一新型显微准弹性激光散射装置

设计并装配了一新型显微准弹性激光散射装置,经比较其主要性能优于同类仪器。利用此装置测量了标准聚苯乙烯球乳液及水溶性聚氨酯高聚物乳液,结果表明测量精确可靠。利用此装置还研究了正常人血红细胞,所测得的正常人红细胞内血红蛋白分子聚集体的线度和膜运动频率与文献报道的基本一致。证明本装置可成功进行单个活细胞的细胞膜以及胞内分子及其聚集体动态特性的非侵入性测量。     

红细胞膜变形能力与胞内分子聚合状态和细胞形态的相关变化

以显微激光散射光谱和图像分析技术,对单个活态红细胞在无扰、在位、实时的情况下,测量了人血红细胞膜的变形能力,以及与之同时对应的胞内血红蛋白的聚合状态和细胞的形态结构。并据此研究了它们的相关关系及随各种生理条件而交互变化的情况。     

对盐和C_8-卵磷脂水溶液的准弹性激光散射研究

本文用自差拍光混频的准弹性激光散射技术,对生物组织重要组成部分的C_a-卵磷脂水溶液内微团的流体力学半径随卵磷脂浓度,温度,添加的单价盐离子类型和强度的变化情况,进行了系统性研究.并根据微团形成的梯级模型,推导出微团的大小和结构在微团形成过程中的变化,微团形成参数以及盐控制微团形成的规律.     

对人眼晶状体α－晶体蛋白聚集体的准弹性激光散射研究

以准弹性激光散射技术,研究了人眼晶状体内α－晶体蛋白聚集体的扩散系数和流体力学半径,以及其弥散性随温度和浓度的变化。所研究的α－晶体蛋白用分离的方法分别取自成人和胚胎眼晶状体。研究结果表明,人眼α－晶体蛋白在一定浓度和温度下可形成聚集体,且其聚集体半径随α－晶体蛋白的浓度近乎线性地增大,随温度的增加而变小。对α－晶体蛋白溶液（５０ｍｍｏｌ／Ｌ磷酸脂缓冲液）的弥散度分析表明,溶液中有两种不同粒径的散     

红细胞膜对NO_2~-通透性的研究

本文采用NO_2~-快速测量红细胞膜对阴离子的通透特性.NO_2~-跨膜运输进红细胞内后,和血红蛋白相互作用,使后者变为高铁血红蛋白,改变了红细胞内血红蛋白的吸收光谱,进而影响细胞悬浮液的光学特性.据此,测量加入NO_2~-后红细胞悬浮液在特定波长处光密度的时间变化,即能追踪阴离子跨膜运输的动态过程.红细胞膜阴离子运输专一性抑制剂DIDS能抑制约70%的NO_2~-跨膜运输.在15-38℃温度范围检测了NO_2~-跨膜通透速率的温度特性,给出活化能为60KJ·mol~(-1).     

蜂毒溶血肽对鸡红细胞及膜的生化作用

本文采用荧光分光光度、薄层层析、原子吸收、荧光显微图像等多种生化技术,系统研究了蜂毒肽作用于鸡红细胞及膜的生化机理。结果表明：蜂毒肽影响红细胞膜上及胞内两种酶的功能。它抑制膜Na⁺-K⁺-ATPase活性,导致胞内外离子转运异常,K⁺浓度失衡;它也抑制细胞内葡萄糖-6-磷酸脱氢酶活性,其正电区域干扰胞内带负电小分子的作用,影响红细胞正常代谢。蜂毒肽干扰膜中阴离子通道的转运功能,使细胞渗透压改变,引起膨胀而溶血。蜂毒肽对有核红细胞核内DNA没有作用,与其他抗微生物多肽作用的靶向不同。据此认为,抗菌蛋白类抗生素对细菌作用的生化机理与传统抗生素不同,这是细菌对其不易产生耐药性的重要原因。     

内钙拮抗剂TMB-8对白皮松花粉管细胞壁构建的影响

应用荧光显微技术、激光共聚焦扫描显微技术、单克隆抗体免疫荧光标记技术以及傅里叶变换显微红外光谱分析(FTIR)等手段,研究了内钙拮抗剂TMB-8对白皮松花粉管胞内Ca2+分布、花粉管生长以及细胞肇构建等的影响.结果表明,白皮松花粉管经TMB-8处理后,胞内的Ca2+浓度下降,花粉管内典型的Ca2+浓度梯度消失,花粉萌发...     

