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PubMed1988

A flow EPR study of deformation and orientation characteristics of erythrocyte ghosts: a possible effect of an altered state of cytoskeletal network.

Using the flow EPR technique, we investigated the resealed ghost deformability in shear flow and the effects of the altered state of cytoskeletal network induced by hypotonic incubation of ghosts. Isotonically resealed ghosts in the presence of Mg-ATP, in which alteration of cytoskeletal network is not effected, have smooth biconcave discoid shapes, and show a flow orientation and deformation behavior similar to that of erythrocytes, except that higher viscosities are required to induce the same degrees of deformation and orientation as in erythrocytes. The flow behavior of resealed ghosts is Mg-ATP dependent, and the shape of the ghosts resealed without Mg-ATP is echinocytic. In contrast, the ghosts resealed by hypotonic incubation show a markedly reduced deformability even with Mg-ATP present. Nonreducing, nondenaturing polyacrylamide gel electrophoresis (PAGE) of the low ionic strength extracts from hypotonically resealed ghosts reveals a shift of the spectrin tetramer-dimer equilibrium toward the dimers. In the maleimide spin-labeled ghosts, the ratios of the weakly immobilized to the strongly immobilized EPR intensities are larger in hypotonically resealed ghosts than in the isotonically resealed ghosts, indicating an enhanced mobility in the spectrin structure in the former. Photomicrographs of hypotonically resealed ghosts show slightly stomatocytic transformations. These data suggest that the shape and the deformability loss in hypotonically resealed ghosts are related to an alteration of the spectrin tetramer-dimer equilibrium in the membrane. Thus, the shift of the equilibrium is likely to affect the regulation of the membrane deformability both in normal and pathological cells such as hereditary elliptocytes.

Adenosine Triphosphate↗
PubMed1983

The interaction of human erythrocyte Band 3 with cytoskeletal components.

Band 3 of the human erythrocyte is involved in anion transport and binding of the cytoskeleton to the membrane bilayer. Human erythrocytes were treated to incorporate varying concentrations of DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) a non-penetrating, irreversible inhibitor of anion transport, and both functions of Band 3 were analyzed. The rate of efflux of 35SO2-4 was measured and the binding of cytoskeletal components to the membrane was evaluated by extracting the membranes with 0.1 N NaOH and analyzing for the peptides remaining with the membrane. It was found that 0.1 N NaOH extracts all the extrinsic proteins from membranes of untreated cells, while, in the case of the membranes from cells treated with DIDS, a portion of the cytoskeletal components, spectrin (Bands 1 and 2) and Band 2.1 (ankyrin, syndein) remain with the membrane. The amount of these cytoskeletal components remaining with the membrane depends on the concentrations of DIDS incorporated. The effect of DIDS on the extractability of the spectrin-Band 2.1 complex correlates well with DIDS inhibition of anion transport (r = 0.91). At DIDS concentrations which completely inhibit anion transport, about 10% of total spectrin-Band 2.1 complex remains unextracted. Another anion-transport inhibitor, pyridoxal phosphate, has no effect on binding of the cytoskeleton to the membrane. On the other hand, digestion of DIDS-pretreated intact erythrocytes with Pronase, chymotrypsin, or trypsin releases the tight binding of Band 3 to cytoskeleton on the inside of the membrane. Since trypsin does not hydrolyze Band 3 the data suggest that a second membrane protein which is trypsin sensitive may be involved with Band 3 in cytoskeletal binding.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1982

Interaction of hemoglobin with the red blood cell membrane. A saturation transfer electron paramagnetic resonance study.

