初次膝关节置换术后放置引流管与否临床疗效的前瞻性对照研究

目的:分析对比初次膝关节置换术后放置和不放置引流管的临床疗效。方法:纳入2017年1月-2017年7月在青岛大学附属医院行初次膝关节双间室置换的患者107例(107膝),其中术后放置引流管组(实验组)54例,术后不放置引流管组(对照组)53例。分别观察比较两组患者的一般临床资料、住院天数。术前和术后第3天血红蛋白、红细胞压积、视觉疼痛模拟评分、膝关节屈曲度数以及两组患者术后出现发热、伤口问题、24 h内辅料渗透、输血情况的例数。结果:两组患者在年龄、性别组成、体重、身高、手术时间等一般临床资料之间以及术前血红蛋白、红细胞压积、视觉疼痛模拟评分、膝关节屈曲度数等方面均无明显差异(P0.05)。术后实验组患者在血红蛋白、红细胞压积、膝关节屈曲度数均高于对照组(P0.05)。在视觉疼痛模拟评分上,实验组患者术后第1天低于对照组(P0.05),但术后第3天两组无明显差异(P0.05)。实验组患者的住院天数、输血患者的数量低于对照组,而发热例数明显高于对照组(P0.05)。结论:初次膝关节置换术后不放置引流管更有利于患者术后的早期康复,并减少患者术后输血率,而且不增加并发症的发生。     

洗涤红细胞输注在早产儿贫血中的应用价值分析

目的:分析洗涤红细胞输注在早产儿贫血中的应用价值。方法:选取2015年10月~2017年11月我院需要输血治疗的贫血早产儿107例,均具有《早产儿管理指南》中的输血指征。采用随机数字表法将患儿分为两组,对照组53例输注红细胞悬浮液,观察组54例输注洗涤红细胞。比较两组患儿输血情况、治疗前后血液流变学相关指标、亚硝基硫醇(RSNO)、超氧化物歧化酶(SOD)和苯二醛(MDA)水平的变化及并发症的发生情况。结果:两组患儿输血量、输血次数、输血后血细胞压积和输血后血红蛋白水平比较均无统计学差异(P>0.05)。输血前,两组患儿全血高切黏度、全血低切黏度、血浆黏度、RSNO、SOD和MDA水平比较无统计学差异(P>0.05);输血后,两组全血高切黏度、全血低切黏度及血浆黏度均较输血前显著下降,且观察组以上指标均显著低于对照组(P<0.05);对照组患儿RSNO、SOD水平显著下降,MDA水平显著升高(P<0.05),观察组患儿以上指标均无显著变化(P>0.05)。观察组患儿呼吸暂停和院内感染的发生率显著低于对照组(P<0.05),两组脑白质损伤及颅内出血的发生率比较均无统计学差异(P>0.05)。结论:洗涤红细胞输注可显著改善贫血早产儿的血液流变学,降低应激反应及呼吸暂停和院内感染的发生率,且输血量、输血次数、输血后血细胞压积和输血后血红蛋白水平与输注红细胞悬浮液相当。     

血凝酶对髋关节置换术低分子肝素抗凝患者出血量及凝血功能的影响

目的:观察注射用血凝酶对髋关节置换术低分子肝素抗凝患者术中出血量及凝血功能的影响。方法:选取收治的髋关节置换术的患者共161例,采用双盲、随机的方法分为实验组83例和对照组78例,所有患者均为我院收治的行髋关节置换术的病例。对照组采用术前给予低分子肝素处理,观察组患者术前给予注射用血凝酶及低分子肝素处理。术后对两组患者术中出血量、术后24 h引流量、术后24 h红细胞(RBC)、血红蛋白(Hb)、纤维蛋白原(Fib)、术后24 h部分凝血活酶时间(APTT)、凝血酶原时间(PT)等情况进行分析。结果:实验组患者术中出血量、术后24 h引流量值均低于对照组,且差异有显著性(P0.05)。两组患者术后24 h红细胞RBC、血红蛋白Hb均下降,但是组间比较差异无统计学意义(P0.05)。术后纤维蛋白原Fib值均上升,组间比较差异无统计学意义(P0.05)。实验患者术后24 h部分凝血活酶时间APTT、凝血酶原时间PT均较术前有所降低,但是于术前及组间比较差异无统计学意义(P0.05)。结论:全髋置换术中使用注射用血凝酶不受低分子肝素的抑制,且安全有效,值得临床进一步研究和应用。     

