禁食与非禁食处理对构建2型糖尿病小鼠模型血糖的影响

为探讨禁食与非禁食处理对构建2型糖尿病小鼠模型血糖变化的影响,分别以普通饲料和高脂饲料喂养3周龄的C57BL/6J雄性小鼠5周,于第5周末采取禁食与非禁食处理,16 h后分别注射链脲佐菌素(streptozotocin,STZ)100 mg/kg体重或相应体积的柠檬酸缓冲液.于第5周末(禁食前)和注射后3周测定非空腹血糖浓度.普通饲料与高脂饲料注射STZ前禁食组血糖水平均显著升高,达到并超过糖尿病小鼠非空腹血糖成模标准(11mmol/L).高脂饲料注射STZ前非禁食组血糖水平表现为缓慢持续升高,其余各组血糖水平均低于糖尿病小鼠非空腹血糖成模标准.结果 表明,高脂饲料联合STZ诱导2型糖尿病小鼠血糖变化是有效的,但注射STZ前的禁食处理不是必需的;单纯注射STZ同样可以诱导糖尿病小鼠血糖升高,但注射前的禁食处理是必要的.     

C57BL/6J小鼠在构建2型糖尿病模型中体重和非空腹血糖浓度的异常变化

由北京市一实验动物生产单位购入近交系C57BL/6J(B6)和封闭群ICR(3周龄)小鼠,分别以高脂饲料、高脂饲料-3%果糖饮水(实验组)和常规饲料(对照组)喂养6周,实验组腹腔注射链脲佐菌素(STZ,100mg/kg体重),然后以相应饲料继续喂养4周。每周测定小鼠体重,于注射STZ前和注射后每周测定非空腹血糖浓度。研究显示,无论是否补充果糖饮水,B6对照组体重显著高于实验组,而相应周龄的ICR小鼠,实验组体重显著高于对照组。两品系小鼠实验组间体重无差异。注射STZ后,B6实验组血糖浓度均没有达到糖尿病小鼠非空腹血糖浓度的成模标准(11mmol/L),而ICR实验组血糖浓度均达到并超过糖尿病小鼠非空腹血糖浓度的成模标准。研究表明,无论补充果糖与否,ICR小鼠均能成功建模,而B6小鼠建模均失败。因此,ICR小鼠仍是目前应用高脂饲料-STZ联合诱导2型糖尿病模型中经济、有效的候选动物,而B6小鼠在体重和血糖浓度上的异常表现很可能是其遗传背景变化的结果,这尚需进一步研究证实。     

高脂饮食加低剂量链脲霉素建立小鼠2型糖尿病模型

目的高脂饮食加低剂量链脲霉素（Streptozotocin,STZ）建立小鼠2型糖尿病模型。方法5周的雄性C57BL/6J小鼠,随机分为正常饲料组、正常饲料加STZ组、高脂饲料组和高脂饲料加STZ组。相应饲料喂养5周后,按照100 mg/Kg的剂量腹腔注射STZ,然后继续喂养4周。在第5周和第9周末测定小鼠的体重、收缩压、血糖、血胰岛素、血甘油三脂和胆固醇水平。结果STZ注射前各组小鼠的体重、血压、血糖、血胰岛素、血脂和血甘油三脂无明显差异（P〉0.05）。STZ注射后4周时,高脂饲料加STZ组小鼠的体重、血糖、血胰岛素、血压和血脂水平明显升高（P〈0.05）;而其他三组的这些指标无明显改变或仅部分升高。结论高脂饮食加低剂量链脲霉素可建立小鼠2型糖尿病模型,该模型具有人2型糖尿病的主要表型特征和相似的发病过程。     

