瓜蒌薤白半夏汤对兔动脉粥样硬化模型主动脉蛋白聚糖的作用

目的从调整动脉壁蛋白聚糖代谢角度观察化痰治法代表方剂——瓜蒌薤白半夏汤抗动脉粥样硬化的药理机制。方法3月龄新西兰兔30只,随机分为正常组、模型组、治疗组。模型组和治疗组均给予高脂饮食造成动脉粥样硬化模型,治疗组同时给予瓜蒌薤白半夏汤灌胃。6周后取材,检测血脂,观察主动脉粥样硬化病变程度,提取主动脉蛋白聚糖,酶解为糖胺多糖,再进行醋酸纤维素薄膜电泳分析,测定各种糖胺多糖组份含量。结果正常组血胆固醇和低密度脂蛋白含量分别为13.43±3.12mmol/L、8.53±1.37mmol/L,动脉壁蛋白聚糖组份硫酸软骨素、硫酸皮肤素含量分别为21.93±1.82mg/g、15.56±1.61mg/g;与正常组比较,模型组表现为典型的动脉粥样硬化病理变化,血总胆固醇和低密度脂蛋白含量明显升高,分别为23.63±4.31mmol/L、15.63±1.27mmol/L(P<0.01),动脉壁蛋白聚糖组份硫酸软骨素、硫酸皮肤素含量明显升高,分别为31.23±1.41mg/g、19.36±1.64mg/g(P<0.01或P<0.05);与模型组比较,治疗组动脉粥样硬化灶病变程度明显减轻,动脉壁蛋白聚糖组份硫酸软骨素、硫酸皮肤素含量明显降低,分别为22.33±1.58mg/g、14.36±1.71mg/g(P<0.01或P<0.05),但血胆固醇和低密度脂蛋白无明显的降低,分别为20.54±3.59mmol/L、14.53±1.32mmol/L。结论瓜蒌薤白半夏汤可降低粥样硬化病变动脉壁硫酸软骨素蛋白聚糖、硫酸皮肤素蛋白聚糖含量,从而减轻动脉粥样硬化病变,但降低血脂作用不明显。

LAMB3、SPOCK1、PRR11在膀胱癌组织中的表达及相关性

目的探究层粘连蛋白亚基B3(laminin subunit B3,LAMB3)、睾丸白聚糖(testicular leukan,SPOCK1)、富含脯氨酸蛋白11基因(proline rich protein 11 gene,PRR11)在膀胱癌组织中的表达及相关性。方法回顾性选取进行膀胱肿瘤根治性切除术的膀胱癌患者53例。使用免疫组化染色方式检测LAMB3、SPOCK1、PRR11,使用Western blot法检测LAMB3、SPOCK1、PRR11表达量,分析对比不同临床特征下LAMB3、SPOCK1、PRR11表达情况,对比三者的相关性。结果癌组织中LAMB3、SPOCK1、PRR11表达高于癌旁组织(P<0.05);不同性别、不同年龄下癌组织LAMB3、SPOCK1、PRR11表达相比(P>0.05),有无吸烟史情况下膀胱癌患者癌组织内LAMB3、SPOCK1、PRR11表达相比(P>0.05);肿瘤最大径>3 cm患者癌组织LAMB3、SPOCK1、PRR11表达高于肿瘤最大径≤3 cm患者(P<0.05);有淋巴结转移膀胱癌患者癌组织LAMB3、SPOCK1、PRR11表达高于无淋巴转移膀胱癌患者(P<0.05);TNM分期T2、T3、T4期膀胱癌患者LAMB3、SPOCK1、PRR11表达高于TNM分期Tis、Ta、T1期膀胱癌患者(P<0.05);低分化膀胱癌患者癌组织中LAMB3、SPOCK1、PRR11表达高于高、中分化膀胱癌患者(P<0.05);膀胱癌组织中LAMB3、SPOCK1呈正相关(r=0.357,P=0.008);膀胱癌组织中LAMB3、PRR11表达呈正相关(r=0.366,P=0.006);膀胱癌组织中SPOCK1、PRR11在膀胱癌组织中呈现出正相关(r=0.534,P=0.001)。结论LAMB3、SPOCK1、PRR11在膀胱癌组织中呈现出高表达,且呈现出正相关关系。

