树突状细胞负载不同转移潜能肝癌的蛋白质谱变化

目的 了解负载不同转移潜能肝癌细胞裂解物和正常肝细胞裂解物后树突状细胞蛋白质表达谱的差异.探索肝癌转移机制.方法 以不同转移潜能人肝癌细胞株(HCCLM6-高转移、MHCC97L-低转移、Hep3B-不转移)制备裂解物与正常肝细胞株(ChangLiver)制备裂解物分别负载DC,并以无裂解物负载DC为对照,对DC全蛋白进行双向电泳、银染、电喷雾质谱分析,Pdquest软件查询IPI上人的非冗余蛋白数据库鉴定差异蛋白.结果 双向电泳图谱匹配率>80%,相关因子平均>0.8;发现最大和最小差异大于3倍的蛋白质点21个,初步鉴定其中的12个.发现这些蛋白涉及细胞骨架(3号点:Ⅰ型角蛋白CK10;16号点:TPMsk3家族蛋白;21号点:β-中心体肌动蛋白)、信号传导(8号点:钙黏蛋白家族成员;11号点:膜联蛋白A2异构体;17号点:内涵体/溶酶体MP1作用蛋白前体)、细胞动力和能量代谢(6号点:细胞色素辅酶Q还原酶复合体核心蛋白;9号点:胆绿素还原酶A前体;13号点:苹果酸脱氢酶;20号点:线粒体前体)等方面及一些未归类蛋白(10号点:抗结直肠癌重链;18号点:Transgelin2).结论 负载不同转移潜能肝癌裂解物后DC蛋白质谱的变化涉及细胞骨架、信号传导、细胞质内物质运输和能量代谢等方面.其中部分蛋白可能与DC2功能和肝癌转移潜能相关.     

Separation and detection of all phosphoinositide isomers by ESI-MS

Phosphoinositides (PIs) play fundamental roles as signalling molecules in numerous cellular processes. Direct analysis of PIs is typically accomplished by metabolic labelling with ³H-inositol or inorganic ³²P followed by deacylation, ion-exchange chromatography and flow scintillation detection. This analysis is laborious, time-consuming, and involves massive amounts of radioactivity. To overcome these limitations we established a robust, non-radioactive LC–ESI–MS assay for the separation and analysis of deacylated PIs that allows discrimination of all isomers without the need for radioactive labelling. We applied the method to various cell types to study the PI levels upon specific stimulation.     

Electron Spectroscopic Imaging

Electron spectroscopic imaging (ESI) is a short term for electron energy loss spectroscopic imaging. ESI is to date the most sensitive electron microscopic analytical technique and has a spatial resolution of 0.5 nm and a mass resolution of less than 50 atoms. ESI takes advantage of filtered, inelastically scattered, transmitted electrons to form spectroscopic images. This analytical mode was developed by F. P. Ottensmeyer of the Ontario Cancer Institute in Toronto, Canada.

Specimens to be analyzed by ESI should not exceed 30 nm in thickness so that multiple scattering of electrons is avoided. Isolated macromolecules constitute ideal specimens. For the analysis of diffusable elements, elaborate techniques involving quick-freezing, drying, and embedding in plastic of the specimens have to be used. An example is given in which calcium was analyzed in the mitochondria of the convoluted proximal tubules in the rat kidney in normal and acute ischemic conditions. The example shows that calcium was not extracted during the preparation of the tissue. The high resolution of ESI allows the localization of calcium inside mitochondria.

Experiments in progress using homogenous standards will soon render ESI entirely quantitative. It is concluded that ESI will become more and more important as a tool for the study of the pathogenesis of conditions in which modification of the distribution and concentration of elements are involved.     

