensional LC for fraction collection. The fractions were then analyzed by nanoLC-MS/MS using a C18 column to acquire collision-activated dissociation (CAD) spectra of the tryptic peptides. It is shown that a PFP column has a different peptide retention specificity from a C18 column. A combination of a PFP column and a C18 column is a viable approach for dispersing peptides in a complex mixture. From the structures of 65 peptides that represented approximately 50% of its sequence, PT was found to have sustained heavy oxidative damages during toxoid preparation. Nearly all methionine, cysteine, and (likely) tryptophan residues were oxidized. Evidence of histidine and tyrosine oxidation was also observed. In addition, a large percentage of asparagine was found hydrolyzed to aspartic acid. These findings corrrelate well with the reduction of PT toxicity by peroxide treatment.     

Enantioselective determination of mexiletine and its metabolites p-hydroxymexiletine and hydroxymethylmexiletine in rat plasma by normal-phase liquid chromatography-tandem mass spectrometry: application to pharmacokinetics

Mexiletine (MEX), hydroxymethylmexiletine (HMM) and p-hydroxymexiletine (PHM) were analyzed in rat plasma by LC-MS/MS. The plasma samples were prepared by liquid-liquid extraction using methyl-tert-butyl ether as extracting solvent. MEX, HMM, and PHM enantiomers were resolved on a Chiralpak(R) AD column. Validation of the method showed a relative standard deviation (precision) and relative errors (accuracy) of less than 15% for all analytes studied. Quantification limits were 0.5 ng ml(-1) for the MEX and 0.2 ng ml(-1) for the HMM and PHM enantiomers. The validated method was successfully applied to quantify the enantiomers of MEX and its metabolites in plasma samples of rats (n = 6) treated with a single oral dose of racemic MEX.     

Phosphatidylglucoside: a novel marker for adult neural stem cells

We investigated the expression of a novel glycophospholipid, phosphatidylglucoside (PtdGlc), in adult mouse brains. Immunohistochemical analysis with DIM21 antibody, a monoclonal anti-PtdGlc antibody, revealed robust PtdGlc staining in the two primary neurogenic regions of the adult rodent brain, the subventricular zone (SVZ) lining the lateral ventricle and the subgranular zone of the dentate gyrus. Intriguingly, the staining pattern of PtdGlc appeared to overlap that of glial fibrillary acidic protein, an adult neural stem cell marker in these regions. Further immunohistochemical analysis revealed that PtdGlc expression on the cell membranes of adult SVZ neural stem cells significantly overlapped with other proposed adult neural stem cell markers. Moreover, PtdGlc(+) cells isolated from adult mouse SVZs by fluorescence-activated cell sorting with anti-PtdGlc antibody efficiently generated neurospheres in cell culture. These cells differentiated into neurons, astrocytes, and oligodendrocytes in vitro, directly demonstrating that PtdGlc-expressing cells possessed multipotency. Our data suggest that PtdGlc could be a useful adult stem cell marker.     

Metabolic flux analysis in Escherichia coli by integrating isotopic dynamic and isotopic stationary 13C labeling data

The novel concept of isotopic dynamic 13C metabolic flux analysis (ID-13C MFA) enables integrated analysis of isotopomer data from isotopic transient and/or isotopic stationary phase of a 13C labeling experiment, short-time experiments, and an extended range of applications of 13C MFA. In the presented work, an experimental and computational framework consisting of short-time 13C labeling, an integrated rapid sampling procedure, a LC-MS analytical method, numerical integration of the system of isotopomer differential equations, and estimation of metabolic fluxes was developed and applied to determine intracellular fluxes in glycolysis, pentose phosphate pathway (PPP), and citric acid cycle (TCA) in Escherichia coli grown in aerobic, glucose-limited chemostat culture at a dilution rate of D = 0.10 h(-1). Intracellular steady state concentrations were quantified for 12 metabolic intermediates. A total of 90 LC-MS mass isotopomers were quantified at sampling times t = 0, 91, 226, 346, 589 s and at isotopic stationary conditions. Isotopic stationarity was reached within 10 min in glycolytic and PPP metabolites. Consistent flux solutions were obtained by ID-13C MFA using isotopic dynamic and isotopic stationary 13C labeling data and by isotopic stationary 13C MFA (IS-13C MFA) using solely isotopic stationary data. It is demonstrated that integration of dynamic 13C labeling data increases the sensitivity of flux estimation, particularly at the glucose-6-phosphate branch point. The identified split ratio between glycolysis and PPP was 55%:44%. These results were confirmed by IS-13C MFA additionally using labeling data in proteinogenic amino acids (GC-MS) obtained after 5 h from sampled biomass.     