Cloning and expression analysis of a long type peptidoglycan recognition protein(PGRP-L) from Xenopus tropicalis

Peptidoglycan recognition proteins（PGRPs） are a family of pattern recognition receptors（PRRs） of the immune system,which bind and hydrolyze bacterial peptidoglycan.Here,a long type PGRP（PGRP-L） was first cloned in the lower vertebrate species Xenopus tropicalis（Xt）.The XtPGRP-L possessed a conserved genomic structure with five exons and four introns.The alignment and phylogenetic analysis indicated that XtPGRP-L might be a type of amidase-like PGRP.The 3-D model showed that XtPGRP-L possessed a conserved structure compared with the Drosophila PGRP-Lb.During embryonic development,XtPGRP-L was not expressed until the 72 h tadpole stage.In adult tissues,it was strongly expressed in the liver,lung,intestine,and stomach.Furthermore,after LPS stimulation,the expression of XtPGRP-L was up-regulated significantly in the liver,intestine and spleen,indicating that XtPGRP-L may play an important role in the innate immunity of Xenopus tropicalis.

Release Model of Water-soluble Chitosan Nanoparticles for Protein Delivery

[Objective] The experiment aimed to explore release rule of water-soluble chitosan (WSC) in vitro. [Method]The bovine serum albumin(BSA) was taken as a model protein drug and some existing release models such as Kinetics model, Gompertz model, Weibull model, Higuchi model and Logistic model were used to fit the BSA release profile from WSC carriers. [Result] Except Higuchi model and Logistic model, other models could fit BSA release profile better. [Conclusion] Gompertz two-order kinetics model could fit the release of WSC nano-particles better and model parameters had practical physical meaning.

Endo-Glycanhydrolases Activities in Artemisia sphaerocephala (Asteraceae) Mucilaginous Achene Germination Process

对白沙蒿 (ArtemisiasphaerocephahaKrasch .)种子萌发不同阶段的种胚提取物中几种可能降解果皮外层粘液物质的多糖内切酶进行了检测。研究结果表明 :多聚半乳糖醛酸酶在干燥胚中已存在并具较高的活性 ,但其活性随着种子吸涨及萌发而降低。此酶并不释放到种胚外。β 甘露聚糖内切酶随着种子的吸涨而增加并被萌发的种胚释放到种子外。纤维素酶在种子萌发的过程中不表现出活性。提取物中的所有多糖内切酶都无法降解果皮外层的粘液物质。

The interaction of versican with its binding partners

Versican belongs to the family of the large aggregating chondroitin sulfate proteoglycans located primarily within the extracellular matrix （ECM）. Versican, like other members of its family, has unique N- and C-terminal globular regions, each with multiple motifs. A large glycosaminoglycan-binding region lies between them. This review will begin by outlining these structures, in the context of ECM proteoglycans. The diverse binding partners afforded to versican by virtue of its modular design will then be examined. These include ECM components, such as hyaluronan, type Ⅰ collagen, tenascin-R, fibulin-1, and -2, fibrillin-1, fibronectin, P- and L-selectins, and chemokines. Versican also binds to the cell surface proteins CD44, integrin β1, epidermal growth factor receptor, and P-selectin glycoprotein ligand-1. These multiple interactors play important roles in cell behaviour, and the roles of versican in modulating such processes are discussed.

In vitro and in vivo protective effects of proteoglycan isolated from mycelia of Ganoderma lucidum on carbon tetrachlorideinduced liver injury

AIM： To investigate the possible mechanism of the protective effects of a bioactive fraction, Ganoderma lucidum proteoglycan （GLPG）isolated from Ganoderma luddum mycelia, against carbon tetrachloride-induced liver injury. METHODS： A liver injury model was induced by carbon tetrachloride. Cytotoxicity was measured by MTY assay. The activities of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were determined with an automatic multifunction-biochemical analyzer and the levels of superoxide dismutase （SOD） and TNF-α were determined following the instructions of SOD kit and TNF radioimmunoassay kit. Uver sections were stained with hematoxylin and eosin （H＆E） for histological evaluation and examined under light microscope. RESULTS： We found that GLPG can alleviate the L-02 liver cells injury induced by carbon tetrachloride （CCh） through the measurements of ALT and AST activities and the administration of GLPG to L-02 cells did not display any toxicity. Furthermore, histological analysis of mice liver injury induced by CCh with or without GLPG pretreatment indicated that GLPG can significantly suppress the toxicity induced by CCh in mice liver. We also found that GLPG reduced TNF-α level induced by CCh in the plasma of mice, whereas increased SOD activity in the rat serum. CONCLUSION： GLPG has hepatic protective activity against CCl4 induced injury both in vitro and in vivo. The possible antihepatotoxic mechanisms may be related to the suppression of TNF-α level and the free radical scavenging activity.