萎缩性肌强直(MYD)患者红细胞膜生物物理特性的研究

作者用荧光方法测定了MYD患者红细胞和正常人红细胞在不同细胞外液K~+浓度下的膜电位,结果MYD患者红细胞膜电位比正常人高;荧光偏振法研究结果表明,MYD患者红细胞膜流动性比正常人高;细胞电泳方法发现MYD患者红细胞电泳迁移率亦较正常人高.这些生物物理特性的改变是相互联系的、反映了MYD患者红细胞膜结构的变化并将直接影响膜的代谢和功能.     

肺癌患者红细胞流变特性研究

采用旋转式粘度计、激光衍射技术和红细胞沉降法对肺癌患者的血液流变特性进行了实验研究。实验结果表明,肺癌患者的血液粘度比正常对照组高,肺癌患者的红细胞变形能力比正常人低,红细胞沉降速度比正常人大。     

对人眼晶状体α－晶体蛋白聚集体的准弹性激光散射研究

以准弹性激光散射技术,研究了人眼晶状体内α－晶体蛋白聚集体的扩散系数和流体力学半径,以及其弥散性随温度和浓度的变化。所研究的α－晶体蛋白用分离的方法分别取自成人和胚胎眼晶状体。研究结果表明,人眼α－晶体蛋白在一定浓度和温度下可形成聚集体,且其聚集体半径随α－晶体蛋白的浓度近乎线性地增大,随温度的增加而变小。对α－晶体蛋白溶液（５０ｍｍｏｌ／Ｌ磷酸脂缓冲液）的弥散度分析表明,溶液中有两种不同粒径的散射体,除开α－晶体蛋白聚集体外,还有一种粒径约为１８．５ｎｍ的估计是α－晶体蛋白单体分子。由于α－晶体蛋白分子的变性聚集在人眼白内障的形成起着重要作用,故本研究的结果对于人眼白内障随年龄及温度变化的发展过程及其机理的认识具有指导意义。     

A study of membrane protein defects and alpha hemoglobin chains of red blood cells in human beta thalassemia

The soluble pool of alpha hemoglobin chains present in blood or bone marrow cells was measured with a new affinity method using a specific probe, beta A hemoglobin chain labeled with [3H]N-ethylmaleimide. This pool of soluble alpha chains was 0.067 +/- 0.017% of hemoglobin in blood of normal adult, 0.11 +/- 0.03% in heterozygous beta thalassemia and ranged from 0.26 to 1.30% in homozygous beta thalassemia intermedia. This elevated pool of soluble alpha chains observed in human beta thalassemia intermedia decreased 33-fold from a value of 10% of total hemoglobin in bone marrow cells to 0.3% in the most dense red blood cells. The amount of insoluble alpha chains was measured by using the polyacrylamide gel electrophoresis in urea and Triton X-100. In beta thalassemia intermedia the amount of insoluble alpha chains was correlated with the decreased spectrin content of red cell membrane and was associated with a decrease in ankyrin and with other abnormalities of the electrophoretic pattern of membrane proteins. The loss and topology of the reactive thiol groups of membrane proteins was determined by using [3H]N-ethylmaleimide added to membrane ghosts prior to urea and Triton X-100 electrophoresis. Spectrin and ankyrin were the major proteins with the most important decrease of thiol groups.     

Influence of sickle hemoglobin polymerization and membrane properties on deformability of sickle erythrocytes in the microcirculation.

