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At least 973 records · Page 54Linked to original sources
PubMed1995

Induction of calpain-mediated spectrin fragments by pathogenic treatments in long-term hippocampal slices.

The use of cultured hippocampal slices for studies of calpain-mediated pathogenesis was investigated. Breakdown products (BDPs), which result from proteolysis of spectrin by calpain I, were assayed with BDP-specific antibodies developed against peptide sequences on either side of the calpain cleavage site. The antibodies recognized either amino- or carboxy-terminal BDPs (147-kD BDPN and 152-kD BDPC, respectively). Various pathogenic manipulations, including trimethyltin, certain snake venoms and agonists for excitatory amino acid receptors, were found to cause rapid and pronounced increases in the proteolytic fragments. These effects were selective, i.e., chemicals or toxins directed at nonglutamatergic neurons had little effect on BDP concentrations. Transient accumulations of spectrin fragments were obtained with brief applications of N-methyl-D-aspartate; longer infusions resulted in lasting increases. Results similar to these have been observed in vivo with ischemic episodes of varying duration. Agonists of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subclass of glutamate receptors produced significant increases in spectrin proteolysis; however, prolonged exposure of the slices to centrally active drugs that enhance the currents passed by the receptors did not. The sensitivity, selectivity and temporal properties of the proteolytic response support the idea that cultured slices can be used to analyze the events leading to and following from calpain activation in the adult brain.

Amino Acid Sequence↗
PubMed1993

Production and characterization of monoclonal antibodies against alpha and beta spectrin subunits.

Twenty monoclonal antibodies against human erythrocyte spectrin were generated using purified native spectrin as the immunogen. Thirteen out of 20 monoclonal antibodies reacted to the alpha subunit, and the remaining seven monoclonal antibodies reacted to the beta subunit in Western blot analysis. One anti-beta subunit and two anti-alpha subunit monoclonal antibodies cross-reacted with the cell lysate of the non-erythroid T. lymphoma, J. Jhan, cell line. Identification of 20 monoclonal antibodies to the respective tryptic domains was also achieved. The specificity of these antibodies to respective tryptic domains of spectrin confirms the alignments and uniqueness of each of the previously identified five domains in the alpha subunit and four domains in the beta subunit, and establishes the identity of proteolytic daughter peptide fragments.

Animals↗
PubMed1993

Identification of the amino acid mutations associated with human erythrocyte spectrin alpha II domain polymorphisms.

Four distinct spectrin alpha II domain polymorphisms are known to occur in several nonwhite populations. Type 1 is essentially the only form found in whites and is also the most common form in nonwhites. In contrast to most other spectrin mutations that are single-base substitutions, two of the alpha II domain polymorphisms, types 2 and 3, are particularly unusual because they appear to involve 4-Kd insertions relative to type 1. We have identified the mutations responsible for these polymorphisms using biochemical approaches and a computer database of spectrin-domain peptides separated by two-dimensional gels. The type 3 mutation is characterized by an apparent 4-Kd increase in alpha II domain peptides with no change in pI. This apparent molecular weight increase is a sodium dodecyl sulfate (SDS) gel artifact resulting from an Arg-->His mutation at residue 22 of the domain. The type 4 polymorphism shows a basic charge shift with no apparent change in molecular weight on gels. This charge shift results from a mutation of Thr-->Arg at position 174 of the domain. This mutation appears to be linked to a "silent" mutation at position 130 from an Ile-->Val. Support for possible linkage was obtained from analysis of three unrelated donors with the type 2 polymorphism. The type 2 polymorphism shows both the charge shift characteristic of the type 4 mutation and the apparent size shift that defines the type 3 polymorphism. Analysis of type 2 peptides confirmed that the two mutations described above for type 4 as well as the mutation at residue 22 observed in type 3 occur simultaneously in type 2. The observation that the type 2 polymorphism is a composite of the type 3 and 4 mutations is especially surprising because the type 2 polymorphism occurs far more frequently than either the type 3 or 4 forms. The basis for apparent linkage between the mutations at residues 130 and 174, which are encoded by different exons, is also not clear. Identification of the mutations described here permits design of genetic screening analyses that can be applied to larger populations to evaluate this potential linkage.