Human hemoglobin has been labeled on cysteine 93(beta) with the maleimide spin label, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl and reassociated with erythrocyte membrane previously stripped of hemoglobin and glyceraldehyde-3-phosphate dehydrogenase. The affinity of hemoglobin for the membrane is not affected by the presence of the label. Saturation transfer electron paramagnetic resonance measurements show that the diffusion rotational movements of hemoglobin are considerably slowed down when it is bound to the erythrocyte membrane. The correlation time of rotation, tau c, is found to be 8 . 10(-6) s as compared with 2 . 10(-8) s when the hemoglobin molecule is in solution. The same values are obtained whether the protein is associated with its high- or low-affinity binding sites. They depend on the viscosity of the solution. The high-affinity sites are presumably located on the segment of the band 3 protein which extends into the cytoplasm and which links through ankyrin, the spectrin-actin cytoskeleton to the membrane. When band 3 is cross-linked into a dimer after reaction with the copper-ortho-phenanthroline chelate, the correlation time of rotation of spin-labelled hemoglobin is unchanged. It is also independent of the presence of the spectrin-actin network and ankyrin. These results show tha the movements of hemoglobin bound by ionic linkage to different part (protein or phospholipid) of the cytoplasmic surface of the membrane are similarly highly restricted by some potential or energetic barrier. They give also evidence for independent movements and flexibility in the assembly of the macromolecules which link the spectrin-actin cytoskeleton to the erythrocyte membrane.

Cyclic N-Oxides↗
PubMed1983

Effect of factors of favism on the protein and lipid components of rat erythrocyte membrane.

Erythrocytes prepared from riboflavin- and tocopherol-deficient (RT-) and from control rats were used to investigate the mechanism of oxidative hemolysis by the factors of favism. RT- erythrocytes have a defense system against the oxidative stress which is blocked either where regeneration of GSH occurs or the scavenging of the radicals from the membrane is prevented. The oxidative factors used were isouramil, divicine and diamide. When RT- erythrocytes were treated with isouramil, GSH decreased to undetectable levels and was not regenerated. Complete hemolysis occurred, but no oxidation of SH groups of membrane proteins or formation of spectrin polymers was detected. A similar effect was observed with diamide. However, SH groups of membrane proteins were completely oxidized and spectrin polymers were formed. Extensive lipid peroxidation was also detected together with a 30% fall in the arachidonic acid level. Control erythrocytes treated with either isouramil or diamide were not hemolyzed. When treated with isouramil, after a fall in the first few minutes, the GSH level was completely regenerated after 20 min. Incubation with diamide caused extensive oxidation of SH groups of membrane proteins and formation of spectrin polymers. No lipid peroxidation was detected after treatment with isouramil, but the same decrease of arachidonic acid occurred as in RT- erythrocytes. These results support the hypothesis that oxidative hemolysis by the factors of favism is caused by uncontrolled peroxidation of membrane lipids.

Animals↗
PubMed1983

Alteration of rheological properties of human erythrocytes by crosslinking of membrane proteins.

The crosslinking of membrane proteins of human erythrocytes by diamide (diazene dicarboxylic acid bis(N,N-dimethylamide) ) was quantified by 4% polyacrylamide gel electrophoresis in 1% sodium dodecyl sulfate. The relation between the crosslinking of membrane proteins and erythrocyte functions (rheological and oxygen transporting) was quantitatively examined. (i) The crosslinking of membrane protein was induced by diamide, without changing the shape and the contents of intracellular organic phosphates (adenylates and 2,3-diphosphoglycerate). The intensity of spectrin 2 in SDS-polyacrylamide gel electrophoresis decreased proportionally to diamide concentration. The percentage decrease in spectrin 2 (using band 3 as an internal standard) was the most appropriate indicator for crosslinking ("% crosslinking'). (ii) The suspension viscosity of erythrocytes increased in proportion to the percentage of crosslinking, in the range of applied shear rates of 3.76-752 s-1. (iii) Erythrocyte deformability (measured by a high-shear rheoscope) was reduced by the crosslinking. The change was detectable even at 5% crosslinking. (iv) Rouleaux formation (measured by a television image analyzer combined with a low-shear rheoscope) was inhibited by the crosslinking. The inhibition was also sensitively detected at more than 5% crosslinking. (v) Hemoglobin in erythrocytes was chemically modified by higher dose of diamide (probably by the binding of diamide with sulfhydryl groups). Also the oxygen affinity of hemoglobin increased and the heme-heme interaction decreased. (vi) The reduction of the crosslinking of membrane proteins by dithiothreitol apparently reversed the intensity of spectrin bands in SDS-polyacrylamide gel electrophoresis and the erythrocyte functions (the suspension viscosity and the deformability), though not completely.