雌性小鼠不适于构建HFD—STZ联合诱导的2型糖尿病模型

目的 探讨雌性小鼠在高脂饲料(HFD)-链脲佐菌素(STZ)联合诱导2型糖尿病模型中的可行性.方法 分别以高脂饲料、高脂饲料-果糖饮水(HFDF)和常规饲料(对照)喂养3周龄的封闭群ICR和近交系C57BL/6J (B6)小鼠6周后,HFD和HFDF组腹腔注射STZ,对照组注射相应体积柠檬酸钠溶液,然后分别以相应饲料继续喂养6周.每周测定小鼠体重,于注射前1周和注射后1～4周测定非空腹血糖浓度.结果 实验结束时各组小鼠体重较初始体重均显著增加.ICR小鼠HFD和HFDF组体重与对照组S无差异,HFD与HFDF组间也无显著变化.虽然B6小鼠HFD与HFDF组体重组间差异不显著,但两组体重均显著低于对照组.注射STZ后1～4周,两品系小鼠HFD与HFDF组血糖水平没有显著升高,组间也没有显著差异,且均没有达到2型糖尿病小鼠成模非空腹血糖标准(11 mmol/L).结论 果糖饮水不能促进高脂饲料诱导的育肥作用,而雌性小鼠也不是HFD-STZ联合诱导2型糖尿病模型的理性选择.     

高脂饮食联合链脲佐菌素对小鼠糖脂代谢及炎症的影响

观察高脂饮食联合链脲佐菌素(STZ)对小鼠糖脂代谢及胰岛功能损伤的影响。将体质量为18~20 g的SPF级雄性C57BL/6J小鼠40只,随机分为对照组和模型组,每组20只。对照组小鼠饲喂对照饲料,模型组小鼠饲喂高脂饲料。1周后,禁食16 h,测量小鼠体质量与空腹血糖;尾静脉采血,分离血清;每周1次,连续4周。4周后,对照组小鼠腹腔注射柠檬酸缓冲液,模型组小鼠腹腔注射STZ,剂量为40 mg·kg~(-1),每天1次,连续3 d。继续饲养2周后,测量随机血糖,以小鼠血糖≥16.7 mmol·L~(-1)者即判定为2型糖尿病。对照组及2型糖尿病小鼠经腹腔注射葡萄糖以进行糖耐量试验。与对照组同一时间的体质量和血糖进行比较,模型组小鼠体质量、血糖均升高,除第1周血糖外,差别均有统计学意义(P0.05)。采用重复测量方差分析,发现喂养时间对体质量(F=200.831)和血糖(F=7.025)均有影响,差异有统计学意义(P0.05);不同喂养时间对低密度脂蛋白(F=30.793)、甘油三酯(F=34.027)和高密度脂蛋白(F=30.793)均有影响,差异有统计学意义(P0.05)。不同饲料喂养与不同喂养时间对体质量和甘油三酯存在交互作用(P0.05)。比较糖耐量曲线发现,成模小鼠较对照组小鼠糖耐量降低。采用ELISA试剂盒检测小鼠血清中TNF-α含量,成模小鼠的TNF-α含量高于对照组,差异有统计学意义(P0.05)。高脂饮食可导致C57BL/6J小鼠糖脂代谢紊乱,给予STZ后引起了小鼠糖耐量降低及炎症损伤,高脂饮食联合STZ可提高小鼠2型糖尿病模型的成模率。     

不同剂量链尿佐菌素诱导2型糖尿病大鼠模型的研究

目的:探讨不同剂量链脲佐菌素(Streptozotocin,STZ)联合高糖高脂饮食对2型糖尿病大鼠模型建立的影响。方法:90只8周龄SD雄性大鼠随机平均分为六组:普通饲料喂养+缓冲液组、高糖高脂饲料喂养+缓冲液(H.E组)、高糖高脂饲料喂养+35mg/kg链尿佐菌素组(H.E+35 mg/kg STZ组)、高糖高脂饲料喂养+45 mg/kg链尿佐菌素组(H.E+45 mg/kg STZ组)、高糖高脂饲料喂养+55 mg/kg链尿佐菌素组(H.E+55 mg/kg STZ组)及高糖高脂饲料喂养+65 mg/kg链尿佐菌素组(H.E+65 mg/kg STZ组),高糖高脂饲料喂养4周后诱导胰岛素抵抗,继之腹腔注射STZ,建立2型糖尿病大鼠模型。检测体重、胰岛素、空腹血糖、血脂、胰岛素敏感指数(ISI)。结果:与常规饮食组相比,高糖高脂饮食各组大鼠出现空腹血浆胰岛素(FINS)、空腹血糖(FBG)、血清甘油三脂(TG)、总胆固醇(TC)、游离脂肪酸(FFA)显著升高(P0.01),ISI显著下降(P0.01)。不同剂量STZ注射,H.E+45 mg/kg STZ组成模率最高且无自愈现象。结论:通过STZ腹腔注射联合高糖高脂饮食可成功复制出实验性2型糖尿病动物模型,45 mg/kg为STZ理想注射剂量。     