Amino Acid Sequence↗
PubMed1993

Tissue-specific alternative splicing of protein 4.1 inserts an exon necessary for formation of the ternary complex with erythrocyte spectrin and F-actin.

Erythrocyte protein 4.1 is an 78- to 80-Kd peripheral membrane protein that promotes the interaction of spectrin with actin protofilaments and links the resulting interlocking network to the integral membrane proteins. There are several isoforms of protein 4.1 that appear to be expressed in a restricted group of tissues. These arise from alternative mRNA splicing events that lead to the combinational insertion or deletion of at least 10 blocks of nucleotides (motifs) within the mature mRNA. One of these, motif I, consists of 63 nucleotides encoding 21 amino acids in the N-terminal region of the putative spectrin/actin-binding domain. The expression of the motif U-containing isoform occurs late in erythroid maturation. We generated recombinant isoforms of protein 4.1 and of the putative 10-Kd spectrin/actin-binding fragment that contain or lack this 21 amino acid sequence and examined their ability to form a ternary complex with erythrocyte spectrin and F-actin. The isoforms of the complete protein and of the 10-Kd fragment that contain the sequence encoded by motif I efficiently form the ternary complex. Isoforms that lack this sequence, but are otherwise identical, do not participate in the formation of the ternary complex. These results, in conjunction with the expression of motif I during late erythroid maturation, suggest that interaction with actin and the erythroid form of spectrin is a specialized property of the erythrocyte form of protein 4.1. Alternative mRNA splicing in developing red blood cells thus plays a key adaptive role in the formation of the highly specialized erythrocyte membrane.

Actins↗
PubMed1996

Spectrin mutations in hereditary elliptocytosis and hereditary spherocytosis.

Hereditary elliptocytosis (HE), its aggravated form hereditary pyropoikilocytosis (HPP), and hereditary spherocytosis (HS) designate a set of congenital hemolytic syndromes. The responsible mutations lie in several genes encoding proteins of the red cell membrane. In particular, they involve the SPTA1 and SPTB genes that encode erythroid spectrin alpha- and beta-chains, respectively. In situ, spectrin is a alpha 2 beta 2 fibrillar tetramer resulting from the head-to-head self-association of two alpha beta dimers. In HE, the 24 known alpha-chain mutations lie in the self-association site or its vicinity, whereas the 17 beta-chain mutations occur in the self-association site itself (record of November 30, 1995). Allele alpha LELY (LELY: Low Expression LYon) is found in ethnic groups remote from one another with a uniform frequency (20-30% of all alpha-alleles). It allows an expanded expression of any HE alpha-allele located in trans and results in severe HE or in HPP. In HS, a number of spectrin mutations have been recorded recently. Allele alpha LEPRA (LEPRA: Low Expression PRAgue) would occur in a recurrent fashion.

Elliptocytosis, Hereditary↗
PubMed1991

Time-dependent elastic extensional RBC deformation by micropipette aspiration: redistribution of the spectrin network?

The time dependence of small elastic extensional RBC deformation by micropipette aspiration has been analyzed. This process shows two-phases which are characterized by time constants of the order of some tenths of seconds and about ten seconds, respectively. The equilibrium tongue length is reached after about 30 s. For the first, fast step we assume that the membrane model of immobilized boundaries holds, i.e., the skeleton is tightly associated with the lipid bilayer and no redistribution of the skeleton with respect to the lipid bilayer is allowed. This lipid-spectrin interaction or anchorage is characterized by some association force density. It has been shown that at a given tongue length the force generated owing to the membrane deformation and acting to redistribute the spectrin, overcomes (in some membrane area) the association force density and results in an additional increase of the sucked membrane length. Equations have been derived to describe this process. From the experimental conditions of an RBC aspiration and the determined tongue length corresponding to the second slow aspiration step, the association force density between the lipid bilayer and the spectrin network may be determined. From literature data and our own results a force density of between 40 and 50 Pa has been estimated.

