PNH病血浆中AchE含量的测定

用ＥＬＩＳＡ方法测定陈发性睡眠性血红蛋白尿症病人血浆和红细胞膜中乙酰胆碱酯酶的含量,结果显示ＰＮＨ病人红细胞膜中的ＡｃｈＥ低于正常,而血浆中的ＡｃｈＥ高于正常。     

人血清补体3b的提纯与鉴定

据报道红细胞膜上有补体3b(C_(3b))的受体。阵发性睡眠性血红蛋白尿症(PNH)是属红细胞的膜病变,对补体较为敏感,易产生溶血,可能是因为膜上C_(3b)受体与正常红细胞不同。为了研究C_(3b),与膜上C_(3b)受体结合情况,并进一步分离膜上C_(3b)受体,我们制备了纯的C_(3b)。一、材料与方法 1.C_3的提纯见文献[2] 2.免疫双扩散见文献[3] 3.C_3的垂直板型聚丙烯酰胺凝胶电泳(PAGE)见献文[2] 4.C_3分子量测定按Pharmacia标准蛋白测分子量法     

流式检测PNH克隆的方法学探讨及临床筛检和意义

目的:探讨流式细胞仪高敏感方法检测PNH克隆的必要性和血细胞减少患者PNH克隆的发生率及临床意义。方法:采用CD59检测红细胞及FLAER联合CD24或CD14检测粒细胞和单核细胞的方法检测了20例健康志愿者和1 095例血细胞减少患者的PNH克隆比例,同时对31例患者采用传统的CD59方法检测粒细胞和单核细胞中CD59~-细胞比例。结果:根据健康志愿者正常背景和患者获取的细胞数,确定粒细胞、单核细胞和红细胞PNH克隆的最低检测限(LOD)分别为0.04%、0.10%和0.05%;827例患者FLAER~-CD24~-粒细胞、FLAER~-CD14~-单核细胞和CD59~-红细胞的中位比例分别为0.02%、0.02%和0.03%;318例(38.45%)患者粒细胞PNH克隆高于LOD,180例FLAER~-CD24~-粒细胞1.00%,粒红和粒单细胞的一致性只有43%～45%。138例FLAER~-CD24~-粒细胞≥1.00%,粒红、粒单和粒单红细胞的一致率分别为91.30%、97.83%和89.86%;比较CD59~-和FLAER~-CD24~-粒细胞、CD59~-和FLAER~-CD14~-单核细胞比例,CD59~-细胞比例明显更低(P0.000 1和P=0.000 9);26.85%和24.54%患者因出现FLAER~-CD24~+粒细胞与FLAER~-CD14~+单核细胞,导致FLAER单阴比例高于FLAER和锚连蛋白双阴比例,对PNH克隆比例低于0.10%的患者影响更大;36例PNH患者和50例MDS或AA患者PNH克隆比例分别为90.30%(44.49%～99.05%)和1.30%(0.10%～96.07%)(P0.000 1);PNH克隆比例等于41.81%时,诊断PNH的敏感度和特异度分别为100.0%和96.0%。结论:在本实验条件下根据正常背景值,确定粒细胞、红细胞的LOD分别为0.04%和0.05%。粒细胞PNH克隆比例≥1.00%,与单核和红细胞诊断PNH克隆阳性结果更一致,PNH克隆比例高于41.81%时PNH疾病可能性更大。     

红细胞膜肌醇磷酯结合蛋白的研究

本文用~8H-肌醇标记红细胞膜,分别用薄层层析及SDS-PAGE对肌醇磷酯在红细胞膜上的分布作了探讨。发现一种在脂双层内,另一种是与膜蛋白结合(即肌醇磷酯结合蛋白)作为膜蛋白的锚(anchor)而存在,并用抗AchE及DAF单克隆抗体作免疫印渍实验,在电泳中~3H-肌醇磷酯所在部位与AchE及DAF相符。这结果与文献中报道AchE及DAF为肌醇磷酯结合蛋白一致。至于这两种形式不同的肌醇磷酯的相互关系还有待研究。     

补体与红细胞膜的结合作用

被激活的补体3(C_3),能与红细胞膜结合;C_(3b)与红细胞膜形成的复合物,又激活一系列补体(C_5-C_9),使细胞溶解。有些溶血性疾病与此种补体溶血有关。我们实验曾证明,阵发性睡眠性血红蛋白尿症(PNH)红细胞对补体溶血敏感。为了探讨PNH补体溶血反应的机理,我们曾用对唾液酸专一结合的鲎血凝集素处理PNH红细胞,表明它能降低补体溶血,并证明它与红细胞膜血型糖蛋白结合。看来鲎血凝集素可能影响红细胞膜与C_(3b)的结     

高胆固醇血症血液流变性的研究

对实验性高胆固醇血症家兔血液流变性、红细胞流变性、红细胞膜脂分析以及与血清胆固醇浓度之间关系的研究指出随血清胆固醇浓度升高,全血表观粘度和血浆粘度升高,红细胞(?)积和(?)降低.进一步分析表明,随血清胆固醇浓度升高,红细胞膜中胆固醇Ch含量增加,引起膜胆固醇和磷脂(P1)的克分子比(Ch,P1)升高,膜的荧光偏振度(P)增大,表示红细胞流动性降低,而对照组各项指标在实验过程中保持稳定.     