The rheological properties of normal erythrocytes appear to be largely determined by those of the red cell membrane. In sickle cell disease, the intracellular polymerization of sickle hemoglobin upon deoxygenation leads to a marked increase in intracellular viscosity and elastic stiffness as well as having indirect effects on the cell membrane. To estimate the components of abnormal cell rheology due to the polymerization process and that due to the membrane abnormalities, we have developed a simple mathematical model of whole cell deformability in narrow vessels. This model uses hydrodynamic lubrication theory to describe the pulsatile flow in the gap between a cell and the vessel wall. The interior of the cell is modeled as a Voigt viscoelastic solid with parameters for the viscous and elastic moduli, while the membrane is assigned an elastic shear modulus. In response to an oscillatory fluid shear stress, the cell--modeled as a cylinder of constant volume and surface area--undergoes a conical deformation which may be calculated. We use published values of normal and sickle cell membrane elastic modulus and of sickle hemoglobin viscous and elastic moduli as a function of oxygen saturation, to estimate normalized tip displacement, d/ho, and relative hydrodynamic resistance, Rr, as a function of polymer fraction of hemoglobin for sickle erythrocytes. These results show the transition from membrane to internal polymer dominance of deformability as oxygen saturation is lowered. More detailed experimental data, including those at other oscillatory frequencies and for cells with higher concentrations of hemoglobin S, are needed to apply fully this approach to understanding the deformability of sickle erythrocytes in the microcirculation. The model should be useful for reconciling the vast and disparate sets of data available on the abnormal properties of sickle cell hemoglobin and sickle erythrocyte membranes, the two main factors that lead to pathology in patients with this disease.     

Mouse beta thalassemia, a model for the membrane defects of erythrocytes in the human disease

The present study describes the pathophysiology, at the cellular level, of the mouse beta thalassemia and shows the pertinence of this model for the human disease. The homozygous state of mouse beta thalassemia is characterized by a clinical syndrome similar to the human beta thalassemia intermedia, but it cannot be explained by the small deficiency in beta chain synthesis. The small pool of unpaired and soluble alpha chains present in mouse reticulocytes contrasts with the large amount of insoluble alpha chains in erythrocytes which is induced by the high instability of mouse alpha chains and the absence of significant proteolysis. The amount of insoluble alpha chains associated with red cell ghosts is similar in human and mouse disease of similar severity. The study of membrane protein defects showed a decreased amount of spectrin (alpha and beta chains) and dramatic changes in the distribution of the most reactive thiol groups of membrane proteins. These results were similar to that previously described in the human disease (Rouyer-Fessard, P., Garel, M. C., Domenget, C., Guetarni, D., Bachir, D., Colonna, P., and Beuzard, Y. (1989) J. Biol. Chem. 264, 19092-19098). Abnormal density distribution curves of erythrocytes and oxidant-induced lysis of red blood cells used as functional tests were similar in the human and mouse beta thalessemia. We conclude from the present study that 1) mouse beta thalassemia is an excellent model for the membrane defects occurring in the human disease; 2) disease expression is not the reflection of the globin chain unbalance only nor of the soluble pool of alpha hemoglobin chain but mainly is a reflection of insoluble alpha chains; and 3) the rate of proteolysis and instability of alpha chains are important factors which must be taken into consideration in the pathophysiology and the clinical heterogeneity of the disease.     

Entrapment of purified alpha-hemoglobin chains in normal erythrocytes. A model for beta thalassemia

Altered membrane proteins have been previously described in beta thalassemia and are thought to play an important role in the shortened erythrocyte survival. To investigate the mechanism by which these changes occur, purified heme-containing alpha-hemoglobin chains were entrapped within normal erythrocytes by reversible osmotic lysis. These resealed cells exhibited normal hemoglobin concentration, cell volume, deformability, and no substantial modifications of membrane proteins. Incubation (37 degrees C; up to 20 h) of the alpha-chain-loaded cells resulted in increasing amounts of membrane-associated alpha-chains. This was associated with concurrent decreases in the protein concentrations and reactive thiol groups of spectrin, ankyrin, and actin as determined by gel electrophoresis. The decreases in membrane protein concentration and reactive thiol groups after 20 h of incubation were closely correlated (R2 = 0.947) in the alpha-chain-loaded cells. Indicative of increased oxidant stress within the alpha-chain-loaded erythrocytes, methemoglobin generation was also significantly increased in the alpha-chain-loaded erythrocytes. In addition, entrapment of alpha-chains led to a progressive and significant decrease in erythrocyte deformability. Thus, the entrapment of purified alpha-chains in normal erythrocytes resulted in structural and functional abnormalities very similar to that observed in beta-thalassemic erythrocytes in vivo. The model described provides a means by which the fate of excess alpha-chains, their pathophysiological effects, as well as possible therapeutic approaches to thalassemias can be examined.     

