3‐O‐Sulfation of Heparan Sulfate Enhances Tau Interaction and Cellular Uptake

Prion‐like transcellular spreading of tau in Alzheimer's Disease (AD) is mediated by tau binding to cell surface heparan sulfate (HS). However, the structural determinants for tau–HS interaction are not well understood. Microarray and SPR assays of structurally defined HS oligosaccharides show that a rare 3‐O‐sulfation (3‐O‐S) of HS significantly enhances tau binding. In Hs3st1^?/? (HS 3‐O‐sulfotransferase‐1 knockout) cells, reduced 3‐O‐S levels of HS diminished both cell surface binding and internalization of tau. In a cell culture, the addition of a 3‐O‐S HS 12‐mer reduced both tau cell surface binding and cellular uptake. NMR titrations mapped 3‐O‐S binding sites to the microtubule binding repeat 2 (R2) and proline‐rich region 2 (PRR2) of tau. Tau is only the seventh protein currently known to recognize HS 3‐O‐sulfation. Our work demonstrates that this rare 3‐O‐sulfation enhances tau–HS binding and likely the transcellular spread of tau, providing a novel target for disease‐modifying treatment of AD and other tauopathies.     

Synthesis of a Systematic 64-Membered Heparan Sulfate Tetrasaccharide Library

Heparan sulfate (HS) has multifaceted biological activities. To date, no libraries of HS oligosaccharides bearing systematically varied sulfation structures are available owing to the challenges in synthesizing a large number of HS oligosaccharides. To overcome the obstacles and expedite the synthesis, a divergent approach was designed, where 64 HS tetrasaccharides covering all possible structures of 2-O-, 6-O- and N-sulfation with the glucosamine-glucuronic acid-glucosamine-iduronic acid backbone were successfully produced from a single strategically protected tetrasaccharide intermediate. This extensive library helped identify the structural requirements for HS sequences to have strong fibroblast growth factor-2 binding but a weak affinity for platelet factor-4. Such a strategy to separate out these two interactions could lead to new HS-based potential therapeutics without the dangerous adverse effect of heparin-induced thrombocytopenia.     

Single‐Molecule Fluorescence Detection of a Synthetic Heparan Sulfate Disaccharide

The first single‐molecule fluorescence detection of a structurally‐defined synthetic carbohydrate is reported: a heparan sulfate (HS) disaccharide fragment labeled with Alexa488. Single molecules have been measured whilst freely diffusing in solution and controlled encapsulation in surface‐tethered lipid vesicles has allowed extended observations of carbohydrate molecules down to the single‐molecule level. The diverse and dynamic nature of HS–protein interactions means that new tools to investigate pure HS fragments at the molecular level would significantly enhance our understanding of HS. This work is a proof‐of‐principle demonstration of the feasibility of single‐molecule studies of synthetic carbohydrates which offers a new approach to the study of pure glycosaminoglycan (GAG) fragments.     

Capillary electrophoresis and enzyme solid phase assay for examining the purity of a synthetic heparin proteoglycan-like conjugate and identifying binding to basic fibroblast growth factor

Interaction of basic fibroblast growth factor (bFGF) with heparin/heparan sulfate proteoglycans protects the growth factor against proteolytic degradation and is essential for its cellular activity. Although the structural requirements of heparin and heparan sulfate for the high-affinity binding to bFGF have been extensively examined, studies on intact heparin proteoglycans are limited. In this report, the purity and the binding ability of a heparin proteoglycan-like molecule-the heparin-bovine serum albumin (heparin-BSA) conjugate-was examined using capillary zone electrophoresis (CZE). Furthermore, the affinity of bFGF binding to the heparin-BSA conjugate was studied using an enzyme solid-phase assay. Chondroitin sulfate, dermatan sulfate, hyaluronan, heparan sulfate and variously sulfated disaccharides derived from heparin and heparan sulfate were also studied for their ability to compete with the binding of bFGF to heparin. Heparin-BSA conjugate was synthesized by reductive amination and, following precipitation with 1.5 vols of ethanol-sodium acetate, it was obtained free of contaminating heparin. Heparin-BSA-bFGF conjugate was obtained following incubation of heparin-BSA with bFGF for 2 h at 37 degrees C. Intact heparin, heparin-BSA and heparin-BSA-bFGF conjugates were completely resolved by CZE using 50 mM phosphate, pH 3.5, as operating buffer, reversed polarity (30 kV) and detection at 232 nm. Competitive solid phase assay showed that, among the glycosaminoglycans tested, heparin exhibits the highest affinity binding to bFGF (IC(50) = 6.4 nM). Heparan sulfate showed a lower affinity as compared with that of heparin, whereas all other glycosaminoglycans and heparin/heparan sulfate-derived disaccharides tested showed minute effects. The developed CZE method is rapid and accurate and can be easily used to identify bFGF-interacting heparin preparations of biopharmaceutical importance.     