铁过载人群外周血相关指标变化分析

目的：探讨铁过载人群外周血相关指标的变化及血清铁蛋白(SF)和铁营养、血常规指标之间的关系,为铁过载的诊断及分类提供科学依据。方法：随机抽取196名铁正常人群(男SF:15~200μg/L、女SF：15~150μg/L)和226名铁过载人群(男SF:＞200μg/L、女SF:＞150μg/L)。采用放射免疫法检测SF浓度,检测铁营养状况和血常规指标,分析血清铁蛋白和铁营养、血常规指标之间的相关性。结果：铁过载人群血清铁浓度22.93μmol/L和转铁蛋白饱和度36%显著高于铁正常组的17.83μmol/L和28%(P<0.05),铁过载组人群不饱和铁结合力水平为40.69μmol/L和转铁蛋白浓度245.67mg/dL,显著低于正常组的46.97μmol/L和264.33 mg/dL(P<0.05),两组间总铁结合力水平的比较无显著差异(P>0.05);铁过载组红细胞计数平均为4.98×1012/L和血红蛋白含量平均为155g/L,显著高于铁正常组的4.82×1012/L和147g/L (P<0.05),铁过载组红细胞压积44%和平均红细胞血红蛋白含量31.17pg,显著高于铁正常组人群的42%和30.61pg (P<0.05),两组之间白细胞计数和血小板计数的比较无显著差异(P>0.05)。相关分析显示,血清铁蛋白与血清铁、转铁蛋白饱和度、红细胞计数、白细胞计数、血红蛋白、红细胞压积和平均红细胞血红蛋白含量呈显著正相关(P<0.05),与不饱和铁结合力、转铁蛋白水平和血小板计数呈显著负相关(P<0.05)。结论：中老年人群铁过载时,机体内血清铁、转铁蛋白饱和度、红细胞计数、血红蛋白、红细胞压积和平均红细胞血红蛋白含量均升高,不饱和铁结合力和转铁蛋白水平均降低;血清铁蛋白和血红蛋白呈显著正相关。因此,采用血常规检查和铁营养指标的联合检测来评价铁过载的程度,可为铁过载的早期发现、早期治疗提供科学依据。     

氨甲环酸联合利伐沙班对单侧全膝关节置换术后患者出血量、凝血功能以及膝关节功能的影响

目的:探讨氨甲环酸联合利伐沙班对单侧全膝关节置换术后患者出血量、凝血功能及膝关节功能的影响。方法:选取2015年1月-2016年1月在解放军425医院骨科初次行单侧全膝关节置换术的患者66例为研究对象,按照随机数字表法分为治疗组与对照组,每组各33例。治疗组患者在止血带释放前向关节腔内注射氨甲环酸,对照组患者则给予氯化钠注射液进行静脉滴注,在术后6-12h内两组患者均口服利伐沙班。记录并对比两组患者总失血量、隐性出血量、输血率、输血量,对比两组患者手术前后凝血功能指标、膝关节功能评分、膝关节活动度、疼痛视觉模拟评分(VAS),并观察两组患者并发症发生情况。结果:与对照组对比,治疗组患者的总失血量、隐性出血量、输血量及输血率均明显降低(P0.05);治疗组与对照组患者术前与术后3 d的活化部分凝血酶时间(APTT)、凝血酶原时间(PT)、纤维蛋白原(FG)水平比较均无显著差异(P0.05);术前两组患者的美国膝关节协会评分(AKSS)及膝关节活动度比较无显著差异(P0.05),术后7 d,治疗组与对照组患者的AKSS评分及膝关节活动度均较术前升高(P0.05),且治疗组患者的AKSS评分及膝关节活动度高于同时期对照组(P0.05);术前两组VAS评分比较无显著差异(P0.05),术后1 d,两组患者的VAS评分比较及与同组术前比较均无显著差异(P0.05),术后7 d,两组患者的VAS评分明显较术前及术后1 d降低(P0.05),但两组之间比较无显著差异(P0.05)。两组患者并发症总发生率对比无显著差异(P0.05)。结论:氨甲环酸联合利伐沙班可有效降低行单侧全膝关节置换术患者的出血量,加快关节功能的恢复,且不影响患者的凝血功能,值得临床推广。     