Azo Compounds↗
PubMed1980

Endogenous phosphorylation of soluble enzymes in human red cells. Cyclic 3',5'-AMP-dependent phosphorylation of phosphofructokinase without detectable regulatory effect.

ATP-depleted human red cells have been incubated in a glucose-containing medium with [32P]orthophosphate in the presence and in the absence of cyclic 3',5'-AMP and dibutyril cyclic 3',5'-AMP. Spectrin, pyruvate kinase, phosphofructokinase, glucose-6-phosphate dehydrogenase and hemoglobin A1 have been purified and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein-bound radioactivity has been measured from the sodium dodecyl sulfate polyacrylamide gels and the trichloroacetic acid-precipitated proteins. In the cytosol, the most intense phosphorylation was found for pyruvate kinase whose, in the presence of cyclic AMP, specific radioactivity was comparable to that of the membrane protein and spectrin. In the absence of cyclic nucleotides it was five times less phosphorylated. Phosphofructokinase was only phosphorylated when the red cells were incubated with cyclic nucleotides; the extent of phosphorylation was four times less than for pyruvate kinase. Hemoglobin, glucose-6-phosphate dehydrogenase and a contaminant protein copurified with phosphofructokinase were not phosphorylated: the 'background' of the radioactivity found for these proteins was 100 times less than for pyruvate kinase and spectrin, and 20 times less than for phosphofructokinase (+cyclic AMP).

Cyclic AMP↗
PubMed1989

2,5-Hexanedione modifies skeletal proteins of the red blood cells and increases the binding of hemoglobin to the membrane.

The effects of 2,5-hexanedione (2,5 HD) on skeletal proteins of red blood cells (RBCs) were investigated both in vitro (human RBCs) and in vivo in male Sprague-Dawley rats which had been treated with the drug for several days. We found that 2,5 HD induced the following major changes in the electrophoretic pattern of the skeletal proteins: (i) the appearance of high-molecular weight bands, (ii) a dose-dependent decrease in spectrin Bands 1 and 2, and (iii) a dose-dependent increase in the amount of hemoglobin (Hb) associated with the membrane. Membranoskeletons, prepared from resealed ghosts which had been previously treated with 2,5 HD, were able to bind an increased amount of Hb from untreated RBCs, thus suggesting a drug-induced modification of the membrane. Extraction of spectrin and actin from ghosts did not remove the membrane-bound Hb and, furthermore, Hb bound to 2,5 HD-treated membranes mainly bearing Band 3 and free of peripheral proteins. These data suggested a 2,5 HD-induced modification of an intrinsic membrane protein, probably Band 3. This hypothesis was consistent with the observation that 2,5 HD also induced a modification of Band 3 aminogroups, as evidenced by a dose-dependent decrease in the binding of eosin probes. Furthermore, RBCs treated in vitro with 2,5 HD bound an increased amount of autologous immunoglobulins (IgG). As reported by Kay and Low et al. the binding of autologous IgG is a phenomenon associated with the aging process of RBCs and may involve a modification of Band 3. Our data show that RBCs treated with 2,5 HD acquired various characteristics of senescent cells such as spectrin cross-linking, Hb-membrane binding and increased IgG binding, and suggest that 2,5 HD treatment might affect RBC survival.

Animals↗
PubMed1990

Turnover of cytoskeletal proteins in vivo.