HIV-1 Tat and Heparan Sulfate Proteoglycans Orchestrate the Setup of in Cis and in Trans Cell-Surface Interactions Functional to Lymphocyte Trans-Endothelial Migration

HIV-1 transactivating factor Tat is released by infected cells. Extracellular Tat homodimerizes and engages several receptors, including integrins, vascular endothelial growth factor receptor 2 (VEGFR2) and heparan sulfate proteoglycan (HSPG) syndecan-1 expressed on various cells. By means of experimental cell models recapitulating the processes of lymphocyte trans-endothelial migration, here, we demonstrate that upon association with syndecan-1 expressed on lymphocytes, Tat triggers simultaneously the in cis activation of lymphocytes themselves and the in trans activation of endothelial cells (ECs). This “two-way” activation eventually induces lymphocyte adhesion and spreading onto the substrate and vascular endothelial (VE)-cadherin reorganization at the EC junctions, with consequent endothelial permeabilization, leading to an increased extravasation of Tat-presenting lymphocytes. By means of a panel of biochemical activation assays and specific synthetic inhibitors, we demonstrate that during the above-mentioned processes, syndecan-1, integrins, FAK, src and ERK_1/2 engagement and activation are needed in the lymphocytes, while VEGFR2, integrin, src and ERK_1/2 are needed in the endothelium. In conclusion, the Tat/syndecan-1 complex plays a central role in orchestrating the setup of the various in cis and in trans multimeric complexes at the EC/lymphocyte interface. Thus, by means of computational molecular modelling, docking and dynamics, we also provide a characterization at an atomic level of the binding modes of the Tat/heparin interaction, with heparin herein used as a structural analogue of the heparan sulfate chains of syndecan-1.     

Synthesis of a Targeted Library of Heparan Sulfate Hexa‐ to Dodecasaccharides as Inhibitors of β‐Secretase: Potential Therapeutics for Alzheimer’s Disease

Heparan sulfates (HS) are a class of sulfated polysaccharides that function as dynamic biological regulators of the functions of diverse proteins. The structural basis of these interactions, however, remains elusive, and chemical synthesis of defined structures represents a challenging but powerful approach for unravelling the structure–activity relationships of their complex sulfation patterns. HS has been shown to function as an inhibitor of the β‐site cleaving enzyme β‐secretase (BACE1), a protease responsible for generating the toxic Aβ peptides that accumulate in Alzheimer’s disease (AD), with 6‐O‐sulfation identified as a key requirement. Here, we demonstrate a novel generic synthetic approach to HS oligosaccharides applied to production of a library of 16 hexa‐ to dodecasaccharides targeted at BACE1 inhibition. Screening of this library provided new insights into structure–activity relationships for optimal BACE1 inhibition, and yielded a number of potent non‐anticoagulant BACE1 inhibitors with potential for development as leads for treatment of AD through lowering of Aβ peptide levels.     

Development and validation of an LC–MS/MS Method for the quantitation of heparan sulfate in human urine

Heparan sulfate is a linear polysaccharide and serves as an important biomarker to monitor patient response to therapies for MPS III disorder. It is challenging to analyze heparan sulfate intact owing to its complexity and heterogeneity. Therefore, a sensitive, robust and validated LC–MS/MS method is needed to support the clinical studies for the quantitation of heparan sulfate in biofluids under regulated settings. Presented in this work are the results of the development and validation of an LC–MS/MS method for the quantitation of heparan sulfate in human urine using selected high‐abundant disaccharides as surrogates. During sample processing, a combination of analytical technologies have been employed, including rapid digestion, filtration, solid‐phase extraction and chemical derivatization. The validated method is highly sensitive and is able to analyze heparan sulfate in urine samples from healthy donors. Disaccharide constitution analysis in urine samples from 25 healthy donors was performed using the assay and demonstrated the proof of concept of using selected disaccharides as a surrogate for validation and quantitation.     

用炼锌厂废泥制取硫酸铜、海绵镉和硫酸锌

研究了以炼锌厂废泥为原料,经氧化、酸浸、中和沉淀、稀硫酸溶解、置换等步骤制取硫酸铜、海绵镉和硫酸锌的工艺.     