A survey of peritonitis and exit-site and/or tunnel infections in Japanese children on PD

To obtain data on peritonitis and exit-site and/or tunnel infections (ESI/TI) in Japanese children undergoing peritoneal dialysis (PD) from January 1999 through June 2003, we surveyed 22 members of the Japanese Study Group of Pediatric Peritoneal Dialysis (JSPPD) by questionnaire. One hundred and thirty patients were eligible. Seventy episodes of bacterial peritonitis occurred in 45 patients (0.17 episodes/patient-year), and 123 ESI/TI occurred in 60 patients (0.29 episodes/patient-year). S. aureus and MRSA were found to be the causative organisms in 39% and 13% of the peritonitis episodes, and in 59% and 20% of the ESI/TI, respectively. Tunnel infection was found in 55% of the MRSA peritonitis episodes. Eleven percent of the peritonitis episodes relapsed, and 19% needed hemodialysis. One patient died due to MRSA peritonitis. The PD catheter was removed in all fungal and 78% of MRSA peritonitis. However, the type of organism did not influence the need for catheter-related surgery for ESI/TI. Neither peritonitis nor ESI/TI was prevented by the use of a swan-neck catheter, a downward-pointing exit site, povidone iodine exit-site care, bathing instruments, or nasal mupirocin. In conclusion, MRSA peritonitis was not uncommon in children in Japan, was frequently associated with tunnel infections, and had a poor outcome. No association was found between the occurrence of infection and preventive measures previously reported as effective. Alternative approaches are needed in children, especially for MRSA.Members of the Japanese Study Group of Pediatric Peritoneal Dialysis (JSPPD) that participated in this survey: Yuko Akioka (Chiba), Kazumoto Iijima (Tokyo), Masahiro Ikeda (Tokyo), Masaaki Ikoma (Kawasaki), Yuhei Ito (Kurume), Osamu Uemura (Ohbu), Yoshiyuki Ohtomo (Iwatsuki), Yoshitsugu Kaku (Fukuoka), Takashi Sakano (Hiroshima), Kenichi Satomura (Osaka), Junzo Suzuki (Fukushima), Eihiko Takahashi (Yokohama), Masafumi Taki (Okayama), Motoshi Hattori (Tokyo), Hitoshi Nakazato (Kumamoto), Shinya Nakamura (Sagamihara), Kandai Nozu (Kobe), Toshio Yanagihara (Niigata), Hiroshi Yoshimura (Uruma)     

Ionization of unconjugated, glycine- and taurine-conjugated bile acids by electrospray ionization mass spectrometry

We investigated the effect of organic anions as spray liquid additives on the ionization efficiency of unconjugated, glycine-conjugated and taurine-conjugated bile acids under electrospray ionization conditions. Addition of organic acids influenced the ionization efficiency of whole bile acids. Use of a stronger acid reduced the peak intensity of unconjugated and glycine-conjugated bile acids, while the use of TFA, the strongest acid tested, improved the intensity of taurine conjugates. The hydroxyl group at the C-12 position of cholic acid and deoxycholic acid easily underwent intra-molecular hydrogen bonding with the side chain carboxyl group, accelerating the ionization efficiency. This intra-molecular hydrogen bond may also affect the formation of product ions in low energy-CID. The addition of ammonium ions to the spray liquid influenced the ionization of all bile acids, specifically enhancing the ionization efficiency of unconjugated bile acids.     

Influence of lipid lowering fibrates on P-glycoprotein activity in vitro

Statin/fibrate combinations are frequently used to treat mixed dyslipidemia. However, these combinations may cause life-threatening drug interactions (e.g. rhabdomyolysis) possibly induced by modifications of cytochrome P450 isozyme activities. Some statins are also transported by P-glycoprotein (Pgp) and may act as inhibitors of this drug efflux pump. So far, nothing is known about possible Pgp modulating effects of fibrates. We tested whether gemfibrozil, fenofibrate, fenofibric acid, and bezafibrate inhibit Pgp in vitro using a calcein acetoxymethylester (calcein-AM) uptake assay and confocal laser scanning microscopy with bodipy-verapamil as substrate in L-MDR1 cells, which overexpress human Pgp. In uptake assays in cells with (L-MDR1) and without (LLC-PK1) human Pgp we also investigated whether these compounds are transported by Pgp. Intracellular concentrations were measured by liquid chromatography tandem mass spectrometry. Of the tested fibrates, only fenofibrate increased calcein-AM uptake into cells indicating an inhibition of Pgp mediated transport by this compound. The potency of fenofibrate (mean+/-SD: 7.1+/-3.2 microM), evaluated by calculating the concentration needed to double baseline fluorescence (f2), was similar to that of simvastatin (5.8+/-1.5 microM), lovastatin (10.1+/-1.0), and verapamil (4.7+/-0.8 microM). For simvastatin and fenofibrate Pgp inhibition was confirmed with confocal laser scanning microscopy. Fenofibrate, fenofibric acid, gemfibrozil, and bezafibrate showed no difference in the cellular uptake between LLC-PK1 and L-MDR1, indicating that the tested fibrates are not Pgp substrates. In conclusion, this study demonstrates that fenofibrate inhibits Pgp in vitro with a potency similar to simvastatin.     