Removing Endotoxin from Protein Solution by Chitosan Modified Affinity Membrane

Affinity membrane was prepared with chitosan immobilized on the hydrophile- modified poly(vinylidene fluoride) (PVDF) membrane. Fourier transform infrared spectroscopy (FTIR.) analysis indicated that the contents of —NH2 and —OH groups increased and fluoride decreased on the membrane surface after modification. Using this kind of affinity membrane, the effects of operation parameters such as pH, ionic strength and flow rate, on the amount of endotoxin removed were investigated. The results showed that the equilibrium adsorption capacity and the dissociation constant of the affinity membrane to endotoxin were 21.4 EU·mg-1 membrane and 0.50EU·ml-1, respectively, at pH 7.0 and ionic strength 0.2 mol·L-1. Adsorption appeared to follow a typical Langmuir adsorption isotherm. At pH 5.0, ionic strength of 0.2 mol·L-1, the removal rate of endotoxin from BSA solution with the chitosan affinity membrane was up to 88.6% (11.50 EU·mg-1 membrane), and the recovery of BSA was 93.4% (0.187mg·mg-1 membrane), while at pH 11.0, ionic strength of 0.2mol·L-1, the removal rate of endotoxin from lysozyme solution was 72.4% (9.92EU·mg-1 membrane), and the recovery of lysozyme was 92.3% (0.104 mg·mg-1 membrane).

Protein adsorption onto diethylaminoethyl dextran modified anion exchanger:Effect of ionic strength and column behavior

Our previous studies on bovine serum albumin （BSA） adsorption to diethylaminoethyl dextran （DEAE dextran, DexD, grafting-ligand） and DEAE （D, surface-ligand） modified Sepharose FF resins found that all the grafted resins （FF-DexD and FF-D-DexD） exhibited extremely fast uptake rate （effective diffusivity, De, De/Do 〉 1.4）, which was six times greater than the ungrafted resins （De/Do 〈 0.3）. In this work, the influence of ionic strength （IS） on 6 typical DEAE dextran-grafted resins was investigated. Bath adsorption equilibria and kinetics, breakthrough, and linear gradient elution experiments were conducted. Commercial DEAE Sepharose FF was used for comparison. It is found that protein adsorption capacities on DEAE dextran-FF resins and the commercial resin decreased with increasing IS, but DEAE dextran-FF resins exhibited much higher capacity sensitivity to salt concentration. Besides, steeper decrease of adsorption capacities could be obtained at higher graftingligand or surface-ligand density. It is worth noting that the facilitating role of surface-ligand to the ＂chain delivery＂ effect was weakened after adding salt, leading to the less improvement in uptake rate by increasing surface-ligand density at higher IS. Although the uptake rates of the DEAE dextran-FF resins increased first and then decreased with increasing fS, they kept the extremely high level of De values （De/Do 〉 1.1 ） at the their working/binding IS range. Moreover, the DEAE dextran-FF resin displayed much higher adsorption capacities and De values than commercial ungrafted resin in their working condition. Furthermore, the column results of DEAE dextran-FF resins presented higher dynamic binding capacities than and similar elution ISs with DEAE Sepharose FF to achieve similar （or even higher） recoveries suggest the excellent chromatographic column performance of the DEAE dextran-FF resins. Finally, both high recovery and purity of BSA and γ-globulin could be easily achieved using the typical DEAE dextran-FF column, FF-D60-DexD160, to separate their binary mixtures, by step gradient elution. The research has provided new insights into the practical application of the series of DEAE-dextran grafted resins in protein chromatography and proved their superiority.