Identification and Structural Characterization of Novel Chondroitin/Dermatan Sulfate Hexassacharide Domains in Human Decorin by Ion Mobility Tandem Mass Spectrometry

Chondroitin sulfate (CS) and dermatan sulfate (DS) are found in nature linked to proteoglycans, most often as hybrid CS/DS chains. In the extracellular matrix, where they are highly expressed, CS/DS are involved in fundamental processes and various pathologies. The structural diversity of CS/DS domains gave rise to efforts for the development of efficient analytical methods, among which is mass spectrometry (MS), one of the most resourceful techniques for the identification of novel species and their structure elucidation. In this context, we report here on the introduction of a fast, sensitive, and reliable approach based on ion mobility separation (IMS) MS and MS/MS by collision-induced dissociation (CID), for the profiling and structural analysis of CS/DS hexasaccharide domains in human embryonic kidney HEK293 cells decorin (DCN), obtained after CS/DS chain releasing by β-elimination, depolymerization using chondroitin AC I lyase, and fractionation by size-exclusion chromatography. By IMS MS, we were able to find novel CS/DS species, i.e., under- and oversulfated hexasaccharide domains in the released CS/DS chain. In the last stage of analysis, the optimized IMS CID MS/MS provided a series of diagnostic fragment ions crucial for the characterization of the misregulations, which occurred in the sulfation code of the trisulfated-4,5-Δ-GlcAGalNAc[IdoAGalNAc]₂ sequence, due to the unusual sulfation sites.     

4-(2-苄氧基乙氧基羰基)氧杂环丁-2-酮的合成及表征

通过16例人肺鳞癌和小细胞肺癌组织中表达蛋白的二维电泳分离和质谱分析,经数据库检索鉴定了53个蛋白,其中24个蛋白与肺癌发病机制相关,4个蛋白在其它癌症中有报道,表达呈现差异的蛋白点有44个,其中34个在表达量上有差异,lO个蛋白在鳞癌和小细胞癌间表现为有和无的关系,蛋白功能分析提示人肺鳞癌与小细胞癌的蛋白质组表达存在差异,分析这些差异蛋白有利于肺癌分型及其生物标志物研究。     

大豆分离蛋白-十二烷基硫酸钠微胶囊的制备与表征

以大豆分离蛋白(SPI)和十二烷基硫酸钠(SDS)为壁材, 以十六烷为芯材, 通过复凝聚法制备了微胶囊. 首先确定了SPI和SDS发生复凝聚的适宜pH、SPI/SDS配比、壁材浓度等. 在确定的实验条件下进行复凝聚, 凝聚物产率可达85%. 改变搅拌转速和芯壁比, 考察它们对微胶囊性能的影响. 用光学显微镜观察了微胶囊形貌. 用气相色谱测定了微胶囊的载药量和包覆率. 芯壁比为2、搅拌转速为400 r/min时所制备微胶囊的载药量可达61%. 随着芯壁比的增大, 微胶囊粒径及载药量都逐渐增大.     

Characterization of scaffolds based on chitosan and collagen with glycosaminoglycans

Scaffolds based on chitosan (CTS), collagen (Coll), and glycosaminoglycans (GAGs) cross-linked by N-(3-dimethylamino propyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) mixture were obtained with the use of the freeze-drying method. They were characterized by different analyses, e.g. mechanical and swelling tests, porosity, and density measurement. Moreover, the scaffolds behavior in cell culture was examined with human osteosarcoma SaOS-2 cells. The results showed that the scaffolds based on CTS, Coll, and GAGs cross-linked by EDC/NHS present physicochemical properties appropriate for biomedical purposes. They show porosity above 90% and are highly swellable. The increasing GAGs content improves the attachment and survival of cells on the obtained scaffolds. It can be assumed that scaffolds based on CTS and Coll, GAGs-enriched and cross-linked by EDC/NHS addition are biocompatible, and have properties appropriate for the tissue engineering purposes.     

血清中硫脂质的提纯及其结构分析

应用离子交换及硅胶柱色谱法分离提纯血清中酸性脂质,并采用薄层色谱法(TLC)、TLC免疫染色法、质谱法及溶化分解等方法分析血清中硫脂质的种类及结构。实验确定了样品的提纯体系,证实胆固醇硫酸及脑硫脂是血清中的主要硫脂质。     

用高效液相色谱法测定热应激时大鼠肺及肝组织中细胞膜磷脂的变化

应用高效液相色谱法（ＨＰＬＣ）测定了正常大鼠肺及肝组织中细胞膜磷脂的含量及热应激时膜磷脂的含量变化。流动相为甲醇∶乙腈∶８５％磷酸（３∶１００∶１，Ｖ／Ｖ／Ｖ），色谱柱为μ－Ｐｏｒａｓｉｌ柱（３．９ｍｍｉ．ｄ．×３００ｍｍ）。通过测定膜磷脂的变化，可以为阐明机体的发病机理提供可靠的数据     