The turnover of the microtubule-associated proteins 1B and 2 (MAP1B and MAP2), tubulin, high molecular weight neurofilament protein (NF-H), and spectrin were studied by in vivo labeling. Radiolabeled [35S]methionine was injected intracranially to 10-day-old rats and the rate of turnover was measured for total and specific brain proteins. The turnover of total brain proteins was biphasic and consisted of a fast and a slow component with half lives of 6.5 +/- 0.4 and 14.2 +/- 0.7 (mean +/- S.E.M.) days, respectively. The turnover of individual cytoskeletal brain proteins was also biphasic. The fast decay rates of MAP1B, MAP2, tubulin and spectrin were 5.8 +/- 0.7, 6.9 +/- 0.3, 4.8 +/- 0.5 and 4.9 +/- 0.4 days, respectively, while the slow decay rates of these proteins were 12.0 +/- 1.3, 12.4 +/- 1.7, 15.0 +/- 0.5 and 16.0 +/- 1.2 days, respectively. In addition, the Triton X-100 insoluble fraction of MAP1B, tubulin, spectrin and NF-H showed monophasic decay rates of 29.0 +/- 2.3, 15.0 +/- 1.4, 16.0 +/- 0.9 and 18.5 +/- 1.5 days, respectively, which were similar to their slow decay rates in whole brain homogenates, suggesting that incorporation of these proteins into the cytoskeletal lattice increases their stability.

Animals↗
PubMed1992

Identification of an amelin isoform located in axons.

A new axonal isoform of amelin, an analogue of the erythrocyte spectrin binding protein termed protein 4.1 has been identified in mouse brain. This new isoform has a molecular weight of 93 kDa, and migrates to a more acidic pH (pH 7.5-8.0) than the previously described amelin E (pH 8.5) on two dimensional NEPHGE-SDS PAGE. The 93 kDa protein looks nearly identical to amelin E on two dimensional chymotryptic iodopeptide mapping, and both share partial homology with rbc protein 4.1. The new isoform is located in axons, and the soma of neurons in mouse cerebellum, while amelin E is located in neuronal soma and dendrites. The axonal amelin antibody detects a 97 kDa protein in embryonic tissue which diminishes during development; and a 93 kDa protein which is first seen at postnatal day 1 of mouse brain ontogeny, increasing constantly to its adult concentrations. This time course of expression is quite different than amelin E, which is present at embryonic day 15 and diminishes constantly reaching its lowest concentration in the adult brain. We hypothesize that axonal amelin and amelin E may play important roles in the interaction of brain spectrin(240/235) and brain spectrin(240/235E) with f-actin and neuronal membranes.

Aging↗
PubMed1990

Transplantation studies in mice with congenital hemolytic anemia.

Sphha/sphha anemic mice have an abnormality in the erythroid membrane protein, alpha spectrin, and exhibit multiple related clinical abnormalities, including spherocytosis, shortened red cell survival, chronic hemolysis, hemosiderosis, and extramedullary hematopoiesis. In addition, these mutant mice exhibit a granulocytosis and lymphocytosis, lymph node hyperplasia, elevated serum immunoglobulins, membranoproliferative glomerulonephritis, and decreased lifespan--abnormalities that are less clearly attributable to a spectrin defect. In order to further elucidate the mechanisms of disease in these animals, we undertook a series of bone marrow transplantation experiments. Transplantation of anemic marrow into lethally irradiated congenic +/+ mice resulted in chronic spherocytosis, hemolytic anemia, peripheral leukocytosis, and extramedullary hematopoiesis. Additionally, transplant recipients of anemic marrow which had received a higher radiation dose (12 Gy) had increased numbers of peripheral blood CD4+ and CD8+ lymphocytes, a hypocellcular thymus, and a severe pneumonitis characterized by nodular areas of consolidation and edema. Mice receiving congenic +/+ marrow and irradiated with the same radiation dose exhibited minimal pulmonary abnormalities. Anemic mice transplanted with congenic +/+ marrow usually died, but the survivors exhibited reversal of some clinicopathological changes. These results would suggest that the clinical abnormalities of sphha/sphha mice are in part attributable to abnormalities of hematopoietic stem cells but may also involve defects in other cell types. The pathogenesis of the accompanying lymphoid abnormalities observed in this mutant anemic mouse and any correlation with the erythroid spectrin defect are presently unknown. The pulmonary disease that develops in the transplant recipients of anemic marrow needs to be characterized further but may represent a unique model of lung injury.