右美托咪定对低血红蛋白全麻患者脑氧饱和度及术后认知功能的影响*

目的:探讨右美托咪定对低血红蛋白全麻患者脑氧饱和度及术后认知功能的影响。方法:选取择期在全麻行开腹妇科手术、血红蛋白8～9 g/dl的患者30例,ASA分级Ⅰ～Ⅱ级,将其随机分成两组:生理盐水组(N组)和右美托咪啶组(D组),每组15例。两组麻醉诱导后,均行七氟醚复合瑞芬太尼静吸复合麻醉维持。D组在麻醉诱导前经15 min静脉输注右美托咪定0.5μg/kg,继之以0.3μg·kg~(-1)·h~(-1)的速率输注至术毕,N组给予等容量生理盐水。分别于入室时(T_0)、吸氧后3 min(T_1)、手术开始即刻(T_2)、手术开始后10 min(T_3)、20 min(T_4)、30 min (T_5)、手术结束即刻(T_6)以及患者苏醒拔管后5 min(T_7)记录平均动脉压(Mean Arterial Pressure,MAP)、心率(Heart Rate,HR)、血氧饱和度(Percutaneous Oxygen Saturation,SpO_2)、呼吸末二氧化碳分压(End-Tidal Carbon DioxidePartial Pressure,PETCO_2)、脑氧饱和度(Regional Cerebral Saturation Of Oxygenation,rSO_2)、脑电双频指数(Bispectral Index,BIS)以及腋温。并于术前1 d、术后1 d以及术后3 d,记录蒙特利尔认知评估量表(Montreal Cognitive Assessment,MoCA)数值以及术后认知功能障碍(Postoperative Cognitive Dysfunction,POCD)的发生率。结果:与N组比较,D组在T_2～T_6时的rSO_2升高,HR降低(P0.05),两组各时点MAP、SpO_2、PETCO_2、BIS以及腋温比较差异无统计学意义(P0.05)。与N组比较,D组术后1天、术后3天MoCA评分均显著升高,且术后认知功能障碍的发生率明显降低(P0.05)。结论:右美托咪定能够提高低血红蛋白患者的脑氧饱和度,改善患者脑氧供需平衡,降低术后认知功能障碍的发生率。     

