Lung alveolar proteomics of bronchoalveolar lavage from a pulmonary alveolar proteinosis patient using high-resolution FTICR mass spectrometry

High-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was developed and applied to the proteome analysis of bronchoalveolar lavage fluid (BALF) from a patient with pulmonary alveolar proteinosis. With use of 1-D and 2-D gel electrophoresis, surfactant protein A (SP-A) and other surfactant-related lung alveolar proteins were efficiently separated and identified by matrix-assisted laser desorption/ionization FTICR mass spectrometry . Low molecular mass BALF proteins were separated using a gradient 2-D gel. An efficient extraction/precipitation system was developed and used for the enrichment of surfactant proteins. The result of the BALF proteome analysis show the presence of several isoforms of SP-A, in which an N-non-glycosylierte form and several proline hydroxylations were identified. Furthermore, a number of protein spots were found to contain a mixture of proteins unresolved by 2-D gel electrophoresis, illustrating the feasibility of high-resolution mass spectrometry to provide identifications of proteins that remain unseparated in 2-D gels even upon extended pH gradients.

High resolution mass spectrometric alveolar proteomics: Identification of surfactant protein SP-A and SP-D modifications in proteinosis and cystic fibrosis patients

In the present study, one- and two-dimensional gel electrophoresis combined with high resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) have been applied as powerful approaches for the proteome analysis of surfactant proteins SP-A and SP-D, including identification of structurally modified and truncation forms, in bronchoalveolar lavage fluid from patients with cystic fibrosis, chronic bronchitis and pulmonary alveolar proteinosis. Highly sensitive micro preparation techniques were developed for matrix-assisted laser desorption/ionization (MALDI) FT-ICR MS analysis which provided the identification of surfactant proteins at very low levels. Owing to the high resolution, FT-ICR MS was found to provide substantial advantages for the structural identification of surfactant proteins from complex biological matrices with high mass determination accuracy. Several protein bands corresponding to SP-A and SP-D were identified by MALDI-FT-ICR MS after electrophoretic separation by one- and two-dimensional gel electrophoresis, and provided the identification of structural modifications (hydroxy-proline) and degradation products.

The encysted dormant embryo proteome of Artemia sinica

The possibility of the brine shrimp Artemia to produce dormant embryo (cysts) in diapause is a key feature in its life history. In the present study, we obtained a proteomic reference map for the diapause embryo of Artemia sinica using two-dimensional gel electrophoresis with a pH range of 4-7 and a molecular weight range of 10-100 kDa. Approximately 233 proteins were detected, and 60 of them were analyzed by capillary liquid chromatography tandem mass spectrometry (LC-MS/MS). Of these, 39 spots representing 33 unique proteins were identified, which are categorized into functional groups, including cell defense, cell structure, metabolism, protein synthesis, proteolysis, and other processes. This reference map will contribute toward understanding the state of the diapause embryo and lay the basis and serve as a useful tool for further profound studies in the proteomics of Artemia at different developmental stages and physiological conditions.

A Proteomic Study on Cell Cycle Progression of Endothelium Exposed to Tumor Conditioned Medium and the Possible Role of Cyclin D-1/E

This study was designed to comprehensively analyze the differential expression of proteins from human umbilical vein endothelial cells (HUVECs) exposed to tumor conditioned medium (TCM) and to identify the key regulator in the cell cycle progression. The HUVECs were exposed to TCM from breast carcinoma cell line MDA-MB-231, then their cell cycle distribution was measured by flow cytometer (FCM). The role of protein in cell cycle progression was detected via two-dimensional polyacrylamide gel electrophoresis (2-DE) and western blotting. Following the stimulation of TCM, HUVECs showed a more cells in the S phase than did the negative control group (ECGF-free medium with 20% FBS), but the HUVECs' level was similar to the positive control group (medium with 25 mug/ml ECGF and 20% FBS). Increased expression of cyclin D-1/E and some changes in other related proteins occurred after incubation with TCM. From our results, we can conclude that breast carcinoma cell line MDA-MB-231 may secrete soluble pro-angiogenic factors that induce the HUVEC angiogenic switch, during which the expression of cell cycle regulator cyclin D-1/E increases and related proteins play an important role in this process.

