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At least 919 records · Page 51Linked to original sources
PubMed1998

Annexin VI-mediated loss of spectrin during coated pit budding is coupled to delivery of LDL to lysosomes.

Previously we reported that annexin VI is required for the budding of clathrin-coated pits from human fibroblast plasma membranes in vitro. Here we show that annexin VI bound to the NH2-terminal 28-kD portion of membrane spectrin is as effective as cytosolic annexin VI in supporting coated pit budding. Annexin VI-dependent budding is accompanied by the loss of approximately 50% of the spectrin from the membrane and is blocked by the cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Incubation of fibroblasts in the presence of ALLN initially blocks the uptake of low density lipoprotein (LDL), but the cells recover after 1 h and internalize LDL with normal kinetics. The LDL internalized under these conditions, however, fails to migrate to the center of the cell and is not degraded. ALLN-treated cells have twice as many coated pits and twofold more membrane clathrin, suggesting that new coated pits have assembled. Annexin VI is not required for the budding of these new coated pits and ALLN does not inhibit. Finally, microinjection of a truncated annexin VI that inhibits budding in vitro has the same effect on LDL internalization as ALLN. These findings suggest that fibroblasts are able to make at least two types of coated pits, one of which requires the annexin VI-dependent activation of a cysteine protease to disconnect the clathrin lattice from the spectrin membrane cytoskeleton during the final stages of budding.

Annexin A6↗
PubMed1984

Spectrin immunofluorescence distinguishes a population of naturally capped lymphocytes in situ.

Immunofluorescence analysis of mammalian lymphocytes using antiserum directed against chicken erythrocyte alpha-spectrin revealed a lymphocyte population in which spectrin antigen was arranged in the form of a discrete cap (hereafter referred to as capped lymphocytes). This subset could be easily distinguished from other lymphocytes in which the spectrin antigen was diffusely distributed near the plasma membrane (noncapped lymphocytes). The subset of capped lymphocytes could be visualized in situ and in isolated cells in the absence of added ligand. Using frozen sections of lymphoid organs that were fixed in formaldehyde prior to the immunofluorescence procedure, capped lymphocytes were found in characteristic locations depending on the tissue examined. In the thymus, the major population of medullary lymphocytes were capped whereas cortical lymphocytes were mostly noncapped. In Peyer's patches, capped lymphocytes were interspersed with noncapped lymphocytes throughout the tissue. In the spleen, capped lymphocytes were concentrated in the periarterial lymphoid sheath of the white pulp and in lymph nodes they were found predominantly in the paracortical and cortical regions. Capped lymphocytes were not visible in the thymus until just before birth and did not appear in the spleen until 3 d after birth. When lymphocytes were isolated from lymphoid organs, fixed in formaldehyde and prepared for immunofluorescence, capped and noncapped lymphocytes were still identifiable and present in the same relative proportions as seen in situ. Results identical to those described above are obtained using antisera directed against guinea pig fodrin. Natural capping of proteins previously shown to co-migrate with a variety of cell surface macromolecules after cross-linking may be a new means of identifying various stages of lymphocyte activation or differentiation.

Animals↗
PubMed1991

Characterization of the lateral interaction between human erythrocyte spectrin subunits.

A technique in which the subunits of human erythrocyte spectrin were immobilized on a nitrocellulose membrane was developed to study which domains of the subunit are able to bind to the counterpart subunit. The limited tryptic digestion of the isolated alpha and beta subunits of human erythrocyte spectrin produced eight fragments in the alpha subunits and nine fragments in the beta subunit. Four fragments of the beta (80, 60, 44, and 18 kDa) and two of the alpha (82 and 33 kDa) bound to alpha and beta subunits which were immobilized on nitrocellulose membrane strips, respectively. The binding affinities of all the fragments to the subunits, however, were remarkably lower than that of the mother proteins. The titration of fluorescence anisotropy of N-(1-anilinonaphthyl-4)maleimide which was covalently attached to the subunit by the trypsin-digested fraction of the counterpart subunit also indicate weak binding of the fragments even in solution. These findings suggest that the high-affinity binding of the alpha subunit to the beta subunit to form spectrin alpha beta dimer occurs only when the binding domains are arrayed along the polypeptide chains at the appropriate positions on the subunits.