Rational Design and Expedient Synthesis of Heparan Sulfate Mimetics from Natural Aminoglycosides for Structure and Activity Relationship Studies

Heparan sulfate (HS) contains variably repeating disaccharide units organized into high- and low-sulfated domains. This rich structural diversity enables HS to interact with many proteins and regulate key signaling pathways. Efforts to understand structure-function relationships and harness the therapeutic potential of HS are hindered by the inability to synthesize an extensive library of well-defined HS structures. We herein report a rational and expedient approach to access a library of 27 oligosaccharides from natural aminoglycosides as HS mimetics in 7–12 steps. This strategy significantly reduces the number of steps as compared to the traditional synthesis of HS oligosaccharides from monosaccharide building blocks. Combined with computational insight, we identify a new class of four trisaccharide compounds derived from the aminoglycoside tobramycin that mimic natural HS and have a strong binding to heparanase but a low affinity for off-target platelet factor-4 protein.     

Metabolism of Pyrene and Chrysene in Epithelial Human Bronchial and Hamster Lung Cells

Abstract

The metabolism of pyrene and chrysene in epithelial human bronchial and in hamster lung cells has been studied and found to be very similar in both systems, although it differs from that observed in rat lung microsomes. Metabolite profiles have been analyzed by means of capillary GC and by GC/MS.     

Isolation,Characterization, and Compositional Analysis of Polysaccharides from Pinot Noir Wines: An Exploratory Study

It has been reported that polysaccharides in wine can interact with tannins and other wine components and modify the sensory properties of the wine. Unfortunately, the contribution of polysaccharides to wine quality is poorly understood, mainly due to their complicated structure and varied composition. In addition, the composition and molecular structure of polysaccharides in different wines can vary greatly. In this study, the polysaccharides were isolated from pinot noir wine, then separated into high-molecular-weight (PNWP-H) and low-molecular-weight (PNWP-L) fractions using membrane-based ultrafiltration. Each polysaccharide fraction was further studied using size exclusion chromatography, UV–Vis, FT-IR, matrix-assisted laser desorption/ionization–high-resolution mass spectrometry, and gas chromatography-mass spectrometry (GC-MS). The results showed that PNWP-L and PNWP-H had different chemical properties and compositions. The FT-IR analysis showed that PNWPs were acidic polysaccharides with α- and β-type glycosidic linkages. PNWP-L and PNWP-H had different α- and β-type glycosidic linkage structures. FT-IR showed stronger antisymmetric and symmetric stretching vibrations of carboxylate anions of uronic acids in PNWP-L, suggesting more uronic acid in PNWP-L. The size exclusion chromatography results showed that over 72% of the PNWP-H fraction had molecular sizes from 25 kDa to 670 kDa. Only a small percentage of smaller molecular polysaccharides was found in the PNWP-H fraction. In comparison, all of the polysaccharides in the PNWP-L fraction were below 25 KDa, with a majority distributed approximately 6 kDa (95.1%). GC-MS sugar composition analysis showed that PNWP-L was mainly composed of galacturonic acid, rhamnose, galactose, and arabinose, while PNWP-H was mainly composed of mannose, arabinose, and galactose. The molecular size distribution and sugar composition analysis suggested that the PNWP-L primarily consisted of rhamnogalacturonans and polysaccharides rich in arabinose and galactose (PRAG). In comparison, PNWP-H were mostly mannoproteins and polysaccharides rich in arabinose and galactose (PRAG). Further research is needed to understand the impacts of these fractions on wine organoleptic properties.     

石墨烯/硫酸铅复合材料的制备及其在铅酸电池中的电化学性能

利用简单的浸渍法制备了石墨烯/硫酸铅复合材料,使得硫酸铅可以直接用作铅酸电池负极材料。该复合材料分别以100 mA.g-1、200 mA.g-1和300 mA.g-1电流密度放电时,平均放电比容量分别可达到110、94和69 mAh.g-1,而硫酸铅仅为49、5和0.5 mAh.g-1,显示出复合材料在高倍率充放电下更好的比容量和再接受充电能力。循环伏安测试表明石墨烯的电容效应随扫描速率增大而增强,同时析氢也变得严重,使得复合材料在充放电过程中充电效率比纯硫酸铅低20%。在充放电过程中,石墨烯能够提高硫酸铅1倍以上的放电容量,并将充电电压提高0.1 V。XRD和SEM结果显示硫酸铅均匀分布在石墨烯片层上,没有出现团聚现象。