Isolation of Prawn(Exopalaemon carinicauda) Lipopolysaccharide and β-1,3-Glucan Binding Protein Gene and Its Expression in Responding to Bacterial and Viral Infections

The pattern recognition proteins （PRPs） play a major role in immune response of crustacean to resist pathogens. In the present study, as one of PRPs, lipopolysaccharide and 13-1, 3-glucan binding protein （LGBP） gene in the ridge tail white prawn （Exopalaemon carinicauda） （EcLGBP） was isolated. The full-length cDNA of EcLGBP was 1338 bp, encoding a polypeptide of 366 amino acid residules. The deduced amino acid sequence of EcLGBP shared high similarities with LGBP and BGBP from other crus- taceans. Some conservative domains were predicted in EcLGBP sequence. EcLGBP constitutively expressed in most tissues at dif- ferent levels, and the highest expression was observed in hepatopancreas. With infection time, the cumulative mortality increased gradually followed by the proliferation of Vibrio parahaemolyticus and white spot syndrome virus （WSSV）. The expression of EcLGBP in response to E parahaemolyticus infection was up-regulated in hemocytes and hepatopancreas, and the up-regulation in hepatopancreas was earlier than that in hemocytes. EcLGBP expression after WSSV infection increased at 3 h, then significantly decreased in both hemocytes and hepatopancreas. The results indicated that EcLGBP was involved in the immune defense against bacterial and viral infections.

Synthesis and anti-shock activity of divalent β-D-galactopyranosyl-(1→4)-β-D-glucopyranosides

Three divalent β-D-galactopyranosyl-（1 → 4）-β-D-glucopyranosides were synthesized using acetylated lactosyl bromide as donor and polyethylene glycols with different polymeric degree （n = 4, 5, 6） as linker, and evaluated for in vivo inhibitory activity to leukocyte-endothelial cell adhesion on severe bum-shock rats. The result showed that the length of linkers had apparent influence on anti cell adhesion activity.

CONSTRUCTING AAV-TGFβ_1 AND COMPARISING ITS BIOLOGICAL EFFECTS ON PROTEOGLYCAN SYNTHESIS OF NUCLEUS PULPOUS CELLS WITH AV-TGFβ_1

Objective To construct adeno-associated virus express system for TGFβ1 (AAV-TGFβ1) and compare its biological effects on proteoglycan synthesis of the rabbit lumbar disc nucleus pulpous (NP) cells with adenovirus (Ad) express system for TGFβ1 (AV-TGFβ1). Methods TGFβ1 gene was obtained by polymerase chain reactions (PCR). The upstream of TGFβ1 contained restriction enzyme site of EcoR Ⅰ, and the restriction enzyme site of Sal Ⅰ was at the downstream of TGFβ1. Using the multiple cloning sites (MCS) in plasmid AAV and the corresponding contained restriction enzyme site in PCR product of TGFβ1, TGFβ1 gene was subcloned into AAV. The recombinant plasmid AAV-TGFβ1 was detected by restriction enzyme digestion and DNA sequencing. Then, AAV-TGFβ1 virus was packaged and TGFβ1 expression mediated by AAV was detected by immunofluence analysis in H293 cells. AAV transfection rate to NP cells was evaluated with AAV-PEGF. After NP cells were respectively transfected by AAV-TGFβ1 virus and AV-TGFβ1 virus, proteoglycan synthesis was detected and compared by using Antonopulos methods. Results DNA sequencing revealed that the PCR-amplified TGFβ1 gene was consistent with NCBI Gene Bank. The recombinant plasmid was proved to be constructed successfully by restriction enzyme digestion. AAV could be transfected into NP cells and mediate an efficient expression of TGFβ1 protein. AV-TGFβ1 virus could quickly enhance the proteoglycan synthesis of the NP cells, but its biological effect was transient. AAV-TGFβ1 virus could enhance stably proteoglycan synthesis. Conclusion AAV-TGFβ1 virus was successful constructed and enhanced stably proteoglycan synthesis of NP cells.

miRNA-133a-UCP2 pathway regulates inflammatory bowel disease progress by influencing inflammation, oxidative stress and energy metabolism