Anisotropy↗
PubMed1991

Specific identification and subcellular localization of three calmodulin-binding proteins in the rat gonadotrope: spectrin, caldesmon, and calcineurin.

In an effort to characterize the second messenger system for LH release, we have previously identified five calmodulin-binding proteins in rat gonadotropes of Mr greater than 205,000, 200,000, 135,000, 60,000, and 52,000. In the present study, we have used a calmodulin overlayer assay combined with Western blotting to determine the molecular identity of three calmodulin-binding proteins in rat gonadotropes: the alpha subunit of spectrin (Mr greater than 205,000), caldesmon (Mr 84,000), and the alpha subunit of calcineurin (Mr 60,000). The Mr greater than 205,000 and Mr 60,000 components or rat pituitary which bind calmodulin are immunoreactive with spectrin and calcineurin antisera, respectively. Rat pituitary also contains an Mr 84,000 component, which is immunoreactive with polyclonal sera and monoclonal antibody raised to chicken gizzard caldesmon (Mr 150,000). Like caldesmon from other sources, the Mr 84,000 component remains soluble after heat treatment and preferentially binds either filamentous actin or calmodulin, depending on the Ca2+ concentration. The three calmodulin-binding proteins were localized specifically in gonadotropes using indirect immunofluorescence microscopy or by Western-blotting cell fractions enriched for gonadotropes. After differential centrifugation of pituitary homogenate, spectrin immunoreactivity was found associated with the nuclear and secretory granule fractions, whereas caldesmon immunoreactivity was seen in the cytosolic fraction and calcineurin in the cytosolic and nuclear fractions. Although the precise role for these proteins remains unknown, the apparent requirement for calmodulin and the small number of calmodulin-binding proteins in the gonadotrope suggest their involvement in mediating GnRH actions.

Animals↗
PubMed1998

Regional calpain and caspase-3 proteolysis of alpha-spectrin after traumatic brain injury.

Activity of calpains and caspase-3 inferred from proteolysis of the cytoskeletal protein alpha-spectrin into signature spectrin breakdown products (SBDPs) was used to provide the first systematic and simultaneous comparison of changes in activity of these two families of cysteine proteases after traumatic brain injury (TBI) in rats. Distinct regional and temporal patterns of calpain/caspase-3 processing of alpha-spectrin were observed in brain regions ipsilateral to the site of injury after TBI, including large increases of 145 kDa calpain-mediated SBDP in cortex (up to 30-fold), and enduring increases (up to 2 weeks) of 145 kDa SBDP in hippocampus and thalamus. By contrast, 120 kDa caspase-3-mediated SBDP was absent in cortex and showed up to a 2-fold increase in hippocampus and striatum at early (hours) after TBI. Future studies will clarify the pathological significance of large regional differences in activation of calpain and caspase-3 proteases after TBI.

Animals↗
PubMed2000

Behavioral efficacy of posttraumatic calpain inhibition is not accompanied by reduced spectrin proteolysis, cortical lesion, or apoptosis.

Administration of the selective calpain inhibitor AK295 has been shown to attenuate motor and cognitive dysfunction after brain trauma in rats. To explore mechanisms underlying the behavioral efficacy of posttraumatic calpain inhibition, we investigated histologic consequences of AK295 administration. Anesthetized Sprague-Dawley rats received lateral fluid percussion brain injury of moderate severity (2.2 to 2.4 atm) or served as uninjured controls. At 15 minutes after injury, animals were randomly assigned to receive a 48-hour infusion of either 2 mmol/L AK295 (120 to 140 mg/kg) or saline via the carotid artery. At 48 hours and 1 week after injury, spectrin fragments generated specifically by calpain were detected by Western blotting and immunohistochemistry, respectively, in saline-treated, brain-injured animals. Interestingly, equivalent spectrin breakdown was observed in AK295-treated animals when cortical and hippocampal regions were evaluated. Similarly, administration of the calpain inhibitor did not attenuate cortical lesion size or the numbers of apoptotic cells in the cortex, subcortical white matter, or hippocampus, as verified by terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick-end labeling and morphology, at 48 hours after injury. These data suggest that an overt reduction in spectrin proteolysis, cortical lesion, or apoptotic cell death at 48 hours or 1 week is not required for behavioral improvements associated with calpain inhibition by AK295 after experimental brain injury in rats.