腰丛-坐骨神经阻滞联合喉罩全麻对老年全髋关节置换术患者血流动力学和术后认知功能的影响

目的:探讨腰丛-坐骨神经阻滞联合喉罩全麻对老年全髋关节置换术患者血流动力学和术后认知功能的影响。方法:选取2017年6月~2019年4月期间我院收治的择期行全髋关节置换术的老年患者121例。采用随机数字表法分为A组(n=60,喉罩全麻)和B组(n=61,腰丛-坐骨神经阻滞联合喉罩全麻),比较两组患者围术期指标、血流动力学指标、简易智能状态量表(MMSE)评分及不良反应情况。结果:两组患者麻醉诱导后(T2)-术后1 h(T5)时间点心率(HR)、平均动脉压(MAP)、血氧饱和度(SpO₂)均呈先降低后升高趋势(P<0.05);B组T2~手术结束即刻(T4)时间点SpO₂、MAP、HR高于A组(P<0.05)。两组术后1 d、术后3 d、术后7 d MMSE评分均呈先降低后升高趋势,但B组MMSE评分高于A组(P<0.05)。两组术中出血量比较差异无统计学意义(P>0.05),B组麻醉维持时间长于A组,术后苏醒时间、呼吸功能恢复时间则短于A组(P<0.05)。两组不良反应发生率对比未见统计学差异(P>0.05)。结论:老年全髋关节置换术患者术中给予腰丛-坐骨神经阻滞联合喉罩全麻,可有效改善围术期指标,稳定患者血流动力学,减少术后认知功能损害。     

自身免疫性溶血性贫血(AIHA)患者的不同输血方法与效果

目的:探讨不同输血方法治疗自身免疫性溶血性贫血(autoimmune hemolytic anemia,AIHA)的效果。方法:2017年1月-2018年12月选择在本院血液科诊治的64例自身免疫性溶血性贫血患儿,根据输血方法的不同分为观察组与对照组,各32例。观察组给予洗涤红细胞输注治疗,对照组给予非洗涤红细胞(悬浮红细胞)输注治疗,记录两组输血效果。结果:治疗后4 w观察组的总有效率显著高于对照组(100.0%vs.87.5%,P0.05)。两组治疗后4 w的红细胞计数与血红蛋白都显著高于治疗前,且观察组显著高于对照组(P0.05)。观察组的吸氧、机械通气、住院时间都显著少于对照组(P0.05)。观察组治疗过程的过敏反应、发热反应、紫癜等不良反应发生率显著低于对照组(3.1%vs. 21.9%,P0.05)。结论:洗涤红细胞输注治疗自身免疫性溶血性贫血患儿能促进机体红细胞计数与血红蛋白恢复正常,减少不良反应的发生,提高治疗效果与促进患儿康复。     

不同输血策略对ICU贫血患者预后的影响

目的:分析输血策略对ICU贫血患者预后的影响。方法:采用限制性输血患者54例纳入A组,采用开放性输血患者50例纳入B组,调取相关资料,进行对比分析,并将患者预后分为死亡(n=6)、恶化(n=31)与好转(n=67)三类,其中死亡与恶化合计为预后不佳,进行多因素Logistic回归分析。结果:A组入院基础、ICU输红细胞前、出院时Hb值低于B组,器官衰竭个数高于B组,差异具有统计学意义(P0.05);APACHEⅡ评分(OR=0.93,95%CI0.89-0.92)、ICU输红细胞前Hb值(OR=0.99,95%CI0.94-1.02)、器官衰竭数量(OR=0.57,95%CI0.34-1.04)成为患者预后不佳独立风险因素,差异具有统计学意义(P0.05)。     