Studies on the osmotic resistance and the viability of amidinated erythrocytes]

The present studies are concerned with properties of amidinated erythrocytes. The reactions of dimethyladipimidate with proteins in solution and red blood cells, respectively, result in an intermolecular cross-linking. Following an amidination of human serum albumin or human gamma-globulin cross-linked products of increased molecular weight have been demonstrated by polyacrylamide gel and immune electrophoresis. Human erythrocytes previously amidinated intensely, exhibit a restricted motility of membrane particles and cross-linked hemoglobin. Intensely amidinated erythrocytes are resistant against distilled water, and they do no longer agglutinate. The findings presumably indicate an increased permeability of the amidinated red cell membrane. The glycolytic activity was found to be normal in moderately amidinated erythrocytes. In comparison with normal red blood cells, previously moderately amidinated erythrocytes of the rat become sequestered more quickly after re-injection into the vascular system.     

Co-localization of the immunoreactivities of corticotropin-releasing factor and arginine vasotocin in the brain and pituitary system of the teleost Catostomus commersoni

Summary Using the label-fracture technique, an ultrastructural comparison was made of the number and distribution of wheat germ agglutinin (WGA)-binding sites between human normal and sickle red blood cells. The WGA was adsorbed to colloidal gold, and quantitative analysis of the electron micrographs revealed that more binding sites were present on the sickle erythrocytes than on the normal erythrocytes. Moreover, the sites were more clustered on the sickle red cells than on the normal red cells. Use of another lectin, Bandieraea simplicifolia-II, revealed that it did not bind to normal or sickle red cells. Because of the affinity of the WGA for sialic acid residues, it is probable that the WGA is binding to a transmembrane sialoglycoprotein, glycophorin A. The conformation and/or distribution of the glycophorin A molecules may be altered by the sickle hemoglobin that binds to the red cell membrane. Hence, as detected by WGA, new surface receptors, which could play a role in the adhesion of sickle cells to endothelium may be exposed.     

Anion transport in normal erythrocytes,sickle red cells,and ghosts in relation to hemoglobins and magnesium

"Band 3," an integral membrane protein of red blood cells, plays a relevant role in anionic transport. The C- and N-terminal portions of band 3 are cytoplasmatics, and the last is the link site for different glycolitic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphofructokinase, and hemoglobin. All or some of these interactions on the CDB3 protein could allow a subtle modulation of anion flux. The interaction among HbA, Mg(2+), and membrane proteins has been sufficiently investigated, but not the effect of Mg(2+) on pathological hemoglobin in relation to the influx of the SO(4)(2-). The aim of this study was to evaluate the involvement of hemoglobin S in sulfate transport. This has been measured with native and increased concentrations of Mg(2+), using normal erythrocytes containing HbA, sickle red cells containing HbS, or ghosts obtained from both erythrocytes and normal erythrocytes ghosts with HbS added. The magnitude of the SO(4)(2-) rate constant measured in normal red blood cells increased markedly when measured in the presence of varied Mg(2+) concentrations. The results show that a low increase of intracellular Mg(2+) concentrations exercises a different HbA modulation on band 3 protein and consequently higher anion transport activity. The same experiments carried out in sickle red cells showed that the SO(4)(2-) rate constant measured in the presence of native concentrations of Mg(2+) was normal, compared to normal red cells, and was not affected by any increase of intracellular Mg(2+). Our suppositions with regard to the importance exercised by the hemoglobin and the Mg(2+) on the SO(4)(2-) influx were confirmed by comparison of the data obtained through measuring SO(4)(2-) influx with native and increased concentrations of Mg(2+) in both normal and sickle red cell ghosts. Both revealed the same sensitivity to Mg(2+) due to withdrawal of hemoglobins. The incorporation of HbS in normal as well as in sickle red cell ghosts reduced the Mg(2+) response to sulfate influx in both the reconstituted ghosts. Our research demonstrated that the different effects exercised on the rate constants of SO(4)(2-) influx in normal (HbA) and sickle red cells (HbS) by the increased intracellular Mg(2+) could be ascribed to the physical-chemical influence exercised either on the hemoglobins or on the intracellular contents of erythrocytes.     