肺叶切除术对红细胞及淋巴细胞膜流动性的影响

目的：研究肺叶切除术对红细胞及淋巴细胞膜流坳性的影响。方法：选择２０例择期开胸手术病人,均作肺叶切除术。分别用微量滴定法、高效液权色谱法和ＤＰＨ荧光探剂法测定血浆和红细胞膜ＰＬＡ２活性,红细胞膜磷脂ＰＳ、ＰＥ、ＰＣ和红细胞、淋巴膜脂流动性。结果：手术１０ｍｉｎ、手术６０ｍｉｎ和手术结束后３０ｍｉｎ血浆和红细胞膜ＰＬＡ２活性均显著高于麻醉诱导前;手术６０ｍｉｎ和手术结束后３０ｍｉｎ 红细胞膜ＰＳ、Ｐ     

肾脏与红细胞生成的关系

很早人们就发现,当机体缺氧或失血时,红细胞生成即加速,这是因为在血浆中有一种活性物质,能够刺激红细胞的生成。1948年这种活性物质被命名为红细胞生成素(Erythropoietin)。后来发现,肾脏是产生红细胞生成素的主要器官。杰克逊(Jacobson)等人首先观察到,当动物切除双侧肾脏后,血浆中红细胞生成素迅速减少,且缺氧或失血时,血浆中也不再出现该物质。临床上,患肾脏肿瘤的病人,有时出现红细胞增多,其血中红细胞生成素也增多。此后,从肾肿瘤组织中     

测定酶活性的电化学方法及其影响因素

作者曾用固定化细胞技术分离了人红细胞膜表面蛋白质。为了能快速、准确、自动连续地测定固定化红细胞膜中乙酰胆碱酯酶(AchE)等各酶的活性,设计组装了一架酶活性电化学测定装置。本文报道试用该装置作测定胆碱酯酶(ChE)纯品的实验结果。早在1962年,Kramer等人就报道了一种半微量测定AchE,ChE,以及各种硫代胆碱酯的电化学方法。以后又将该仪器应用于测定高毒性有机磷化物,葡萄糖氧化酶、黄嘌呤氧化酶,以及过氧化物酶与过氧化氢酶类。并设计了能酶反应连续分析的装置。在此基     

金属硫蛋白与红细胞的相互作用

根据金属硫蛋白(MT)对标记在膜上的马来酰亚胺自旋标记物的ESR波谱的影响,研究了MT与红细胞膜的相互作用,发现不同种属的MT对膜构象的影响不同.体外实验表明,MT可以吸附在红细胞的表面,用CdCl₂诱导家兔,对血浆和红细胞溶血液中的MT组分进行色谱分离,发现血液中的MT主要存在于血细胞中.进而对血液MT的来源、分布及其重要的生物学意义进行了讨论.     

阵发性睡眠性血红蛋白尿症红细胞乙酰胆碱酯酶的研究

用化学方法测定了乙酰胆碱脂酶（ＡｃｈＥ）活性,阵发性睡眠性血红蛋白尿症（ＰＮＨ）红细胞远低于正常红细胞。为了进一步研究ＰＮＨＡｃｈＥ（—）的红细胞,采用Ｐｒｏｔｅｉｎ　Ａ　Ｓｅｐｈａｒｏｓｅ　６ＭＢ结合ＡｃｈＥ单抗亲和层析法分离出ＰＮＨＡｃｈＥ（—）的红细胞。用间接免疫荧光流式细胞术检测,ＰＮＨ细胞ＡｃｈＥ低于正常,而ＰＮＨＡｃｈＥ（—）红细胞未能检出ＡｃｈＥ。３Ｈ－肌醇标记实验证明,正常红细胞膜区带４．１处有较高的放射活性,而ＰＮＨ红细胞极低,ＰＮＨＡｃｈＥ（—）红细胞完全无放射活性。用ＡｃｈＥ抗体做免疫印渍实验证明了ＡｃｈＥ存在区带４．１部位。ＤＭＰＣ诱导正常和ＰＮＨ红细胞,检测二者囊泡化的程度,发现ＰＮＨ病人红细胞远比正常人红细胞易于囊泡化。     