麻醉方式对老年髋关节置换术患者的影响

目的:观察和比较腰硬联合麻醉与全麻对行择期髋关节置换术老年患者的生命体征、简易智力状况检查量表(Mini-mental State Examinatlon,MMSE)评分、认知功能障碍(postoperative cognitive dysfunction,POCD)发生率的影响。方法:选取2015年1月-2017年6月于我院行择期髋关节置换术的80例老年患者为研究对象,随机分为腰硬联合麻醉组和全麻组,每组各40例。全麻组患者术前应用全身麻醉,腰硬联合麻醉组患者术前应用腰硬联合麻醉。观察两组患者麻醉前后的生命体征、MMSE评分变化及POCD的发生情况。结果:腰硬联合麻醉组患者麻醉后收缩压(Systolic pressure,SP)、舒张压(diastolic pressure,DP)、心率(heart rate,HR)、呼吸频率(Respiratory rate,RR)均低于全麻组(P0.05),两组患者血氧饱和度(Pulse Oxygen Saturation,Sp O2)比较差异无统计学意义(P0.05)。腰硬联合麻醉组患者麻醉起效时间、运动阻滞恢复时间以及麻醉药用量均低于全麻组(P0.05)。术后6 h、24 h、72 h,腰硬联合麻醉组的MMSE评分均高于全麻组患者(P0.05)。术后1 d,全麻组的患者出现19例POCD,腰硬联合麻醉组出现4例,发生率显著低于全麻组(P0.05);两组在术后3 d的POCD发生率比较差异无统计学意义(P0.05)。结论:腰硬联合麻醉用于择期行髋关节置换术的老年患者具有良好的临床效果,麻醉起效快,缩短了完全阻滞时间,明显改善了患者的生命体征,降低术后认知功能障碍的发生,麻醉药物用量少。     

Effects of interleukin-8 on the developing human intestine

The human fetal/neonatal gastrointestinal tract is exposed to biologically significant concentrations of interleukin (IL)-8 swallowed with amniotic fluid and human milk. We hypothesized that IL-8 has a physiologic function in the developing human intestine. IL-8 was measured in preterm and term human milk, tested for stability under conditions simulating neonatal gastric and proximal small intestinal digestion, and its receptors were sought in human fetal bowel. The effect of IL-8 was then measured on intestinal cells in vitro. We observed that IL-8 is present in significant concentrations in human milk and that it is stable under conditions simulating digestion. Both IL-8 receptors, CXCR1 and CXCR2, are expressed extensively in the fetal intestine. When human fetal and adult intestinal cells are treated with rhIL-8 in vitro, there is a consistent increase in cell migration, proliferation, and differentiation. IL-8 also protects intestinal cells against chemical injury. These results suggest that besides its better-known role as a neutrophil chemoattractant, IL-8 has a trophic function in the developing human intestine.     

Effects of the sugar headgroup of a glycoglycerolipid on the phase behavior of phospholipid model membranes in the dry state

Glycolipids are important components of almost all biological membranes. They possess unique properties that have only been incompletely characterized so far. The plant glycolipid digalactosyldiacylglycerol (DGDG) strongly influences the physical behavior of phospholipid model membranes in both the dry and hydrated state. It was, however, unclear whether the strong effect of DGDG on the gel to liquid-crystalline phase transition temperature (Tm) in dry phosphatidylcholine (PC) bilayers is mainly due to the high degree of unsaturation of the DGDG fatty acyl chains or to interactions between the DGDG and PC headgroups. Also, no information on the relative effectiveness of membrane bound and free sugars on membrane phase behavior was available. We have used Fourier-transform infrared spectroscopy (FTIR) to investigate the phase properties and H-bonding patterns in dry membranes made from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) containing one saturated and one monounsaturated (16:0/18:1) fatty acid and different fractions of DGDG or 1,2-dilinolenoyl-sn-glycero-3-phosphatidylcholine (DLPC) (18:3/18:3). This was compared to the effects of galactose (Gal) and digalactose (diGal). All additives depressed Tm of the dry membranes, but DGDG was much more effective than DLPC or Gal. diGal had a similar effect as DGDG, pointing to the sugar headgroup as the component with the strongest influence on membrane phase behavior. A combination of DLPC and diGal, which should theoretically be equivalent to DGDG, was much more effective than the galactolipid. H-bonding interactions with the P = O group of PC were also stronger for free diGal than for DGDG, indicating that the free sugar may be structurally more flexible to adopt an optimal conformation for interactions with the PC headgroup.     

