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PubMed1993

Red cell-reactive non-specific immunoglobulins and autoantibodies in the sera of normal and anaemic dogs.

The levels of red blood cell (RBC) membrane-reactive IgG were measured in sera from normal and anaemic dogs using an enzyme-linked immunosorbent assay with erythrocyte ghosts as the target antigen (g-ELISA). The anaemic dogs were classified as cases of primary autoimmune haemolytic anaemia (AIHA), other anaemias with elevated levels of RBC-bound immunoglobulin detected by a direct enzyme-linked antiglobulin test (DELAT), or DELAT-negative anaemias. The g-ELISA detected IgG capable of binding RBC membranes in all the serum samples tested, and the levels were significantly higher (P < 0.05) in dogs with AIHA than in cases of DELAT-negative anaemia or in healthy animals. When sera were preabsorbed with RBC ghosts, g-ELISA readings were reduced in two of three AIHA cases that were tested, but not in dogs from any other group. There was no correlation between g-ELISA results and serum IgG levels in the cases of AIHA, but there was a significant relationship (rs = 0.74, P < 0.001) between these parameters in the dogs with other DELAT-positive anaemias. When spectrin(s) from the internal RBC membrane skeleton was used as the target antigen in the s-ELISA, sera from all groups of anaemic dogs yielded a wide range of values. Although the levels of spectrin-reactive IgG were significantly higher (P < 0.05) in AIHA cases than normal dogs, suggesting that haemolysis provoked production of anti-membrane skeleton antibodies, the highest reading was recorded in a healthy animal. It is concluded that all canine sera contain IgG non-specifically reactive with RBC membranes and that serum antibodies against the erythrocyte surface can also be detected in some dogs with primary AIHA. Distinct spectrin-reactive serum IgG antibodies can also be demonstrated in normal and AIHA positive dogs.

Anemia, Hemolytic, Autoimmune↗
PubMed1994

The Plasmodium falciparum protein RESA interacts with the erythrocyte cytoskeleton and modifies erythrocyte thermal stability.

The ring-infected erythrocyte surface antigen (RESA) associates with spectrin in the erythrocyte membrane (Foley, M., Tilley, L., Sawyer, W. H. and Anders, R. F. (1991) Mol. Biochem. Parasitol., 46, 137-148). A fragment of the RESA protein, which was expressed in Escherichia coli, was found to bind to inside-out vesicles of erythrocyte membranes in an apparently saturable manner. Upon extraction of inside-out vesicles with Triton X-100, the RESA fragment remained associated with the erythrocyte cytoskeleton. Using the technique of steady-state fluorescence polarisation, we have studied the thermal denaturation of fluorescein-labelled spectrin in the presence of recombinant RESA. We found that the RESA fragment partially protected spectrin against heat-induced conformational changes. Furthermore, erythrocytes infected with a RESA (-) laboratory strain (FCR3) were shown to be more susceptible to heat-induced fragmentation than erythrocytes infected with a RESA (+) strain of the parasite. RESA does not, however, appear to play an essential role in the invasion process per se as erythrocytes resealed to contain anti-RESA antibodies were efficiently invaded.

Animals↗
PubMed1987

Immunological detection of high molecular weight proteins by gel and blot overlay.

The sensitivity and specificity of the gel overlay and western blot methods of immunodetection are compared for spectrins, typical high molecular weight proteins. The gel overlay method is more sensitive and specific for the immunodetection of brain spectrin (240/235) and rbc spectrin. As the western blot technique will remain the method of choice for many applications because of its relative speed, we discuss methods for optimizing its sensitivity and selectivity.

Animals↗
PubMed1988

Pyruvate kinase-deficiency anemia: membrane approach.

Low ionic strength extraction (37 degrees C, 30 min) of ghosts from PK-deficient erythrocytes provided crude spectrin extract. No significant differences in the extract composition compared to normal donors were observed. The reticulocyte-dependent spectrin extractability was found among the subjects with PK-deficiency anemia. Likewise ATP-depletion affects spectrin extractability and also leads to the adsorption of cytoplasmic protein MW 50,000 to the reticulocyte membrane. The measurement of membrane fluidity using the fluorescence probe DPH did not reveal significant alterations in the moiety of integral membrane constituents.

Adenosine Triphosphate↗
PubMed1987

Effects of inherited membrane abnormalities on the viscoelastic properties of erythrocyte membrane.