Anemia, Hemolytic, Congenital↗
PubMed1984

The patterns of expression of two ankyrin isoforms demonstrate distinct steps in the assembly of the membrane skeleton in neuronal morphogenesis.

We have identified in chicken neurons two membrane-bound isoforms of goblin (ankyrin), the specific membrane attachment protein for spectrin in avian erythrocytes, which exhibit distinct patterns of expression and assembly during neuronal morphogenesis. Early in cerebellar and retinal development, neither goblin isoform is detected in mitotic cells. Subsequently, beta-goblin (Mr 237,000) is expressed in postmitotic cells, and gradually accumulates with alpha gamma (brain) spectrin on the neuronal plasma membrane during the phase of cell growth. At the onset of synaptogenesis and the cessation of cell growth, their levels of synthesis decline sharply while the assembled proteins remain as stable membrane components. Concomitantly, there is a dramatic induction in the accumulation of alpha-goblin (erythroid goblin; Mr 260,000) and alpha beta (erythroid) spectrin, whose assembly is limited to the plasma membrane of perikarya and dendrites.

Aging↗
PubMed1990

Calcium-calmodulin-dependent phosphorylation of cytoskeletal proteins from adrenal cells.

We have identified a highly active Ca2+ calmodulin-dependent protein kinase in the cytoskeletons of normal (bovine fasciculata) and transformed (Y-1 mouse tumor) adrenal cells. In view of evidence for the involvement of calmodulin and microfilaments in the regulation of cholesterol transport and hence steroidogenesis, it is likely that this kinase is important in this process. The kinase activity was examined for its capacity to phosphorylate endogenous proteins analyzed by one- and two-dimensional gel electrophoresis, in the presence of saturating amounts of Ca2+ (5 mM) and calmodulin (5 microM). Three inhibitors of calmodulin (trifluoperazine, pimozide and W-7) inhibit steroidogenesis and Ca2(+)-calmodulin-dependent phosphorylation kinase activity with similar values for EC50 for the two processes. All three inhibitors inhibit the increased transport of cholesterol to mitochondria in response to ACTH. Two substrates for the kinase (alpha-spectrin and beta-tubulin) were identified and two others (51,000 and 60,000 molecular weight) were tentatively identified as the subunits of the kinase itself in cytoskeletons of both cell types. Calmodulin-binding proteins analyzed by [125I]iodocalmodulin overlay and calmodulin-Sepharose affinity chromatography were also identified in the same cytoskeletons including alpha-spectrin, the Ca2+ calmodulin-dependent phosphatase calcineurin and three that were tentatively identified as the two subunits of the kinase itself and myosin light chain kinase. It is concluded that calmodulin, by binding to the kinase and phosphatase, is capable of influencing the degree of phosphorylation of specific substrates in the cytoskeleton and of forming complexes with spectrin, actin and tubulin. These events may be involved in the regulation of the rate-limiting step of steroidogenesis, i.e. transport of cholesterol to mitochondria.

Adrenal Gland Neoplasms↗
PubMed1998

The effect of oxidative stress on structural transitions of human erythrocyte ghost membranes.

Differential scanning microcalorimetry was used to study the effect of oxidative stress induced by cumene hydroperoxide (CHP) and Fe2+ on structural transitions of membranes of human erythrocyte ghosts. The CHP homolysis was shown to cause: (a) reduction of the intensity of all structural transitions with the disappearance of B1- and D-transitions; (b) decrease in the enthalpy of oxidized membrane denaturation; (c) negative slope of thermograms; (d) anomalous growth of heat absorption by membranes above 72 degreesC. All these changes occurred until the ratio Fe2+/CHP/membranes<0.02:0.05:1 was reached, i.e., prior to the moment of maximal level of TBA-RS in membrane ghosts. We interpret changes in the character of heat absorption by oxidized membranes as perturbations in the structural organization and interactions inside the spectrin-actin-protein 4.1 domains, the spectrin-protein 4.2 domain, as well as inside the domain of spectrin-ankyrin-cdB3 and the domain formed by the msdB3. These perturbations are associated mainly with the decrease in the concentration of native protein in the domains because of oxidative aggregation of proteins, as evidenced by SDS electrophoresis of oxidized membranes. Preincubation of membranes with tocopherol did not block the aggregation of proteins in electrophoresis and the decrease in the intensity of structural transitions, whereas it blocked completely the formation of TBA-RS, changes in the thermogram slope and the sharp rise in the heat absorption above 72 degreesC. This proves that these processes are determined by the thermotropic properties of the oxidized lipid bilayer of membranes and also provides evidence that the degradation of PUFA of phospholipids modifies both the structure of protein domains and the physical properties of the lipid bilayer of membranes.

