黏液性/浆液性胰腺囊性肿瘤囊液蛋白质糖基化差异表达研究

随着影像学技术的进步,胰腺囊性病变(恶性胰腺囊性病变包括黏液性囊腺瘤(mucinous cystic neoplasm,MCN)和导管内乳头状黏液瘤(intraductal papillary mucinous neoplasm,IPMN)以及黏液性囊腺癌(mucinous cystic adenocarcinoma,MCA)的检出率有所提高,但是区分囊性病变的良恶性仍然是一个难题.本研究根据外科手术病理结果及囊液液基细胞结果,从120例经CT或MRI方法诊断为胰腺囊性肿瘤患者中选取35例样本,其中胰腺黏液性囊腺瘤(MCN)组17例与浆液性囊腺瘤(serous cystic neoplasm,SCN)组18例,通过超声内镜下细针穿刺(endoscopic ultrasonography-guided fine needle aspiration,EUS-FNA)方法吸取囊液,采用凝集素芯片分析蛋白质糖链谱差异.经t检验结果表明,MCN组与SCN组相比,6种凝集素,STL、WGA、BPL、DBA、PTL-Ⅰ及MAL-Ⅰ,识别的糖链结构二者之间存在明显差异(P0.05),其中凝集素BPL、DBA、WGA、STL识别的糖链结构在MCN囊液中呈高表达(R2.0),例如DBA特异识别的Tn抗原表达的增多,可能与肿瘤上皮细胞分泌的黏蛋白增多密切相关.而凝集素PTL-Ⅰ、MAL-Ⅰ特异识别Galβ-1、4Glc NAc结构以及Gal NAcα-1、3Gal结构在MCN囊液中表达降低(R0.5).通过凝集素印记法检测了STL和BPL分别于MCN、SCN囊液中蛋白的结合情况,结果表明STL和BPL与囊液中的糖蛋白结合明显强于SCN组.本文通过比较MCN和SCN糖蛋白糖谱表达差异,寻找胰腺囊性肿瘤诊断和肿瘤良恶性评估的新方法,同时为探索胰腺囊性肿瘤发生发展机制和治疗的潜在靶点奠定基础.     

Purification and molecular characterization of a novel mannose‐specific lectin from Dioclea reflexa hook seeds with inflammatory activity

A novel lectin present in Dioclea reflexa seeds (DrfL) was discovered and described in this study. DrfL was purified in a single step by affinity chromatography in a Sephadex G‐50 column. The lectin strongly agglutinated rabbit erythrocytes and was inhibited by α‐methyl‐d ‐mannoside, d ‐mannose, and d ‐glucose. The hemagglutinating activity of DrfL is optimum at pH 5.0–7.0, stable up to 50 °C, and dependent on divalent cations. Similar to other lectins of the subtribe Diocleinae, the analysis by mass spectrometry indicated that DrfL has three chains (α, β, and γ) with masses of 25 562, 12 874, and 12 706 Da, respectively, with no disulfide bonds or glycosylation. DrfL showed inflammatory activity in the paw edema model and exhibited low cytotoxicity against Artemia sp. Copyright © 2015 John Wiley & Sons, Ltd.     

Vibrio cholerae hemolysin: The β-trefoil domain is required for folding to the native conformation

Vibrio cholerae cytolysin/hemolysin (VCC) is a 65 kDa β-pore-forming toxin causing lysis and death of eukaryotic cells. Apart from the core cytolysin domain, VCC has two lectin domains with β-trefoil and β-prism folds. The β-prism domain binds to cell surface carbohydrate receptors; the role of the β-trefoil domain is unknown. Here, we show that the pro-VCC mutant without the β-trefoil domain formed aggregates highly susceptible to proteolysis, suggesting lack of a properly folded compact structure. The VCC variants with Trp532Ala or Trp534Ala mutation in the β-trefoil domain formed hemolytically inactive, protease-resistant, ring-shaped SDS-labile oligomers with diameters of ~19 nm. The Trp mutation induced a dramatic change in the global conformation of VCC, as indicated by: (a) the change in surface polarity from hydrophobic to hydrophilic; (b) movement of core Trp residues to the protein-water interface; and (c) decrease in reactivity to the anti-VCC antibody by >100-fold. In fact, the mutant VCC had little similarity to the wild toxin. However, the association constant for the carbohydrate-dependent interaction mediated by the β-prism domain decreased marginally from ~3×10⁸ to ~5×10⁷ M^?1. We interpret the observations by proposing: (a) the β-trefoil domain is critical to the folding of the cytolysin domain to its active conformation; (b) the β-prism domain is an autonomous folding unit.     

