Molecular basis of spectrin deficiency in hereditary pyropoikilocytosis

Hereditary pyropoikilocytosis (HPP) is a recessively inherited hemolytic anemia characterized by severe poikilocytosis and red blood cell fragmentation. HPP red blood cells are partially deficient in spectrin and contain a mutant alpha or beta-spectrin that is defective in terms of spectrin self-association. Although the nature of the latter defect has been studied in considerable detail and many mutations of alpha-spectrin and beta spectrin have been identified, the molecular basis of spectrin deficiency is unknown. Here we report two mechanisms underlying spectrin deficiency in HPP. The first mechanism involves a thalassemia-like defect characterized by a reduced synthesis of alpha-spectrin as shown by studies involving synthesis of spectrin in two unrelated HPP probands and their parents: One parent carries the elliptocytogenic spectrin mutation, whereas the other parent is fully asymptomatic. Peripheral blood mononuclear cells as a source of erythroid burst-forming unit (BFUe) were cultured in a two-phase liquid culture system that gives rise to terminally differentiated erythroblasts. Pulse-labeling studies of an equal number of erythroblasts or morphologically identical maturity showed that the synthesis of alpha-spectrin as well as the mRNA levels as measured by the competitive polymerase chain reaction (PCR) method are markedly reduced in the presumed asymptomatic carriers and the HPP probands. In contrast, the synthesis and mRNA levels of beta-spectrin were normal. These results constitute a direct demonstration of an alpha-spectrin synthetic defect in a subset of asymptomatic carriers of HPP and HPP probands. The second mechanism underlying spectrin deficiency involves increased degradation of mutant spectrin before its assembly on the membrane. This is evidenced by pulse labeling studies of erythroblasts from a patient with HPP associated with a homozygous state for spectrin alpha I/46 mutation (leu-pro mutation at AA 207 of alpha-spectrin). These studies showed that although spectrin is synthesized in the cytosol in normal amounts, the rate of turnover of alpha-spectrin is faster resulting in about 40% to 50% reduced assembly of alpha-spectrin and beta-spectrin on the membrane. Thus, spectrin deficiency in this case is at least in part caused by increased susceptibility of the mutant spectrin to degradation before its assembly on the membrane. We conclude that at least two separate mechanisms underlie the molecular basis of spectrin deficiency in HPP.     

Antibodies to human T-cell leukemia virus type III in hemophiliacs from Spain

We tested serum samples from 50 hemophiliacs from Sevilla, Spain, for antibody to HTLV-III by indirect membrane immunofluorescence (IMI) and radioimmunoprecipitation with SDS polyacrylamide gel electrophoresis (RIP-SDS/PAGE). All had received commercial clotting factors from the United States with the exception of one hemophiliac who had never been transfused. Thirty-four (68%) reacted with HTLV-III-infected cells (H9/HTLV-III) by both methods, but not with the uninfected line (H9). Of 41 hemophilia-A patients tested, 28 (68%) were positive, and of nine hemophilia-B patients, six (66%) were positive. The nontransfused hemophilia-B patient was negative for antibody to HTLV-III by both methods. One patient with clinical AIDS tested positive as did six of seven with chronic unexplained lymphadenopathy. The eight individuals with AIDS or lymphadenopathy all had hemophilia A. We conclude that exposure to HTLV-III is widespread among asymptomatic hemophiliacs in Spain.     

Expression of embryonic zeta-globin and epsilon-globin chains in a 10- year-old girl with congenital anemia

A 10-year-old Danish girl with congenital anemia is described. At birth, she had severe anemia and erythroblastosis and was transfused a number of times during the first year. The need for transfusions has since declined steadily. Her reticulocyte counts varied between 2% and 15%, and her bone marrow aspirate showed some dyserythropoietic features. Her hemoglobin F level was consistently elevated, up to as much as 41%. Her erythrocytes had a normal level of I antigen but an undetectable level of i antigen. Moreover, embryonic zeta-globin and epsilon-globin chains were present in some of her circulating erythrocytes. These findings may represent the manifestations of a new variant of congenital anemia.     

Allelic loss of IRF1 in myelodysplasia and acute myeloid leukemia: retention of IRF1 on the 5q- chromosome in some patients with the 5q- syndrome

Acquired interstitial deletions of the long arm of chromosome 5 occur frequently in the myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Recently IRF1, a putative tumor suppressor gene localized to the long arm of chromosome 5, has been shown to be deleted from the 5q- chromosome in a group of patients with MDS and AML. It has been suggested that the loss of IRF1 may be critical to the development of the 5q- syndrome. We have investigated the allelic loss of IRF1 in a group of 12 patients with MDS and a 5q deletion and 2 patients with AML and a 5q deletion. Gene dosage experiments demonstrated that 12 of 14 patients had loss of one allele of the IRF1 gene but no evidence of homozygous loss and that 2 patients with 5q- syndrome retained both copies of the gene. The retention of IRF1 on the 5q- chromosome in these two cases has been confirmed by fluorescent in situ hybridization localization using an IRF1 cosmid. Pulsed field gel electrophoresis was used to determine whether there was any evidence for structural rearrangement in the region encompassing the IRF1 gene in these two patients. No aberrant bands were detected with a range of rare cutter enzyme digests. We conclude that IRF1 maps outside the commonly deleted segment of the 5q- chromosome and that loss of IRF1 is not solely responsible for the development of the 5q- syndrome.     

