红细胞膜骨架收缩蛋白及其基因突变

血影收缩蛋白通过自身聚合由异二聚体形成四聚体（ＳＰＴ），参与构成红细胞膜蛋白质网状骨架，并与膜骨架蛋白其它成分及膜固有蛋白质相互作用，维持正常红细胞的形态和功能。收缩蛋白基因突变导致红细胞收缩蛋白自身聚合位点及其它结构区域的异常，使ＳＰＴ形成障碍，均使红细胞膜结构与功能出现异常。     

红细胞膜骨架收缩蛋白及其基因突变

血影收缩蛋白通过自身聚合由异二聚体形成四聚体(SPT),参与构成红细胞膜蛋白质网状骨架,并与膜骨架蛋白其它成分及膜固有蛋白质相互作用,维持正常红细胞的形态和功能。收缩蛋白基因突变导致红细胞收缩蛋白自身聚合位点及其它结构区域的异常,使SPT形成障碍,均使红细胞膜结构与功能出现异常。     

尿症病人红细胞变形能力与红细胞膜硒浓度的关系

从硒与红细胞膜收缩蛋白的关系探讨尿毒症病人和４０例健康人ＥＤ，红细胞膜硒浓度，红细胞膜脂质过氧化物，膜收缩蛋白二聚体和四聚体的变化。结果尿毒症病人红细胞滤过指数，Ｅ－ＬＰＯ，ＳＰ－Ｄ／ＳＰ－Ｔ明显增高，Ｅ－Ｓｅ，ＳＰ－Ｔ明显降低２，与对照组比较差异有极显著性。尿毒症病人ＥＦＩ与Ｅ－Ｓｅ，ＳＰ－Ｄ呈负相关，与Ｅ－ＬＰＯ，ＳＰ－Ｄ，ＳＰ－Ｄ／ＳＰ－Ｔ呈正相关；Ｅ－Ｓｅ与Ｅ－ＬＰＯ，ＳＰ－Ｄ，ＳＰ－Ｄ／     

肿瘤抑制基因p53·p16与脑肿瘤

目的：探索肿瘤抑制基因ｐ５３、ｐ１６变异在脑肿瘤发病中的意义。方法：对２３例脑肿瘤病人原发肿瘤标本用免疫组化ＬＳＡＢ法行肿瘤抑制基因ｐ５３、ｐ１６蛋白检测。结果：星形细胞瘤Ⅰ级９例，ｐ１６蛋白４例表达３０％以下，５例不表达，ｐ５３蛋白３例表达３０％以下，６例不表达；Ⅱ～Ⅳ级７例，ｐ１６蛋白１例表达３０％以下，６例不表达，ｐ５３蛋白４例表达３０％～６０％，３例不表达；脑膜瘤、垂体腺瘤７例，ｐ１６蛋白３例表达３０％以下，４例不表达，ｐ５３蛋白２例表达３０％以下，５例不表达；结论：ｐ５３、ｐ１６蛋白缺失、变异是肿瘤发生的重要原因之一，ｐ５３变异主要见于恶性胶质细胞瘤，偏良性的胶质细胞瘤和脑膜瘤、垂体瘤，ｐ５３蛋白轻度阳性表达，提示肿瘤可能处于早期恶性变过程中。ｐ１６蛋白缺失多见于恶性胶质瘤，也见于部分良性肿瘤。     

一种人FLT3配基同源异型蛋白在组织中的分布

目的研究人ＦＬＴ３配基膜外区缺失１３９ｂｐ的同源异型蛋白在组织中的分布特点。方法采用聚合酶链或逆转录聚合酶链反应法对ＦＬＴ３配基在人肝脏、脑、骨髓、肌肉、肾脏、正常人外周血及４种白血病细胞系中的分布进行了研究,并利用ＤＮＡ测序法鉴定ＰＣＲ产物。结果除脑中未见ＦＬＴ３配基表达外,在肝脏、肌肉、骨髓、肾脏、胎肝、正常人外周血细胞及白血病细胞系中均可检测到ＦＬＴ３配基,而膜外区缺失１３９ｂｐ的ＦＬＴ３配基同源异型蛋白只分布于骨髓、胎肝中。结论ＦＬＴ３配基膜外区缺失１３９ｂｐ的同源异型蛋白分布于造血组织中,可能与造血调控有关。     

川崎病3例报告

川崎病３例报告我院近年收治川崎病３例，现报告如下：３例中男２例，女１例，年龄２～２．５岁。共同的临床表现：高热５～１５天，用抗生素无效；双结膜充血；口唇鲜红皲裂；全身多形性皮疹，皮疹无水疮、无糜烂、无痂皮；颈淋巴结肿大；恢复期指端膜样脱皮。其中１例还...     

