Association state of human red blood cell band 3 and its interaction with ankyrin

We have studied the association state of band 3, the anion channel and predominant transmembrane protein of the human red blood cell, and the anomalous stoichiometry and dynamics of its interaction with ankyrin, which acts as a link to the spectrin of the membrane skeletal network. Band 3 exists in benign nonionic detergent solutions as a dimer. Tetramer is formed irreversibly in the course of manipulations, particularly in ion-exchange chromatography. The dimer in solution binds ankyrin without self-associating. In ankyrin-free inside-out membrane vesicles and when incorporated into phosphatidylcholine liposomes, only some 10% to 15% of band 3 chains bind ankyrin at saturation. Moreover, in liposomes this was independent of protein:lipid ratio between 1:2 and 1:40. The bound fraction of band 3 remains with the detergent-extracted membrane cytoskeleton, but is released if the ankyrin has been cleaved with chymotrypsin before detergent treatment; thus, the attachment to the membrane cytoskeleton is entirely through ankyrin and not through other constituents such as protein 4.1. The ratio of band 3 to ankyrin in this complex implies that it consists of two chains of band 3 and one chain of ankyrin, at least after detergent extraction. The bound and free populations of band 3 exchange freely in the membrane. In the artificial liposome membrane binding of ankyrin to band 3 dimers cause association of the band 3 into higher aggregates, as seen in freeze-fracture electron microscopy. Successive manipulations of the red blood cell membrane, which are involved in the preparation of ghosts, of inside-out vesicles, and of inside-out vesicles stripped of peripheral proteins are accompanied by progressive aggregation of intramembrane particles, as judged by freeze-fracture electron microscopy. Thus the intramembrane particles are evidently stabilized in the intact cell by the peripheral protein network and the cytosolic milieu. Aggregation may be expected to limit the number of functional ankyrin binding sites. However, although extraneous ankyrin binds to the unoccupied binding site on the spectrin tetramers in intact ghost membranes, little or no ankyrin can bind to the unoccupied band 3 dimers in situ, perhaps by reason of occlusion of binding sites by the membrane skeletal network.     

A donor-derived asialo-GM1+ cell induces depression of B-cell genesis during systemic graft-versus-host disease

The nature of the effector cell(s) responsible for the depression of B- cell genesis in the bone marrow of mice undergoing systemic graft- versus-host disease (GVHD) has been examined. Donor C57BL/6 (B6) mice were treated in vivo with either a single injection of anti-asialo GM1 antibody (anti-ASGM1) to eliminate naturally occurring (endogenous) ASGM1+ cells or B6xAF1 (B6AF1) lymphoid cells followed by anti-ASGM1 to eliminate both endogenous and "induced" ASGM1+ cells. Lymphoid cells from donor mice after the elimination of endogenous ASGM1+ cells produced severe GVHD and concomitant depression of B-cell genesis when injected into B6AF1 recipients. In contrast, cells from donors depleted of both the endogenous and inducible ASGM1+ populations did not cause GVHD or depletion of B lineage cells in B6AF1 recipients but did depress B-cell genesis in B6C3F1 mice. The "induced" ASGM1+ cells were Thy 1+, but their elimination did not significantly alter either overall T-cell function or specific cytotoxic T-cell (CTL) reactivity against the sensitizing (B6AF1) strain. The results suggest that the effector cell responsible for the depression of B-cell genesis during systemic GVHD can be induced to express ASGM1, is strain-specific and Thy 1+; but is not a conventional CTL.     

Frequent expression of the cell death-inducing gene Bax in Reed- Sternberg cells of Hodgkin's disease

The expression of a cell death-inducing gene, Bax, was investigated in 52 cases of Hodgkin's disease in parallel with Epstein-Barr virus and was compared with the immunodetection of other apoptosis-regulating proteins, Mcl-1, Bcl-2, and Bcl-x. Bax immunostaining was found in 92% of the cases, among them 28% with a strong signal in more than 75% of the Reed-Sternberg cells. Mcl-1 was positive in 80% of the cases, whereas Bcl-2 and Bcl-x were found in 53% and 88% of the cases, respectively. Of 48 (89%) Bax-positive tumors, 43 were found to express apoptosis-inhibiting proteins such as Mcl-1 or Bcl-2. With the exception of 1 case, all Bax-positive tumors also expressed either Bcl- 2, Bcl-x, Mcl-1, or combinations of these anti-apoptotic proteins. No correlation was found between Bax expression and the presence of apoptotic cells as detected by morphology and the in situ 3' OH-DNA end- labeling technique. Our findings show that the apoptosis-inducing gene Bax expression is frequently expressed in Hodgkin's disease, providing a potential explanation for the good chemoresponses generally obtained for patients with this neoplastic disorder.     