Biochemical and functional consequences of dissociation of the platelet membrane glycoprotein IIb-IIIa complex

The platelet membrane glycoproteins, IIb and IIIa, form a Ca2+- dependent heterodimer complex that functions as the fibrinogen receptor in activated platelets to mediate platelet aggregation. Little is known about factors that affect the IIb-IIIa complex within the platelet membrane. It has been observed that platelets incubated with ethylene glycol tetra-acetic acid (EGTA) at 37 degrees C are unable to aggregate or to bind monoclonal antibodies specific for the IIb-IIIa complex. To determine whether this is due to a dissociation of IIb from IIIa, we developed a method for quantitating the complex on nondenaturing, polyacrylamide gradient gels. Platelets were surface-labeled with 125I and then solubilized and electrophoresed in 0.2% Triton and 10 mmol/L CHAPS. Under these conditions and in the presence of 1 mmol/L Ca2+, glycoproteins IIb and IIIa migrated on the gels as a discrete band at Rf = 0.33. Protein that was eluted from this band bound to an immunoaffinity column specific for the IIb-IIIa complex. In contrast, when the IIb-IIIa complex was solubilized and then dissociated with EGTA, the discrete band at Rf = 0.33 was no longer present, and IIb and IIIa were now found in a broad band at Rf = 0.45 to 0.50. To study IIb and IIIa within the surface membrane, the 125I-labeled platelets were first incubated with 0.5 mmol/L EGTA (1 nmol/L free Ca2+) at 22 degrees C and then solubilized in the absence of EGTA. The IIb and IIIa from these platelets migrated at Rf = 0.33, indicating the presence of the intact IIb-IIIa complex. In contrast, when the platelets were incubated at 37 degrees C for one hour with the EGTA, the discrete band at Rf = 0.33 representing the IIb-IIIa complex gradually disappeared. This phenomenon could not be reversed by adding Ca2+ back to the platelets before solubilization and electrophoresis. This loss of the IIb-IIIa complex from intact platelets was accompanied by (a) a progressive and irreversible decrease in adenosine diphosphate (ADP)-induced platelet aggregation and (b) decreased binding of a complex-dependent monoclonal antibody to the platelets. These studies demonstrate that when platelets are exposed to low Ca2+ at 37 degrees C, the IIb-IIIa heterodimer complexes in their surface membranes are irreversibly disrupted. Because intact IIb-IIIa complexes are required for platelet aggregation, the loss of these complexes may account for the failure of these platelets to aggregate in response to ADP.     

骨髓干细胞动员治疗脑缺血损伤

学术背景：近来的研究表明，来源于骨髓的干细胞具有横向分化潜能和成神经分化特性，对脑缺血损伤后神经功能恢复和神经细胞的修复具有保护作用。 目的：通过回顾近年来骨髓干细胞动员对脑缺血损伤保护作用的文献，对骨髓干细胞的特性及动员过程、途经,可能的作用机制、影响因素进行阐述，以期完善骨髓干细胞动员防治脑缺血损伤的基础与临床研究。 检索策略：应用计算机检索Medline1997—01／2007—01与骨髓干细胞动员和脑缺血相关的文章。检索词“bone marrow stem cells，cerebral ischemia”并限定语言种类为English，同时计算机检索CNKI数据库1997—01／2007—01相关文章，并限定文章语言种类为中文，检索词为“骨髓干细胞，脑缺血”。就检索到的300余篇文献进行筛选，选择以骨髓干细胞动员和脑缺血损伤的基础研究为主要内容的文献63篇，其中研究内容相似的，以近5年且发表在较权威杂志者优先。 文献评价：将入选的30篇文献进行分类，11篇研究骨髓干细胞的生物学特性，19篇与骨髓干细胞动员治疗脑缺血损伤的过程．途经,可能的作用机制、影响因素相关。 资料综合：来源于骨髓的干细胞具有很强的“可塑性”，自我更新和高度增殖能力，其可能通过修复和替代脑缺血损伤后的神经细胞，促进或直接参与缺血区新生血管生成，分泌神经营养因子等，对脑缺血损伤后神经功能恢复发挥保护作用，其作用机制和作用方式目前尚不确切，有待于建立完整的动物模型技术平台，通过基础研究进行解决。 结论：骨髓干细胞对脑缺血损伤具有修复作用，采用骨髓干细胞动员的干预策略，方法简便、安全有效,无创伤且可避免干细胞移植的免疫排斥反应，具有较好的发展潜力，有望成为治疗脑缺血性疾病的研究重点。     

Screening with monoclonal anti-Fy3 to provide blood for phenotype- matched transfusions for patients with sickle cell disease

BACKGROUND: In the United States, there is a shortage of blood group phenotype-matched red cells (RBCs) for patients with sickle cell disease (SCD). A protocol designed to supply phenotype-matched RBCs for these patients by combining the recruitment of African American blood donors and automated testing of RBCs for these patients for the presumptive Fy(a-b-) phenotype using monoclonal anti-Fy3 was evaluated. STUDY DESIGN AND METHODS: African American donors were recruited, to increase the likelihood of phenotype matches in the donor population. Samples of RBCs were tested for the presumptive Fy(a-b-) phenotype by using monoclonal anti-Fy3 and an automated blood typing analyzer. RBCs confirmed to be Fy(a-b-) were retyped for selected Rh, MNS, Kell, Duffy, and Kidd blood system antigens. The extended phenotypes were matched with those of 41 SCD patients requiring transfusions. RESULTS: Of 8323 blood donations during the study, approximately 40 percent (3329) were made by African Americans. Approximately 22 percent (737) of African Americans were identified as Fy(a-b-) by this protocol and 12 percent (410) were phenotype matches for the 41 SCD patients. CONCLUSION: Combining the recruitment of African American blood donors and automated phenotyping using monoclonal anti-Fy3 offers a practical, relatively low-cost strategy for supplying phenotype-matched RBCs for SCD patients. This protocol increases the options for addressing the shortage of phenotype-matched RBCs for SCD patients.