AIM To investigate the role of the miR-133a-UCP2 pathway in the pathogenesis of inflammatory bowel disease (IBD) and to explore the potential downstream mechanisms with respect to inflammation, oxidative stress and energy metabolism. METHODS C57BL/6 mice were fed dextran sulfate sodium (DSS) liquid for 7 consecutive days, followed by the administration of saline to the DSS group, UCP2 siRNA to the UCP2 group and a miR-133a mimic to the miR-133a group on days 8 and 11. Body weight, stool consistency and rectal bleeding were recorded daily, and these composed the disease activity index (DAI) score for the assessment of disease severity. After cervical dislocation was performed on day 14, the length of the colon in each mouse was measured, and colonic tissue was collected for further study, which included the following: haematoxylin and eosin staining, UCP2 and miR-133a detection by immunohistochemical staining, western blot and quantitative real-time PCR, measurement of apoptosis by TUNEL assay, and the assessment of inflammation (TNF-alpha, IL-1 beta, IL-6 and MCP1), oxidative stress (H2O2 and MDA) and metabolic parameters (ATP) by ELISA and colorimetric methods. RESULTS An animal model of IBD was successfully established, as shown by an increased DAI score, shortened colon length and specific pathologic changes, along with significantly increased UCP2 and decreased miR-133a levels. Compared with the DSS group, the severity of IBD was alleviated in the UCP2 and the miR-133a groups after successful UCP2 knockdown and miR-133a overexpression. The extent of apoptosis, as well as the levels of TNF-alpha, IL-1 beta, MDA and ATP, were significantly increased in both the UCP2 and miR-133a groups compared with the DSS group. CONCLUSION The miR-133a-UCP2 pathway participates in IBD by altering downstream inflammation, oxidative stress and markers of energy metabolism, which provides novel clues and potential therapeutic targets for IBD.

The Role of the Charged Residues of the GP2 Helical Regions in Ebola Entry

The glycoprotein （GP） of Ebola is the sole structural protein that forms the spikes on the viral envelope. The GP contains two subunits, GPI and GP2, linked by a disulfide bond, which are responsible for receptor binding and membrane fusion, respectively. In this study, the full length of GP gene of Ebola Zaire species, 2028 base pairs in length, was synthesized using 38 overlapping oligonucleotides by multiple rounds of polymerase chain reaction （PCR）. The synthesized GP gene was shown to be efficiently expressed in mammalian cells. Furthermore, an efficient HIV-based pseudotyping system was developed using the synthetic GP gene, providing a safe approach to dissecting the entry mechanism of Ebola viruses. Using this pseudotyping system and mutational analysis, the role of the charged residues in the GP2 helical regions was examined. It was found that substitutions of the most charged residues in the regions did not adversely affect GP expression, processing, or viral incorporation, however, most of the mutations greatly impaired the ability of GP to mediate efficient viral infection. These results demonstrate that these charged residues of GP2 play an important role in GP-mediated Ebola entry into its host cells. We propose that these charged residues are involved in forming the intermediate conformation（s） of GP in membrane fusion and Ebola entry.

Effect of Discontinuous Administration of β -glucan and Glycyrrhizin on the Growth and Immunity of Pacific White Shrimp Litopenaeus Vannamei

A six-week growth trial was conducted to compare the effects of different feeding strate- gies of dietary immunostimulants on the growth and immunity of white shrimp Litopenaeus vannamei （4.70 ±0.20g）. Shrimps were fed with diet containing glycyrrhizin continuously, containing β -glucan continuously, discontinuously （seven days with diet containing β -gluseven days with diet without -glucan; two days with diet containing β-glucan following five days with diet without -glucan）,

Partial characterization of the N-linked oligosaccharide structures on P-selectin glycoprotein ligand-1 (PSGL-1)

PSGL-1, a specific ligand for P-, E- and L-selectin, was isolated from in vivo [3H]-glucosamine labeled HL- 60 cells by a combination of wheat germ agglutinin-agarose and P- or E-selectin-agarose chromatography. N- linked oligosaccharides were released from the purified, denatured ligand molecule by peptide: N-glycosidase F treatment and, following separation by Sephacryl S-200 chromatography, partially characterized using lectin, ion-exchange and size-exclusion chromatography in combination with glycosidase digestions. The data obtained suggest that the N-glycans on PSGL-1 are predominantly core-fucosylated, multiatennary complex type structures with extended, poly- N- acetyllactosamine contaniing outer chains. A portion of the outer chains appears to be substituted with fucose indicating that the N-glycans, in addition to the O-glycans on PSGL-1, may be involved in selectin binding.