Syntheses of phosphatidyl-beta-D-glucoside analogues to probe antigen selectivity of monoclonal antibody 'DIM21'

Herein, we report the chemical syntheses of a series of phosphatidyl-beta-D-glucoside (PtdGlc) analogues, including 6-O-Ac, sn-2-O-Me, phosphorothioate as well as phosphatidylgalactoside and -mannoside derivatives. In the key step, beta-glycosyl H-phosphonate was condensed with enantiomerically pure diacylglycerol. Comparison of spectroscopic data with mono-acetylated PtdGlc from natural source confirmed the presence of an acetyl moiety at position 6. Furthermore, the reactivity of PtdGlc and its analogues toward monoclonal antibody 'DIM21' (MAb DIM21) was evaluated, revealing the crucial structural antigen features for successful MAb DIM21 binding.     

自噬在声动力疗法抑制C6胶质瘤细胞增殖中的作用

目的:探讨自噬在血卟啉单甲醚(Hematoporphyrin monomethyl ether,HMME)介导的声动力疗法(Sonodynamic therapy,SDT)抑制C6胶质瘤细胞增殖中的作用。方法:选取对数期生长的C6胶质瘤细胞并随机分为四组:对照组(未予处理)、超声组(单独超声照射)、HMME组(单独加入HMME)、SDT组(超声照射+HMME)。透射电镜观察SDT处理的C6胶质瘤细胞中自噬体数量的改变。应用qRT-PCR和免疫印迹分析SDT处理对C6胶质瘤细胞中的LC3、Beclin1、Bcl-2 m RNA及蛋白表达水平的影响。MTT检测C6胶质瘤细胞的活力变化。结果:透射电子显微镜显示SDT组自噬体数量较对照组明显增多。SDT组C6胶质瘤细胞中微管相关蛋白1轻链3 (Microtubule associated protein 1 light chain 3, LC3)、Beclin1 m RNA和蛋白水平高于对照组,B细胞淋巴瘤-2(B cell lymphoma-2, Bcl-2) m RNA和蛋白水平低于对照组。与对照组相比,SDT组C6胶质瘤细胞存活率从0 h至6 h逐渐下降,从12 h至72 h逐渐升高。3-甲基腺嘌呤(3-Methyladenine,3-MA)+SDT、氯喹(Chloroquine,CQ)+SDT处理后C6胶质瘤细胞存活率较SDT组明显降低。结论:SDT可能通过诱导自噬抑制C6胶质瘤细胞增殖。     

Tandem mass spectrometric assay of succinylacetone in urine for the diagnosis of hepatorenal tyrosinemia

We describe an isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of succinylacetone (SA) in urine for the diagnosis of hepatorenal tyrosinemia (HT1). The method used 15N-labeled 5(3)-methyl-3(5)-isoxazole propionic acid as internal standard. Urine samples were oximated with hydroxylamine hydrochloride at 80 degrees C, extracted by solvent-solvent extraction, and followed by derivatization of the butyl ester. The butylated isoxazole derivatives of SA and its internal standard were detected and quantified using positive ion electrospray LC-MS/MS with selected reaction monitoring. The turnaround time between injections was 10 min. Calibration curves were linear over the range of 0.0633-63.3 micromol/L. The intra- and interday assay variations were less than 7%. Mean recoveries of SA at three different concentrations ranged from 96 to 109%. During the course of this study, we identified 12 new patients with HT1 and applied this method to follow up the treatment of 4 of these patients as well as previously diagnosed HT1 patients.     

Identification of phosphorylation sites on phosducin-like protein by QTOF mass spectrometry.

Post-translational modifications are used by cells to control the functions of proteins. Phosducin-like protein (PhLP) is a regulator of G-protein signaling that is post-translationally modified via phosphorylation. Phosphorylation of PhLP initiates its degradation by the 26S proteasome in serum-stimulated cells. In this report, we show that PhLP is phosphorylated in serum-stimulated Chinese hamster ovary (CHO) cells. Through the use of tandem mass spectrometry (MS/MS), the specific amino acids phosphorylated can be identified. A PhLP-myc-His construct was purified and phosphorylated by serum-stimulated CHO extract. The resulting protein was digested with trypsin and the peptides were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Automated collison-induced dissociation data acquisition was compared with LC-MS/MS of manually chosen parents. In general, LC-MS/MS is superior for parent ions chosen manually, with the notable exception that automated fragmentation employs dynamic collision energy, which can result in higher quality collison-induced dissociation. Using the LC-MS/MS methods, four phosphorylation sites on PhLP were positively identified.     