高脂喂养联合链脲佐菌素注射的糖尿病大鼠模型特征

目的观察高脂喂养联合低剂量STZ注射的SpragueDawley(SD)大鼠2型糖尿病模型的代谢特征、病理学以及胰岛分子生物学变化。方法4周龄雄性SD大鼠36只随机分为三组(1)正常对照组(Control)9只,普通饲料喂养。(2)高脂组(HighFatchow,HE)9只,高脂饲料喂养,为普通饲料中添加20%脂肪(猪油和蛋黄粉各50%)和20%蔗糖。(3)糖尿病组(DM)18只。喂养4周后腹腔注射STZ(40mg/kg)。所有大鼠做灌胃葡萄糖耐量(OGTT)试验。放免法测定血清胰岛素,免疫组化染色观察胰岛β细胞的形态学特点,彩色图像分析系统测定胰岛素表达量,RT-PCR测定胰腺β细胞胰岛素mRNA表达水平。结果糖尿病大鼠空腹血糖(FBG)、胰岛素水平(FINS)显著高于Control组和HE组大鼠(P<0.01),空腹血清甘油三酯(TG)和游离脂肪酸(FFA)水平显著高于Control组(P<0.05);胰岛β细胞吸光度(A)显著低于高脂组大鼠(P<0.05),降低11.6%。胰岛素免疫反应阳性区占胰岛百分比显著低于Control组和HE组,分别下降31.9%(P<0.05)和43.1%(P<0.01)。胰岛素mRNA表达水平显著低于HE组(P<0.05)。STZ注射后48h(基线值)大鼠FBG水平的分布情况为A组(FBG<10.0mmol/L)占7/18;B组(FBG10~19.9mmol/L)占5/18;C组(FBG≥20mmol/L)占6/18。STZ注射后9d的OGTT结果与基线值相比,B组OGTT值总体变化最小,A组FBG的变异最大,达到25%。结论高脂喂养联合低剂量STZ注射的糖尿病大鼠模型模拟2型糖尿病发生的主要病理生理过程,具有高血糖、高胰岛素血症以及血脂异常等基本特征。     

2型糖尿病大鼠模型制备的影响因素及其特点

目的探讨高脂喂养联合低剂量链脲佐菌素(Streptozotocin,STZ)制备2型糖尿病大鼠模型的造模方法和影响因素。方法4周龄雄性Sprague Dawley(SD)大鼠45只随机分为三组:(1)正常组(normal control,NC),9只,普通饲料喂养。(2)高脂组(high fat,HF),9只,高脂饲料喂养。(3)糖尿病模型组,根据高脂喂养时间差异和STZ剂量不同设计了3种模型制备方法:A组,9只,高脂喂养满4周,注射STZ 30 mg/kg;B组,9只,高脂喂养满8周,注射STZ 20 mg/kg;C组,9只,高脂喂养满8周,注射STZ 30 mg/kg。所有大鼠于48h、2周和4周后行灌胃葡萄糖耐量试验(OGTT)评价成模率和血糖波动情况。实验结束时测定血清胰岛素、甘油三酯(TG)和胆固醇(TC),RT-PCR测定胰腺内胰岛素mRNA表达水平,免疫组化染色观察胰岛细胞形态学特点,用彩色图像分析系统进行定量比较。结果糖尿病C组血糖显著升高,成模后2周血糖下降,4周后又上升到基线水平,成模率100%。糖尿病A组、B组在4周后血糖逐渐降低到接近正常水平,成模率分别为55.6%、11.1%。C组与HF组相比,胰岛素敏感性显著下降(P<0.01)。β细胞内胰岛素水平下降39.3%(P<0.01),胰岛内β细胞所占比例下降了79.2%(P<0.01),胰腺内胰岛素mRNA表达水平减少19.2%(P<0.01),α细胞升高了1倍(P<0.01)。结论高脂喂养8周后腹腔注射低剂量STZ(30 mg/kg)制备的2型糖尿病大鼠模型,成模率高,模型稳定。     