ABC免疫电泳对膀胱癌P21蛋白表达的观察

用ＡＢＣ免疫电泳检测了２９例膀胱移行细胞癌、６例癌旁粘膜、３例膀胱炎和３例正常膀胱粘膜的Ｐ２１蛋白表达，发现２５例（８６．２％）膀胱癌、３例癌旁组织、１例正常膀胱膜、２例胶胱炎组织有Ｐ２１蛋白表达。随肿瘤分级增高，Ｐ２１蛋白条带的光密度积分增加；肿瘤组织Ｐ２１蛋白光密度积分高于癌旁组织，癌旁组织又高于正常膀胱粘膜。     

海南黎族地中海贫血患者红细胞膜收缩蛋白和带3蛋白mRNA的表达

我们观察了 32例海南黎族地中海贫血 (珠蛋白生成障碍性贫血 ,地贫 )患者红细胞膜骨架收缩蛋白 (ｓｐｅｃｔｒｉｎ)和ｂａｎｄ 3(带 3蛋白 )ｍＲＮＡ表达水平的变化 ,探讨了红细胞膜收缩蛋白和带 3蛋白在地贫发病机制中的作用。病例和方法1　病例　 32例地贫患者均为三代以上世居在海南岛的黎族居民 ,经临床和实验室检查诊断为地贫 ,其中血红蛋白Ｈ病7例 ,α地贫杂合子 12例和 β地贫纯合子 5例、杂合子 (中间型 ) 8例。 30名正常对照者均为世居在海南岛的健康黎族居民。2　全血制备血膜及ＳＤＳ ＰＡＧＥ　全血制备血膜方法同文献[1];红细…     

针灸治疗产后尿潴留的探讨

研究２６例产后尿潴留患者的治疗情况，１５例采用针灸加留置导尿管进行治疗（以下简称治疗组），另１１例采用留置导尿管进行治疗（以下简称对照组），结果表明，治疗组疗效优于对照组，Ｐ＜０．０５。治疗组比对照组恢复自行排尿提早４．２２天，其中针灸１次，拨除导尿管，自行排尿者占６１％；针灸２次者占２６％；针灸３次者占１３％；总有效率１００％。因此采用针灸治疗，缩短了产后尿潴留时间，减少了尿路逆行感染、子宫收缩不良、产后出血并发症的发生，有利产妇分娩后的康复。     

巨幼细胞贫血、骨髓增生异常综合征及白血病患者叶酸结合蛋白的研究

为研究叶酸结合蛋白特性，用放射受体结合技术及ＳＤＳ－聚丙烯酰胺凝胶电泳和同位素扫描结合的方法，分析了巨幼细胞贫血（ＭＡ）、骨髓增生异常综合征（ＭＤＳ）和白血病患者血清可溶性叶酸受体（ｓＦＲ）及骨髓单核细胞膜叶酸受体（ＦＲ）的分子量。发现白血病细胞膜上有两种叶酸结合蛋白：分子量为３．５万的ＦＲ以及分子量为４．５万的还原叶酸载体（ＲＦＣ）。ＭＤＳ组中５例ＲＡＥＢ、ＲＡＥＢ－ｔ患者骨髓单个核细胞膜上亦出现ＦＲ和ＲＦＣ；７例ＲＡＳ和ＲＡ以及ＭＡ患者骨髓单核细胞膜叶酸结合蛋白仅为ＦＲ。ＲＦＣ是一种幼稚型蛋白，在ＭＤＳ患者骨髓细胞膜上出现，可能是白血病前期表现。     

Spectrin extractability from erythrocyte in Duchenne muscular dystrophies and the effect of proteases on erythrocyte ghosts

We studied the erythrocyte membrane proteins from patients with Duchenne muscular dystrophy (DMD) using SDS-polyacrylamide gel electrophoresis. Our observations were the following: (1) The electrophoretic densitogram of freshly prepared DMD-ghosts was similar to that of controls. After the extraction of spectrin from ghosts with 1 mmol/l EDTA, pH 8.0, the unextractable spectrin remained more firmly bound in the DMD ghost residues than in controls. Extractability of spectrin from DMD ghosts was decreased about 20%. In addition, several minor bands were detected between spectrin and Band 3 in the DMD ghost residues (treated ghosts). (2) Ca2+-activated, neutral protease reacted more effectively with spectrin of DMD ghosts and ATP-depleted ghosts than with that of controls. (3) Erythrocyte actin (Band 5) of DMD ghosts was more fragile than that of controls and of ATP-depleted actin in the EDTA extracts. Gradually, partial degradation of actin was observed for three weeks at 4 degrees C. (4) The intracellular ATP level and the activities of membrane-bound (Mg2+--Ca2+) ATPases in DMD erythrocytes were unchanged. We suggest that spectrin from DMD ghosts as well as actin may be subject to increased degradation.     