Sorting of the human folate receptor in MDCK cells

The human folate receptor (hFR) is a glycosylphosphatidy-linositol (GPI) linked plasma membrane protein that mediates delivery of folates into cells. We studied the sorting of the hFR using transfection of the hFR cDNA into MDCK cells. MDCK cells are polarized epithelial cells that preferentially sort GPI-linked proteins to their apical membrane. Unlike other GPI-tailed proteins, we found that in MDCK cells, hFR is functional on both the apical and basolateral surfaces. We verified that the same hFR cDNA that transfected into CHO cells produces the hFR protein that is GPI-linked. We also measured the hFR expression on the plasma membrane of type III paroxysmal nocturnal hemoglobinuria (PNH) human erythrocytes. PNH is a disease that is characterized by the inability of cells to express membrane proteins requiring a GPI anchor. Despite this defect, and different from other GPI-tailed proteins, we found similar levels of hFR in normal and type III PNH human erythrocytes. The results suggest the hypothesis that there may be multiple mechanisms for targeting hFR to the plasma membrane.     

Characterization of the enhanced susceptibility of paroxysmal nocturnal hemoglobinuria erythrocytes to complement-mediated hemolysis initiated by cobra venom factor

When whole serum C is activated by cobra venom factor complexes (CoFBb), paroxysmal nocturnal hemoglobinuria (PNH) III E (the most C-sensitive type) are hemolyzed, but normal and PNH II E (the intermediately sensitive type) are not. Previous studies have shown that after exposure to CoFBb and serum, PNH III E bind relatively large amounts of the trimolecular C complex, C5b67, whereas normal and PNH II E bind virtually none. In the studies reported herein, we have observed that when normal and PNH III E are incubated with isolated C5, C6, and 125I-C7 in the presence CoFBb, the normal E bind more C5b-7 than the PNH cells. When C7-deficient serum is included in the reaction mixture, however, the PNH E are once again observed to bind much greater amounts of C5b-7. These observations suggest that plasma and membrane factors act in concert to restrict the assembly of the trimolecular C5b-7 complex on human E. PNH III E appear to be deficient in the membrane component of this inhibitory system.     

Lysosomal breakdown of erythrocytes in the sheep placenta

Summary The breakdown of erythrocytes within the lysosomal apparatus of trophoblastic epithelial cells of the sheep placenta was studied at the ultrastructural level. Acid phosphatase activity could be demonstrated in the interspace between the erythrocyte membrane and the lysosomal membrane, but not inside ingested erythrocytes. The erythrocyte plasma membrane remained observable until the final stage of the breakdown process. Together with a peripheral layer of indigestible hemoglobin it might form a barrier for further penetration of lysosomal enzymes into the ingested erythrocyte. The hemoglobin of the erythrocyte is suggested to diffuse through the erythrocyte plasma membrane into the interspace between this membrane and the lysosomal membrane. Subsequently, the hemoglobin is digested in the interspace or in fragments pinched off from erythrocyte-containing lysosomes (=erythrolysosomes). The fragmentation of erythrolysosomes is considered to be the most efficient mechanism for the breakdown of red blood cells in the trophoblastic epithelium of the sheep placenta. The method of entry of hydrolytic enzymes into erythrocyte-containing phagosomes is discussed.     

Asymmetry in the renewal of molecular classes of phosphatidylcholine in the rat-erythrocyte membrane

1. 1. Rat-blood phospholipids were labeled in vivo with [³²P]phosphate. The erythrocytes were treated with phospholipase A₂ plus sphingomyelinase to discriminate between the labeling patterns of the phospholipids from the inner and outer layer of the membrane.

2. 2. The specific activities of the more unsaturated classes of phosphatidylcholine were higher in the outer layer of the erythrocyte membrane than in the inner layer. The disaturated class, however, had the highest specific activity in the inner layer.

3. 3. After incubating ³²P-labeled erythrocytes in unlabeled plasma, the labeling pattern recovered in the molecular classes of plasma phosphatidylcholine was very similar to that of the phosphatidylcholines in the outer layer of the erythrocyte membrane.

4. 4. It is proposed that the exchange of phosphatidylcholines between plasma and the outer layer of the erythrocyte is mainly responsible for the renewal of the unsaturated phosphatidylcholines of the erythrocyte, and that the acylation activity of the erythrocyte is directed towards the formation of disaturated phosphatidylcholines at the inside of the membrane.

A soluble form of the glycolipid-anchored receptor for urokinase-type plasminogen activator is secreted from peripheral blood leukocytes from patients with paroxysmal nocturnal hemoglobinuria.