油菜内源和外源蛋白的表达及环境因素的影响

植物与昆虫的互作关系是个长期进化的过程，虫害给农业生产带来巨大损失。本研究以甘蓝型油菜（Brassica napus）为例，研究了不同环境条件和遗传背景下外源基因的表达与效用，同时利用蛋白质组技术，研究了虫害损伤模拟条件下植物可能存在的内源抗性机制。甘蓝型油菜中转入了人工合成的Bt（Bacillus thuringiensis）杀虫基因，能使植物产生抗虫蛋白抵御虫害。我们在湖北湖南两个实验点进行了大田实验，按植株生长发育的4个不同时期从转基因植株的叶片上采样，研究抗虫蛋白在植物体内的表达动态。植株顶部第三片展开叶的Bt毒蛋白浓度在结荚期前随植物生长而不断增加，而在结荚期出现或增或减的现象。采样叶片的可溶性总蛋白浓度含量一直呈增加的趋势，直到结荚以后出现含量的明显降低。同时，收集了转基因油菜与湘油15号在田间自然杂交形成的杂交后代种子用于栽培，用GFP仪检测杂交后代的绿色荧光蛋白（green fluorescent protein），并用聚合酶链式反应（polymerase chain reaction, PCR）检测并确认带有转基因的杂交植株。为了检测带有转基因的杂交后代油菜中Bt毒蛋白的杀虫效率，用对Bt毒蛋白敏感的试虫品系——初孵棉铃虫幼虫（Helicoverpa armigera）进行杀虫活性检测实验。结果表明，携带Bt基因的杂交湘油及其转基因亲本对试虫的体重增长量均产生了负面影响，可以推断在调查取样的植株生长发育阶段，转基因杂交后代与其转基因亲本植株的杀虫效率没有显著差异。转基因植物及其杂交后代中抗虫蛋白的持续表达及田间带有转基因的自播植物的出现会使害虫产生耐受抗性的潜在可能性增加。 相对于人为增加的抗虫基因，植物在长期对抗昆虫的过程中也进化形成了自我防御机制，能够产生特异的抗性蛋白来应对昆虫的取食。本研究用机械损伤模拟害虫取食，对比了油菜受到物理损伤前后可溶性总蛋白的含量变化并试图通过蛋白质组学技术来检测可能发生变化的蛋白质。Bradford定量测定发现，同一植株同一叶片损伤前后可溶性总蛋白含量差异显著，损伤后蛋白表达量显著增高。蛋白质组双向凝胶电泳及其差异分析显示，损伤前后有8个蛋白质点发生明显的上调或下调。选择其中2个差异蛋白点经过MALDI-TOF质谱鉴定，它们分别是Rubisco小亚基前体以及果糖－1，6－二磷酸醛缩酶和粪卟啉－3－氧化酶的混合物，这些蛋白质在其他植物的抗逆研究中也有报道，它们可能在油菜叶片应答机械损伤过程中对维持植物的生理功能也有重要作用。

Characterization and cloning of a novel phospholipase A(2) from the venom of Trimeresurus jerdonii snake

A phospholipase A(2) (PLA(2)) called jerdoxin, was isolated from Trimeresurus jerdonni snake venom and partially characterized. The protein was purified by three chromatographic steps. SDS-polyacrylamide gel electrophoresis in the presence or absence of dithiothreitol showed that it had a molecular mass of 15 kDa. Jerdoxin had an enzymatic activity of 39.4 mumol/min/mg towards egg yolk phosphatidyl choline (PC). It induced edema in the footpads of mice. In addition, jerdoxin exhibited indirect hemolytic activity. About 97% hemolysis was observed when 2 mug/ml enzyme was incubated for 90 min in the presence of PC and Ca2+. No detectable hemolysis was noticed when PC was not added. Ca2+ was necessary for jerdoxin to exert its hemolytic activity, since only 52% hemolysis was seen when Ca2+ was absent in the reaction mixture. Furthermore, jerdoxin inhibited ADP induced rabbit platelet aggregation and the inhibition was dose dependent with an IC50 of 1.0 muM. The complete amino acid sequence of jerdoxin deduced from cDNA sequence shared high homology with other snake venom PLA(2)s, especially the D49 PLA(2)s. Also, the residues concerned to Ca2+ binding were conserved. This is the first report of cDNA sequence of T jerdonii venom PLA(2). (C) 2002 Elsevier Science Ltd. All rights reserved.

One-step immobilization of tris(2,2 '-bipyridyl)ruthenium(II) via vapor-surface sol-gel deposition towards solid-state electrochemiluminescence detection

A novel method for immobilization of tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)(3)Cl-2) on electrode surfaces based on the vapor-surface sol-gel deposition strategy is first demonstrated in this paper. Ru(bpy)(3)Cl-2 immobilized sol-gel (Ru(bpy)(3)Cl-2/sol-gel) films were characterized by UV-vis spectroscopy and field-emitted scanning electron microscopy (FE-SEM). These results showed that Ru(bpy)(3)Cl-2 was successfully incorporated into the silica sol-gel film. it was found that many irregular Ru(bpy)(3)Cl-2/sol-gel clusters were formed on surfaces through one deposition and thick sol-gel films were observed after further deposition.