Reductions of erythrocyte membrane viscoelastic coefficients reflect spectrin deficiencies in hereditary spherocytosis.

Hereditary spherocytosis is a common hemolytic anemia associated with deficiencies in spectrin, the principal structural protein of the erythrocyte membrane-skeleton. We have examined 20 different individuals from 10 spherocytosis kindreds and 2 elliptocytosis kindreds to determine the effects of different levels of spectrin deficiency on the viscoelastic properties of the erythrocyte membrane. Micropipettes were used to perform single-cell micromechanical measurements of approximately 1,000 individual cells to determine the membrane elastic shear modulus, the apparent membrane bending stiffness, and whole cell recovery time constant for the different cell populations. The membrane viscosity was calculated by the product of the shear modulus and the recovery time constant. Results show correlation between the fractional reduction in shear modulus and the fractional reduction in spectrin content (determined by spectrin radioimmunoassay) and spectrin density (determined by the ratios of spectrin to band 3 on electrophoresis gels) suggesting that membrane shear elasticity is directly proportional to the surface density of spectrin on the membrane (P less than 0.001). The apparent membrane bending stiffness is also reduced in proportion to the density of spectrin (P less than 0.001). The membrane viscosity is reduced relative to control (P less than 0.001), but the nature of the relationship between spectrin density and membrane viscosity is less clearly defined. These studies document striking relationships between partial deficiencies of erythrocyte spectrin and specific viscoelastic properties of the mutant membranes.     

Defective binding of spectrin to ankyrin in a kindred with recessively inherited hereditary elliptocytosis

The interaction of spectrin with spectrin-depleted inside-out membrane vesicles was studied in a kindred with an atypical variant of hereditary elliptocytosis inherited in a recessive manner. The probands are characterized by prominent elliptocytosis, decreased erythrocyte thermal stability, an altered limited tryptic peptide pattern of spectrin digested at low ionic strength, and defective spectrin dimer-dimer association. The parents are normal. The spectrin/band 3 ratio determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of isolated membranes of the probands was decreased to approximately 70% of control values, and total erythrocyte spectrin content in one proband was also decreased on SDS-PAGE. When a monospecific antispectrin antibody was used, a faintly labeled fragment of molecular weight approximately 28,000 was detected on immunoblots of whole cell lysates of one proband and a control, but could not account for the decreased erythrocyte spectrin content of the proband on SDS-PAGE. Binding and competitive inhibition studies revealed an alteration in the spectrin-ankyrin interaction due to an abnormality of spectrin in the probands. No defect was found in the mother; the father's spectrin showed decreased binding affinity, although it was not so severe as in the probands. Separation of bound and unbound spectrin dimers from one proband and subsequent conversion to tetramers showed that the self-association defect was detectable only on the bound subpopulation of her spectrin. These findings demonstrate a hitherto undescribed functional abnormality of spectrin in this kindred which could result in decreased stability of the membrane skeleton and contribute to the elliptocytic shape of these erythrocytes.     

Altered erythrocyte spectrin extractability in Duchenne muscular dystrophy

Spectrin extractability was measured in the erythrocyte membranes obtained from patients with Duchenne muscular dystrophy (DMD) and normal matched controls. After the extraction of spectrin by adding deionized water, an aliquot of the pellet was applied to 6% SDS-polyacrylamide gels. Spectrin/Band 3 ratio on the gels was used as an indicator of spectrin extractability. Our data dis- closed that spectrin extractability is significantly reduced in DMD patients when compared with that of controls and these results suggest that spectrin from DMD patients is more tightly bound to the membranes. Our data may also be applicable to the discussion of the relationship of spectrin to other membrane properties as well as to previously described membrane abnormali- ties in DMD erythrocytes.     

Two elliptocytogenic alpha I/74 variants of the spectrin alpha I domain. Spectrin Culoz (GGT----GTT; alpha I 40 Gly----Val) and spectrin Lyon (CTT----TTT; alpha I 43 Leu---Phe).