Anion Exchange Protein 1, Erythrocyte↗
PubMedDate not supplied

Cytoskeleton and tissue origin in the anterior cynomolgus monkey eye.

We studied cytoskeletal proteins and other markers for embryologic origin in the outflow pathways of the aqueous humor, cornea, sclera, and ciliary muscle of the cynomolgus monkey. The corneal endothelium and trabecular cells stained with markers for vimentin, smooth muscle cell alpha-actin, F-actin, spectrin, vinculin, and talin. The endothelium of Schlemm's canal stained with markers for vimentin, spectrin, and F-actin. These results suggest that trabecular cells are a kind of myofibroblast and support the belief that the endothelial cells of Schlemm's canal are vascular in origin. Fibrillary staining with antibodies to vimentin, spectrin, neurofilament protein, and glial acid fibrillary protein was observed along and between the ciliary muscle cells. Cells in the deep sclera adjacent to the supraciliary space stained with antibodies to smooth muscle alpha-actin, alpha-vinculin, talin, and desmin. These cells may anchor ciliary muscle cells into the sclera or may be developmental remnants of ciliary muscle cells. Leu 19 immunoreactivity was found in the corneal endothelium, in all trabecular cells, in ciliary muscle cells, and in keratocytes and fibroblasts in the superficial part of the cornea and sclera. All of these cells are therefore likely to express neural cell adhesion molecules indicating neuroectodermal origin.

Animals↗
PubMed1990

A mechanism of formation of protein-free regions in the red cell membrane: the rupture of the membrane skeleton.

The process of rupture and redistribution of the red cell membrane skeleton is analyzed theoretically. Following the emergence of the rupture the spectrin-actin network is redistributed on the cytoplasmic surface of the membrane bilayer. Due to the interaction of the membrane skeleton and integral proteins the redistribution of the spectrin-actin network leads to the release of purely lipid regions of the membrane. The scale of the protein redistribution caused by the rupture of the membrane skeleton and the size of the lipid domains produced depend on the shape of the membrane and the value of the electrical interaction of the membrane proteins. The lipid domains occurring as a result of the rupture and relaxation of the spectrinactin network can spontaneously increase or decrease its area. The criteria determining the conditions which result in the system's evolutions leading to the domain growth have been obtained. The character of the evolution is determined by the shape of the membrane region in which the rupture occurs as well as the relation between the effective linear tension of the rupture boundary and the modulus of elasticity of the spectrin-actin network.

Animals↗
PubMed1999

Antigen specificity in canine autoimmune haemolytic anaemia.

Autoimmune haemolytic anaemia (AIHA) is the most common clinical manifestation of autoimmunity in the dog and generally presents as a profound, regenerative, Coombs' positive anaemia of acute or chronic onset. The disease pathogenesis involves formation of erythrocyte-specific autoantibodies of the IgG and IgM class that may fix complement resulting in intra- or extravascular haemolysis. Western blotting and immunoprecipitation studies using autoantibody eluted from the erythrocytes of dogs with AIHA have demonstrated specificity for erythrocyte glycophorins and the membrane anion-exchange molecule (band 3). Autoantibodies specific for the cytoskeletal molecule spectrin have been identified in serum by ELISA. The specificity of autoreactive T-cells has been examined in vitro using bulk cultures stimulated with a panel of autoantigens including intact erythrocyte membranes, purified glycophorin and spectrin fractions and a panel of overlapping 15-mer glycophorin peptides. Control responses to ConA and recall (vaccine antigens) and non-recall (KLH) antigens were measured in the same system. PBMC obtained from dogs that had recovered from AIHA consistently proliferated in response to erythrocyte membranes, with occasional responses to spectrin or glycophorin. PBMC from sone clinically normal dogs also responded to erythrocyte membranes. PBMC obtained from dogs closely related to AIHA cases gave the most consistent responses, including proliferation when stimulated by the glycophorin peptides. These data suggest that normal dogs harbour erythrocyte autoreactive lymphocytes, and that these cells may be primed in dogs recovered from AIHA or having genetic susceptibility to the disease.