Animals↗
PubMed2004

Accumulation of calpain and caspase-3 proteolytic fragments of brain-derived alphaII-spectrin in cerebral spinal fluid after middle cerebral artery occlusion in rats.

Preclinical studies have identified numerous neuroprotective drugs that attenuate brain damage and improve functional outcome after cerebral ischemia. Despite this success in animal models, neuroprotective therapies in the clinical setting have been unsuccessful. Identification of biochemical markers common to preclinical and clinical cerebral ischemia will provide a more sensitive and objective measure of injury severity and outcome to facilitate clinical management and treatment. However, there are currently no effective biomarkers available for assessment of stroke. Nonerythroid alphaII-spectrin is a cytoskeletal protein that is cleaved by calpain and caspase-3 proteases to signature alphaII-spectrin breakdown products (alphaII-SBDPs) after cerebral ischemia in rodents. This investigation examined accumulation of calpain- and caspase-3-cleaved alphaII-SBDPs in cerebrospinal fluid (CSF) of rodents subjected to 2 hours of transient focal cerebral ischemia produced by middle cerebral artery occlusion (MCAO) followed by reperfusion. After MCAO injury, full-length alphaII-spectrin protein was decreased in brain tissue and increased in CSF from 24 to 72 hours after injury. Whereas alphaII-SBDPs were undetectable in sham-injured control animals, calpain but not caspase-3 specific alphaII-SBDPs were significantly increased in CSF after injury. However, caspase-3 alphaII-SBDPS were observed in CSF of some injured animals. These results indicate that alphaII-SBDPs detected in CSF after injury, particularly those mediated by calpain, may be useful diagnostic indicators of cerebral infarction that can provide important information about specific neurochemical events that have occurred in the brain after acute stroke.

Animals↗
PubMed1983

Inhibition of malarial invasion of red cells by chemical and immunochemical linking of spectrin molecules.

The invasion of resealed human red cell ghosts by Plasmodium falciparum, and those from monkey cells by P. knowlesi, was strongly inhibited by anti-spectrin antibodies introduced into their cytoplasm. Univalent F(ab)1 fragments gave no such effect, but a combination of these fragments and goat anti-rabbit immunoglobulin, to restore bifunctionality, caused perceptible inhibition of invasion. Disulphide cross-links introduced between spectrin molecules in intact red cells by the membrane-permeant oxidizing agent, diamide, again led to inhibition of invasion. This effect was largely reversed by reduction of the cross-links. Gel electrophoresis was used to confirm that cross-linking was essentially confined to spectrin, and that extended covalent networks were not formed. It follows that local formation of bridges, whether by antibodies or oxidation of thiol groups, functions by inhibiting a local rearrangement of the cytoskeleton that forms a step in the invasion process.

Animals↗
PubMed1989

Spectrin autoantibodies in normal human serum and in polyclonal blood grouping sera.

Naturally occurring spectrin autoantibody was detected in normal human sera and in polyclonal blood grouping sera by the immunoblotting technique. The autoantibody seems to be IgG, stable, of high titre and low affinity. It was detected in all sera tested. Preparations of monoclonal antibodies directed against some blood group antigens and anti-A1 lectin reagent were devoid of spectrin autoantibody as expected. This autoantibody may be instrumental in clearing red cell membrane components from the circulation during haemolysis. Care must be exercised in studies designed to determine the association of some blood group antigens with the red cell membrane skeleton, when using polyclonal sera which contain spectrin autoantibodies in addition to the antibodies against the blood group antigen in question.

Antibodies, Monoclonal↗
PubMed1983

Acceleration of coagulation by spectrin-free erythrocytic vesicles. Evidence for lipid flip-flop during vesicle formation.

Spectrin-free erythrocytic vesicles isolated from outdated liquid-preserved blood samples show a distinct increase of the clot-promoting activity compared with membrane phospholipid equivalents of intact erythrocytes. We suppose that, among other remodelling processes, local spectrin detachment from the inner membrane surface may trigger a flip-flop-mediated disturbance of the membrane lipid asymmetry. The interactions of spectrin with the cytoplasmic membrane surface seem to be essential for the structural membrane integrity including the lipid asymmetry.