Conformational studies on the MUC1 tandem repeat glycopeptides: implication for the enzymatic O-glycosylation of the mucin protein core

The tandem repeat of the MUC1 protein core is a major site of O-glycosylation that is catalyzed by several polypeptide GalNAc-transferases. To define structural features of the peptide substrates that contribute to acceptor substrate efficiency, solution structures of the 21-residue peptide AHGVTSAPDTRPAPGSTAPPA (AHG21) from the MUC1 protein core and four isoforms, glycosylated with alpha-N-acetylgalactosamine on corresponding Thr residues, AHG21 (T5), AHG21 (T10), AHG21 (T17), and AHG21 (T5,T17), were investigated by NMR spectroscopy and computational methods. NMR studies revealed that sugar attachment affected the conformational equilibrium of the peptide backbone near the glycosylated Thr residues. The clustering of the low-energy conformations for nonglycosylated and glycosylated counterparts within the VTSA, DTR, and GSTA fragments (including all sites of potential glycosylation catalyzed by GalNAc-T1, -T2, and -T4 transferases) showed that the glycosylated peptides display distinct structural propensities that may explain, in part, the differences in substrate specificities exhibited by these polypeptide GalNAc-transferases.     

Plasmodium falciparum BAEBL Binds to Heparan Sulfate Proteoglycans on the Human Erythrocyte Surface

Erythrocyte invasion is critical to the pathogenesis and survival of the malarial parasite, Plasmodium falciparum. This process is partly mediated by proteins that belong to the Duffy binding-like family, which are expressed on the merozoite surface. One of these proteins, BAEBL (also known as EBA-140), is thought to bind to glycophorin C in a sialic acid-dependent manner. In this report, by the binding assay between recombinant BAEBL protein and enzyme-treated erythrocytes, we show that the binding of BAEBL to erythrocytes is mediated primarily by sialic acid and partially through heparan sulfate (HS). Because BAEBL binds to several kinds of HS proteoglycans or purified HS, the BAEBL-HS binding was found to be independent of the HS proteoglycan peptide backbone and the presence of sialic acid moieties. Furthermore, both the sialic acid- and HS-dependent binding were disrupted by the addition of soluble heparin. This inhibition may be the result of binding between BAEBL and heparin. Invasion assays demonstrated that HS-dependent binding was related to the efficiency of merozoite invasion. These results suggest that HS functions as a factor that promotes the binding of BAEBL and merozoite invasion. Moreover, these findings may explain the invasion inhibition mechanisms observed following the addition of heparin and other sulfated glycoconjugates.     

A tetraantennary glycan with bisecting N-acetylglucosamine and the Sd(a) antigen is the predominant N-glycan on bovine pregnancy-associated glycoproteins

Pregnancy-associated glycoproteins (PAGs) are major secretory proteins of trophoblast cells in ruminants. Binucleate trophoblast giant cells (BNCs) store these proteins in secretory granules and release them into the maternal organism after fusion with maternal uterine epithelial cells. By matrix assisted laser desorption ionisation-mass spectrometry (MALDI-MS) analysis and linkage analysis, we show that by far, the most abundant N-glycan of PAGs in midpregnancy is a tetraantennary core-fucosylated structure with a bisecting N-acetylglucosamine (GlcNAc). All four antennae consist of the Sd(a)-antigen (NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc-). Immunohistochemistry with the mono- clonal antibody CT1, which recognizes the Sd(a)-antigen, shows that BNC granules contain the Sd(a)-antigen from gestation day (gd) 32 until a few days before parturition. Lectin histochemistry with Maackia amurensis lectin (MAL), which binds to alpha2-3sialylated lactosamine, shows that BNC granules are MAL-positive prior to gd 32 and also at parturition. The observed tetraantennary glycan is a highly unusual structure, since during the synthesis of N-glycans, the insertion of a bisecting GlcNAc inhibits the activity of the GlcNAc-transferases that leads to tri- and tetraantennary glycans. The study defines the substantial changes of PAG N-glycosylation in the course of pregnancy. This promotes the hypothesis that PAGs may have different carbohydrate-mediated functions at different stages of pregnancy.     