Spectrin alpha I/74 elliptocytosis results from abnormalities involving the "head" region of spectrin dimer. Increased susceptibility to trypsin enhances cleavage of the alpha spectrin chain, yielding an increased amount of the alpha I 74-kD fragment at the expense of the alpha I 80-kD parent fragment. Recently we showed that the mutations causing the Sp alpha I/74 abnormality may lie in the alpha- or the beta-chain, and that spectrin Culoz and spectrin Lyon were two (alpha I/74) alpha-variants, respectively. We now show that the spectrin Culoz alpha I domain undergoes prominent tryptic cleavage after Lys 42, whereas cleavage prevails after Arg 39 in spectrin Lyon. Applying the polymerase chain reaction (PCR) technique to exon 2 of the spectrin alpha I domain, we have established that the mutation responsible for spectrin Culoz is alpha I 40 Gly----Val; GGT----GTT. Applying the PCR technique to the cDNA derived from reticulocyte mRNA, we have shown that the mutation responsible for spectrin Lyon is alpha I 43 Leu----Phe; CTT----TTT. Studies of normal controls and of family members using dot blot hybridization with allele-specific oligonucleotide probes confirmed these results. Variants such as spectrin Culoz and spectrin Lyon should provide insight into a region that participates in spectrin dimer self-association and whose susceptibility to proteolysis must reflect subtle conformational changes.     

Molecular Defect of Spectrin in Hereditary Pyropoikilocytosis: ALTERATIONS IN THE TRYPSIN-RESISTANT DOMAIN INVOLVED IN SPECTRIN SELF-ASSOCIATION

In hereditary pyropoikilocytosis (HPP) the erythrocyte membrane skeleton exhibits mechanical instability that can be correlated to defective self-association of spectrin heterodimers. To detect structural changes in the functional domains of HPP spectrin we have examined the peptide pattern produced by limited tryptic digestion of spectrin extracts from two families that contain three HPP patients. Limited tryptic digestion of all three HPP patients revealed a similar and reproducible decrease in the staining intensity of an 80,000-, and 22,000-, and an 88,000-dalton polypeptide with a concomitant increase in a 74,000- and a 90,000-dalton polypeptide as compared with controls. Only changes in the 80,000-, and 74,000-, and 22,000-dalton polypeptides could be correlated to defective spectrin self-association and the amount of spectrin dimers in 0°C extracts of the HPP patients and their affected kindred. Similar results were obtained when the tryptic digests were analyzed by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the affected 74,000- and 80,000-dalton polypeptides focusing into multiple spots ranging in isoelectric point from 5.3-5.4. When HPP spectrin dimers and tetramers were separated and subjected to trypsin digestion, changes in the 80,000-, 74,000-, and 22,000-dalton polypeptides were found predominantly in the spectrin dimer pool. Similar results were obtained for spectrin from two of the probands' mother, whom we have identified as an HPP carrier. We conclude that these HPP patients contain a population of normal, (principally tetrameric) and mutant (principally dimeric) spectrin. The latter is characterized by a defective spectrin dimer self-association due to conformational changes that affect the 80,000-dalton domain.     

Abnormal degradation of erythrocyte membrane proteins in hereditary spherocytosis

The degradation rate of erythrocyte membrane proteins by membrane-bound proteases was compared in healthy controls and in patients with hereditary spherocytosis (HS). An increased degradation rate of spectrin and a decreased digestion rate of Band 3 were found in HS patients. These differences may be due to altered accessibilities of membrane proteins to proteases and/or to changes in the pattern of membrane-associated proteases and may be connected with the decrease in the spectrin: Band 3 ratio reported for erythrocyte membranes in HS.     

A molecular defect of spectrin in a subset of patients with hereditary elliptocytosis. Alterations in the alpha-subunit domain involved in spectrin self-association.