Anemia, Hemolytic, Autoimmune↗
PubMed2002

Immunohistochemical and biochemical assessment of caspase-3 activation and DNA fragmentation following transient focal ischemia in the rat.

In the present study, we evaluated the time-course of caspase-3 activation, and the evolution of cell death following focal cerebral ischemia produced by transient middle cerebral artery occlusion in rats. Ischemia-induced active caspase-3 immunoreactivity in the striatum but not the cortex at 3 and 6 h time points post-reperfusion. Furthermore, using a novel approach to visualize enzymatic activity, deltaC-APP, a C-terminal cleavage product of APP generated by caspase-3, was found to immunolocalize to the same areas as active caspase-3. Double-labeling studies demonstrated co-localization of these two proteins at the cellular level. Further double-labeling experiments revealed that active caspase-3 was confined to neuronal cells which were still viable and thus immunoreactive for NeuN. DNA fragmentation, assessed histologically by terminal dUTP nick-end labeling (TUNEL), was observed in a small number of cells in the striatum as early as 3 h, but only began to appear in the cortex by 6 h. DNA fragmentation was progressive, and by 24 h post-reperfusion, large portions of both the striatum and cortex showed TUNEL positive cells. However, double-labeling of active caspase-3 with TUNEL showed only minimal co-localization at all time-points. Thus, caspase-3 activation is an event that appears to occur prior to DNA fragmentation. As a confirmation of the histological TUNEL data, 24 h ischemia also induced the generation of nucleosome fragments, evidenced by cell death enzyme-linked immunosorbent assay. Using a novel ischemia-induced substrate cleavage biochemical approach, spectrin P120 fragment, a caspase-specific cleavage product of alpha II spectrin, a cytoskeletal protein, was shown to be elevated by western blotting. Brain concentrations of both nucleosomes and spectrin P120 correlate with the degree of injury previously assessed by triphenyltetrazolium chloride staining and infarct volume calculation. Together, our findings suggest a possible association between caspase-3 activation and ischemic cell death following middle cerebral artery occlusion brain injury.

Animals↗
PubMed2000

Plasmodium falciparum histidine-rich protein 1 associates with the band 3 binding domain of ankyrin in the infected red cell membrane.

Infection of erythrocytes by the malaria parasite Plasmodium falciparum results in the export of several parasite proteins into the erythrocyte cytoplasm. Changes occur in the infected erythrocyte due to altered phosphorylation of proteins and to novel interactions between host and parasite proteins, particularly at the membrane skeleton. In erythrocytes, the spectrin based red cell membrane skeleton is linked to the erythrocyte plasma membrane through interactions of ankyrin with spectrin and band 3. Here we report an association between the P. falciparum histidine-rich protein (PfHRP1) and phosphorylated proteolytic fragments of red cell ankyrin. Immunochemical, biochemical and biophysical studies indicate that the 89 kDa band 3 binding domain and the 62 kDa spectrin-binding domain of ankyrin are co-precipitated by mAb 89 against PfHRP1, and that native and recombinant ankyrin fragments bind to the 5' repeat region of PfHRP1. PfHRP1 is responsible for anchoring the parasite cytoadherence ligand to the erythrocyte membrane skeleton, and this additional interaction with ankyrin would strengthen the ability of PfEMP1 to resist shear stress.

Animals↗