Cytological Techniques↗
PubMed1976

The influence of salt on the aggregation state of spectrin from bovine erythrocyte membranes.

Sedimentation velocity and gel filtration experiments have been performed with bovine spectrin over a wide range of neutral salt concentrations. Increasing salt concentrations tend to increase both the sedimentation coefficient of spectrin and the elution volume of the protein from 4% agarose columns. No conformation change can be detected by means of optical rotation measurements as the salt concentration is raised. The results are incompatible with the hypothesis that salt causes the aggregation of spectrin, but are consistent with the existence of marked charge effects operative at low salt concentrations. In support of the charge effect hypothesis, acidic groups have been detected on the agarose gels, and ion-exclusion behaviour on the column has been observed with other proteins of similar size.

Animals↗
PubMed1982

Calmodulin-dependent spectrin kinase activity in resealed human erythrocyte ghosts.

Membrane protein phosphorylation has been studied in resealed human erythrocyte ghosts by measuring the incorporation of 32P into spectrin and band 3. Norepinephrine- and Ca2+-stimulated phosphate incorporation was diminished in ghosts depleted of calmodulin. Ghosts prepared with endogenous calmodulin showed Ca2+- and norepinephrine-stimulated protein phosphorylation only when the ghosts had been resealed in the presence of (gamma-32P)ATP. Ghosts resealed with or without calmodulin in the presence of unlabeled ATP showed no net gain or loss of 32P when exposed to norepinephrine or a Ca2+-specific ionophore. These observations suggest that Ca2+ and norepinephrine stimulation of membrane protein phosphorylation is mediated by calmodulin-dependent spectrin kinase activity, and not by increased turnover of spectrin ATPase or by inhibition of phosphospectrin phosphatase.

Calcimycin↗
PubMed1992

A model of spectrin as a concertina in the erythrocyte membrane skeleton.

To maintain its distinctive biconcave shape, the erythrocyte has a skeleton composed largely of the protein spectrin, which associates closely and exclusively with the cell membrane. Although the membrane skeleton forms through specific protein-protein interactions of defined stoichiometry, it has a flexible structure and organization due to the unusual molecular properties of spectrin. Here we describe these properties and propose a model to account for the extensibility of spectrin and for its organization in the skeleton.

Journal Article↗
PubMed2006

Calpain- and caspase-mediated alphaII-spectrin and tau proteolysis in rat cerebrocortical neuronal cultures after ecstasy or methamphetamine exposure.

Abuse of 3,4-methylenedioxymethamphetamine (MDMA or Ecstasy) and methamphetamine (Meth or Speed) is a growing international problem with an estimated 250 million users of psychoactive drugs worldwide. It is important to demonstrate and understand the mechanism of neurotoxicity so potential prevention and treatment therapies can be designed. In this study rat primary cerebrocortical neuron cultures were challenged with MDMA and Meth (1 or 2 mM) for 24 and 48 h and compared to the excitotoxin N-methyl-D-aspartate (NMDA). The neurotoxicity of these drugs, as assessed by microscopy, lactate dehydrogenase release and immunoblot, was shown to be both dose- and time-dependent. Immunoblot analysis using biomarkers of cell death showed significant proteolysis of both alphaII-spectrin and tau proteins. Breakdown products of alphaII-spectrin (SBDPs) of 150, 145, and 120 kDa and tau breakdown products (TBDPs) of 45, 32, 26, and 14 kDa were observed. The use of the protease inhibitors calpain inhibitor SJA6017 and caspase inhibitors z-VAD-fmk and Z-D-DCB, attenuated drug-induced alphaII-spectrin and tau proteolysis. The calpain inhibitor reduced the calpain-induced breakdown products SBDP145 and TBDP14, but there was an offset increase in the caspase-mediated breakdown products SBDP120 and TBDP45. The caspase inhibitors, on the other hand, decreased SBDP120 and TBDP45. These data suggest that both MDMA and Meth trigger concerted proteolytic attacks of the structural proteins by both calpain and caspase family of proteases. The ability of the protease inhibitors to reduce the damage caused by these drugs suggests that the treatment arsenal could include similar drugs as possible tools to combat the drug-induced neurotoxicity in vivo.