Detection of factor XIIIa (active fibrin-stabilizing factor) in normal plasma

Factor XIIIa (active fibrin-stabilizing factor) generated in heat- defibrinated plasma by the addition of thrombin can be measured by 14C- putrescine incorporation into casein. Modification of this assay be substituting 3H-putrescine of high specific activity as the donor amine permits measurement of amine incorporation by plasma even in the absence of added thrombin. Incorporation is calcium dependent, inhibited by iodoacetamide, and absent from congenital factor XIII- deficient plasma and from normal platelets. The transamidating activity detected by radioenzymatic assay catalyzed the formation of gamma-gamma dimers and alpha polymers of fibrin and was thus biologically functional. This fibrin cross-linking activity was absent from factor XIII-deficient plasma. These experiments show (1) some factor XIII is present in plasma as factor XIIIa; (2) this factor XIIIa can cross-link fibrin and thus has biologic activity as well; and (3) this activity is not present in factor XIII-deficient plasma. Factor XIIIa in normal plasma is possibly activated in vivo, perhaps by circulating thrombin, factor Xa, or other proteolytic enzymes.     

Marrow- and spleen-seeding efficiencies of all murine hematopoietic stem cell subsets are decreased by preincubation with hematopoietic growth factors

The cobblestone-area forming cell (CAFC) assay permits a direct measurement of the seeding of primitive and more mature murine hematopoietic stem cell subsets by comparing the number of CAFC in the original transplant with the number of CAFC retrieved from bone marrow (BM) and spleen after transplantation. We found no differences in seeding efficiency between the more mature and primitive CAFC subsets, nor between seeding efficiencies of stem cells from low-density (LD) fractions of normal and day-6 post-5-fluorouracil BM. The data show that 18% to 20% of all intravenously transplanted stem cell subsets seed to the BM, whereas 8% to 10% seed to the spleen. In addition, similar seeding efficiencies were found for day-12 spleen colony- forming unit (CFU-S-12) as was determined by retransplantation. Previously, it has been reported that a 2- to 3-hour preincubation of BM with interleukin-3 (IL-3) enhances the in vivo repopulating ability of a graft. To test whether hematopoietic growth factors affected this increased engraftment by enhancing the seeding of the transplanted marrow, we assessed the 16- to 18-hour seeding efficiency of short- and long-term in vivo repopulating stem cell subsets to BM and spleen using the CAFC assay, after preincubation with or without hematopoietic growth factors. A 2- to 3-hour preincubation with IL-3, or a combination of IL-3, IL-12, and steel factor, at 37 degrees C, led to a substantial decrease in seeding compared with control (which was kept on ice) of all hematopoietic subsets measured, both in spleen and BM. In concert with these data, the long-term in vivo repopulating ability of growth-factor incubated BM was also decreased when compared with control. In conclusion, we have been unable to observe a beneficial effect of growth factor preincubation on the repopulating ability of a graft.     

Recombinant gibbon interleukin 3 supports formation of human multilineage colonies and blast cell colonies in culture: comparison with recombinant human granulocyte-macrophage colony-stimulating factor

The genetic sequences encoding the gibbon and human interleukin 3 (IL 3) proteins were molecularly cloned. The amino acid sequence of the mature gibbon IL 3 protein proved to share 93% homology with the corresponding human protein. We examined the effects of biosynthetic (recombinant) gibbon IL 3 on the proliferation and differentiation of an enriched population of human hematopoietic progenitors and compared the results with the effects of recombinant human granulocyte- macrophage colony-stimulating factor (GM-CSF). Gibbon IL 3 supported the formation of various types of single lineage as well as multilineage colonies by My-10+ bone marrow cells in the presence of human erythropoietin (Ep). In contrast, recombinant human GM-CSF supported the formation of single-lineage colonies and only a small number of multilineage colonies. Both IL 3 and GM-CSF had significant erythroid burst-promoting activity (BPA). Delayed addition of gibbon IL 3 to low serum culture of My-10+ marrow cells supported the formation of blast cell colonies with variable but high replating capability. Human GM-CSF was less effective than IL 3 in support of multipotential blast cell colonies. These results are analogous to the effects of murine IL 3 and GM-CSF on murine progenitors and support the notion that the primary factor for multipotential progenitors is IL 3.