Blood Coagulation↗
PubMed1995

The C-terminal domain of alpha-spectrin is structurally related to calmodulin.

An alignment of amino acid sequences suggests that the spectrin domain, which contains two EF-hand calcium-binding motifs, is structurally related to calmodulin. It is possible to align approximately 160 residues at the C-terminus of alpha-spectrin with the entire calmodulin sequence. We have expressed this domain in Escherichia coli and purified it. Circular dichroic and nuclear magnetic resonance spectroscopy show that the protein is folded and mostly helical. The conformation of the protein, as monitored spectroscopically, is sensitive to calcium at 0.1-1.0 mM. Equilibrium dialysis shows that there are two binding sites within this domain, with affinities in the 0.5 mM range. The domain can be split into N-terminal and C-terminal halves which fold independently. Only the N-terminal subdomain binds calcium. These data suggest that the C-terminus of alpha-spectrin has a domain with a calmodulin fold and two calcium-binding sites. Sequence alignments suggest that the related domains in alpha-actinin, and possibly in dystrophin, may share the same calmodulin-like structure. However, only non-muscle alpha-actinins appear to have one or two EF-hand(s) with the calcium-binding consensus sequence, and a strict consensus is not found in the muscle alpha-actinins or dystrophins.

Amino Acid Sequence↗
PubMed2006

Methamphetamine-induced spectrin proteolysis in the rat striatum.

Methamphetamine (METH) is a widely abused psychostimulant. Multiple high doses of METH cause long-term toxicity to dopamine (DA) and serotonin (5-HT) nerve terminals in the brain, as evidenced by decreases in DA and 5-HT content, decreases in tyrosine and tryptophan hydroxylase activities, decreases in DA and 5-HT re-uptake sites, and nerve terminal degeneration. Multiple high doses of METH are known to elicit a rapid increase in DA release and hyperthermia. Although METH also produces a delayed and sustained rise in glutamate, no studies have shown whether METH produces structural evidence of excitotoxicity in striatum, or identified the receptors that mediate this toxicity directly, independent of alterations in METH-induced hyperthermia. These experiments investigated whether METH can cause excitotoxicity as evidenced by cytoskeletal protein breakdown in a glutamate receptor-dependent manner. METH increased calpain-mediated spectrin proteolysis in the rat striatum 5 and 7 days after METH administration without affecting caspase 3-dependent spectrin breakdown. This effect was completely blocked with the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, GYKI 52466, but not the NMDA receptor antagonist, MK-801. However, AMPA or NMDA receptor antagonism did not attenuate the METH-induced depletions of the dopamine transporter (DAT). Independent mechanisms involved in mediating spectrin proteolysis and DAT protein loss are discussed.

Animals↗
PubMedDate not supplied

Detection and immunolocalization of human erythrocyte spectrin immunoanalogues in Toxoplasma gondii (Protozoan, Parasite).

We demonstrated here the presence of proteins antigenically related to human erythroid spectrin in the parasitic protozoan Toxoplasma gondii. A high molecular weight doublet (M(r) 245-240,000), present in equimolar ratio, and low molecular weight poly-peptides (M(r) 75,000) were reacted with monoclonal and polyclonal anti-human erythroid spectrin antibodies on electroblotted nitro-cellulose sheets. Indirect immunofluorescence assay clearly showed that these proteins were localized in the anterior pole of the organism. Immunogold staining further revealed specific labeling of conoid, rhoptries, micronemes, and dense granules of the apical complex. The presence of the M(r) 245-240,000 doublet and the M(r) 75,000 spectrin-like proteins in the anterior pole of T. gondii may probably be consistent with a structural stabilizer function in its organelles which are suspected to be involved in the process of host cell invasion.

Animals↗
PubMed1987

Defective spectrin dimer self-association in thalassemic red cells.