Amino Acid Chloromethyl Ketones↗
PubMed1976

Erythrocyte spectrin. Purification in deoxycholate and preliminary characterization.

Erythrocyte spectrin, isolated by aqueous extraction of erythrocyte ghosts, may be freed from contaminating membrane lipids and small amounts of other proteins by gel chromatography in 5 or 10 mM deoxycholate. The purified protein, in deoxycholate, is a mixture of monomers and dimers, both highly asymmetric molecules. The hydrodynamic properties of the dimer closely resemble those of muscle myosin, and spectrin and myosin also have similar circular dichroism spectra. The proportion of dimer to monomer in the purified protein varies from one preparation to another, an observation for which there is no simple explanation. In the absence of deoxycholate, spectrin associated beyond the dimer stage, possibly by loose end-to-end aggregation involving hydrophobic forces.

Binding Sites↗
PubMed1975

The isolation of aggregates of spectrin from bovine erythrocyte membranes.

Aggregated states of spectrin from bovine erythrocyte membranes can be detected in sedimentation velocity experiments. These aggregates have been isolated by means of gel filtration on columns of 4% agarose. They appear to be stable over a wide range of pH and ionic strength, although they are dissociated by sodium dodecyl sulphate. Sedimentation equilibrium measurements yielded values of 960 000 and 480 000 for the molecular weights of the major aggregates, corresponding to a tetramer and dimer, respectively. The presence of different aggregated states in spectrin preparations may explain the wide variation in the reported physical properties of spectrin.

Animals↗
PubMed1992

A new member of the spectrin superfamily may participate in the formation of embryonic muscle attachments in Drosophila.

Myotube migration and the formation of muscle attachments are crucial events for the proper development of muscle patterning in the Drosophila embryo. This paper describes the identification of a new embryonic muscle-specific protein, MSP-300, in Drosophila. This protein is initially expressed by muscle precursors at muscle-ectoderm and muscle-muscle attachment sites. As development continues, MSP-300 becomes associated with muscle myofibrillar network. Studies of the subcellular localization of this muscle-specific protein in primary embryonic cultured myotubes show that MSP-300 decorates actin filaments, and that it is specifically enriched in sites where actin microfilaments are linked to the plasma membrane. Migrating myotubes exhibit high levels of this protein at their leading edge while, in myotubes that have already developed sarcomeric architecture, the protein is localized mainly at the Z-discs. Sequence of a partial 3.9 kb cDNA clone and molecular analysis of the predicted protein sequence of this protein indicates that it encodes a high relative molecular mass protein (approximately 300 x 10(3), which exhibits at least five spectrin-like repeats. Several properties are shared by MSP-300 and members of the spectrin superfamily: it is associated with actin microfilaments, its sequence exhibits spectrin-like repeats and it is localized at sites where actin is linked to the plasma membrane. This protein could have a developmental role in the formation of muscle-ectoderm attachments and may be involved in myotube migration on the ectoderm.

Amino Acid Sequence↗
PubMed2000

Human spectrin Src homology 3 domain binding protein 1 regulates macropinocytosis in NIH 3T3 cells.