小鼠胰岛素抵抗哮喘模型的建立与评估

目的:探讨小鼠胰岛素抵抗哮喘模型的建立方法,并进行评估。方法:C57BL/6J小鼠随机分为4组:正常对照组(HC)、哮喘组(NIRA)均给予普通饲料喂养;胰岛素抵抗组IRNA)、胰岛素抵抗+哮喘组(IRA)均给予高脂饲料(D12492)喂养。每周称重,第6-14周,每周检测各组小鼠空腹血糖(FPG)、空腹血清胰岛素(FINS)水平,计算稳态模型胰岛素抵抗评价指数(HOMA-IR)评估胰岛素抵抗程度;小鼠胰岛素抵抗模型建立成功后在其基础上诱导哮喘模型,NIRA组和IRA组小鼠给予卵清蛋白(OVA)致敏、激发;HC组和IRNA组小鼠给予生理盐水作为对照,末次激发24后,制作肺病理切片,计数肺泡灌洗液(BALF)中白细胞总数及分类,检测血清和BALF中相关炎性因子的水平,比较各组小鼠胰岛素抵抗指数与哮喘评价指标,评估模型。结果:(1)第9周末,IRNA组、IRA组小鼠的HOMA-IR值均2.5,表明胰岛素抵抗小鼠模型建立成功;(2)肺组织病理切片中,HC组、IRNA组小鼠肺组织无炎症改变,NIRA组、IRA组炎细胞浸润明显,尤以IRA组更甚。(3)与HC组比较,NIRA组(P0.01)、IRA组(P0.01)BALF中白细胞总数、嗜酸性粒细胞比例明显增高;(4)血清中抗OVA特异性Ig E(P0.01)和Ig G1(P0.05)水平显著升高;(5)血清和BALF中IL-4(P0.01)、IL-17(P0.05)的水平明显升高,且IRA组(P0.05)明显高于NIRA组;IFN-γ(P0.05)的水平明显降低,且IRA组(P0.05)明显低于NIRA组。结论:用高脂饲料喂养C57BL/6J小鼠9周,可建立稳定的胰岛素抵抗模型,从第10周开始用OVA致敏、激发诱发哮喘,可成功建立稳定的胰岛素抵抗哮喘小鼠模型,为进一步研究胰岛素抵抗与哮喘相关机制奠定基础。     

人脐带间充质干细胞对猕猴糖尿病治疗效果的研究

目的通过用人脐带间充质干细胞(huMSCs)治疗猕猴糖尿病模型观察其血糖的变化。方法猕猴9只,分为对照组3只和模型组6只,模型组通过高糖高脂饮食及静脉注射链尿佐菌素(STZ)诱导成糖尿病模型,两组间比较用t检验。12周时再分为模型对照组3只和治疗组3只,治疗组每只每周静脉回输huMSCs 1×10~6个/kg,连续3周,每周检测各组血糖变化,三组间比较用方差分析。结果两组猕猴高糖高脂饮食喂养第8周时模型组空腹血糖值[(22.00±3.00)mmol/L]与对照组[(4.75±0.20)mmol/L]相比差异有统计学意义(t=9.94,P0.01)。40周时1只猕猴因糖尿病死亡,病理切片证实心、肝、脾、肺、肾和胰腺均有病变,符合糖尿病特征。huMSCs治疗后,3周血糖连续下降,到第4周对照组、模型组、治疗组空腹血糖分别为(4.85±0.35)mmol/L、(18.20±1.00)mmol/L、(4.09±0.50)mmol/L,3组差异有统计学意义(F=388.10,P0.01)。两两比较结果表明对照组和模型组比较差异有统计学意义(t=24.173,P0.01),模型组与治疗组比较差异有统计学意义(t=25.549,P=0.014)。结论 STZ联合高糖高脂饲料能成功诱导出猕猴糖尿病模型,用huMSCs能有效降低血糖,使糖尿病猕猴恢复正常血糖,方法简便。     