The relative proportions of spectrin tetramer and dimer forms extrated from red cell membranes in a low ionic strength buffer at 4 degrees C were determined for 15 normal subjects, 27 subjects with alpha-thalassemia (7 alpha-thalassemia trait, 9 Hb H disease (alpha-thal 1/alpha-thal 2) and 11 Hb H with Hb Constant Spring (CS), 23 subjects with beta-thalassemia (6 beta-thalassemia trait, 5 homozygous beta-thalassemia, 11 beta(0)-thalassemia with Hb E and 1 beta(+)-thalassemia with Hb E), 6 subjects with Hb E (2 homozygous and 4 carriers) and 1 subject with combined alpha-thal 1/Hb CS and Hb E (AE Bart's disease). In all subjects (except carriers of Hb E and 1 splenectomized case of beta(0)-thal/Hb E) spectrin dimer forms were elevated when compared to levels in normal controls, but there were no significant differences between carrier and disease forms. Conversion of spectrin dimers to tetramers at 30 degrees C was reduced in the thalassemic subjects with disease but was within normal range for thalassemic carriers.

Adolescent↗
PubMed1991

Uncoupling of the spectrin-based skeleton from the lipid bilayer in sickled red cells.

The distribution of spectrin and band 3 in deoxygenated reversibly sickled cells was visualized by immunofluorescence and immunoelectron microscopy. Antibodies against band 3, the major lipid-associated transmembrane protein, labeled the entire cell body, including the entire length of the long protruding spicule, whereas antibodies against spectrin labeled only the cell body and the base region of the spicules. The results suggest that the formation of long spicules during sickling is associated with a continuous polymerization of hemoglobin S polymers, presumably through gaps in the spectrin-actin meshwork, and a subsequent uncoupling of the lipid bilayer from the submembrane skeleton.

Anemia, Sickle Cell↗
PubMed1998

Substitution of the human beta-spectrin promoter for the human agamma-globin promoter prevents silencing of a linked human beta-globin gene in transgenic mice.

During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human beta-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Agamma-globin gene linked to a 576-bp fragment containing the human beta-spectrin promoter. In these mice, the beta-spectrin Agamma-globin (betasp/Agamma) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, betasp/Agamma-, psibeta-, delta-, and beta-globin genes showed no developmental switching and expressed both human gamma- and beta-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Agamma-globin gene promoter showed developmental switching and expressed Agamma-globin mRNA in yolk sac and fetal liver erythroid cells and beta-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the gamma-globin promoter with the beta-spectrin promoter allows the expression of the beta-globin gene. We conclude that the gamma-globin promoter is necessary and sufficient to suppress the expression of the beta-globin gene in yolk sac erythroid cells.

Animals↗
PubMed1999

Immunohistochemical identification of erythroid precursors in paraffin embedded bone marrow sections: spectrin is a superior marker to glycophorin.

AIM: To investigate whether spectrin can be used as an immunohistochemical marker for erythroid precursors in routinely processed paraffin embedded bone marrow sections. METHODS: Bone marrow biopsies and clot sections were stained with rabbit antihuman erythrocyte spectrin antibodies, specific for erythroid cells as shown by western blotting and bone marrow smears, and compared to sections stained with antiglycophorin monoclonal antibodies (JC159 and Ret49f). RESULTS: Antispectrin antibodies resulted in diffuse cytoplasmic staining of early erythroblasts and membranous staining of late erythroblasts as well as erythrocytes. In haematopathological samples, immature erythroid cell clusters were clearly identified. In contrast, antiglycophorin monoclonal antibodies resulted in only membranous staining of late erythroblasts, and faint staining of early erythroblasts. CONCLUSIONS: Spectrin may be a superior marker to glycophorin for the identification of erythroid precursors in paraffin embedded sections.

Biomarkers↗
PubMed1979

Effect of heat on the circular dichroism of spectrin in hereditary pyropoikilocytosis.

Hereditary pyropikilocytosis is a hemolytic anemia in which the erythrocytes show increased sensitivity to heat-induced fragmentation. Circular dichroism measurements were employed to study the effect of heat on the secondary structure of pyropoikilocyte membrane proteins. The magnitude of the ellipticity at 222 nm over the temperature range from 25 degrees to 70 degrees C was determined for erythrocyte ghosts, spectrin, and ghost residue after extraction. In pyropoikilocyte ghosts, protein denaturation began at a lower temperature and the midpoint of the structural transition was displaced from 49 degrees C (the value for normal ghosts) to 44 degrees C. This thermal transition was present in spectrin, but not in the ghost residue after extraction. We conclude that an abnormality in the spectrin molecule alters the physical and morphologic properties of the erythrocyte membrane in pyropoikilocytosis.

Anemia, Hemolytic, Congenital↗