Several workers have identified molecular abnormalities associated with inherited blood disorders. The present work examines how these alterations in molecular structure affect the viscoelastic properties of the red blood cell membrane. Changes in the membrane shear modulus, the membrane viscosity, and the apparent membrane bending stiffness were observed in cells of eight patients having a variety of disorders: Two had reductions in the number of high-affinity ankyrin binding sites, two had abnormalities associated with the protein band 4.1, and six were known to be deficient in spectrin. The data suggest that the membrane shear modulus is proportional to the density of spectrin on the membrane and support the view that spectrin is primarily responsible for membrane shear elasticity. Although membranes having abnormalities associated with the function of ankyrin or band 4.1 exhibited reduced elasticity, the degree of mechanical dysfunction was quantitatively inconsistent with the extent of the molecular abnormality. This indicates that these skeletal components do not play a primary role in determining membrane shear elasticity. The membrane viscosity was reduced in seven of the eight patients studied. The reduction in viscosity was usually greater than the reduction in shear modulus, but the degree of reduction in viscosity was variable and did not correlate well with the degree of molecular abnormality.

Ankyrins↗
PubMed1997

Influence of network topology on the elasticity of the red blood cell membrane skeleton.

A finite-element network model is used to investigate the influence of the topology of the red blood cell membrane skeleton on its macroscopic mechanical properties. Network topology is characterized by the number of spectrin oligomers per actin junction (phi a) and the number of spectrin dimers per self-association junction (phi s). If it is assumed that all associated spectrin is in tetrameric form, with six tetramers per actin junction (i.e., phi a = 6.0 and phi s = 2.0), then the topology of the skeleton may be modeled by a random Delaunay triangular network. Recent images of the RBC membrane skeleton suggest that the values for these topological parameters are in the range of 4.2 < phi a < 5.5 and 2.1 < phi s < 2.3. Model networks that simulate these realistic topologies exhibit values of the shear modulus that vary by more than an order of magnitude relative to triangular networks. This indicates that networks with relatively sparse nontriangular topologies may be needed to model the RBC membrane skeleton accurately. The model is also used to simulate skeletal alterations associated with hereditary spherocytosis and Southeast Asian ovalocytosis.

Actins↗
PubMed1998

Structure of the erythrocyte membrane skeleton as observed by atomic force microscopy.

The structure of the membrane skeleton on the cytoplasmic surface of the erythrocyte plasma membrane was observed in dried human erythrocyte ghosts by atomic force microscopy (AFM), taking advantage of its high sensitivity to small height variations in surfaces. The majority of the membrane skeleton can be imaged, even on the extracellular surface of the membrane. Various fixation and drying methods were examined for preparation of ghost membrane samples for AFM observation, and it was found that freeze-drying (freezing by rapid immersion in a cryogen) of unfixed specimens was a fast and simple way to obtain consistently good results for observation without removing the membrane or extending the membrane skeleton. Observation of the membrane skeleton at the external surface of the cell was possible mainly because the bilayer portion of the membrane sank into the cell during the drying process. The average mesh size of the spectrin network observed at the extracellular and cytoplasmic surfaces of the plasma membrane was 4800 and 3000 nm2, respectively, which indicates that spectrin forms a three-dimensionally folded meshwork, and that 80% of spectrin can be observed at the extracellular surface of the plasma membrane.

Cell Fractionation↗
PubMed1999

Direct measures of large, anisotropic strains in deformation of the erythrocyte cytoskeleton.

The erythrocyte's spectrin-actin membrane skeleton is directly shown to be capable of sustaining large, anisotropic strains. Photobleaching of an approximately 1-micrometer stripe in rhodamine phalloidin-labeled actin appears stable up to at least 37 degrees C, and is used to demonstrate large in-surface stretching during elastic deformation of the skeleton. Principal extension or stretch ratios of at least approximately 200% and contractions down to approximately 40%, both referenced to an essentially undistorted cell, are visually demonstrated in micropipette-imposed deformation. Such anisotropic straining is seen to be consistent at a qualitative level with now classic analyses (Evans. 1973. Biophys. J. 13:941-954) and is generally nonhomogeneous though axisymmetric down to the submicron scale. Local, direct measurements of stretching prove quantitatively consistent (within approximately 10%) with integrated estimates that are based simply on a measured relative density distribution of actin. The measurements are also in close agreement with direct computation of mean spectrin chain extension in full statistical mechanical simulations of a coarse-grained network held in a micropipette. Finally, as a cell thermally fragments near approximately 48 degrees C, the patterned photobleaching demonstrates a destructuring of the surface network in a process that is more readily attributable to transitions in spectrin than in F-actin.

Actins↗
PubMed1998

Temporal ordering of pathogenic events following transient global ischemia.