Macropinocytosis is an endocytic process that occurs through non-clathrin coated vesicles larger than 0.2 microm in diameter. Although macropinocytic vesicles are readily visualized in cultured cells by the introduction of fluorescent, water-soluble dyes into the culture medium, protein markers associated with this type of vesicles have not yet been well defined. Here, we report that human spectrin SH3 domain binding protein 1, or Hssh3bp1, associates with macropinosomes in NIH 3T3 fibroblasts. Hssh3bp1 macropinosomes are heterogeneous in morphology and size, do not endocytose transferrin and are resistant to brefeldin A treatment. Cytochalasin D, and wortmannin block endocytosis of fluorescent dyes into the Hssh3bp1 macropinosomes and dramatically affect their morphology. Overexpression of Hssh3bp1-green fluorescent protein abolished fusion of vesicles resulting in a decreased endocytosis of fluorescence dyes, thus suggesting a potential regulatory role of Hssh3bp1 in macropinocytosis. In the macropinosomes of NIH 3T3 cells, Hssh3bp1 associates with a 200-kDa protein that crossreacts with a monoclonal antibody to the erythroid alpha-spectrin SH3 domain. Thus macropinosomes in cells may contain a spectrin-like protein.

3T3 Cells↗
PubMed1998

Identification of elf1, a beta-spectrin, in early mouse liver development.

Beta-spectrins play essential roles in cell-cell interactions and in the maintenance of cell polarity. Our aim was to identify beta-spectrin genes important for the establishment of hepatocyte polarity and differentiation. Using subtractive screening of cDNA libraries from early embryonic mouse livers (post-coital days 10, 11, and 12), we have isolated elf1 (embryonic liver fodrin 1), a differentially expressed beta-spectrin or fodrin (betaSpIIsigmaI). Elf1 encodes a 220-amino acid protein with an NH2 terminal actin-binding domain. In situ hybridization studies demonstrate elf1 expression initially in day 10 embryonic heart tissue, then in day 11-11.5 hepatic tissue. These studies suggest that elf1 may play a role in the emergence of hepatocyte polarity during liver development.

Amino Acid Sequence↗
PubMed1997

Genetic basis of the polymorphisms of the alphaI domain of spectrin.

Defects of alpha spectrin have been identified in many cases of hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP). To aid in the genetic analysis of families with these disorders, the locations of three alpha-spectrin gene polymorphisms were mapped, the genetic basis of these polymorphisms identified, and PCR-based assays designed for their identification. The frequencies of these polymorphisms were determined in two populations and in patients with alphaI/50a HE and HPP. These studies identified two distinct haplotypes and provided evidence that two HE/HPP mutations associated with the alphaI/50a protein phenotype, L207P and L260P, arose on separate chromosomal backgrounds.

Alleles↗
PubMed2001

Spectrin: the ghost in the machine.

It has long been speculated that spectrin, the actin crosslinking and molecular scaffold protein, is involved in the development of apicobasal polarity in epithelia. While spectrins can undoubtedly influence the protein content of specific membrane domains, recent genetic evidence indicates that this activity is not necessary for the establishment or maintenance of this axis. Instead, these studies point to critical roles in tissue stability and morphogenesis. A possible role in cellular contractility is highlighted in this review.

Animals↗
PubMed1988

Impaired echinocytic transformation of ankyrin- and spectrin-deficient erythrocytes in mice.

The membrane skeleton of the red blood cell plays an important role in the determination of cell deformability and cell shape. Under various in vitro conditions, red blood cells undergo an echinocytic or stomatocytic shape transformation. The mechanism of this fundamental process is not well understood. We have studied the red cell shape transformation in normoblastic anemia mice (nb/nb) and spherocytic anemia mice (sph/sph), which are deficient in ankyrin and spectrin, respectively. We found that both ankyrin-deficient cells (nb/nb) and spectrin-deficient cells (sph/sph) have a reduced capacity to undergo echinocytic transformation with various echinocytogenic treatments, that is, incubation with sodium salicylate (40 and 120 mM), calcium loading (50 microM A23187 + 2.2 mM Ca2+), or metabolic depletion (24 hr at 37 degrees C). These results suggest that the functional integrity of the membrane skeleton is essential for the maintenance and transformation of the red cell shape.

Anemia↗