甲型H1N1流感病毒在季流病毒、禽流感病毒共感染时变异可能性的验证

目的 甲型H1N1流感病毒A/California/7/2009分别与A/Brisbane/10/07和A/ShenZhen/406H/06共感染小型香猪,预测甲流病毒在与季流H3N2病毒/甲流病毒与禽流感病毒共感染时是否会发生变异.方法 分别将A/California/7/2009(CA7)与A/Brisbane/10/07(H3N2),A/California/7/2009与A/Shenzhen/406H/06(H5N1)对5～6月龄小型猪共感染,小型猪经复方氯胺酮0.1 mL/kg麻醉后进行滴鼻感染,感染后第5天安乐死动物,取动物肺组织作病毒测序分析.结果 A/California/7/2009(CA7)与A/Brisbane/10/07(H3N2)共感染后,A/California/7/2009病毒PB1基因993位G→A突变,PA基因1659位G→A突变,没有氨基酸的变异.A/California/7/2009与A/Shenzhen/406H/06(H5N1)共感染后A/California/7/2009病毒PB2基因1711位T→C突变.碱基的突变未引起氨基酸的变异.结论 A/California/7/2009(CA7)与A/Brisbane/10/07(H3N2),A/California/7/2009与A/Shenzhen/406H/06(H5N1)共感染后在猪的体内没有发生病毒重组、变异.     

Pathogenesis of primary defects in mitochondrial ATP synthesis

Maternally inherited mutations in the mtDNA-encoded ATPase 6 subunit of complex V (ATP synthase) of the respiratory chain/oxidative phosphorylation system are responsible for a subgroup of severe and often-fatal disorders characterized predominantly by lesions in the brain, particularly in the striatum. These include NARP (neuropathy, ataxia, and retinitis pigmentosa), MILS (maternally inherited Leigh syndrome), and FBSN (familial bilateral striatal necrosis). Of the five known pathogenic mutations causing these disorders, four are located at two codons (156 and 217), each of which can suffer mutations converting a conserved leucine to either an arginine or a proline. Based on the accumulating data on both the structure of ATP synthase and the mechanism by which rotary catalysis couples proton flow to ATP synthesis, we propose a model that may help explain why mutations at codons 156 and 217 are pathogenic.     

A new kind of carbohydrate array,its use for profiling antiglycan antibodies,and the discovery of a novel human cellulose-binding antibody

In this study, we use a novel glycan array to analyze the glycan-binding antibody repertoire in a pool of affinity-purified IgG collected from a healthy human population. The glycan array used is based on mono- and oligosaccharides covalently linked to the surface via a long linker at their reducing ends. They are thus presented to the medium with a well-defined orientation and are accessible for specific binding by glycan-binding proteins, such as antibodies and lectins. A novel anticellulose antibody was detected that binds specifically to beta4-linked saccharides with a preference for glucopyranose over galactopyranose residues. We also found previously known antiglycan antibodies against mono- and oligosaccharides that are constituents of commonly occurring bacterial polysaccharides. We propose that this array can facilitate high-throughput screening of glycan-binding proteins and the search for biomarkers for personalized medicine.     

Analysis of the two active sites of the hyaluronan synthase and the chondroitin synthase of Pasteurella multocida

Type A Pasteurella multocida produces a hyaluronan (HA) capsule to enhance infection. The 972-residue HA synthase, pmHAS, polymerizes the linear HA polysaccharide composed of alternating beta3N-acetylglucosamine (GlcNAc)-beta4glucuronic acid (GlcUA). We demonstrated previously that pmHAS possesses two independent glycosyltransferase sites. Here we further define the sites and putative motifs. Deletion of residues 1-117 does not affect HA polymerizing activity. The carboxyl-terminal boundary of the GlcUA-transferase resides within residues 686-703. Both transferase sites contain a DXD motif essential for HA synthase activity. D247N or D249N mutants possessed only GlcUA-transferase activity, whereas D527N or D529N mutants possessed only GlcNAc-transferase activity, further confirming our assignment of the two active sites within the synthase polypeptide. A potential role of the DXD motif in substrate binding was supported by experiments utilizing high UDP-sugar concentrations that partially rescued the activity of certain mutants. The WGGED sequence motif is involved in GlcNAc-transferase activity because mutants with substitutions at E369 or D370 possessed only GlcUA-transferase activity. Type F P. multocida synthesizes an unsulfated chondroitin (beta3GalNAc-beta4GlcUA) capsule. A chimeric enzyme consisting of residues 1-427 of pmHAS and residues 421-704 of pmCS, the homologous chondroitin synthase, was an active HA synthase. The converse chimeric enzyme consisting of residues 1-420 of pmCS and residues 428-703 of pmHAS was a functional chondroitin synthase. Analyses of a panel of pmHAS/pmCS chimeric enzymes identified a 44-residue region, corresponding to pmHAS residues 225-265, involved in UDP-hexosamine selectivity. Overall, these findings further support the model of two independent transferase sites within a single polypeptide.     