Rats were subjected to transient global ischemia (four vessel occlusion) and time-related changes in the selectively vulnerable hippocampal field CA1 were characterized. The assessment included ex vivo field responses to afferent stimulation, silver staining, calpain-induced spectrin breakdown, chromatolysis, and cell death, beginning at 6 h post-ischemia and continuing until total disintegration of the pyramidal cells occurred several days later. The earliest change observed was a modest increase in the slope and amplitude of field CA1 potentials (at 6 h). The hyperresponsiveness was most apparent at higher stimulation currents and persisted unchanged at 16 h post-ischemia. Three effects became detectable within 24 h, post-ischemia: (a) an increase in concentrations of calpain-mediated, spectrin breakdown products; (b) enhanced silver staining in the deep pyramidal neurons of the field CA1 with lesser, though still apparent, staining of stratum radiatum, and (c) a decrease in amplitude and slope of field CA1 responses to afferent stimulation. Both the concentration of spectrin breakdown products and the intensity of silver staining progressively increased to a maximum at four days post ischemia, while the amplitude and slope of the field responses dropped to a very low level between 24 and 48 h. Disturbances of Nissl staining were finally evident at 48 h, with nearly complete disappearance of staining at five days post-ischemia. This study provides the first demonstration of a close and early temporal relationship between calpain proteolysis, subcellular damage to the pyramidal cells and their loss of function following global ischemia, prior to their eventual death.

Animals↗
PubMed1983

Mapping the fodrin molecule with monoclonal antibodies. A general approach for rod-like multidomain proteins.

Fodrin is an axonally transported F-actin crosslinking rod-shaped multidomain protein having a length of 200 nm. It is though to be related to erythroid spectrin (220,000 and 240,000 Mr) as it is built as a tetramer derived from a heterodimer comprised of two large subunits (235,000 and 240,000 Mr). A panel of 24 monoclonal antibodies has been used to probe the fodrin structure by direct microscopical decoration and identification of large tryptic fragments retaining antigenicity. The combined results unambiguously define the fodrin organization and show it to be strongly related to erythrocyte spectrin. They demonstrate the heterodimer organization, the head-to-head association of dimers, the protease-resistant domain structure of each subunit and indicate that different termini of the polypeptides are localized at the free F-actin binding ends and the heterodimer association sites. They also show that within the same mammalian species even the highly related 240,000 Mr subunits of fodrin and spectrin are immunologically distinct, most likely reflecting different gene products.

Animals↗
PubMed1997

Perineurium talin immunoreactivity decreases in diabetic neuropathy.

We studied the immunolocalization of Dp116 (a 116 kDa protein product of the dystrophin gene), vinculin, talin, vimentin, desmin, spectrin and titin in the sural nerve biopsies of 25 patients with peripheral neuropathies of different origin. 4 patients presented with HMSN type 1, 4 with HMSN type 2, 2 with HNPP, 4 with CIDP, 5 with chronic axonal neuropathy of unknown origin, 3 with vasculitic neuropathy, 3 with diabetic neuropathy. Expression and localization of Dp116, vinculin, vimentin, desmin, spectrin and titin did not differ from normal control cases. Spectrin and titin immunoreactivities were absent and desmin was occasionally found in few epineurial vessels. A thin rim of Dp116 binding surrounded the outermost layer of myelin sheaths. Perineurium and epineurial vessels stained deeply for vinculin. Vimentin immunoreactivity was seen in all endoneurial, perineurial and epineurial cells. Immunoreactivity for talin was normally found at endoneurial and epineurial vessel walls, perineurial cells and epineurial fibroblasts in all the sural nerves except diabetic nerves. In the latter, whereas talin binding was normal in the vessel walls and epineurial fibroblasts, it was markedly reduced in the perineurium. On immunoblot, two bands at 235 and 190 kDa were found in the sural nerves with the antibody anti-talin, and both were reduced only in the patients with diabetic neuropathy. We postulate that decreased perineurium talin in diabetic polyneuropathy may be related to the known alterations of the tight junctions of the perineurial cells, which have been proposed to be a contributory factor to impaired permeability barrier properties.

Case-Control Studies↗
PubMed1987

Model of red blood cell membrane skeleton: electrical and mechanical properties.

A theoretical membrane skeleton model of erythrocyte has been developed and successfully applied to interpret electrical and mechanical properties of the red blood cell spectrin-actin network. The model is based on the structure of the membrane skeleton that is comprised of unit cells each containing an actin protofilament and shooting forth a few spectrin heterodimers. The loose ends of the heterodimers of adjacent cells can form bonds with each other giving rise to an integrated network. The number of bonds depends on the temperature. The bond length being excessive (2.6 times the distance between the centers of adjacent cells), the bonds are flexible, and can thus be regarded as entropy springs. The advanced model has been employed to calculate the shear modulus of the membrane skeleton as well as to establish its temperature dependence. In a wide range of temperatures mu(T) is a decreasing function well fitting the experimental data. The relationship between the membrane bilayer-free size of the skeleton and the ionic strength of the solution has been derived to appear in good agreement with the results obtained previously. Experimental data combined with the advanced theory yield the average number of heterodimers per unit cell, m0, as equal to ca. 5; the spectrin heterodimer charge has been estimated.