Muramyl dipeptide bound to poly-l-lysine substituted with mannose and gluconoyl residues as macrophage activators

Poly-l-lysine modified with mannose derivatives, the residual cationic charges of which being neutralized byN-acylation, were synthesized and used as carriers of a macrophage activator (N-acetylmuramyl dipeptide, MDP). The influence of the acylating agent on the targeting efficiency was investigated: a hydrosolubilizing group such as a gluconoyl moiety led to very efficient carrier conjugates, while an acetyl group did not. The effect of sugar and acyl content of the polymers was assessed using these compounds as inhibitors of red blood cell agglutination by Concanavalin A. The binding and specific endocytosis of poly-l-lysine substituted with several mannose derivatives and gluconoyl residues (GlcA_x-, Man_y-PLK) have been determined by a quantitative flow cytometry analysis. MDP bound to these conjugates was much more efficientin vitro than free MDP in macrophage cytostasis assays.Abbreviations MDP N-acetylmuramyl dipeptide - PLK poly-l-lysine - BSA bovine serum albumin - BOC t-butyloxycarbonyl - DMF dimethylformamide - DCHU N, N-dicyclohexylurea - DCCl N, N-dicyclohexylcarbodiimide - TEA triethylamine - Su succinimidyl - DMSO dimethylsulfoxide - FITC fluoresceinyl isothiocyanate - RPMI Roswell Park Memorial Institute - PBS phosphate buffered saline - Fl fluoresceinyl - GG glycyl-glycyl     

Immunocytochemical localisation of phloem lectin from Cucurbita maxima using peroxidase and colloidal-gold labels

Antibodies were raised against lectin purified from the sieve-tube exudate of Cucurbita maxima. Immunocytochemistry, using peroxidase-labelled antibodies and Protein A-colloidal gold, was employed to determine the location of the lectin within the tissues and cells of C. maxima and other cucurbit species. The anti-lectin antibodies bound to P-protein aggregates in sieve elements and companion cells, predominantly in the extrafascicular phloem of C. maxima. This may reflect the low rate of translocation in these cells. Under the electron microscope, the lectin was shown to be a component of P-protein filaments and was also found in association with the sieve-tube reticulum which lines the plasmalemma. The anti-lectin antibodies reacted with sieve-tube proteins from other species of the genus Cucurbita but showed only limited reaction with other genera. We suggest that the lectin serves to anchor P-protein filaments and associated proteins to the parietal layer of sieve elements.Abbreviation SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Regulation of processing of a plant glycoprotein in the Golgi complex: A comparative study usingXenopus oocytes

The synthesis of phytohemagglutinin (PHA), the major seed lectin ofPhaseolus vulgaris, was investigated inXenopus oocytes injected with RNA isolated from developing bean cotyledons. As is the case for normal PHA, oocyte-synthesized PHA polypeptides were found to contain two asparagine-linked oligosaccharide chains, one of which was of the high-mannose type and the other one of the Golgi-modified type, being largely resistant to endo--N-acetylglucosaminidase H digestion and containing fucose. The modified oligosaccharide chain of oocyte-synthesized PHA appeared to be much larger and more heterogeneous with respect to the modified chain normally present on PHA. When the oocytes were injected with purified mRNA for PHA, isolated by hybrid-selection using a PHA complementary-DNA clone, the results were the same as those obtained by injecting total cotyledonary RNA. On the whole, these results indicate that plant glycoproteins are directed to the Golgi complex even when synthesized in an animal cell, and that correct sorting of the oligosaccharide chains to be processed is independent of the cell-type in which protein synthesis occurs. The form of processing is however cell-type specific.Abbreviations endo H endo H--N-acetylglucosaminidase H - ER endoplasmic reticulum - PHA phytohemagglutinin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