Heparin sequencing

Heparin is a highly sulfated glycosaminoglycan widely used as an anticoagulant. Modifications in its relatively uniform structure appear to be key to its recognition and modulation of serine proteases, growth factors, chemokines, and extracellular proteins, as has been most clearly demonstrated in the antithrombin binding site. We sequenced the major oligosaccharides released from mastocytoma heparin by partial nitrous acid using a highly sensitive technique tailored for sequencing of metabolically radiolabeled heparin. It utilizes partial nitrous acid cleavage to allow simultaneous sequencing of the internal components of the oligosaccharide under investigation by specific lysosomal exoenzymes. Sequencing revealed that although the majority of the heparin disaccharides are N-, 2-O-, and 6-O-sulfated, the less sulfated disaccharides (lacking 2-O- or 6-O-sulfates) seem to be spaced out along the chain. The technique may be particularly useful for characterizing heparin from novel sources, such as the glial progenitor cells and Ascidia, as well as for sequencing protein binding sites.     

Glycoprofiling with micro-arrays of glycoconjugates and lectins

To facilitate deciphering the information content in the glycome, thin film-coated photoactivatable surfaces were applied for covalent immobilization of glycans, glycoconjugates, or lectins in microarray formats. Light-induced immobilization of a series of bacterial exopolysaccharides on photoactivatable dextran-coated analytical platforms allowed covalent binding of the exopolysaccharides. Their specific galactose decoration was detected with fluorescence-labeled lectins. Similarly, glycoconjugates were covalently immobilized and displayed glycans were profiled for fucose, sialic acid, galactose, and lactosamine epitopes. The applicability of such platforms for glycan profiling was further tested with extracts of Caco2 epithelial cells. Following spontaneous differentiation or on pretreatment with sialyllactose, Caco2 cells showed a reduction of specific glycan epitopes. The changed glycosylation phenotypes coincided with altered enteropathogenic E. coli adhesion to the cells. This microarray strategy was also suitable for the immobilization of lectins through biotin-neutravidin-biotin bridging on platforms functionalized with a biotin derivatized photoactivatable dextran. All immobilized glycans were specifically and differentially detected either on glycoconjugate or lectin arrays. The results demonstrate the feasibility and versatility of the novel platforms for glycan profiling.     

Differentiating N-linked glycan structural isomers in metastatic and nonmetastatic tumor cells using sequential mass spectrometry

In an effort to understand the role of molecular glycosylationin cancer a murine model has been used to characterize and fingerprintmalignancies in established cell lines that manifest all thehallmarks of metastatic disease: spontaneous development, localinvasion, intravasation, immune system survival, extravasation,and secondary tumor formation involving liver, kidney, spleen,lung, and brain. Using astrocyte cell controls, we comparedN-linked glycosylation from a nonmetastatic brain tumor cellline and two different metastatic brain tumor cells. Selectedions in each profile were disassembled by ion trap mass spectrometry(MSⁿ) which exhibited multiple structural differences betweeneach tissue. These unique structures were identified withinisomeric compositions as pendant nonreducing termini of di-and trisaccharide fragments, probably transparent to a tandemMS approach but distinctively not to sequential ion trap MSⁿdetection.     