Structure and role of sialic acids on the surface of Aspergillus fumigatus conidiospores

Aspergillus fumigatus is an opportunistic fungal pathogen that causes a life-threatening invasive fungal disease (invasive aspergillosis, IA) in immunocompromised individuals. The first step of pathogenesis is thought to be the attachment of conidia to proteins in lung tissue. Previous studies in our laboratory have shown that conidia adhere to basal lamina proteins via negatively charged sugars on their surface, presumably sialic acids. Sialic acids are a family of more than 50 substituted derivatives of a nine-carbon monosaccharide, neuraminic acid. The purpose of this study was 2-fold: (1) to determine the structure of sialic acids and the glycan acceptor on A. fumigatus oligosaccharides and (2) to determine the effect on the removal of sialic acids from conidia on conidial binding to the extracellular matrix protein fibronectin and phagocytosis of conidia by cultured macrophages and type 2 pneumocytes. Surface sialic acids were removed using Micromonospora viridifaciens sialidase or using acetic acid, mild acid hydrolysis. Lectin binding studies revealed that the majority of conidial sialic acids are alpha2,6-linked to a galactose residue. High-pressure liquid chromatography of derivatized sialic acids released from conidia revealed that unsubstituted N-acetylneuraminic acid is the predominant sialic acid on the surface of conidia. Enzymatic removal of sialic acid significantly decreased the binding of conidia to fibronectin by greater than 65% when compared with sham-treated controls. In addition, removal of sialic acids decreased conidial uptake by cultured murine macrophages and Type 2 pneumocytes by 33% and 53%, respectively. Hence, sialylated molecules on A. fumigatus conidia are ligands for both professional and nonprofessional phagocytes.     

Nucleolin: acharan sulfate-binding protein on the surface of cancer cells

Glycosaminoglycans (GAGs) are complex polysaccharides that participate in the regulation of physiological processes through the interactions with a wide variety of proteins. Acharan sulfate (AS), isolated from the giant African snail Achatina fulica, primarily consists of the repeating disaccharide structure alpha-D-N-acetylglucosaminyl (1-->4) 2-sulfoiduronic acid. Exogenous AS was injected subcutaneously near the tumor tissue in C57BL/6 mice that had been implanted with Lewis lung carcinoma cells (LLCs). The location of AS in the tumor was assessed by staining of sectioned tissues with alcian blue and periodic acid-Schiff (PAS) reagent. In vitro assays indicated binding of cells to 50 microg/ml AS (or heparin) after a 5-h incubation. Immunofluorescence assays, using anti-AS antibody, detected AS at the cell surface. The outer-surface of LLCs were next biotinylated to identify the AS-binding proteins. Biotinylated cells were lysed, and the lysates were fractionated on the AS affinity column using a stepwise salt gradient (0, 0.1, 0.3, 0.5, 0.7, 1.0, and 2.0 M). The fractions were analyzed by SDS-PAGE with silver staining and western blotting. We focused on the proteins with high affinity for AS (eluting at 1 M NaCl) and detected only two bands by western blotting. ESI Q-TOF MS analysis of one of these bands, molecular weight approximately 110 kDa, showed it to be nucleolin. A phosphorylated form of nucleolin on the surface of cells acts as a cell surface receptor for a variety of ligands, including growth factors (i.e., basic fibroblast growth factor) and chemokines (i.e., midkine). These results show that nucleolin is one of several AS-binding proteins and suggest that AS might demonstrate its tumor growth inhibitory activity by binding the nucleolin receptor protein on the surface of cancer cells.     