A new anti-H lectin from the seeds ofGalactia tenuiflora

A new anti-blood group H lectin was isolated from the seeds ofGalactia tenuiflora. This lectin is mostly specific for the H type 2 trisaccharide but it shows some cross-reactivity with the H type 4 and H type 3 trisaccharides. Differences between this lectin and lectin 1 fromUlex europaeus are described. These differences concern the respective abilities of the lectins to recognize erythrocytes from some H deficient phenotypes, the inhibitions by salivas and the tissue distribution of the antigens recognized by the two lectins. The most important differences were noted in the surface epithelium of the stomach. This area is known to express ABH antigens under the control of theSe gene as defined by theUlex europaeus lectin 1, yet it is always strongly labelled by theGalactia tenuiflora lectin irrespective of the secretor status of the tissue donor.     

A single step purification of a sialic acid binding lectin (AchatininH) from Achatina fulica snail

A sialic acid binding lectin, Achatinin_H, was purified in single step from the hemolymph of the land snail, Achatina fulica, by the affinity chromatography on sheep submaxillary mucin coupled to Sepharose 4B. The yield of the lectin was found to be 3 mg from 100 ml of hemolymph. The homogeneity of the lectin was established by alkaline gel electrophoresis, immunodiffusion, immunoelectrophoresis and analytical isoelectrophoresis. The molecular weight of the native protein was 242000, having identical subunits of Mr 15000. The lectin agglutinated rabbit erythrocytes in the presence of Ca^2–. The inhibition study clearly suggests that the binding site of the lectin recognizes sialic acid as the immunodominant sugar. This was further confirmed by the observation that there was a marked decrease of agglutinating activity of the lectin with neuraminidase treated rabbit erythrocytes and asialofetuin was unable to inhibit the activity of Achatinin_H. Among the inhibitors used the glycoconjugate containing 2-6 linkages of N-acetylneuraminic acid with subterminal galactopyranose or 2-acetamido-2-deoxy-galactopyranose residue was found to be better inhibitor than that containing 23 linkages of N-acetyl neuraminic acid. Besides that sialoglycoprotein containing both N and O type of glycosidic linkages plays an important role in binding with the lectin. Fetuin was found to be the best inhibitor.     

Lectin binding sites on the plasma membrane of epididymal and ejaculated chimpanzee sperm

Lectins have been used to analyze variations in the distribution and density of exposed saccharides of the sperm plasma membrane during physiologic maturation and after ejaculation. Studies have been conducted in a number of nonprimate species but have been conducted to only a limited extent in nonhuman primates. In this study, pure suspensions of chimpanzee sperm from the caput and cauda epididymis and from the ejaculate were labeled with lectins conjugated to fluorescein isothiocyanate in order to visualize changes in the distribution of exposed membrane glycocomponents. The lectins used were Con A, DBA, RCA-I, and WGA. Con A binding showed minimal change during epididymal transit, with an increased binding to the flagellum after ejaculation. DBA binding was relatively constant in all specimens. RCA-I showed distinct changes in binding pattern between epididymal and ejaculated sperm. On ejaculated sperm strong fluorescence was limited to the posterior head and to the midpiece. WGA binding increased during epididymal passage and decreased after ejaculation. There appears to be a wide variety of saccharide groups available for lectin binding on the surface of epididymal and ejaculated chimpanzee sperm. The general similarity in binding patterns of caput and cauda epididymal chimpanzee sperm exposed to Con A and DBA might reflect the fact that sperm morphology does not change during epididymal transit in this species, thus implying a more stable membrane structure than is present in other primates so far studied.