Hereditary elliptocytosis (HE) is a clinically and biochemically heterogenous group of diseases characterized by elliptically shaped erythrocytes and an autosomal dominant mode of inheritance. Whereas the self-association of spectrin heterodimers to tetramers is defective in a subpopulation of HE patients, designated HE[SpD-SpD], it is normal in others. We have examined the peptide pattern produced by limited tryptic digestion of spectrin extracts from patients with HE[SpD-SpD] to determine if the functional defects in spectrin self-association could be correlated with structural changes in the spectrin molecule. Although the peptide pattern produced by limited tryptic digestion of spectrin extracts from those HE patients with normal spectrin self-association was indistinguishable from the pattern from control normal volunteers, digestion of the spectrin extracts from the HE[SpD-SpD] patients showed a reproducible diminution in the 80,000-D domain of the alpha-subunit, which is involved in spectrin dimer self-association. The decrease in the 80,000-D fragment was associated with an increase in a 74,000-D fragment in eight of nine families, or, in one family, with an increase of fragments at 46,000 and 17,000 D. These atypical peptide patterns were similar to those previously reported in two variants of hereditary pyropoikilocytosis (HPP), which also had defective self-association of spectrin. These data indicate that two distinct structural variants of spectrin alpha-subunit are associated with the defective spectrin heterodimer self-association in a subpopulation of HE patients.     

Diminished spectrin extraction from ATP-depleted human erythrocytes. Evidence relating spectrin to changes in erythrocyte shape and deformability.

We measured spectrin "extractability" in erythrocytes which were metabolically depleted by incubation at 37 degrees C in plasma or glucose-free buffers. Membranes were extracted with 1 mM EDTA (pH 8, 40 h, 4 degrees C) and analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This procedure solubilized 85--90% of the spectrin, actin, and residual hemoglobin from ghosts of fresh erythrocytes. In incubated erythrocytes, inextractable spectrin rapidly accumulated when ATP concentrations fell below 0--15% of normal. In severely depleted cells, 60--90% of the total ghost spectrin became inextractable. Inextractability was not abolished by physically disrupting the ghost before extraction, but was reversed when erythrocyte ATP was replenished with adenosine. The accumulation of inextractable spectrin correlated temporally with the increase in apparent membrane deformability and the increases in erythrocyte vicosity, calcium content, sodium gain, and potassium loss characteristic of ATP-depleted erythrocytes. No change in integral membrane protein topography (assessed by the distribution of intramembranous particles and concanavalin A surface-binding sites) was detected in depleted cells. Analogous changes were observed in erythrocytes exposed to extremes of pH and temperature. When the pH in the erythrocyte interior fell below 5.5, a pH where spectrin was aggregated and isoelectrically precipitated, erythrocyte and ghost viscosity increased coincident with a marked decrease in spectrin extractability. Similarly above 49 degrees C, a temperature where spectrin was denatured and precipitated, erythrocyte viscosity rose as inextractable spectrin accumulated. These observations provide direct evidence of a change in the physical state of spectrin associated with a change in erythrocyte shape and deformability. They support the concept that erythrocyte shape and deformability are largely determined by the shape and deformability of the spectrin-actin protein meshwork which laminates the inner membrane surface.     

Leukocyte-platelet interaction. Release of hydrogen peroxide by granulocytes as a modulator of platelet reactions.

Irreversibly sickled cells (ISC's) are circulating erythrocytes in patients with sickle cell disease that retain a sickled shape even when oxygenated. Evidence points to a membrane defect that prevents the return of these cells to the normal biconcave shape. The erythrocyte membrane protein spectrin is believed to help control erythrocyte shape and deformability. Recent studies suggest that normally spectrin and an erythrocyte actin form a self-supporting, fibrillar, lattice-like network on the cytoplasmic membrane surface. When normal erythrocyte ghosts are extracted with Triton X-100 all the integral membrane proteins and most of the membrane lipids are removed, leaving a ghost-shaped residue composed principally of spectrin and actin. We concentrated ISC's from patients with sickle cell anemia and compared the morphology and protein composition of ghosts and Triton-extracted ghost residues prepared from these ISC's with similar preparations of reversibly sickable cells and normal cells. (a) Many ISC's formed ISC-shaped ghosts. (b) All ISC-shaped ghosts formed ISC-shaped Triton residues. (c) Spectrin, erythrocyte actin (Band 5), an unidentified Band 3 component, and Band 4.1 were the major protein components of the Triton residues. All membrane-associated sickle hemoglobin was removed by the Triton treatment. (d) No ISC-shaped ghosts or ISC-shaped Triton residues were formed when deoxygenated, sickled RSC's were lysed or Triton-extracted. ISC-shaped ghosts and Triton residues were never formed from normal cells. These observations suggest that a defect of the "spectrin-actin lattice" may be the primary abnormality of the ISC membrane. Since ISC's are rigid cells, the data support the postulate that spectrin is a major determinant of membrane deformability. Finally, they provide direct evidence that spectrin is important in determining erythrocyte shape.