The effects of ethanol on the glycosylation of human transferrin

Appearance of a hyposialylated transferrin fraction in the plasma during chronic alcohol exposure is a well-known phenomenon, and it represents the best available marker of chronic alcohol consumption. The mechanisms of its appearance are still not well understood and are extremely complex, involving biosynthesis and catabolism alterations, although the only structural abnormality described corresponds to the loss of an entire glycan chain. We analyzed and compared the oligosaccharides present on the different isoforms of purified transferrin isolated from control and patients with severe alcohol abuse by fluorescent carbohydrate electrophoresis and matrix-assisted laser desorption ionization mass spectrometry. Our data indicate that the major modification observed is the loss of an entire oligosaccharide chain; we also demonstrate that there is a modification of terminal sialylation. Carbohydrate-deficient transferrin (CDT) is the result of multiple alterations of glycosylation. These results give a partial explanation to the poor sensitivity of the measurement of CDT and its controversial use as a marker of chronic alcohol consumption.     

Structural and topological studies on the lipid-mediated assembly of a membrane-associated lipomannan in Micrococcus luteus

The biosynthesis of three mannolipids and the presence of a membrane-associated lipomannan in Micrococcus luteus (formerly Micrococcus lysodeikticus) were documented over 30 years ago. Structural and topological studies have been conducted to learn more about the possible role of the mannolipids in the assembly of the lipomannan. The major mannolipid has been purified and characterized as alpha-D-mannosyl-(1 --> 3)-alpha-D-mannosyl-(1 --> 3)-diacylglycerol (Man2-DAG) by negative-ion electrospray-ionization multistage mass spectrometry (ESI-MSn). Analysis of the fragmentation patterns indicates that the sn-1 position is predominantly acylated with a 12-methyltetradecanoyl group and the sn-2 position is acylated with a myristoyl group. The lipomannan is shown to be located on the exterior face of the cytoplasmic membrane, and not exposed on the surface of intact cells, by staining of intact protoplasts with fluorescein isothiocyanate (FITC)-linked concanavalin A (Con A). When cell homogenates of M. luteus are incubated with GDP-[3H]mannose (GDP-Man), [3H]mannosyl units are incorporated into Man1-2-DAG, mannosylphosphorylundecaprenol (Man-P-Undec) and the membrane-associated lipomannan. The addition of amphomycin, an inhibitor of Man-P-Undec synthesis, had no effect on the synthesis of Man1-2-DAG, but blocked the incorporation of [3H]mannose into Man-P-Undec and consequently the lipomannan. These results strongly indicate that GDP-Man is the direct mannosyl donor for the synthesis of Man1-2-DAG, and that the majority of the 50 mannosyl units in the lipomannan are derived from Man-P-Undec. Protease-sensitivity studies with intact and lysed protoplasts indicate that the active sites of the mannosyltransferases catalyzing the formation of Man1-2-DAG and Man-P-Undec are exposed on the inner face, and the Man-P-Undec-mediated reactions occur on the outer surface of the cytoplasmic membrane. Based on all of these results, a topological model is proposed for the lipid-mediated assembly of the membrane-bound lipomannan.     

Processing of the Matricellular Protein Hevin in Mouse Brain Is Dependent on ADAMTS4

The matricellular SPARC family member hevin (SPARC-like 1/SPARCL-1/SC1/Mast9) contributes to neural development and alters tumor progression in a range of mammalian models. The distribution of hevin in mouse tissues was reexamined with a novel monoclonal antibody that discriminates between hevin and its ortholog SPARC. We now report proteolysis of hevin in many tissues, with the most extensive processing in the brain. We demonstrate a cleavage site within the hevin sequence for the neural tissue proteinase ADAMTS4. Digestion of hevin by ADAMTS4 in vitro produced fragments similar to those present in brain lysates. Monoclonal antibodies revealed a SPARC-like fragment generated from hevin that was co-localized with ADAMTS4 in vivo. We show that proteolysis of hevin by ADAMTS4 in the mouse cerebellum is important for the normal development of this tissue. In conclusion, we have identified the fragmentation of hevin by ADAMTS4 in the mouse brain and propose that this specific proteolysis is integral to cell morphology and extracellular matrix deposition in the developing brain.