Actins↗
PubMed1997

In vivo oxidative modification of erythrocyte membrane proteins in copper deficiency.

Oxidative stress has been postulated to contribute to the pathology associated with dietary copper deficiency. In vivo, erythrocytes are probable targets of oxidative damage because they are exposed to high concentrations of oxygen and contain heme iron that can autoxidize, which results in the formation of superoxide anions. Activity of the important antioxidant enzyme, copper, zinc superoxide dismutase, decreases markedly in erythrocytes during copper deficiency. The effect of dietary copper deficiency on indicators of oxidative stress was examined in erythrocyte membranes of rats maintained on a purified copper-deficient diet for 35 days after weaning. Erythrocytes were separated into young and old populations on a Percoll gradient prior to membrane isolation and quantification of lipid peroxides and protein carbonyls. Protein carbonyls, determined by Western blot immunoassay, were detected predominantly in both the alpha and beta chains of spectrin. Alpha and beta subunits of spectrin in erythrocyte membranes from copper-deficient rats contained higher amounts of carbonyls than controls, regardless of the population of erythrocytes studied. This study suggests that spectrin may be a specific target for oxidative damage when erythrocyte copper, zinc superoxide dismutase activity is reduced by copper deficiency.

Animals↗
PubMed1998

Contribution of METRO pathway localized molecules to the organization of the germ cell lineage.

To elucidate the potential role of localized components in the specification of the germ cell lineage we analyzed the composition of the germ plasm in Xenopus laevis oocytes and early embryos with respect to the vegetally-localized RNAs. We focused on Xlsirts, Xcat2, and Xwnt11 transcripts that are localized to the vegetal cortex through a region of the mitochondrial cloud called the messenger transport organizer (METRO) that also contains the nuage or germ plasm. At the ultrastructural level Xcat2 mRNA was detected on germinal granules while Xlsirts and Xwnt11 were associated with a fibrillar network of the germ plasm in stage-1 and stage-4 oocytes. In embryos, we found that all three RNAs remained associated with the germ plasm. Vg1 mRNA, a transcript localized through the late pathway, was excluded from the germ plasm in oocytes and embryos. Addtionally, we detected the protein spectrin within 16 cell nests of germ cells, in a structure reminiscent of the Drosophila spectrosome. Spectrin was detected in the mitochondrial cloud and was found in the germ plasm during embryogenesis. These data indicate that the various RNAs found within METRO and the protein spectrin are integral components of the Xenopus germ plasm with the RNAs being associated with different subcellular structures. They also suggest that the pathway through which RNAs are localized during oogenesis may be an important factor in biasing their distribution into specific cell lineages. The presence of Xwnt11 in the germ cell lineage suggests that a wnt-directed signaling pathway may be involved in germ cell specification. differentiation or migration.

Animals↗
PubMed1993

7Li NMR relaxation study of Li+ binding in human erythrocytes.

We used 7Li NMR spin-lattice (T1) and spin-spin (T2) relaxation time measurements to investigate the binding of Li+ in human red blood cell (RBC) suspensions. In RBCs containing 1.4 mM Li+, the intracellular 7Li NMR T2 relaxation value (0.30 +/- 0.03 s) was much smaller than the corresponding T1 value (6.0 +/- 0.1 s), yielding a ratio of T1 to T2 of 20. For 1.5 mM LiCl solutions whose viscosities were adjusted to 5 cP with glycerol, the values of the T1/T2 ratios were as follows: 49 for unsealed RBC membrane (2.0 mg of protein/mL); 4.4 for spectrin (1.9 mg/mL); 1.5 for 5.4 mM 2,3-bisphosphoglycerate (BPG); 2.2 for 2.7 mM carbonmonoxyhemoglobin (COHb); 1.6 for 2.0 mM ATP; and 1.2 for a 50/50% (v/v) glycerol-water mixture. Intracellular viscosity and the electric field gradients experienced by Li+ when traversing the spectrin-actin network therefore are not responsible for the large values of the T1/T2 ratios observed in Li(+)-loaded RBCs. We conclude that the RBC membrane is the major Li+ binding site in Li(+)-loaded RBCs (Kb = 215 +/- 36 M-1) and that the binding of Li+ to COHb, BPG, spectrin-actin, or ATP is weak. Partially relaxed 7Li NMR spectra of Li(+)-containing RBC membrane suspensions indicated the presence of two relaxation components, one broad and one narrow.(ABSTRACT TRUNCATED AT 250 WORDS)

2,3-Diphosphoglycerate↗
PubMed1991

Bilayer/cytoskeleton interactions in lipid-symmetric erythrocytes assessed by a photoactivable phospholipid analogue.