Advanced glycosylation end products induce nitric oxide synthase expression in C6 glioma cells: involvement of a p38 MAP kinase-dependent mechanism.

The mitogen-activated protein kinase (MAPK) pathway is believed to function as an important mediator of inducible nitric oxide synthase (iNOS) expression. In the present study, we investigated the role of the p38 MAPK signaling pathway in advanced glycosylation end products (AGEs)-induced iNOS expression in C6 glioma cells. AGEs caused a dose-dependent increase of nitrite accumulation in C6 glioma cells. The AGEs-stimulated nitrite production from C6 glioma cells was inhibited by actinomycin D, cyclohexamide, and the NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME), suggesting that the increase of AGEs-induced nitrite release is due to iNOS up-regulation. Consistently, treatment of C6 glioma cells with AGEs induced iNOS protein expression. AGEs-stimulated nitrite production was inhibited by pretreatment of C6 glioma cells with anti-AGEs antibodies (1:100 or 1:50). The tyrosine kinase inhibitor (genistein and tyrphostin), the Ras-farnesyl transferase inhibitor (FPT inhibitor-II), or the p38 MAPK inhibitor (SB203580) suppressed AGEs-induced iNOS expression and nitrite release from C6 glioma cells. AGEs activated p38 MAPK in C6 glioma cells, and this effect was blocked by genistein (20 microM), tyrphostin (30 microM), FPT inhibitor-II (20 microM), and SB203580 (10 microM). Taken together, our data suggest that AGEs may activate the pathways of tyrosine kinase and Ras to induce p38 MAPK activation, which in turn induces iNOS expression and NO production in C6 glioma cells.     

Effects of chlorogenic acid,caffeine, and coffee on behavioral and biochemical parameters of diabetic rats

Mesenchymal stem cells (MSCs) represent a potential therapeutic target for glioma. We determined the molecular mechanism of inhibitory effect of human umbilical cord-derived MSCs (hUC-MSCs) on the growth of C6 glioma cells. We demonstrated that hUC-MSCs inhibited C6 cell growth and modulated the cell cycle to G0/G1 phase. The expression of β-catenin and c-Myc was downregulated in C6 cells by conditioned media from hUC-MSCs, and the levels of secreted DKK1 were positively correlated with concentrations of hUCMSCs-CM. The inhibitory effect of hUC-MSCs on C6 cell proliferation was enhanced as the concentration of DKK1 in hUCMSCs-CM increased. When DKK1 was neutralized by anti-DKK1 antibody, the inhibitory effect of hUC-MSCs on C6 cells was attenuated. Furthermore, we found that conditioned media from hUC-MSCs transfection with siRNA targeting DKK1 mRNA or pEGFPN1-DKK1 plasmid lost or enhanced the abilities to regulate the Wnt signaling in C6 cells. Therefore, hUC-MSCs inhibited C6 glioma cell growth via secreting DKK1, an inhibitor of Wnt pathway, may represent a novel therapeutic strategy for malignant glioma.     

Determination of dansylated polyamines in red blood cells by liquid chromatography-tandem mass spectrometry

The concentration of polyamines in red blood cells (RBCs) is considered to be an index of cell proliferation. This index has been demonstrated to be of clinical importance for the follow-up and treatment of some cancer patients. The concentration of polyamines in RBCs is usually determined by high-performance liquid chromatography (HPLC) with fluorescence detection. In the current work, we present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of putrescine, spermidine, and spermine, the three major polyamines in RBCs. The polyamines were dansylated and analyzed by an LC gradient of 20-min duration on a C18 column on-line with a tandem mass spectrometer. An internal standard (1,8-diaminooctane) was used for quantification. This method exhibited excellent linearity for the three polyamines with regression coefficients higher than 0.99. The limits of detection for putrescine, spermidine, and spermine were 0.10, 0.75, and 0.50 pmol/ml, respectively. The intrarun precision values for putrescine, spermidine, and spermine all were better than 10%, and the interrun precision values were 13%, 9%, and 20%, respectively. The LC-MS/MS method is sufficiently simple and reliable enough to replace the currently used HPLC method with fluorescence detection in which putrescine is not always detectable.