The cellular urokinase-type plasminogen-activator (uPA) receptor (uPAR) is a glycolipid-anchored membrane protein thought to be involved in pericellular proteolysis during cell migration and tumor invasion. In the present study, we have identified and characterized two soluble forms of uPAR which have retained their ligand-binding capability. One variant was generated in vitro by treatment of intact normal cells with either a phosphatidylinositol-specific phospholipase C (PLC) or endoproteinase Asp-N. The other soluble uPAR variant was secreted in vivo from peripheral blood leukocytes affected by the stem-cell disorder paroxysmal nocturnal hemoglobinuria (PNH), and was found in the plasma from these PNH patients as well as in the conditioned medium from cultured PNH leukocytes. Under normal conditions, we find no evidence for any shedding or secretion of a soluble uPA-binding counterpart to human uPAR in plasma. Unlike normal leukocytes, the PNH-affected cells do not express uPAR on the cell surface, although they do contain apparently normal levels of uPAR-specific mRNA. The secreted uPAR derived from PNH cells has a mobility in SDS/PAGE that is slightly higher than that of uPAR solubilized by PtdIns-specific PLC or detergent, but resembles that of a truncated, recombinant uPAR variant, which has its C-terminus close to the proposed glycolipid-attachment site, suggesting that the secreted protein has been proteolytically processed for glycolipid attachment. The presence in plasma from PNH patients of such a secreted, hydrophilic form of uPAR lends support to the hypothesis that the lesion underlying the PNH disorder resides either in glycolipid biosynthesis or in the function of an as-yet-unidentified transamidating enzyme assumed to cleave and assemble the truncated uPAR with the preformed glycolipid moiety.     

竹红菌甲素对红细胞膜上几种酶光敏失活作用的研究

Hypocrellin A (HA)-sensitized photoinactivation of enzymes in human erythrocyte membrane, including AchE, GPDH, Na(+)-K+ ATPase, Ca2(+)-Mg2+ ATPase were studied in this paper. The sensitivity of these four enzymes inactivated by HA and light are as following order: Ca2(+)-Mg2+ ATPase greater than Na(+)-K+ ATPase greater than GPDH greater than AchE. The relationship among ATPase inactivation, sulfhydryl photoinactivation and lipid peroxidation was also investigated. Results show that SH group photooxidation probably is one of the major reasons of enzyme inactivation whereas lipid peroxidation has little effect. The isolated GPDH was less sensitive than that membrane-bound, GSH, NAD acted protectively on GPDH and ATPase respectively. The evidence of electrophoresis and protein intrinsic fluorescence showed that protein structure did not change significantly even though most activity had lost in case of GPDH.     

Evidence for trafficking of PfEMP1 to the surface of P. falciparum-infected erythrocytes via a complex membrane network

The human malarial parasite Plasmodium falciparum exports virulence determinants, such as the P. falciparum erythrocyte membrane protein 1 (PfEMP1), beyond its own periplasmatic boundaries to the surface of its host erythrocyte. This is remarkable given that erythrocytes lack a secretory pathway. Here we present evidence for a continuous membrane network of parasite origin in the erythrocyte cytoplasm. Co-localizations with antibodies against PfEMP1, PfExp-1, Pf332 and PfSbpl at the light and electron microscopical level indicate that this membrane network is composed of structures that have been previously described as tubovesicular membrane network (TVM), Maurer's clefts and membrane whorls. This membrane network could also be visualized in vivo by vital staining of infected erythrocytes with the fluorescent dye LysoSensor Green DND-153. At sites where the membrane network abuts the erythrocyte plasma membrane we observed small vesicles of 15-25 nm in size, which seem to bud from and/or fuse with the membrane network and the erythrocyte plasma membrane, respectively. On the basis of our data we hypothesize that this membrane network of parasite origin represents a novel secretory organelle that is involved in the trafficking of PfEMP1 across the erythrocyte cytoplasm.     

Erythrocyte membrane fluidity and changes in plasma lipid composition: a possible relationship in childhood obesity

We have studied plasma lipid patterns and erythrocyte membrane fluidity in 60 obese children and 20 normal children. Plasma levels of total cholesterol and associated low-density lipoproteins were significantly increased in 20 obese patients with respect to controls. A significant decrease in membrane fluidity, measured as an increase in the fluorescence polarization value of the probe 1,6-diphenyl-1,3,5-hexatriene, associated with an increase in the cholesterol/protein ratio has been shown in obese patients. The study of the correlation between erythrocyte membrane fluidity and plasma cholesterol has indicated that significant changes in fluidity and membrane lipid composition also occur in erythrocytes of obese patients with normal plasma lipid levels. These findings confirm that the erythrocyte membrane responds very early to modifications of plasma lipoproteins and suggest that in childhood obesity a modified transfer of cholesterol from plasma to erythrocyte membrane may take place.