Carbohydrate-recognition domains of galectin-9 are involved in intermolecular interaction with galectin-9 itself and other members of the galectin family

Galectin-9 (Gal-9) is a tandem-repeat-type member of the galectin family associated with diverse biological processes, such as apoptosis, cell aggregation, and eosinophil chemoattraction. Although the detailed sugar-binding specificity of Gal-9 has been elucidated, molecular mechanisms that underlie these functions remain to be investigated. During the course of our binding study by affinity chromatography and surface plasmon resonance (SPR) analysis, we found that human Gal-9 interacts with immobilized Gal-9 in the protein-protein interaction mode. Interestingly, this intermolecular interaction strongly depended on the activity of the carbohydrate recognition domain (CRD), because the addition of potent saccharide inhibitors abolished the binding. The presence of multimers was also confirmed by Ferguson plot analysis of result of polyacrylamide gel electrophoresis and matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Moreover, this intermolecular interaction was observed between Gal-9 and other galectin members, such as Gal-3 and Gal-8, but not Gal-1. Because such properties have not been reported yet, they may explain an unidentified mechanism underlying the diverse functions of Gal-9.     

Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis

The fundamental importance of correct protein glycosylation is abundantly clear in a group of diseases known as congenital disorders of glycosylation (CDGs). In these diseases, many biological functions are compromised, giving rise to a wide range of severe clinical conditions. By performing detailed analyses of the total serum glycoproteins as well as isolated transferrin and IgG, we have directly correlated aberrant glycosylation with a faulty glycosylation processing step. In one patient the complete absence of complex type sugars was consistent with ablation of GlcNAcTase II activity. In another CDG type II patient, the identification of specific hybrid sugars suggested that the defective processing step was cell type-specific and involved the mannosidase III pathway. In each case, complementary serum proteome analyses revealed significant changes in some 31 glycoproteins, including components of the complement system. This biochemical approach to charting diseases that involve alterations in glycan processing provides a rapid indicator of the nature, severity, and cell type specificity of the suboptimal glycan processing steps; allows links to genetic mutations; indicates the expression levels of proteins; and gives insight into the pathways affected in the disease process.     

Interaction profile of galectin-5 with free saccharides and mammalian glycoproteins: probing its fine specificity and the effect of naturally clustered ligand presentation

Cell-surface glycans are functional docking sites for tissue lectins such as the members of the galectin family. This interaction triggers a wide variety of responses; hence, there is a keen interest in defining its structural features. Toward this aim, we have used enzyme-linked lectinosorbent (ELLSA) and inhibition assays with the prototype rat galectin-5 and panels of free saccharides and glycoconjugates. Among 45 natural glycans tested for lectin binding, galectin-5 reacted best with glycoproteins (gps) presenting a high density of Galbeta1-3/4GlcNAc (I/II) and multiantennary N-glycans with II termini. Their reactivities, on a nanogram basis, were up to 4.3 x 10(2), 3.2 x 10(2), 2.5 x 10(2), and 1.7 x 10(4) times higher than monomeric Galbeta1-3/4GlcNAc (I/II), triantennary-II (Tri-II), and Gal, respectively. Galectin-5 also bound well to several blood group type B (Galalpha1-3Gal)- and A (GalNAcalpha1-3Gal)-containing gps. It reacted weakly or not at all with tumor-associated Tn (GalNAcalpha1-Ser/Thr) and sialylated gps. Among the mono-, di-, and oligosaccharides and mammalian glycoconjugates tested, blood group B-active II (Galalpha1-3Gal beta1-4GlcNAc), B-active IIbeta1-3L (Galalpha1-3Galbeta1-4GlcNAc beta1-3Galbeta1-4Glc), and Tri-II were the best. It is concluded that (1) Galbeta1-3/4GlcNAc and other Galbeta1-related oligosaccharides with alpha1-3 extensions are essential for binding, their polyvalent form in cellular glycoconjugates being a key recognition force for galectin-5; (2) the combining site of galectin-5 appears to be of a shallow-groove type sufficiently large to accommodate a substituted beta-galactoside, especially with alpha-anomeric extension at the non-reducing end (e.g., human blood group B-active II and B-active IIbeta1-3L); (3) the preference within beta-anomeric positioning is Galbeta1-4 > or = Galbeta1-3 > Galbeta1-6; and (4) hydrophobic interactions in the vicinity of the core galactose unit can enhance binding. These results are important for the systematic comparison of ligand selection in this family of adhesion/growth-regulatory effectors with potential for medical applications.