Determination of limonin in rat plasma by liquid chromatography-electrospray mass spectrometry

A simple, sensitive and selective liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI/MS) method for determination of limonin (LM) in rat plasma has been developed and validated. The method had advantages of a single liquid–liquid extraction with ether and high sensitivity. Analyses were conducted at a flow rate of 0.2 ml/min by a gradient elution. The detection utilized selected ion monitoring in the negative ion mode at m/z 460.00 and 423.15 for the deprotonated molecular ions of LM and the internal standard, respectively. The quantitation limit for LM in rat plasma was 1.0 ng/ml. The linearity was also excellent over the concentration range of 1.9–500 ng/ml of LM. The intra- and inter-day precision (relative standard deviation (R.S.D.%)) was lower than 10% and accuracy ranged from 90 to 110%, showing a good reproducibility. This developed method was successfully applied to analysis of LM in biological fluids.     

人参皂苷Rb1在大鼠肠内菌代谢物吸收入血成分的研究

目的:研究口服人参皂苷Rb₁(G-Rb₁)被吸收入血的成分。方法:给大鼠igG-Rb₁500mg·kg^-1后,于不同时间采集粪、尿及血清样品,用电喷雾质谱(ESI-MS)检测吸收入血成分。结果:在血与尿中发现分子量为1108,946及784amu的代谢产物。经ESI-MS2级质谱分析,上述分子量的化合物分别为G-Rb₁,Rd和F₂。结论:G-Rb₁给大鼠ig后,原形及中间代谢产物Rd及F₂被吸收入血。     

Identification of IY81149 and its metabolites in the rat plasma using the on-line HPLC/ESI mass spectrometry

Reversed-phase high-performance liquid chromatography/mass spectrometry (HPLC/MS) with an electrospray ionization (ESI) interface was applied to the identification of metabolites of IY 81149 in the rat plasma. Fragments obtained using collision-induced dissociation (CID) in both positive and negative modes were utilized to elucidate the structure of metabolites. The eluent from the conventional HPLC column was split and directly introduced into an ESI-mass spectrometer for the identification of the structures. The CID technique allowed the sensitive identification of sulfonyl-IY81149 and hydroxy-IY81149 from the rat plasma.     

液质联用鉴定重组人血管内皮抑制素

目的：用液质联用技术鉴定重组人血管内皮抑制素（rhEndostatin）。方法：利用ESI—Q—TOF—MS法测定rhEndostatin的精确相对分子质量，通过HPLC—ESI—Q—TOF—MS／MS法测定其胰蛋白酶酶切后肽段的部分氨基酸序列并结合数据库检索进行结构鉴定。结果：重组人血管内皮抑制素的实测相对分子质量为21114．5，与理论相对分子质量21113．8相比非常接近。HPLC—ESI—Q—TOF—MS／MS测定m／z802．0的肽段的部分氨基酸序列为Ala—Pro—Ser—Ala-Thr—Gly—Gin—Ala—Ser—Ser—Leu—Leu。将其m／z和氨基酸序列在MASCOT数据库检索，结果表明重组人血管内皮抑制素的结构正确。结论：液质联用是鉴定蛋白质的灵敏、快速、准确的新方法。