Relationships between high-molecular-weight glutenin subunit （HMW-GS）, pentosan and various component contents in different wheat varieties

The composition of HMW-GS and pentosan and its various component contents in different wheat varieties were analyzed. According to scores of these subunits in several wheat varieties, the correlation relationship between the composition of HMW-GS, subunit combination types and pentosan, various components and component ratio were ascertained. The results showed that HMW-GSs were very diverse which had 11 types. In different varieties, the variation of Ara was obvious while Xyl was not; pentosan and its various component contents were very different, and their coefficient of variability was quite high. The majority of parameters had a significant negative correlation with Glu-1 where Glu-D1 was stronger than other two loci. N, 7, 17＋18 and 2＋12 subunits had influence on pentosan and its various component contents. But some subunits such as 1 and 5＋ 10 which are good for baking quality had little contribution to pentosan and various component contents. The pentosan and various component contents were higher in the varieties which had （N, 7, 2＋12） subunit combinations. Additionally, the 21 varieties were classified into 4 categories according to HMW-GS scores and pentosan content.

Polyvinyl Alcohol-Collagen Composite with Glycosaminoglycan as Scaffolds for Tissue Engineering

The aim of this study is to prepare a PVA-GAG-COL composite material by polyvinyl alcohol (PVA),glycosaminoglycan (GAG) and collagen (COL),and to investigate the feasibility of serving as a scaffold for tissue engineering. PVA was blended with various amounts of GAG and COL. Different proportional scaffolds could be obtained with different molecular weight and alcoholysis degree of PVA and different amounts of GAG,which exhibited high water content (60%-95%) and showed different inner configuration with swelling ratio (120%-620%). SEM proved that different composite materials had different porous structures.

Moisture Sorption Isotherms of Soy Protein IsolatelCarboxymethyl Chitosan Blend Films

Soy protein isolate/carboxymethyl chitosan （SPI/CMCH） blended films incorporated with glycerol were prepared using solution casting to investigate the effects of the SPI and CMCH ratios （100：0, 88：12, 67：33, 50：50, 33：67, 12：88, 0：100） on the water sorption isotherm. The moisture sorption isotherm of the SPI/CMCH blended films was determined using various relative humidity＇s （16%, 35%, 55% and 76% RH） at 25 ± 1℃. The isotherms showed that the equilibrium moisture content （EMC） of the films increased with increasing CMCH content and the EMC value sharply increased above aw = 0.55. Understanding of sorption isotherms is important for prediction of moisture sorption properties of films via moisture sorption empirical models. The Guggenheim-Oswin, Brunauer-Emmett-Teller （BET）, and Anderson-de Boer （GAB） sorption model predictions were tested against the experimental data. The root mean square （RMS） values from the Oswin, BET, and GAB models respectively ranged from 698.54 to 1,557.54, 38.85 to 58.30, and 52.52 to 95.95. Therefore, the BET model was found to be the best-fit model for SPI/CMCH blended films at 25 ± 1 ℃.

The Antioxidant Effects of Complexes of Tilapia Fish Skin Collagen and Different Marine Oligosaccharides

An excess of reactive oxygen species（ROS）leads to a variety of chronic health problems.As potent antioxidants,marine bioactive extracts containing oligosaccharides and peptides have been extensively studied.Recently,there is a growing interest in protein-polysaccharide complexes because of their potential uses in pharmaceutical and food industries.However,only few studies are available on the antioxidant activities of such complexes,in terms of their ROS scavenging capability.In this study,we combined and superoxide radicals,and to evaluate the influences on the activities of superoxide dismutase（SOD）,glutathione peroxidase（GSH-Px）and the level of malondialdehyde（MDA）in UV-induced photoaging models.The results indicated that the antioxidant activities of all the complexes were stronger than those of their individual components.Among the 11 complexes tested,two complexes,namely MA1000＋CP and κ-ca3000＋CP,turned out to be highly effective antioxidants.Although the detailed mechanisms of this improved scavenging ability are not fully understood,this work provides insights into the design of highly efficient peptide-oligosaccharide complexes for potential applications in pharmaceutical,cosmetics and food industries.