The structural organization of skeletal proteins influences lipid translocation across erythrocyte membrane

In order to define the influence of skeletal protein organization on transmembrane phospholipid movement in erythrocyte membranes, we measured the translocation rate of lysophosphatidylcholine in pathologic red cells. A simple method based on the differential extraction of lysophosphatidylcholine from the red cell membrane by saline and albumin solutions was used to quantitate the translocation rate. Two groups of pathologic red cells were chosen for these studies: red cells with quantitative deficiencies of the skeletal proteins, spectrin and protein 4.1, and sickle erythrocytes in which controlled reorganization of the membrane was induced by hemoglobin polymerization. Marked increase in lipid translocation rate was seen in red cells having quantitative deficiencies of spectrin and protein 4.1. The magnitude of the increase in translocation rate in spectrin-deficient red cells was related to the magnitude of protein deficiency. Translocation rate in sickle erythrocyte membranes increased by 50% upon deoxygenation as a result of sickle hemoglobin polymerization. No increase in translocation rate was seen in normal cells upon deoxygenation. By manipulating the extent of membrane reorganization that occurred following deoxygenation of sickle cells, we have been able to show that skeletal reorganization induced by hemoglobin polymerization and not hemoglobin polymerization per se is responsible for the increase in translocation rate. Together, these findings imply that the structural organization of membrane skeletal proteins plays an important role in regulating the rate of transbilayer movement of lipids across the erythrocyte membrane.     

Effect of sickling on dimyristoylphosphatidylcholine-induced vesiculation in sickle red blood cells

To study the effect of sickling on dimyristoylphosphatidylcholine (DMPC)-induced vesiculation, sickle (SS) red blood cells were incubated with sonicated suspensions of DMPC under either room air or nitrogen. Like normal red cells, when sickle cells were incubated with DMPC under oxygenated conditions, incorporation of DMPC into the erythrocyte membrane occurred, followed by echinocytic shape transformation and subsequent release of membrane vesicles. On the other hand, when SS cells were induced to sickle by deoxygenation, DMPC-induced vesiculation of these cells was dramatically reduced. However, upon reoxygenation, release of vesicles from these sickle erythrocytes occurred immediately. When SS cells were incubated under hypertonic (500 mosM) and deoxygenated conditions (where hemoglobin polymerization occurs but red cells do not show the typical sickle morphology), a similar decrease in the extent of vesiculation was observed. Experiments with radiolabelled lipid vesicles indicated that incorporation of DMPC into erythrocyte membranes occurred in all cases and therefore was not the limiting factor in the reduction of vesiculation in deoxygenated SS cells. Taken together, these results indicate that cellular viscosity and membrane rigidity, both of which are influenced by hemoglobin polymerization, are two important factors in process of vesicle release from sickle erythrocytes.     

Eryptosis in hereditary spherocytosis and thalassemia: role of glycoconjugates

The present work is aimed to study the mechanism of faster erythrocyte clearance in hereditary spherocytosis (HS), a heterogeneous disorders characterized by alterations in the proteins of the red cell membrane skeleton along with different kinds of thalassemia. The maximum exposure of phosphatidylserine (PS) is found in HS compared to those in both α- and β-thalassemia. Interestingly, in HS more PS exposed cells were found in younger erythrocytes compared to normal and the thalassemics where aged cells showed higher loss of PS asymmetry. Loss of sialic acid and GlcNAc bearing glycoconjugates, presumably the glycophorins, was also found upon aging. The loss of PS asymmetry together with the cell surface glycoproteins mediated by membrane vesiculation, seemed to play key role in early clearance of erythrocytes from circulation following a mechanism similar to HbEβ-thalassemia.