Two mechanisms have been proposed for maintenance of transbilayer phospholipid asymmetry in the erythrocyte plasma membrane, one involving specific interactions between the aminophospholipids of the inner leaflet of the bilayer and the cytoskeleton, particularly spectrin, and the other involving the aminophospholipid translocase. If the former mechanism is correct, then erythrocytes which have lost their asymmetric distribution of phospholipids should display altered bilayer/cytoskeleton interactions. To test this possibility, normal erythrocytes, erythrocytes from patients with chronic myelogenous leukemia or sickle disease, and lipid-symmetric and -asymmetric erythrocyte ghosts were labeled with the radioactive photoactivable analogue of phosphatidylethanolamine, 2-(2-azido-4-nitrobenzoyl)-1-acyl-sn-glycero-3-phospho[14C]ethanolamine ([14C]AzPE), previously shown to label cytoskeletal proteins from the bilayer. The labeling pattern of cytoskeletal proteins in pathologic erythrocytes and lipid-asymmetric erythrocyte ghosts was indistinguishable from normal erythrocytes, indicating that the probe detects no differences in bilayer/cytoskeleton interactions in these cells. In contrast, in lipid-symmetric erythrocyte ghosts, labeling of bands 4.1 and 4.2 and actin, and to a lesser extent ankyrin, by [14C]AzPE was considerably reduced. Significantly, however, labeling of spectrin was unaltered in the lipid-symmetric ghosts, suggesting that its relationship with the bilayer is normal in these lipid-symmetric cells. These results do not support a model in which spectrin is involved in the maintenance of an asymmetric distribution of phospholipids in erythrocytes.

Affinity Labels↗
PubMed1977

Spontaneous, reversible protein cross-linking in the human erythrocyte membrane. Temperature and pH dependence.

Changes in pH significantly affect the morphology and physical properties of red cell membranes. We have explored the molecular basis for these phenomena by characterizing the pattern of protein disulfide cross-linkages formed spontaneously in ghost exposed to acid pH or elevated temperature (37 degrees C). Protein aggregation was analyzed by two-dimensional polyacrylamide gel electrophoresis in sodium dodecyl sulfate. incubation of ghosts at pH 4.0 to 5.5 (0-4 degrees C) yielded (i) complexes of spectrin and band 3, (ii) complexes of actin and band 3, (iii) band 3 complexes, i.e. dimer and trimer, and (iv) heterogeneous aggregates involving spectrin, band 3, band 4.2, and actin in varying proportions. Aggregation was maximal near the isoelectric points of the major membrane proteins, and appeared to reflect (i) the aggregation of intramembrane particles including band 3 and (ii) more intimate contact between spectrin-actin meshwork and band 3.

Actins↗
PubMed1977

Selective phosphorylation of erythrocyte membrane proteins by the solubilized membrane protein kinases.

This report describes the substrate and phosphoryl donor specificities of solubilized erythrocyte membrane cyclic adenosine 3',5'-monophosphate (cAMP)-independent protein kinases toward human and rabbit erythrocyte membrane proteins. Three types of substrate preparations have been utilized: heat-inactivated ghosts, isolated spectrin, and 2,3-dimethylmaleic anhydride (DMMA)-extracted membranes. A 30 000-dalton protein kinase, extracted from either human or rabbit erythrocyte membranes, catalyzes the phosphorylation of heat-inactivated membranes in the presence of ATP. The resulting phosphorylation profile is analogous to that of the autophosphorylation of membranes with ATP (in the absence of cAMP). These kinases also phosphorylate band 2 of isolated spectrin and band 3, but not glycophorin, in the DMMA-extracted ghosts. The ability of the 30 000-dalton kinases to use GTP as a phosphoryl donor appears to be related to the substrate or some other membrane factor. A second kinase, which is 100 000 daltons and derived from rabbit erythrocyte membranes, uses ATP or GTP to phosphorylate membrane proteins 2, 2.1, 2.9-3 in heat-inactivated ghosts, band 2 in isolated spectrin, glycophorin, and to a lesser extent, band 3 in the DMMA-extracted ghosts.

Adenosine Triphosphate↗