Kinetic study of paracetamol on prolidase activity in erythrocytes by capillary electrophoresis with Ru(bpy)(3)(2+) electrochemiluminescence detection

We explored the CE with Ru(bpy)(3)(2+) electrochemiluminescence detection for the kinetic study of drug-enzyme interaction. Effects of four nonsteroidal anti - inflammatory drugs including aspirin, paracetamol, sodium salicylate and phenacetin on prolidase (PLD) activity in erythrocytes were investigated. Aspirin enhanced PLD activity whereas the other three had inhibiting effects. This may reveal their different effects on the collagen biosynthesis and catabolism that influence tumor invasiveness. Kinetic study of paracetamol on PLD showed that the value of Michaelis constant Km for PLD was 1.23 mM. The mechanism of PLD inhibition by paracetamol is noncompetitive inhibition, and the inhibitor constant K-i value obtained in our research was 9.73 x 10(3) mu g/L.

Novel hydrogen-bonded three-dimensional networks encapsulating one-dimensional covalent chains: [M(4,4 '-bipy)(H2O)4](4-abs)2 center dot nH(2)O (4,4 '-bipy=4,4 '-Bipyridine; 4-abs=4-aminobenzenesulfonate) (M=Co, n=1; M=Mn, n=2)

Two novel compounds, [Co(4,4'-bipy)(H2O)(4)](4-abS)(2).H2O (1) and [Mn(4,4'-bipy)(H2O)(4)](4-abs)(2).2H(2)O (2) (4,4'-bipy = 4,4'-bipyridine; 4-abs = 4-aminobenzenesulfonate), have been synthesized in aqueous solution and characterized by single-crystal X-ray diffraction, elemental analyses, UV-vis and IR spectra, and TG analysis. X-ray structural analysis revealed that 1 and 2 both possess unusual hydrogen-bonded three-dimensional (3-D) networks encapsulating one-dimensional (1-D) covalently bonded infinite [M(4,4'-bipy)(H2O)(4)](2+) (M = Co, Mn) chains. The 4-abs anions in 1 form 1-D zigzag chains through hydrogen bonds. These chains are further extended through crystallization water molecules into 3-D hydrogen-bonded networks with 1-D channels, in which the [Co(4,4'-bipy)(H2O)(4)](2+) linear covalently bonded chains are located. Crystal data for 1: C22H30CoN4O11S2, monoclinic P2(1), a = 11.380(2) Angstrom, b = 8.0274(16) Angstrom, c = 15.670(3) Angstrom, alpha = gamma = 90degrees, beta = 92.82(3)degrees, Z = 2. Compound 2 contains interesting two-dimensional (2-D) honeycomb-like networks formed by 4-abs anions and lattice water molecules via hydrogen bonding, which are extended through other crystallization water molecules into three dimensions with 1-D hexagonal channels. The [Mn(4,4'-bipy)(H2O)(4)](2+) linear covalent chains exist in these channels. Crystal data for 2: C22H32WN4O12S2, monoclinic P2(1)/c, a = 15.0833(14) Angstrom, b = 8.2887(4) Angstrom, c = 23.2228(15) Angstrom, alpha = gamma = 90degrees, beta = 95.186(3)degrees, Z = 4.

The AFM characterization of Ni(phen)(3)(2+) fixing effects on DNA

In this paper, the fixing and stretching effect of Ni(phen)(2+)(3) with different concentrations on DNA had been studied by Tapping mode AFM. Under an ambient condition, the high-resolution DNA images were obtained, the average height, width and length of well spread DNA molecules were measured. The results showed that because of the variations of ionic concentration, the density and topography of DNA molecules on substrate had a great difference. The AFM and gel electrophoresis results also showed that, under our experimental condition, DNA molecules kept intact, Ni(phen)(2+)(3) did not catalyze the cleavage activity of EcoRI, therefore, Ni (phen)(2+)(3) would be used to make high-resolution physical mapping of DNA by AFM.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of phospholipase A(2) and fibrinolytic enzyme, two enzymes obtained from Chinese Agkistrodon blomhoffii Ussurensis venom

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to analyze two enzymes, phospholipase AZ and fibrinolytic enzyme isolated from Chinese Agkistrodon blomhoffii Ussurensis venom. Using sinapinic acid as the matrix, positive ion mass spectra of the enzymes were obtained, In addition to the dominant protein [M+H](+) ions, multimeric and multiply charged ions were also observed in the mass spectra, The higher the concentration of the enzymes, the more multiply charged polymer and multimeric ions were detected, Our results indicate that MALDI-TOFMS can provide a rapid and accurate method for molecular weight determination of snake venom enzymes, Mass accuracies of 0.1 and 0.3 % were achieved by analysis of highly dialyzed phospholipase A2 and fibrinolytic enzyme, and these results are much better than those obtained using sodium dodecyl sulfate-palyacrylamide gel electrophoresis. MALDI-TOFMS thus provides a reliable method to determine the purity and molecular weight of these enzymes, which are of potential use as therapeutants, Copyright (C) 1999 John Wiley & Sons, Ltd.

Molecular cloning and response to laminarin stimulation of arginine kinase in haemolymph in Chinese shrimp, Fenneropenaeus chinensis

Arginine kinase (AK) was previously reported as a phosphagen-ATP phosphotransferase found in invertebrates. In this study, an 1184 bp cDNA was cloned and sequenced. It contained an open reading frame of 1068 bp that coded for 356 deduced amino acids of AK in Fenneropenaeus chinensis. The calculated molecular mass of AK is 40129.73 Da and pI is 5.92. The predicted protein showed a high level of identity to known AK in invertebrates and creatine kinase from vertebrates, which belong to a conserved family of ATP:guanidino phospho-transferases. In addition, AK protein in plasma of F. chinensis was identified using two-dimensional electrophoresis (2DE) and electrospray ionization mass spectrometry (ESI-MS) according to the calculated molecular mass and pI. AK was significantly decreased in the plasma of F. chinensis at 45 min and recovered at 3 It after laminarin injection as confirmed by 2DE and ESI-MS. The results showed that AK was one of the most significantly changed proteins on two-dimensional gel in the plasma proteins of F. chinensis at 45 min and 3 It after simulation. (c) 2005 Elsevier Ltd. All rights reserved.

Effects of azadirachtin on hemolymph protein expression in Ostrinia furnacalis (Lepidoptera : Crambidae)

The botanical insecticide azadirachtin affects a variety of biological processes. Our early work indicated that protein level and type are significantly influenced by azadirachtin in pupae of Osttiniafumacalis (Guenee) (Lepidoptera: Crambidae) because a correlation exists between protein content and azadiraebtin concentration. By use of proteomic techniques, we analyzed changes in hemolymph protein expression of 48-h-old pupae in O. furnacalis induced by azadirachtin treatment. After feeding by third instars on an artificial diet containing 10 ppm azadirachtin until pupation, 48-b-old pupae were collected, and hemolymph protein samples were prepared. They were separated by two-dimensional polyacrylamide gel electrophoresis, and six proteins were significantly affected by azadiracbtin treatment compared with an untreated control. Two of these proteins were identified by database searching with peptide mass fingerprinting by using matrix-assisted laser desorption/ time-of-flight mass spectrometry after in-gel trypsin digestion. They belong to the insect apolipophorin-III and phospboribosyltransferase family, respectively. These two proteins may function on lipid metabolism in insect hemolymph. Furthermore, fat body is the center of synthesis and secretion of hemolymph proteins. We suggest that the azadirachtin exerts its insecticidal effects on the fat body of O. furnacalis by interfering with protein expression related to hemolymph lipid metabolism.

Highly sensitive determination of the methylated p16 gene in cancer patients by microchip electrophoresis

The p16 tumor suppressor gene is inactivated by promoter region hypermethylation in many types of tumor. Recent studies showed that aberrant methylation of the p16 gene is an early event in many tumors, especially in lung cancer, and may constitute a new biomarker for early detection and monitoring of prevention trials. We detected tumor-associated aberrant hypermethylation of the p16 gene in plasma and tissue DNA from 153 specimens using a modified semi-nested methylation-specific PCR (MSP) combining plastic microchip electrophoresis or slab gel electrophoresis, respectively. Specimens were from 79 lung cancer patients, 15 abdominal tumor patients, 30 positive controls and 30 negative controls. The results showed that the positive rate obtained by microchip electrophoresis was more than 26.6% higher and the same speciticity was kept when compared with slab gel electrophoresis. The microchip electrophoresis can rapidly and accurately analyze the PCR products of methylated DNA and obviously improve the positive rate of diagnosis of cancer patients when compared with gel electrophoresis. This method with the high assay sensitivity might be used for detection of methylation of p16 gene and even to facilitate early diagnosis of cancer patients. (C) 2004 Elsevier B.V. All rights reserved.

Capillary zone electrophoresis characterization of low molecular weight heparin binding to interleukin 2

A method based on capillary zone electrophoresis (CZE) was used to study the interaction between low molecular weight heparin (LMWH) and interleukin 2 (IL-2). The results showed that the increase of the concentration of LMWH led to the decrease of the peak height and the increase of the peak width of IL-2, but the peak areas were kept constant. The binding constant of IL-2 with LMWH was calculated as 1.2 x 10(6) M(-1) by Scatchard analysis, which is in good agreement with the results found in the references using enzyme-linked immunosorbent assay (ELISA). The results demonstrated that the interaction between IL-2 and LMWH is of fast on-and-off kinetic binding reaction. CZE might be used to study not only slow on-and-off rates interactions, but also fast on-and-off rates ones. The binding constant can be calculated easily, and the method can be applied to study a wide range of heparin-protein interactions. (c) 2005 Elsevier B.V. All rights reserved.